Dra. Laura Silva Rosales

Profesor Investigador

CINVESTAV U. Irapuato

A. Postal 629

36821 Irapuato, GTO. México.

Raleigh, NC Marzo 1, 2009

Apreciable Dra. Silva Rosales:

Antes que nada le envío un saludo deseando se encuentre bien y esperando que siga cosechando logros académicos.

El motivo de la presente misiva es participarle mi intención de aplicar para una de las posiciones abiertas en CINVESTAV para Profesor-Investigador.

Tengo interés en regresar a México luego de realizar mis estudios de Doctorado y posición Postdoctoral en USA. CINVESTAV es uno de los pocos lugares en México que ofrece investigación de vanguardia y provee de las facilidades para realizarla. Por ese motivo y por su posición geográfica es el lugar ideal para mi en cuanto a investigación se refiere.

La experiencia que he acumulado durante mi trabajo y estudios me coloca en una posición privilegiada para realizar investigación en su Institución. Poseo una fuerte experiencia en Biología y Bioquímica y he trabajado intensamente con la interacción planta-patógeno. Inicialmente con bacterias infectando frijol, más adelante con viroides en tomate, virus de RNA en papa, tabaco, tomatillo, chile y cucurbitáceas y más recientemente con geminivirus infectando solanáceas y arabidopsis.

He trabajado en epidemiología, reportando por primera vez un devastador virus en México, así como he trabajado en la evolución de los virus y su adaptación a hospederos.

Para mis estudios de doctorado, estudié la respuesta de arabidopsis a la infección por begomovirus, cerrando con esto un hueco que tenía en cuanto al conocimiento de virología de campo comparado con biología celular de la infección viral. Mis estudios se han centrado en cómo los geminivirus afectan el ciclo celular del hospedero, encontrando varios genes involucrados en el proceso que aún necesitan ser estudiados en más detalle.

Tengo la certeza de que mis conocimientos, combinados con las facilidades que ofrece el CINVESTAV, así como sus estudiantes y la planta de profesores me permitirán desarrollar un programa de investigación de excelencia.

Agradezco de antemano la atención que preste a la presente y me permito ponerme a su disposición si requiere de más información.

Atte

Dr. José Trinidad Ascencio Ibáñez

LOGROS ACADEMICOS José T. Ascencio-Ibáñez, PhD

PRESENTACION Estudié la carrera de Biología (UAG, Guadalajara) porque tengo una fascinación por entender la vida. Al terminar mi carrera, realicé mi Servicio Social en el Centro Regional de Estudios de Roedores, Aves y Moluscos (CRERAM) en Zamora, Michoacán. Durante mi estancia en Zamora dí clases en la Normal Primaria, la Normal Superior y la preparatoria. De ahí proseguí a hacer mi tesis de licenciatura en mutantes Tn5 de Pseudomonas syringae pv phaseolicola en CINVESTAV, Irapuato, bajo la dirección de los Dres. Ariel Alvarez Morales y Ana María Bailey Moreno. Al término de los trabajos experimentales, recibí una invitación del Dr. Rafael Rivera Bustamante para trabajar con él en el laboratorio de virología en el CINVESTAV, Irapuato. Trabajé con el Dr. Rivera por una década, durante la cual se establecieron sistemas de transformación de variedades mexicanas de papa que fueron llevados hasta la realización de pruebas de campo (de las primeras en México). Para mi preparación, pasé un año en la compañía Monsanto (Saint Louis Missouri) en sus laboratorios de Chesterfield. Además del trabajo en resistencia a virus en papa, trabajé en el diagnóstico y caracterización molecular de geminivirus. Durante mi tiempo en Cinvestav, una de mis actividades favoritas fué dar cursos. Tuve la oportunidad de participar en muchos de ellos. Algunos como instructor y en otros como co-instructor. Entre los que considero más interesantes están los impartidos en varias Universidades Agrícolas, titulados Introducción a la Ingeniería Genética de Plantas. También participé como instructor en los cursos internacionales que se imparten cada dos años en Cinvestav. Luego de un intento fallido de proseguir mis estudios de postgrado (en 1994), donde la crisis me impidió iniciar mi doctorado en Cinvestav, Irapuato, tuve la oportunidad de salir del país y hacer mi doctorado en la North Carolina State University en el Departamento de Bioquímica Molecular y Estructural bajo la dirección de la Dra. Linda Hanley-Bowdoin, quien dirige uno de los laboratorios más prestigiosos del departamento. Me becaron durante todo el doctorado y me gané un premio al mejor Asistente de Profesor por mi desempeño bajo la tutela del Dr. Dennis Brown, el jefe del departamento. Con tal fortuna que cuando se requirió me solicitaron como Profesor para el curso en el que fuí su asistente (BCH452), el cual impartí durante 2008. Mi postdoctorado ha consistido en la elucidación de las diferencias respecto al ciclo celular entre dos geminivirus que infectan arabidopsis utilizando una batería de herramientas genómicas, moleculares y bioquímicas.

PROPUESTA DE INVESTIGACION PARA CINVESTAV José Trinidad Ascencio Ibáñez, PhD

PROPUESTA

Durante mis estudios de doctorado trabajé con un sistema de microarreglos para detectar cambios en Arabidopsis durante la infección con begomovirus (Cabbagge leaf

curl virus, CaLCuV). Primeramente buscamos genes interesantes y rutas metabólicas que hubieran sido afectados durante la infección. Descubrimos que tanto la ruta del ácido acetil salicílico como la del etileno son reguladas a la alza, mientras que la ruta del metil-jasmonato es claramente reprimida. De ahí proseguimos para entender major cómo es que genes involucrados en senescencia son inducidos por el virus. También exploramos genes involucrados en la replicación del DNA y en mecanismos de reparación de DNA. Finalmente, utilizando líneas sobre-expresoras y mutantes mostramos que el CaLCuV induce a las células a proseguir con un endociclo activamente, mientras reprime la transición mitótica afectando el nodo Cyclinas D-Retinoblastoma -E2F (Ascencio-Ibáñez et al, 2008).

Este trabajo me ha dado mucha información de la bioquímica de la planta y ha incrementado mi conocimiento sobre cómo el virus la altera. De esta experiencia propongo las bases para realizar investigación en Arabidopsis infectada con geminivirus.

Luego de mucha consideracion, hay tres avenidas que propongo sean estudiadas. Estudios de meristemos, dianas de miRNAs y los genes de kinasas dependientes de ciclinas que no han sido caracterizados (CKLs). Meristemo apical

No se sabe con precision cómo es que los geminivirus afectan el meristemo de arabidopsis, dado que no lo alcanzan (Ascencio-Ibáñez, datos no publicados). Solo podemos observar que la productividad es reducida cuando la planta es infectada pero no cómo el virus cambia la naturaleza de los meristemos de una forma que ya no producen más hojas o tejido floral. Los virus normalmente no alcanzan a las células meristemáticas, luego debe haber entonces un bloqueo metabólico o una molécula viajera que le impide al meristemo funcionar apropiadamente.

Algunos genes involucrados en el mantenimiento de meristemo manifestaron alteraciones en la infección geminiviral (CLV1, CLV2, ARR7, entre otros), sugiriendo que los geminivirus directa o indirectamente afectan la expresión de estos genes. Sin embargo, dado que los datos generados fueron obtenidos de muestras de hojas maduras, es interesante averiguar si dichos genes están afectados en el meristemo en sí.

La estrategia posee varios niveles y estoy proponiendo esto como uno de las posibilidades para solicitar apoyo a Conacyt u otra agencia de financiamiento.

Objetivos específicos son: a) Elucidar el patrón de expression de genes seleccionados, entre ellos

CLV1, CLV2 y ARR7 en meristemos apicales de Arabidopsis y, de ser factible, otras plantas infectadas con geminivirus.

b) Detección y localización de los cognados proteicos de estos factores meristemáticos.

c) Determinar la interaccion entre proteínas involucradas en la función del meristemo y proteínas virales o proteínas inducidas por la infección viral.

Datos preliminares ya han sido producidos para ARR7 (Arabidopsis response regulator 7), una proteína que es reprimida por CaLCuV, mientras que otros 5 virus de RNA la inducen. Se utilizarán fusiones ARR7-GFP (ya obtenidas) para estudiar el proceso en el meristemo.

Dianas de miRNAs Otra área sumamente interesante es la que involucra a los miRNAs. El CaLCuV

aparenta ser dependiente de algunos de los genes que está involucrados en el procesamiento de los miRNAs (AGO1, DCL1 y DCL2, particularmente, Ascencio-Ibañez y Hanley-Bowdoin, datos no publicados). Muchos de los genes controlados por miRNAs son afectados en la infección, lo que sugiere una intervención del virus en el control de las dianas de miRNAs para su beneficio. Esto va un poco contracorriente de la literatura en donde se plantea que los geminivirus simplemente actúan como anti-silenciadores, no como promotores de RNAs pequeños.

Particularmente genes involucrados en la respuesta a auxinas son inducidos o reprimidos por los geminivirus, tengo interés en determinar si es a través de los miRNAs o es otro el mecanismo que altera la expression de éstas proteínas. CKLs, (CDK-like) Este grupo de genes incluye 15 miembros. Ninguno de ellos ha sido estudiado con detenimiento. Manifiestan diferencias en su expression temporal e histológica, lo que sugiere que están involucrados en diferentes procesos en el ciclo celular. Dos de ellos (CKL5 y CKL6) son altamente inducidos durante la infección con geminivirus en hoja madura de arabidopsis. Esto nos permitirá iniciar los estudios del grupo. Objetivos específicos:

a) Determinación de los promotores que gobiernan CKL5 y CKL6 y clonaje de vectores de expression con dichos promotores para la producción de plantas transgénicas con genes marcadores.

b) Producción de plantas trasngénicas con promotores de los genes CKL5 y CKL6 y determinación de sus patrones de expression en diferentes condiciones de crecimiento, desarrollo y estrés.

c) Obtención de proteína purificada de CKL5 y CKL6 para su utilización en la producción de anticuerpos y en ensayos de fosforilación.

Otras posibilidades que me interesa explotar es la investigación de las proteínas que responden al virus mediante un análisis proteómico. Utilizando geles de dos dimensiones e inmunoprecipitaciones con proteínas virales pretendo identificar proteínas que están involucradas en la replicación y la respuesta al virus. El Cinvestav posee toda la infraestructura para llevar a cabo estos procesos exitosamente. Mis aportaciones a los estudios geminivirales seguramente fortalecerán y complementarán los del grupo del Dr. Rivera Bustamante. El interés, materiales y técnicas que he acumulado me permiten proponer sobre todo un enfoque desde el punto de vista del ciclo celular, que es un modelo muy interesante que puede expandirse a las solanáceas y otras plantas infectadas por geminivirus y que tienen sus genomas en proceso de ser secuenciados. Estoy seguro que si soy elegido para su Instituto, mi aportación será excelente tanto en investigación como en docencia. Atte Trino Ascencio Ibáñez, PhD

REFERENCES

1) Dr. Linda Hanley-Bowdoin, linda_hanley-bowdoin@ncsu.edu North Carolina State University, Professor of Biochemistry and Genetics

2) Dr. Niki Robertson, niki_robertson@ncsu.edu

North Carolina State University, Professor of Plant Biology

3) Dr. Dennis Brown, dennis_brown@ncsu.edu North Carolina State University, Head of Molecular and Structural Biochemistry Department

4) Dr. Gerardo Arguello-Astorga, grarguel@ipicyt.edu.mx

IPICyT (Instituto Potosino de Investigación Científica y Tecnológica), San Luis Potosí, Mexico. Profesor-Investigador

5) Dr. Luis Herrera-Estrella, lherrera@ira.cinvestav.mx

LANGEBIO-CINVESTAV, Irapuato, Mexico. Director.

JOSE TRINIDAD ASCENCIO-IBAÑEZ

CONTACT INFORMATION

José Trinidad Ascencio-Ibáñez (Trino Ascencio)

Viral Genomics Group. North Carolina State University. Campus Box 7651, Partners III, Rm 804. Raleigh NC 27695.

Phone: (919) 5155736 Fax: (919) 5131209

Cell: (919) 3323226

Email: trino_ascencio@ncsu.edu

Alternative e-mail: trinogen@gmail.com

EDUCATION

2001-2006 North Carolina State University, Raleigh NC. PhD on Biochemistry. 1981–1985 Universidad Autónoma de Guadalajara, Guadalajara, Jalisco. Mexico. Biology BSc. Degree

EXPERIENCE January 2008 to date. Lecturer. North Carolina State University, Molecular and Structural Biochemistry Department, Viral Genomics Group.

2006-2007: Postdoctoral Researcher. Viral Genomics Group. North Carolina State University. 2001-2006: North Carolina State University. Biochemistry Department. Teaching Assistant. Geminivirus Research, Microarrays (DNA Chips). Plant-pathogen interactions. Plant Cell Cycle. PhD in Biochemistry. Protein purification Southern and Northern blots Western blots ELISA Immunohistochemistry RT-PCR and qPCR Microarray hybridizations.

Microarray data analyisis. Affymetrix chips and sofware GeneSpring data analysis miRNA detection (targets and miRNAs) Silencing Viral inoculations Arabidopsis maintenance and analysis FACS nuclei analysis Confocal microscopy FPLC

1989-2006 CINVESTAV (Center for Research and Advanced Studies, Plant Genetic Engineering Department) Irapuato, Mexico Research Assistant (Category I, highest possible). On leave from 2001 to 2006. Field trials of virus resistant transgenic potato lines (resistant to

PVX, PVY and PLRV). Plant Virus Maintenance (Inoculation and Purification). Plant Virus Monitoring by ELISA and DAS-ELISA Procedures. DNA bombardment (Gene gun) on pepper and tobacco plants

for plant DNA virus inoculation. Epidemiology for RNA and DNA plant viruses. Viral DNA Purifications. Plant DNA Purifications. Molecular Biology standard protocols RNA and DNA Virus interactions assays (In planta) Scientific Photography Course, Instructor. Polymerase Chain Reaction Course. Instructor. Greenhouse and Growth Chambers plant assays. Arabidopsis inoculation and maintenance. Molecular Biology Techniques: Dot Blot, Southern Blot, Squash

Blot, Gel electrophoresis (DNA and protein) etc. Cloning. Lab Managing and research duties. Instructor for the International Course in Plant Molecular

Biology, held every 2 years at Cinvestav, Irapuato. 1991-1992 MONSANTO. Saint Louis, MO. 1 year of training on

molecular virology to obtain transgenic potato plants resistant to viruses. Included training on transgenic plant field trials. Brokered by ISAAA and supported by Rockefeller Foundation.

1987-1989 CINVESTAV (Center for Research and Advanced Studies, Plant Genetic Engineering Department) Irapuato, Mexico BSc Thesis work. Tn5 Mutants of Pseudomonas syringae pv phaseolicola with Pathogenicity deficiencies.

TEACHING EXPERIENCE

2008 Lecturer for BCH452 in the Biochemistry Department, NCSU. 2003 TA for BCH452 (Biochemistry lab) 1987-1989 Instituto Irapuato. Irapuato, GTO. Mexico. Biology and Human Physiology Professor. High School level. 1985-1987 Centro Escolar Juana de Asbaje. Zamora,

Michoacán. México. Biochemistry and Earth Sciences Professor at BSc. level and Human Physiology, at High School level. Undergraduate Training:

2008 Diana Vu Nguyen

2008 Yaditza Narvaez

2006-2008 Krystina Geiger

2007 Amber Smith

2004-2007 Jon Belton

1998-1999 Mariana Franco, director of BSc thesis entitled: “Proximal analysis of transgenic potato mexican varieties (Alpha, Rosita and Norteña) with resistance to PVX and PVY”. Cum Laude.

1998 Adrian Solis

High School Training

2008 Jennifer Li and Anand Komepati

1999-2000 Eva Cortes and Victoria N. Romero, they pursued Biology

and finished their college degrees. Nancy is pursuing a MSc program in Experimental Biology.

DISTINCTIONS

2005. Award for best poster to undergrad student Jon Belton, tutored by JT Ascencio-Ibáñez and L. Hanley-Bowdoin during the 4th Annual NC State University Undergraduate Research Summer Program. Immunolocalization of AL1, Geminivirus Replication Factor, in Arabidopsis Plants Infected with Cabbage Leaf Curl Virus 2003. Samuel Towe Award for best Teaching Assistant.

2002 to date. Associate Editor for the Mexican Journal of Phytopathology.

2001. Teaching Assistanship from North Carolina State University, to study PhD on the Department of Molecular and Structural Biochemistry.

1991-1992. Rockefeller funded project “PVX and PVY resistant potatoes”. Transfer of Technology brokered by ISAAA. 1 year of experience at Monsanto, Saint Louis, Missouri. 1988. Best professor of Biology. Instituto Irapuato.

RECENT INVITED TALKS

2008 Geminivirus infection in Arabidopsis alters expression of cell cycle genes and promotes endocycling. American Society of Plant Biologists Annual Meeting, Merida Yucatan, Mexico. 2007 The pathogen response to Cabbage leaf curl virus in Arabidopsis. 5th International Geminivirus Symposium and 3th International ssDNA Comparative Virology Workshop. Ouro Preto, Brasil.

PUBLICATIONS AND ABSTRACTS

Ascencio-Ibañez, José Trinidad, Rosangela Sozzani, Tae-Jin Lee, Tzu-Ming Chu, Russell D. Wolfinger, Rino Cella and Linda Hanley-Bowdoin. 2008. Global analysis of Arabidopsis gene expression uncovers a complex array of changes impacting the pathogen response and cell cycle controls during geminivirus infection. Plant Physiology. July 23, 2008; 10.1104/pp.108.121038 Gerardo Arguello-Astorga, J. Trinidad Ascencio-Ibáñez, Beverly M. Orozco, Linda Hanley-Bowdoin. 2007. High frequency reversion of geminivirus replication protein mutants during infection. Journal of Virology 81:29, 11005-11015. Ascencio-Ibáñez, JT and Settlage, S. 2007. DNA abrasion onto plants is an effective method for geminivirus infection and virus-induced gene silencing. Journal of Virological Methods. 142, 198-203. Ascencio-Ibáñez, JT. 2006. Transcriptional profiling of geminivirus infection in Arabidopsis thaliana Col-0. PhD Thesis. Department of Molecular and Structural Biochemistry, North Carolina State University. L. Pérez-Moreno, E. Rico-Jaramillo, R. Ramírez-Malagón y J.R. Sánchez-Pale. J.T. Ascencio Ibáñez y R.F. Rivera-Bustamente. 2004. Identification of chile pepper pytopathogenic viruses in Guanajuato state, Mexico. First World Pepper Convention, Mexico. Ramos, P. L., Guevara-Gonzalez, R. G., Peral, R., Ascencio-Ibañez, J. T., Polston, J., Argüello-Astorga, G. R., Vega-Arreguín, J. C., and Rivera-Bustamante, R. F. 2003. Tomato mottle Taino virus pseudorecombines with PYMV but not with ToMoV: Implications for the delimitation of cis- and trans-acting replication specificity determinants. Archives of Virology 148:1697-1712 Ascencio-Ibanez, J. T., Arguello-Astorga, G. R. Mendez-Lozano, J. and Rivera-Bustamante, R. F. (2002) First report of Rhynchosia golden mosaic virus (RhGMV) infecting tobacco in Chiapas, Mexico. Plant Disease 86: 692. Ramos, P. L., Fernandez, A., Castrillo, G., Diaz, L., Echemendia, A. L., Fuentes, A., Peral, R., Pujol, M., Ascencio-Ibanez, J. T., and Rivera-Bustamante, R. F. (2002) Macroptilium yellow mosaic virus, a new begomovirus infecting Macroptilium lathyroides in Cuba. Plant Disease 86: 1049 Potyvirus detection in garlic in Guanajuato State. Detección de virus del

grupo potyvirus en el cultivo de ajo en el estado de Guanajuato, México. 2002. Luis Pérez-Moreno, Esteban Rico-Jaramillo, Jesús R. Sánchez-Pale, Rafael Ramírez-Malagón y José T. Ascencio Ibáñez. Mexican Phytopatology Meeting. De la Torre-Almaraz, R., Valverde, R. Méndez-Lozano, J., Ascencio-Ibáñez, JT and Rivera-Bustamante, R. Preliminary Characterization of a Geminivirus in Tomatillo (Physalis ixocarpa) in the Central Region of México. 2002. Agrociencia, Vol 32:004, 471-481. Díaz-Plaza, R., Ascencio-Ibáñez, J.T., Monsalve-Fonnegra, Z.I., Avilés-Baeza, W., Santamaría-Basulto, F., Peña-Ramírez, R., Méndez-Lozano, J. y Rivera-Bustamante, R.F. 2000. TYLCV distribution in the Yucatán Peninsula. XXVII Congress of Mexican Society of Phytopathology. Puerto Vallarta, JAL. Mexico. Méndez-Lozano, J., Ascencio-Ibáñez, J.T., Franco-Ruiz, M., Mexicano-Ojeda, M. y Rivera-Bustamante, R.F. Sinergic interactiion between PVX and PVY interfere with the movement of Pepper Huasteco Yellow Vein geminivirus. 2000. XXVII Congress of Mexican Society of Phytopathology. Puerto Vallarta, JAL. Mexico. Ascencio-Ibáñez, J.T., Pruna-Camacho, M.B. y Rivera-Bustamante, R.F. 2000. Método rápido para detectar la presencia de geminivirus en plantas infectadas: extracción a pequeña escala (Quick Method to Detect Geminiviruses in Infected Plants: Small Scale Purification). XXVII Congress of Mexican Society of Phytopathology. Puerto Vallarta, JAL. Mexico. Pruna Camacho, MB and Ascencio Ibáñez, JT. 2000. Quick Method for DNA purification from sotf tissue. IX State Health Research Council . Guanajuato, Mexico. Ramos González, PL, Arguello-Astorga, GR, Ascencio-Ibáñez, JT y Rivera Bustamante RF. 1999. Replicative complementation analysis among geminiviruses with identical and different iterons. Análisis de Complementación Replicativa entre Geminivirus con Iterones Diferentes o Idénticos. Taller Internacional de Biotecnología Vegetal. Ciego de Avila, Cuba. Ascencio-Ibañez, J. T., Monsalve-Fonnegra, Z. I., Pruna-Camacho, M. B., Díaz-Plaza, R. & Rivera-Bustamante , R. F. (1999). Los Geminivirus (The Geminiviruses). Revista Mexicana de Fitopatología (Mexican Journal of Phytopathology) 17, 113-127. Ascencio-Ibañez, J. T., Díaz-Plaza, R., Méndez-Lozano, J., Monsalve-Fonnegra, Z. I., Argüello-Astorga, G. R. & Rivera-Bustamante, R. F. (1999). First report of tomato yellow leaf curl geminivirus in Yucatán, México. Plant Disease 83: 1178. G.M. Bonilla-Ramírez, R.G. Guevara-González, J.A. Garzón-Tiznado,

J.T. Ascencio-Ibáñez, I. Torres-Pacheco and R.F. Rivera-Bustamante. 1997. “Analysis of the infectivity of monomeric clones of pepper huasteco virus”. Journal of General Virology. 78:947-951. Juan Pablo Martínez-Soriano, Rafael Rivera-Bustamante, Diana Leal-Klevezas and J. Trinidad Ascencio-Ibáñez. “The Polymerase Chain Reaction and Plant Pathogen Detection”. In “International Course on Plant Biotechnology”. Cinvestav, IPN. U. Irapuato. 1997. Alba Jofre y Garfias, Luis Herrera-Estrella, Rafael Rivera-Bustamante, Jose Luis Cabrera-Ponce and J. Trinidad Ascencio-Ibáñez. “Plant Genetic Transformation by the Biolistic Process”. In “International Course on Plant Biotechnology”. Cinvestav, IPN. U. Irapuato. 1997. Becerra-Flora, A., Murillo-Chávez, A., Garzón-Tiznado., J. A., Ascencio-Ibañez, J. T., Peña-Ramírez, R. y Rivera-Bustamante, R. 1996. "Variantes del Virus Huasteco del chile (PHV).". Memorias del XXIII Cong Nal Soc Mex Fitopatología, A. C. y XXXVI Annual Meeting Am Phy Soc Caribb. D. Ascencio-Ibañez, JT., A. Becerra-Flora, R. Peña-Ramírez, V. B. Nikoleava y R. Rivera-Bustamante. 1995. "Detección y Caracterización molecular de geminivirus que infectan tabaco (Nicotiana tabacum) en Chiapas.". Memorias del XXII Congreso Nacional de Fitopatología. Garzón-Tiznado, J. Antonio, Torres-Pacheco Irineo, Ascencio-Ibáñez J. Trinidad, Herrera-Estrella Luis and Rivera-Bustamante Rafael. 1993. “Inoculation of Peppers with Infectious Clones of a New Geminivirus by a Biolistic Procedure”. Phytopathology 83:514-521. Rafael Rivera, Laura Silva, Rafael Gutiérrez, J. A. Garzón, Irineo Torres, Ramón Guevara, Manuel Bonilla, Guido van der Broek, Patricio Arce, Margarita Rafael, Roberto Ruiz, J. Trinidad Ascencio-Ibáñez, Rosa Ma. Rangel, Alicia Becerra, Lourdes Rojas y Carmen Man "La Ingeniería de Plantas y la Resistencia Antiviral."1991. Sociedad Mexicana de Fitogenética, A. C. (SOMEFI). Arce Johnson Patricio, Ascencio Ibáñez José Trinidad y Rafael Rivera Bustamante. 1991. “Plant Virus Control”, in Introducción a la Biología Molecular e Ingeniería Genética de Plantas. (Introduction to Plant Molecular Biology and Plant Genetic Engineering). Edited by Rivera-Bustamante et al. Celaya, Guanajuato.

1625 Roanoke St * RALEIGH, NC 27606 * PHONE (919) 8517407 CELL (919) 3323226

E-MAIL: trinogen@gmail.com, trino_ascencio@ncsu.edu

JOURNAL OF VIROLOGY, Oct. 2007, p. 11005–11015 Vol. 81, No. 200022-538X/07/$08.00�0 doi:10.1128/JVI.00925-07Copyright © 2007, American Society for Microbiology. All Rights Reserved.

High-Frequency Reversion of Geminivirus Replication ProteinMutants during Infection�

Gerardo Arguello-Astorga,† J. Trinidad Ascencio-Ibanez, Mary Beth Dallas,Beverly M. Orozco,‡ and Linda Hanley-Bowdoin*

Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina 27695-7622

Received 30 April 2007/Accepted 24 July 2007

The geminivirus replication protein AL1 interacts with retinoblastoma-related protein (RBR), a key regu-lator of the plant division cell cycle, to induce conditions permissive for viral DNA replication. Previous studiesof tomato golden mosaic virus (TGMV) AL1 showed that amino acid L148 in the conserved helix 4 motif iscritical for RBR binding. In this work, we examined the effect of an L148V mutation on TGMV replication intobacco cells and during infection of Nicotiana benthamiana plants. The L148V mutant replicated 100 times lessefficiently than wild-type TGMV in protoplasts but produced severe symptoms that were delayed compared tothose of wild-type infection in plants. Analysis of progeny viruses revealed that the L148V mutation revertedat 100% frequency in planta to methionine, leucine, isoleucine, or a second-site mutation depending on thevaline codon in the initial DNA sequence. Similar results were seen with another geminivirus, cabbage leaf curlvirus (CaLCuV), carrying an L145A mutation in the equivalent residue. Valine was the predominant aminoacid recovered from N. benthamiana plants inoculated with the CaLCuV L145A mutant, while threonine was themajor residue in Arabidopsis thaliana plants. Together, these data demonstrated that there is strong selectionfor reversion of the TGMV L148V and CaLCuV L145A mutations but that the nature of the selected revertantsis influenced by both the viral background and host components. These data also suggested that high mutationrates contribute to the rapid evolution of geminivirus genomes in plants.

Geminiviruses constitute the largest and most diverse andeconomically important family of plant DNA viruses (56).They infect a broad range of plants and cause devastating cropdiseases, particularly in tropical and subtropical regions of theworld (36, 39, 41). Geminiviruses are characterized by twinicosahedral capsids and small, single-stranded DNA (ssDNA)genomes (56) that display high levels of genetic variability (62).Several studies have indicated that recombination contributesto geminivirus diversity (48, 60, 71). However, unlike double-stranded DNA (dsDNA) viruses, there is also mounting evi-dence that ssDNA viruses are subject to high nucleotide mu-tation rates similar to the levels reported previously for RNAviruses (34, 35, 53). Thus, geminiviruses represent a uniqueopportunity to examine the processes that contribute to thegenetic variation of ssDNA viruses as well as the mechanismsunderlying virus evolution in plants.

The family Geminiviridae is classified into four genera, Be-gomovirus, Curtovirus, Topocuvirus, and Mastrevirus, based ontheir genome organization, host range, and insect vectors (19).The largest genus corresponds to the begomoviruses, whichhave one- or two-genome components (designated DNA-Aand DNA-B), infect dicots, and are transmitted by Bemisiatabaci. Over the past 20 years, there has been a significant

increase in the frequency and severity of begomovirus diseases.During this time, agricultural intensification and changes in theinsect vector facilitated the expansion of begomovirus popula-tions and their movement into new plant hosts and contributedto the emergence of new, more virulent viruses. Sequenceanalysis of emerging viruses implicated recombination and re-assortment in begomovirus evolution. Both processes dependon the formation of mixed infections and the presence ofmultiple viral genome components in a single plant cell (54).Recombinant begomoviruses have been associated with se-vere epidemics in cassava, cotton, and tomato (22, 23, 27, 40,43, 60, 71) and divergence of the viruses indigenous to theIndian subcontinent (58). Reassortment is a contributingfactor to cassava mosaic disease (50), and there are exam-ples of monopartite begomoviruses acquiring DNA-B com-ponents (61). In addition, many begomoviruses are associ-ated with DNA satellites that increase virulence and alterhost range (7, 37). The satellite DNAs can recombine withthemselves and viral genome components (1), further in-creasing variability.

Nucleotide misincorporation during viral DNA replicationalso contributes to genome diversity. Studies of bacterial andanimal systems indicated that the mutation rates of dsDNAand ssDNA viruses differ significantly. The mutation rates fordsDNA phages range from 10�7 to 10�8, while ssDNA phagesdisplay rates of approximately 10�6 (15, 53). Like dsDNAphages, polyomavirus and papillomavirus genomes display lowmutation rates (10�8 to 10�9), similarly to their hosts (26). Incontrast, high mutation rates (ca. 10�4) have been reported forparvoviruses (35, 64, 65) and circoviruses (6, 21). Like gemi-niviruses, these viruses have ssDNA genomes that replicate viarolling-circle mechanisms. Thus, the high levels of sequence

* Corresponding author. Mailing address: Department of Molecularand Structural Biochemistry, North Carolina State University, Raleigh,NC 27695-7622. Phone: (919) 515-6663. Fax: (919) 515-2047. E-mail:linda_hanley-bowdoin@ncsu.edu.

† Present address: Division de Biologıa Molecular, Instituto Poto-sino de Investigaciones Cientıficas y Tecnologicas, 78216 San LuisPotosı, SLP, Mexico.

‡ Present address: Talecris Biotherapeutics, Clayton, NC 27520.� Published ahead of print on 1 August 2007.

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heterogeneity reported for begomoviruses and mastreviruses(9, 24, 28, 44, 59) may reflect replication errors.

Tomato golden mosaic virus (TGMV) and cabbage leaf curlvirus (CaLCuV) are begomoviruses with two-component ge-nomes. Both viruses encode a replication protein designatedAL1 (also named AC1, C1, or Rep), which is required for theinitiation and termination of viral DNA synthesis (20, 32, 45)and acts as a DNA helicase (11, 12). The AL1 protein alsoreprograms mature plant cells to create a permissive environ-ment for viral replication through interactions with the hostretinoblastoma-related protein (RBR), which regulates cell di-vision and differentiation in plants (14, 17, 31). TGMV andCaLCuV AL1 interact with RBR via a unique 11-amino-acidsequence (2, 31). Alanine substitutions across the helix 4 se-quence of TGMV AL1 differentially impacted RBR binding inyeast two-hybrid studies and suggested that residue L148 in themiddle of the motif provides a critical binding contact (2, 31).In the experiments reported here, we examined the impact ofvarious amino acid substitutions at TGMV AL1 L148 and theequivalent CaLCuV AL1 L145 on viral replication and infec-tivity. These studies showed that some mutations reverted at100% frequency during infection and provided evidence forthe capacity of geminivirus populations to evolve rapidly toamend deleterious changes in their genomes.

MATERIALS AND METHODS

AL1 mutants and PCR. The construction of the TGMV AL1 L148A, L148V,L148M, L148G, and L148I mutations was described previously (2). The muta-tions are designated by the wild-type residue and its position number followed bythe mutant amino acid. TGMV AL1 L148V* and E146A L148V were generatedusing pNSB148 (46) and primers 5�-TAATTATCTGaAcGGCTTCTTCTTTGGAAGAAGCATTTAAC and 5�-ATTATCTGCAcGGCcgCTTCTTTGGAAGAAGCATTTAA, respectively (lowercase type indicates mutant nucleotides.).TGMV A replicons encoding the mutant AL1 proteins were generated by sub-cloning SalI/NheI fragments corresponding to AL1 amino acids 120 to 312 fromthe mutagenesis clones into the same sites of the wild-type replicon pMON1565(45) to give pNSB919 (E146A/L148V), pNSB979 (L148V), pNSB997 (L148A),pNSB1000 (L148G), and pNSB1031 (L148V*).

TGMV AL1 mutants were also constructed from variants generated duringinfection with TGMV AL1 L148V, E146A/L148V, or L148V* mutants. TotalDNA from systemically infected, symptomatic leaves was amplified by PCR usingprimers 5�-CGACAAAGACGGAGATACTC and 5�-GTCTCATCTCGTCTGGCACG to give a 281-bp fragment corresponding to TGMV A positions 2006 to2287. The PCR products were digested with SalI/NcoI and subcloned into thesame sites of a modified pBlueScript SK(�) plasmid (Stratagene, Inc.) to gen-erate the intermediate plasmids pNBS1076, pNSB1111, pNSB1077, andpNSB1078. SalI/NheI fragments from these plasmids were then subcloned intothe same sites of pMON1565 to generate replicons carrying the AL1 mutationsL148I (pNSB1082), L148M (pNSB1113), C128W L148V (pNSB1083), andR125G L148V I155L (pNSB1084).

Wild-type CaLCuV replicons pCpCLCV A.003 and pCpCLCV B.003 contain1.6 copies of the A and B genomes, respectively (68). The CaLCuV AL1 muta-tion CaL145A in pNSB1097 (2) was subcloned as an AatII/NsiI fragment (AL1amino acids 132 to 332) into the equivalent sites of pCpCLCVA.003 to generatethe corresponding replicon pNSB1101. Mutant CaLCuV replicons were alsogenerated by PCR of variants produced during infection with the CaLCuVL145A mutant. Total DNA from systemically infected symptomatic leaves wasamplified by PCR using primers 5�-GTGAATCCGGGCAGTACAAGGTGTC-3� and 5�-CCCAGATAAAAACGGAATTCTCTGCC-3 to give an 854-bpfragment between positions 1425 and 2279. The PCR products were digestedwith AatII/EcoRI and subcloned into the same sites of pCpCLCV A.003 toproduce replicons carrying the CaLCuV L145V (pNSB1104), L145A/I167L(pNSB1005), and L145T (pNSB1008) mutations.

Replication and infectivity assays. Transient replication assays were per-formed using protoplasts isolated from Nicotiana tabacum (BY2) suspensioncells, electroporated, and cultured as described previously (20). Cells were trans-fected with 5 �g of wild-type or mutant A component DNA from TGMV or

CaLCuV and 25 �g of sheared salmon sperm DNA. Total DNA was extracted72 h after transfection, digested with either DpnI/XhoI (TGMV) or DpnI/EcoRI(CaLCuV), and examined for double- and single-stranded viral DNA accumu-lation by agarose gel blot analysis using 32P-radiolabeled virus-specific probesagainst A-component DNA. Double-stranded viral DNA was quantified by phos-phorimager analysis. Each replication assay was performed in at least threeindependent experiments.

Nicotiana benthamiana plants were infected by bombardment or agroinocula-tion (16, 42), while Arabidopsis thaliana Col-0 rosettes were infected by agroin-oculation (66). For bombardment, wild-type or mutant replicon DNA (10 �g) foreither TGMV A or CaLCuV A was precipitated onto 1-mm gold microprojec-tiles in the presence of the corresponding wild-type B-replicon DNA. The wild-type TGMV A and B plasmids were pMON1565 (45) and pTG1.4B (20), whilethe wild-type CaLCuV A and B plasmids were pCPCBLCVA.003 andpCPCbLCVB.002 (68). For agroinoculation, Agrobacterium tumefaciens culturescarrying a wild-type CaLCuV A (pNSB1090) or a mutant A replicon were mixedwith a culture carrying a wild-type CaLCuV B replicon (pNSB1091) and syringeinoculated immediately below the plant apex. Total DNA was extracted fromyoung leaf tissue of individual plants at the indicated times after bombardment(13) and linearized with XhoI (TGMV) or EcoRI (CaLCuV). Total DNA (2.5�g/lane) was resolved on 1% agarose –Tris-acetate-EDTA gels, transferred ontonylon, and hybridized with a 32P-radiolabeled probe specific for A-componentDNA.

Total DNA from infected plants was also amplified by PCR using the primersdescribed above, and the AL1 coding region was sequenced directly. The DNAsequencing chromatograms were examined directly to assess the heterogeneity ofthe population sequence at individual nucleotide positions.

RESULTS

Virus replication is differentially affected by substitutions atL148. Previously, we showed that valine and glycine substitu-tions at TGMV AL1 amino acid L148 in the helix 4 motif (Fig.1A) reduce RBR binding activity to 31 and 36% of wild-typebinding in yeast two-hybrid assays (2). To better understandthe role of L148 in AL1 function in planta, we examined theimpact of valine and glycine mutations on TGMV replicationand infectivity assays. We compared the replication of an ala-nine (E146A L148A), a glycine (L148G), and three valine(L148V, L148V* and E146A L148V) mutants to that of awild-type replicon (Fig. 1B). L148V and L148V* encode iden-tical proteins but contain one or two nucleotide changes incodon 148, respectively. E146A L148A and E146A L148V aredouble mutants that also carry alanine substitutions at positionE146. E146A L148V has a single nucleotide change at codon148, like L148V. These mutations were subcloned into the AL1open reading frame of a replicon plasmid carrying a partialtandem copy of TGMV A with two common regions (55). Toensure maintenance of the mutations, they were subclonedinto the unique copy region of the plasmid.

We analyzed the transient replication of wild-type TGMV Aand the mutant replicons in N. tabacum BY-2 protoplasts at72 h posttransfection on agarose gel blots probed with radio-labeled TGMV A DNA. The double-stranded form of theE146A L148A mutant (Fig. 1C, lane 2) accumulated to 12% ofwild-type levels (lane 1), similar to the 13% level reportedpreviously for an L148A mutant (2). The L148G mutant (Fig.1C, lane 3) failed to replicate to detectable levels (Fig. 1C, lane3). All three valine mutants (Fig. 1C, lanes 4 to 6) were se-verely impaired for replication, accumulating to ca. 1% ofwild-type TGMV A DNA levels. The same TGMV DNA ac-cumulation patterns were observed when tobacco protoplastswere cotransfected with a TGMV B replicon and plant expres-sion cassettes corresponding to the mutant AL1 proteins andwild-type AL3 (data not shown).

11006 ARGUELLO-ASTORGA ET AL. J. VIROL.

The L148 valine mutants develop symptoms and accumulateviral DNA at variable times after inoculation. Previous studiesshowed that N. benthamiana plants infected with the TGMVAL1 helix 4 mutants KEE146 and L148A develop mild chlo-rosis along the veins 2 to 3 weeks later than plants inoculatedwith wild-type virus, which showed severe stunting, chlorosis,and leaf curling (2, 31). We asked if the glycine and valinesubstitutions at position L148 also impact symptoms in infec-tivity assays. N. benthamiana plants were cobombarded withwild-type TGMV B replicon DNA and either wild-type ormutant A-component DNA, and symptoms were monitoreduntil the plants flowered and set seed at ca. 45 days postinocu-lation (dpi).

Plants bombarded with wild-type TGMV began to show

symptoms at 4 to 5 dpi, with all of the plants displaying severesymptoms by 6 to 7 dpi (Fig. 2). In contrast, none of the plantsinoculated with the glycine or valine mutants showed symp-toms at 6 dpi in three independent experiments. Consistentwith its inability to replicate in tobacco protoplasts, the L148Gmutant did not induce any disease symptoms by 45 dpi (datanot shown). The three valine mutants were infectious but dis-played different kinetics of symptom appearance (Fig. 2).Plants infected with the L148V mutant developed symptomsbetween 8 and 14 dpi, while plants infected with the E146AL148V mutant began to show symptoms between 9 and 21 dpi.Plants infected with the L148V* mutant showed the greatestdelay, with symptoms appearing between 13 and 27 dpi. Theaverage time of symptom appearance was 11.8 dpi for L148V,15.5 dpi for E146A L148V, and 23.2 dpi for L148V*. In allcases, plants inoculated with the valine mutants eventuallydeveloped severe symptoms that were indistinguishable fromthose induced by wild-type TGMV.

To determine if the time of symptom appearance corre-sponded to DNA accumulation for the valine mutants, totalDNA was isolated from newly emerging leaves of inoculatedplants at 7, 14, and 19 dpi and analyzed on agarose gel blotsusing a radiolabeled TGMV A probe. At 7 dpi, high levels ofviral DNA were observed in all plants inoculated with wild-type TGMV (Fig. 3A, lanes 1 and 2), while only one plantinfected with the L148V mutant contained detectable levels ofTGMV A DNA (lanes 3 to 5), and no plants inoculated withL148V* (lanes 8 to 12) or E146A L148A (lanes 13 to 17) had

FIG. 1. L148 mutants are impaired for TGMV AL1 replication.(A) Schematic of the TGMV AL1 protein. Solid boxes mark thelocations of the three motifs conserved among rolling-circle replicationinitiator proteins, the oval indicates a predicted pair of �-helices, andthe stippled box shows the location of the ATP binding motif. Helix 4residues (E146 and L148) that were mutated are indicated. (B) Thesequence between TGMV AL1 amino acids 144 and 154 (helix 4) isshown. E146 and L148 substitutions are shown for the five AL1 mu-tants below the sequence. The codons specifying residues E, A, and Lof helix 4 and the mutations introduced into the three codons areshown on the right (modified nucleotides are indicated by lowercasetype). Mutants L148V and L148V* differ only in the third position ofthe codon. (C) Replication of TGMV AL1 mutants was analyzed intobacco protoplasts by agarose gel blot hybridization. Lanes 1 to 6 aretransfections with TGMV A replicons with either wild-type (wt) (lane1) or mutant AL1 genes corresponding to E146A L148A (lane 2),L148G (lane 3), L148V (lane 4), E146A L148V (lane 5), and L148V*(lane 6). The positions of double-stranded (ds) and single-stranded (ss)forms of TGMV A DNA are marked on the left. An overexposedimage (magnification, �20) of lanes 4 to 6 is shown on the right. Thelevels of replication of the different mutants relative to wild-typeTGMV (100) are indicated at the bottom.

FIG. 2. Symptom appearance is delayed in plants infected withTGMV L148 valine mutants. N. benthamiana plants cobombardedwith either wild-type or mutant TGMV A and wild-type TGMV Breplicons were examined daily for the appearance of symptoms in newgrowth. The dpi when plants displayed unequivocal symptoms (yellowveins and leaf curling) are plotted for each construct. The symbolsrepresent when individual plants displayed symptoms for wild type(�), L148V (E), L148V* (�), and E146A L148V (†). The total num-ber of plants was 12 for the wild type, 8 for L148V, 10 for L148V*, and9 for E146A L148V. The arrows indicate the average time of symptomappearance for the plants infected with each construct. The data sum-marize results from two independent experiments.

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detectable viral DNA. By 14 dpi, all plants bombarded with theL148V mutant accumulated wild-type levels of viral DNA (Fig.3B, lanes 3 to 7). At 14 dpi, plants infected with the E146AL148V mutant exhibited variable levels of TGMV DNA intheir tissues, ranging from higher-than-wild-type levels (Fig.3B, lanes 15 and 16) to barely detectable levels (lanes 13 and14). In contrast, only one of five plants inoculated with theL148V* mutant accumulated substantial amounts of viralDNA at 14 dpi (Fig. 3B, lane 9), although TGMV A DNA wasdetected at very low levels in two other plants (lanes 8 and 12).At 19 dpi, all plants infected with the E146A L148V mutant(Fig. 3C, lanes 13 to 17) and four of five plants inoculated withthe L148V* mutant exhibited high levels of viral DNA inleaves (lanes 8 to 12). In general, plants with detectableTGMV DNA were symptomatic, with the only exception beingplants with very low DNA levels (Fig. 3B, lanes 12 and 14).These results were unexpected because of the low replicationefficiencies observed for the valine mutants in protoplasts. Theresults also differed significantly from those reported previ-ously for the KEE146 and L148A mutants, which never accu-mulated high levels of viral DNA in infected plants over time(2, 31).

The L148 valine mutations are unstable in infected plants.The variability in symptom appearance and viral DNA accu-mulation and the subsequent development of severe symptomsand high viral DNA levels in plants infected with the L148

valine mutants are consistent with the selection and propaga-tion of a more fit viral variant. We tested this idea by examiningthe mutated region of the AL1 open reading frame in totalDNA extracts from symptomatic young leaves isolated fromplants infected with the mutant viruses. A 280-bp fragmentencoding TGMV AL1 amino acids 106 to 198, which encom-passes the RBR-binding domain (31), was amplified from eightplants infected with the L148V mutant and sequenced directly(Fig. 4A). The L148V codon was modified in all eight plants. Insix plants, a G-A transition resulted in a methionine codon atposition 148 (Fig. 4A). In the remaining two plants, a G-C orG-T transversion was associated with a reversion of the L148Vmutation to leucine.

The bias towards methionine substitutions at L148V wasalso seen in plants inoculated with the E146A L148V mutant(Fig. 4A). In 12 of 13 plants, sequencing uncovered a transitionevent in which GTG was changed to ATG. Reversion of theL148V mutation to leucine as a consequence of a G-T trans-version was seen in only one plant. Interestingly, the E146Amutation in the double mutant was unaltered in all 13 plants(Fig. 4A), indicating that variant selection was highly specificfor the L148V codon.

The L148V* codon was also altered at high efficiency during

FIG. 3. Viral DNA accumulation is delayed in plants infected withTGMV L148 valine mutants. N. benthamiana plants were bombardedwith DNA corresponding to TGMV A and B replicons. The AL1 geneeither was the wild type (wt) (lanes 1 and 2) or carried the L148V(lanes 3 to 7), L148V* (lanes 8 to 12), or E146A L148V (lanes 13 to 17)mutation. For each construct, total DNA was isolated from youngleaves of the same five plants at 7, 14, and 19 dpi and analyzed byagarose gel blot hybridization. The positions of single-stranded (ss)and double-stranded (ds) forms of TGMV A DNA are marked on theleft. ND, not determined.

FIG. 4. TGMV L148 valine mutants revert at high frequency. TotalDNA was isolated from symptomatic leaves of N. benthamiana plantsinfected with mutant TGMV A and wild-type TGMV B replicons at 19dpi. The AL1 coding region between amino acids 120 and 180 wasamplified from individual plants and sequenced directly. (A) Modifi-cations recovered at codon 148 for L148V, L148V*, and E146A L148Vmutants. The original mutations are designated by lowercase type, andthe nucleotide changes in the revertants are shown by uppercase,boldface type. The resulting amino acid, the number of plants, andtime of symptom appearance (dpi) are shown on the right for each typeof revertant. The dagger corresponds to the second-site revertants inB. The total number of plants analyzed for each TGMV mutant isindicated. (B) The TGMV AL1 sequence between amino acids 115and 156 is shown, with the locations of the predicted �-helices and aconserved sequence marked. Mutations in the second-site revertantsare listed on the left, and the amino acid changes are shown below thecorresponding positions.

11008 ARGUELLO-ASTORGA ET AL. J. VIROL.

infection, but the sequence changes differed from those de-tected for the L148V codon (Fig. 4A). The G-A transitionfound at high frequency in the viral progeny of the L148V andE146A L148V mutants occurred only twice in the 10 L148V*mutant-infected plants. In this case, the GTT codon was al-tered to ATT to specify an isoleucine. In six plants, a G-Ctransversion in the first nucleotide of codon 148 took place,resulting in a reversion to leucine (Fig. 4A). Surprisingly, twophenotypic revertants maintained the original L148V* muta-tion but were altered at other nucleotides in adjacent se-quences. One of these second-site revertants displayed a pointmutation at codon 128 that substituted a cysteine residue fortryptophan, whereas the other phenotypic revertant with anintact L148V* codon had two changes that generated the sub-stitutions R125G and I155L (Fig. 4B).

Because only a minority of the valine mutants reverted tothe wild-type leucine residue, we asked if the mutations in thesequenced region of the recovered TGMV AL1 mutants wereresponsible for the observed revertant phenotypes. A fragmentencoding TGMV AL1 amino acids 119 to 180 from cloned andsequenced PCR products corresponding to each revertant classwas subcloned in place of the homologous region in the wild-type TGMV A replicon. The mutant replicons were tested intransient replication assays (Fig. 5). The L148M mutant (Fig.5A, lane 2) replicated more efficiently than wild-type TGMV A(lane 1) in tobacco protoplasts, resulting in four times moreviral DNA. Viral DNA levels corresponding to the L148I (Fig.5A, lane 4) and C128W L148V (lane 5) mutations were lower(32% and 8%, respectively) than those of the wild type (lane 3)but significantly higher than those of the original L148V* mu-tant (Fig. 1C, lane 6). The R125G L148V I155L triple mutant(Fig. 5A, lane 6) did not replicate to readily detected levels inprotoplasts, suggesting that other compensatory mutationsoutside of the subcloned region were responsible for the re-vertant phenotype.

The revertant replicons were also assayed for symptom pro-duction in N. benthamiana infectivity assays (data not shown).Plants inoculated with the L148M and L148I mutants dis-played severe symptoms at 6 dpi. One plant infected with theC128W L148V mutant also showed symptoms at 6 dpi, whilethree plants displayed symptoms by 8 dpi. Plants inoculatedwith the R125G L148V I155L mutant developed wild-typesymptoms at variable times ranging from 13 to 31 dpi, like theother L148V* mutants (Fig. 2). To verify that the observedsymptoms corresponded to viral DNA accumulation, totalDNA was isolated from plants at 7 dpi and analyzed on agarosegel blots using a radiolabeled TGMV A probe. High levels ofdsDNA and ssDNA were seen in plants inoculated with wild-type TGMV (Fig. 5B, lanes 1 to 3) and the L148M mutant(lanes 4 to 6), while lower amounts of viral DNA were detectedfor the L148I (lanes 7 to 9) and C128W L148V (lanes 10 to 12)mutants. None of the plants inoculated with the R125G L148VI155L mutant (Fig. 5B, lanes 13 to 15) had detectable amountsof viral DNA at this time point. Together, these results showedthat the L148M, L148I, and C128W L148V mutations re-stored, at least partially, virus replication and infectivity to thevaline mutants. The absence of detectable viral DNA early ininfection and the development of delayed severe symptoms incombination with the protoplast data suggested that the

R125G L148V I155L mutant replicates poorly and undergoesreversion during infection.

The CaLCuV L145A mutant also reverts at high frequency.CaLCuV is a begomovirus that is distantly related to TGMV.A previous study showed that the mutation of L145 in the helix4 motif of the CaLCuV AL1 protein also impairs RBR inter-actions (2). We asked if an alanine substitution at positionL145 negatively impacts CaLCuV infectivity (Fig. 6A), as re-ported previously for the equivalent TGMV L148A mutation(2). N. benthamiana plants were cobombarded with a wild-typeCaLCuV B replicon and either a wild-type CaLCuV A repli-con or a mutant A component carrying the L145A substitution.By 4 to 5 dpi, plants inoculated with wild-type virus developedclear symptoms that included leaf curling, vein yellowing, andstunting of new growth (data not shown). The symptoms weremore severe than those observed for TGMV-infected plants.In contrast, the five plants inoculated with the CaLCuV L145Amutant did not exhibit any sign of disease at that time. Instead,symptoms appeared in two plants at 15 to 16 dpi, and theremaining plants showed symptoms after 21 dpi. There was a

FIG. 5. Replication analysis of TGMV revertants. (A) Replicationof TGMV A replicons encoding AL1 revertants was analyzed in to-bacco protoplasts by agarose gel blot hybridization. Lanes 1 to 6 aretransfections with TGMV A replicons with either wild-type (wt) (lanes1 and 3) or mutant AL1 genes corresponding to L148M (lane 2), L148I(lane 4), C128W L148V (lane 5), and R125G L148V I155L (lane 6).The position of the double-stranded (ds) TGMV A DNA is marked onthe left, and levels of replication relative to wild-type TGMV (100) ateach exposure are indicated at the bottom of each lane. (B) N.benthamiana plants were cobombarded with wild-type or mutantTGMV A and wild-type TGMV B DNA. At 7 dpi, total DNA wasisolated from three individual plants infected with TGMV B and eitherwild-type TGMV A (lanes 1 to 3) or mutant replicons carrying theL148M (lanes 4 to 6), L148I (lanes 7 to 9), C128W L148V (lanes 10 to12), or R125G L148V I155L (lanes 13 to 15) mutations. DNA accu-mulation was monitored by agarose gel blot hybridization. The posi-tions of single-stranded (ss) and double-stranded forms of TGMV Aare marked on the left.

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statistically significant reduction in the overall height of CaLCuV-infected plants (Fig. 6B) compared to mock-inoculated plants.Plants inoculated with CaLCuV L145A were less stunted thanthose infected with wild-type virus but displayed shorter inter-nodes than mock-inoculated plants (Fig. 6B).

The delay observed for the CaLCuV L145A mutant is rem-iniscent of the TGMV L148A mutant, but unlike TGMVL148A, all of the CaLCuV L145A-inoculated plants eventuallydeveloped strong symptoms. To better understand this differ-ence, we examined viral DNA accumulation in plants infectedwith the CaLCuV L145A mutant on agarose gel blots using aradiolabeled CaLCuV A probe (Fig. 6C). At 7 dpi, high levelsof viral DNA were detected in plants inoculated with wild-typevirus (Fig. 6C, lane 1), while none of the plants infected withthe L145A mutant contained detectable levels of viral DNA(lanes 3 to 7). By 16 dpi, three plants infected with the L145Amutant contained detectable levels of viral DNA (Fig. 6C,lanes 8, 9, and 11), and all of the plants were positive for viral

DNA by 25 dpi (lanes 13 to 17). The level of viral DNA at 16 and25 dpi was variable, ranging from high amounts to trace amounts.These results resembled the TGMV DNA accumulation patternsseen for the L148 valine substitutions (Fig. 3), suggesting that theCaLCuV L145A mutation is not stable in plants.

To determine if the L145A mutation is stable, we amplifiedan 854-bp fragment comprising part of the CaLCuV AL1 andAL2 genes out of total DNA from symptomatic young leaves offive plants infected with the mutant. Sequencing of the PCRproducts revealed that the original alanine codon was nolonger present in four plants (Fig. 6D). In three plants, a C-Ttransition had occurred to give a valine codon at position 145.In the fourth plant, a GG-AA double transition produced asynonymous mutation at alanine codon 144 and a nonsynony-mous mutation at position 145, resulting in a threonine substi-tution (Fig. 6D). In a fifth plant, the L145A mutation wasmaintained, but an A-C transversion changed L167 to isoleu-cine (Fig. 6A and D).

FIG. 6. Reversion of the CaLCuV L145A mutation in N. benthamiana. (A) The sequence of wild-type CaLCuV AL1 from amino acids 141 to177 is shown. The location of helix 4 and the L145A mutation is indicated. The † symbol marks the amino acid modified in the second-site revertantL145A I167L in D and E. (B) Comparison of the heights of mock-, CaLCuV (wild-type [wt])-, or L145A (mutant)-inoculated N. benthamiana plantsat 25 dpi. Each point represents an individual plant, with the mean height for each treatment shown at the top. The means of the three treatmentswere statistically different (P � 0.01 in a two-tailed Student’s t test). (C) N. benthamiana plants were cobombarded with a wild-type CaLCuV Breplicon and wild-type CaLCuV A (lanes 1 and 2) or an L145A mutant replicon (lanes 3 to 17). Total DNA from the same five plants at 7, 16,and 25 dpi was analyzed by DNA blot hybridization. The positions of single-stranded (ss) and double-stranded (ds) forms of CaLCuV A DNA aremarked on the left. Viral DNA was detected only in plants displaying symptoms. (D) The AL1 coding region between amino acids 132 and 349was amplified from individual plants and sequenced directly. The original mutation is designated by lowercase type, and the nucleotide changesin the revertants are shown by uppercase, boldface type. Numbers of plants are shown on the right for each type of revertant for bombardment(gun) and agroinoculation (agro) experiments. The total number of plants analyzed for each inoculation protocol is indicated below. (E) Repli-cation of CaLCuV A mutants in tobacco protoplasts was analyzed by agarose gel blot hybridization. Lanes 1 to 6 correspond to transfections withCaLCuV A replicons with either wild-type (lane 1 and 3) or mutant AL1 genes corresponding to L145A (lane 2), L145V (lane 4), L145A I167L(lane 5), and L145T (lane 6). The relative accumulation of viral DNAs is given at the bottom of each lane, with the wild type set at 100 for eachexposure.

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The preferential reversion of CaLCuV L145A to valine wasunexpected because a valine substitution at L148 in TGMV AL1severely impaired replication and infectivity. The CaLCuV andTGMV AL1 proteins show significant divergence in the resi-dues flanking the helix 4 motif, and substitutions at the con-served leucine may differentially impact the replication of thetwo viruses. To test this possibility, a fragment containingCaLCuV AL1 amino acids 132 to 349 derived from PCR prod-ucts of each revertant class was subcloned in place of thehomologous region in the wild-type CaLCuV A replicon andsequenced prior to transient replication assays (Fig. 6E). TheCaLCuV L145V (Fig. 6E, lane 4) and L145A I167L (lane 5)mutants replicated similarly in tobacco protoplasts, accumulat-ing to ca. half of wild-type DNA levels (lane 3). The CaLCuVL145T mutant (Fig. 6E, lane 6) replicated less efficiently, re-sulting in only 8% of wild-type levels. The L145A (Fig. 6E, lane6) mutant was the most severely impaired for replication, ac-cumulating at 1% of wild-type levels. This value is significantlyless than that reported previously for TGMV L148A, whichreplicates to 14% of wild-type levels (2), and more similar tothat seen for the TGMV L148 valine mutations (Fig. 1C).Consistent with their replication phenotypes, the subclonedCaLCuV L145V, L145A I167L, and L145T replicons wereinfectious on plants (data not shown).

N. benthamiana plants agroinoculated with wild-typeCaLCuV developed severe symptoms indistinguishable fromthose seen with bombardment (data not shown). The timing ofsymptom appearance differed between the two protocols, withthe agroinoculated plants displaying symptoms 10 to 12 dpi,compared to 4 to 5 dpi for bombarded plants. However, like

the bombardment experiments, plants agroinoculated with theCaLCuV L145A mutant were delayed relative to the wild-typecontrol. None of the L145A-inoculated plants exhibited symp-toms at 12 dpi, two plants showed signs of disease at 21 dpi,four additional plants displayed symptoms at 23 to 25 dpi, andall nine plants exhibited severe symptoms by 33 dpi.

PCR amplification of viral DNA followed by direct sequenc-ing revealed that the engineered alanine codon was altered inmost plants (Fig. 6D). In four plants, the original GCC alaninecodon was changed to a GCT valine codon, while a mixture ofthe two codons was detected in two plants. One plant con-tained a mixture corresponding to the original GCC codon anda new GTC codon specifying threonine. Another plant re-tained the original GCC codon but had a second-site L167Imutation, the same second-site reversion found in the bom-bardment experiment. These results indicated that CaLCuVL145A reversion is not dependent on the inoculation protocolfor N. benthamiana.

Different CaLCuV L145A reversions are recovered fromArabidopsis plants. Although CaLCuV infects N. benthamiana,its natural hosts are members of the family Brassicaceae. Wetook advantage of the ability of CaLCuV to infect Arabidopsisthaliana to ask if the plant host can influence the frequency orthe nature of reversion. Arabidopsis plants were agroinoculatedwith a wild-type CaLCuV B replicon and either a wild-typeCaLCuV A replicon or a mutant A component carrying theL145A substitution. At 12 to 15 dpi, all of the plants infectedwith wild-type CaLCuV developed strong symptoms character-ized by yellowing, leaf curling, and severe stunting of newgrowth. In contrast, none of the 12 plants inoculated with the

FIG. 7. Reversion of the CaLCuV L145A mutation in Arabidopsis plants. A. thaliana plants agroinoculated with a wild-type CaLCuV B repliconand wild-type CaLCuV A or an L145A mutant replicon are shown. (A) Mock (left), wild-type (middle), and L145A (right) symptoms at 20 dpi.(B) L145A symptoms at 29 dpi. (C) The AL1 coding region between amino acids 132 and 349 was amplified from individual plants and sequenceddirectly. The original mutations are designated by lowercase type, and the nucleotide changes in the revertants are shown by uppercase, boldfacetype. The altered amino acid and the numbers of plants are shown on the right for each type of revertant. The total number of plants analyzedis indicated below. (D) Total DNA was isolated from plants at 29 dpi and analyzed by agarose gel blot hybridization. The reversion at L145A isindicated at the top of each lane. The position of double-stranded (ds) CaLCuV A DNA is marked on the left.

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L145A mutant exhibited symptoms at this time. By 20 dpi, oneplant showed mild symptoms, and three additional plants dis-played signs of disease by 25 dpi (Fig. 7A). Over the next 8days, all of the L145A-inoculated plants developed strongsymptoms in leaves and flowers (Fig. 7B). These results sug-gested that the CaLCuV L145A mutation is also unstable inArabidopsis plants.

An 854-bp viral fragment was amplified from total DNAisolated from symptomatic young leaves of CaLCuV L145A-inoculated Arabidopsis plants at 29 dpi, and the PCR productswere sequenced directly. In six plants, a G-A transition at thefirst nucleotide position changed the engineered alanine codon(GCC) to a threonine codon (ACC) (Fig. 7C). In one plant, aC-T transition at the second nucleotide position resulted in avaline codon (GTC). A double substitution changing GCC tothe leucine codon CTC was recovered from a single plant. Infour cases, the recovered sequence was a mixture of the engi-neered GCC codon and the revertant ACC codon, indicativeof the presence of two viral variants in the same plant. To-gether, these results demonstrated a preference for the A145Treversion (10 out 12), with the A145V and A145L reversionsoccurring at reduced frequency (Fig. 7C). This is in contrast todata for N. benthamiana (Fig. 6D) showing that the A145Vreversion occurs more frequently (9/14) than the A145T event(3/14).

The same DNA samples were analyzed by agarose gel blot-ting using a radiolabeled CaLCuV A-specific probe. A bandcorresponding to the double-stranded form of CaLCuV A wasobserved in 10 of the 12 plants (Fig. 7D), while no single-stranded DNA was detected (data not shown). The levels ofdouble-stranded viral DNA varied between plants and did notcorrelate with revertant type. CaLCuV replication assays incultured Arabidopsis cells were not successful (data notshown), so it was not possible to distinguish the impact ofreplication efficiency and the time of reversion on the fre-quency and accumulation of the revertants. However, theCaLCuV data demonstrated that the plant host influencesthe outcome of the reversion process but not the overallfrequency, which was 100% in all instances.

DISCUSSION

It is generally thought that nucleotide misincorporation doesnot contribute significantly to the genomic variation of smallDNA viruses that are replicated by cellular DNA polymerases(15). This assumption is supported by long-term mutation ratesfor dsDNA viruses, which are low and comparable to thosemeasured for cellular genes (5). There are numerous reports ofthe emergence of geminivirus strains with altered pathogenic-ity (62), indicative of rapid genetic change that has been at-tributed to recombination or reassortment among differentviral genomes. However, our finding that mutations in the helix4 motif of the AL1 gene of two distantly related begomovirusesrevert at 100% frequency suggests that nucleotide substitutionsoccur with high incidence and are under strong selective pres-sure during geminivirus infection. Thus, in agreement withrecent reports of high mutation rates for other ssDNA virusesinfecting vertebrates and bacteria (51, 64, 65), nucleotide sub-stitution events are likely to contribute to the diversity andrapid evolution of geminivirus ssDNA genomes.

Analysis of TGMV and CaLCuV variants with mutations atthe equivalent L148 and L145 residues in their respective helix4 motifs revealed several features of the nucleotide substitu-tion process during geminivirus infection. First, the process ishighly efficient, with reversions occurring in 100% of plantsinfected with either one of the TGMV L148 valine mutants(L148V, L148V*, and E146A L148V) or the CaLCuV L145Amutant. Second, the process is selective for mutations thatimpair protein function. The E146A mutation, which has nodetectable impact on AL1 function (2), was stable even whenthe valine residue reverted in the E146A L148V mutant. Last,the frequency of a reversion event reflected the number ofnucleotide changes required to generate a given amino acidcodon. L148V, which has a GTG valine codon, reverted to anATG methionine codon via a single nucleotide change in 18 of21 events. The generation of an ATG codon from L148V*,with a GTT valine codon, would have required two changesand was not recovered. Instead, the most common change (6 of10 events) was to a CTT leucine codon, which also involved asingle nucleotide change. Similarly, the low recovery of leucineor methionine revertants (1 of 25) from CaLCuV L145A-in-fected plants most likely reflected the need for multiple nucle-otide changes to generate the requisite codons.

Comparison of TGMV and CaLCuV revertants also uncov-ered some important differences. The CaLCuV L145A mutantwas unstable during infection, while the equivalent L148A mu-tant of TGMV AL1 was stable (2). Valine was the most fre-quent reversion recovered from N. benthamiana plants inocu-lated with the CaLCuV L145A mutant, while TGMV L148Vmutants were unstable during infection. These differences can-not be attributed to host effects because N. benthamiana servedas the host for both viruses. CaLCuV AL1 is representative ofa small group of replication proteins in the SLCV group thatlack the conserved element DGRSARGG(C/Q)Q (3). Inter-estingly, the second-site mutation C125W mapped to this se-quence in TGMV AL1. The CaLCuV L145V mutant repli-cated efficiently in cultured cells (46% of wild-type levels),while a TGMV L148V mutant replicated poorly (1% of wild-type levels). An I167L second-site revertant of CaLCuVL145A, which was recovered twice, also replicated efficiently inculture. Leucine occurs at the equivalent position in TGMVand is the most common residue at this site in other begomo-virus replication proteins except for members of the SLCVgroup, which have branched aliphatic residues (our unpub-lished observation). However, a TGMV L148A mutant accu-mulates to only 13% of the wild-type level in cultured cells,while a CaLCuV L145A I167L mutant accumulates to 47% ofwild-type levels, suggesting that different sequence constraintson TGMV and CaLCuV AL1 impact virus stability and rever-tant selection during infection.

The different fates of the CaLCuV L145A mutant in N.benthamiana and Arabidopsis indicated that the plant host alsoinfluences the reversion outcome. Like the experiments with N.benthamiana plants, the CaLCuV L145A mutant was unstablein Arabidopsis and reverted at 100% frequency. However, mostrevertants (10 of 12) contained a threonine substitution atcodon 145 in CaLCuV AL1, in contrast to the valine revertantsisolated from N. benthamiana plants. The L145T mutant accu-mulated to 7% of wild-type levels in BY-2 protoplasts, suggest-ing that the threonine mutant does not replicate efficiently in

11012 ARGUELLO-ASTORGA ET AL. J. VIROL.

Nicotiana species, consistent with it being isolated only oncefrom N. benthamiana. The preferential isolation of CaLCuVL145T from Arabidopsis may be indicative of efficient replica-tion in this host. The selection of a valine or a threoninerevertant of CaLCuV L145A may reflect different sequencerequirements for an AL1/host protein interaction in N.benthamiana versus Arabidopsis. Although no studies havelinked the begomovirus AL1 protein to host range (49, 52), thereversion of a tomato yellow leaf curl virus C4 mutant intomato but not in N. benthamiana has been attributed to dif-ferent host constraints on systemic movement (29). Mastrevi-rus replication proteins have also been implicated in hostadaptation (33). Interestingly, partial reversion of a three-nu-cleotide mutation in the RepA coding sequence also occurs at100% frequency during maize streak virus (MSV) infection(67). Unlike the TGMV and CaLCuV reversions, the MSVreversion was restricted to a single nucleotide transversionevent that restored nucleic acid folding but not RepA function.

The 100% reversion frequencies for TGMV, CaLCuV, andMSV mutants and the isolation of second-site revertants implythat the family Geminiviridae is subject to high rates of nucle-otide substitution. The high rates may reflect a failure of gemi-nivirus infection to activate the mismatch repair system, whichis responsible for the excision and replacement of misincorpo-rated nucleotides during chromosomal replication. Methylatedviral DNA is not a good template for replication and transcrip-tion (8, 18), and geminiviruses actively interfere with DNAmethylation pathways in infected cells (70). As a consequence,viral DNA is undermethylated, making it difficult for the mis-match repair machinery to distinguish between parental andnascent DNA strands. In addition, gene profiling experimentsindicated that although other DNA repair pathways are up-regulated during geminivirus infection, the expression of mis-match repair machinery is not increased in CaLCuV-infectedArabidopsis plants (J. T. Ascencio-Ibanez and L. Hanley-Bow-doin, unpublished result). Together, these results indicatedthat geminivirus replication products are not corrected by mis-match repair, increasing the likelihood that a mutation will befixed. Greater genetic variability might facilitate geminivirusadaptation to new hosts and changing environments, ultimatelyleading to increased viral fitness (57). The failure to recoverthe TGMV L148V and CaLCuV L145A mutants from mostplants suggested that the less fit mutant A component is lostrandomly during viral movement, ultimately leading to its dis-appearance from the population. The 7 of 26 plants carryingmixtures of the CaLCuV L145A mutant and various revertantsmay reflect intermediates in this process.

TGMV L148 is located in an 80-amino-acid region of AL1known to mediate oligomerization and binding to AL3, RBR,and other host factors (4, 10, 30, 31, 47, 63). There was noobvious correlation between the effects of the various L148mutations on RBR binding and viral replication, as illustratedby comparisons of the relative RBR binding (25, 31, and 36%)and replication (13, 1, and 0%) activities of L148A, L148V andL148G, respectively (2). In addition, AL1 oligomerization ac-tivity, which is required for viral replication, is only moderatelyreduced for the L148A, L148V, and L148G mutants (2). Thus,the instability of the valine mutants is not due to reducedoligomerization or RBR binding and instead may reflect thedestabilization of the AL1 protein or impaired interactions

with a host factor required for viral replication. Strikingly, onlyleucine, methionine, and isoleucine revertants were recovered,indicating that only a few amino acids are permissible at theL148 position. All three amino acids have large hydrophobicside chains and high probabilities of occurring in �-helices.TGMV AL1 proteins carrying either leucine or methioninedisplay similar functional properties, while the isoleucine vari-ant is moderately reduced in activity. Interestingly, leucine andmethionine are the only amino acids found at the equivalentposition in the helix 4 motif of all characterized begomovirusand curtovirus replication proteins (data not shown).

An important consequence of high mutation and recombi-nation rates is the continuous production of genetic variationin geminivirus populations. This variability is balanced by acomplex set of selection pressures including those associatedwith intrinsic properties of the virus, such as the maintenanceof essential nucleotide structures and replication signals, andselection pressures to maintain crucial interactions with planthosts and insect vectors. Thus, despite their variation potential,geminiviruses populations exhibit significant genetic stabilityover time and space, as has been documented for plant RNAviruses that also display high mutation rates (25, 57). None-theless, the evolutionary potential of geminiviruses needs to beconsidered in long-term control strategies, because any diseasemanagement effort will result in selective pressure on the viruspopulation to adapt to new circumstances (38, 57). A recentmathematical analysis of the potential impact of disease con-trol strategies concluded that the use of resistant cultivars withreduced within-plant virus titers puts pressure on the targetvirus to evolve towards a higher multiplication rate (69). Theresults reported here demonstrated experimentally that gemi-nivirus variants with residual replication capabilities are understrong selective pressure to generate variants that replicate tohigh titers. Given the large size and genetic heterogeneity ofgeminivirus populations and their capacity to rapidly changetheir genomes by recombination and mutation, it will be nec-essary to devise resistance strategies that prevent virus repli-cation and not simply reduce it because of the risk of gener-ating more harmful variants that overcome resistance.

ACKNOWLEDGMENTS

This research was supported by a grant from the National ScienceFoundation (MCB-0110536 to L.H.-B.) and a postdoctoral fellowshipfrom the PEW Foundation (P0291SC to G.A.-A.).

We thank Marilyn Roossinck (The Samuel Roberts Noble Founda-tion), Dominique Robertson, Sharon Settlage, and Luisa Lopez-Ochoa(all at NCSU) for their critical comments on the manuscript.

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54. Rasheed, M. S., L. A. Selth, A. M. Koltunow, J. W. Randles, and M. A.Rezaian. 2006. Single-stranded DNA of Tomato leaf curl virus accumulatesin the cytoplasm of phloem cells. Virology 348:120–132.

55. Rogers, S. G., D. M. Bisaro, R. B. Horsch, R. T. Fraley, N. L. Hoffman, L.Brand, J. S. Elmer, and A. M. Lloyd. 1986. Tomato golden mosaic virus Acomponent DNA replicates autonomously in transgenic plants. Cell 45:593–600.

56. Rojas, M. R., C. Hagen, W. J. Lucas, and R. L. Gilbertson. 2005. Exploitingchinks in the plant’s armor: evolution and emergence of geminiviruses.Annu. Rev. Phytopathol. 43:361–394.

57. Roossinck, M. J. 1997. Mechanisms of plant virus evolution. Annu. Rev.Phytopathol. 35:191–209.

58. Rothenstein, D., D. Haible, I. Dasgupta, N. Dutt, B. L. Patil, and H. Jeske.2006. Biodiversity and recombination of cassava-infecting begomovirusesfrom southern India. Arch. Virol. 151:55–69.

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VOL. 81, 2007 AL1 REVERTANTS 11015

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Journal of Virological Methods 142 (2007) 198–203

DNA abrasion onto plants is an effective method forgeminivirus infection and virus-induced gene silencing

Jose Trinidad Ascencio-Ibanez ∗, Sharon B. SettlageNorth Carolina State University, Department of Molecular and Structural Biochemistry, Viral Genomics Group,

CB 7651 Partners III, Room B04, Raleigh, NC 27695, USA

Received 20 September 2006; received in revised form 8 December 2006; accepted 25 January 2007Available online 6 March 2007

bstract

Geminiviruses belong to a rapidly growing group of plant pathogens that contribute to crop losses in tropical and subtropical areas of the world.eminivirus infection is a model for plant DNA replication and virus/host interactions. Geminiviruses are also used as vectors to induce silencingf endogenous genes in several plant species. A method was analyzed for inoculating geminiviruses using plasmid DNA rubbed onto leaves in theresence of an abrasive (DNA abrasion). Although the use of DNA abrasion to inoculate geminiviruses has been described previously, the techniqueas fallen out of favor and has not been systematically optimized. However, consistent efficiencies of 100% infection rates can be achieved byNA abrasion. The symptoms of Tomato Golden Mosaic Virus or Cabbage Leaf Curl Virus infection on Nicotiana benthamiana were similar in

iming and appearance to the symptoms observed in plants inoculated using Agrobacterium as the delivery method. More importantly, silencingf an endogenous gene was highly efficient when a geminivirus silencing vector was inoculated by the DNA abrasion method. Other plant speciesuccessfully inoculated with geminiviruses by DNA abrasion were Nicotiana tabacum, Capsicum annuum and Nicandra physalodes. Unfortunately,

rabidopsis thaliana could not be infected with Cabbage Leaf Curl Virus using leaf abrasion, demonstrating limitation of the method. However,

eaf abrasion to inoculate geminiviruses is an easy and inexpensive method that should be considered as an accessible technique to the growingumber of researchers using geminiviruses.ublished by Elsevier B.V.

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eywords: Direct DNA inoculation; Geminivirus; Mechanical transmission; Si

. Introduction

Geminiviruses are widely distributed single-stranded DNAlant pathogens, infecting crops in tropical and sub-tropicalreas of the world (Brown, 2001). Four genera (Mastrevirus,urtovirus, Topocuvirus and Begomovirus) are included in theeminiviridae family based on genome organization, insect vec-

or and host range (Rybicki et al., 2000). Among them, theegomovirus genus includes a growing number of members

hat infect many dicotyledonous crops and are transmitted byemisia tabaci. Begomoviruses can have one or two genome

omponents coding for 5–7 genes. Replication occurs by aombination of rolling circle and recombination-dependenteplication (Hanley-Bowdoin et al., 2000; Preiss and Jeske,

∗ Corresponding author. Tel.: +1 919 5155736; fax: +1 919 5131209.E-mail address: trino ascencio@ncsu.edu (J.T. Ascencio-Ibanez).

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166-0934/$ – see front matter. Published by Elsevier B.V.oi:10.1016/j.jviromet.2007.01.031

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003). Plant biologists have become increasingly interested ineminiviruses as tools to study gene silencing (Carrillo-Tripp etl., 2006).

Currently, most experimental inoculations of geminivirusesre accomplished by the following methods depending on theirus and host combination: (a) direct virus transmission fromn infected plant through an insect vector, (b) the engineeringnd cloning of hemidimers or dimers into Agrobacterium tume-aciens vectors for agroinoculation, (c) the use of biolistics ord) grafting. However, in early geminivirus studies, mechani-al inoculation of virus purified from infected plants was thenly method to re-inoculate a field-isolated geminivirus in thebsence of its insect vector. With appropriate host/virus combi-ations, efficiencies of up to 100% were reported (Elmer et al.,

988; Etessami et al., 1988; Gilbertson et al., 1993; Hamiltont al., 1983; Hayes et al., 1988; Liu et al., 1997; Stanley et al.,990; Sunter et al., 2001). Some geminiviruses, such as maizetreak virus, were never transmitted successfully mechanically

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nd required cloning into agrobacterium to be experimentallynoculated (Davies et al., 1987; Grimsley and Bisaro, 1987),hile others such a Beet Curly Top Virus and Squash Leafurl Virus could be mechanically inoculated with extremely

ow efficiency (Lazarowitz and Lazdins, 1991; Stanley et al.,986). Mechanical inoculation was largely replaced by agro- oriolistic methods because of a general conclusion that mechan-cal inoculation was inefficient (Grimsley and Bisaro, 1987).s a consequence, many newer researchers are unaware of

he possibility of using mechanical inoculation with gemi-iviruses.

Biolistics, insect transmission, grafting and agro-inoculationequire at least minimal laboratory equipment and/or technicalkills, restricting the ability of the many researchers withoutccess to the requisite resources or experience. In addition,grobacterium is a pathogen and may contribute to backgroundffects, independent of geminivirus infection. Because of theseifficulties, mechanical inoculation of cloned geminivirus repli-on DNA was revisited and shown to be viable for infectionnd virus induced gene silencing (VIGS). With certain consid-rations, such as the use of the appropriate leaf stage, DNAmount and plant/host combination, any researcher can simplynd quickly use DNA abrasion to test a geminivirus derivedeplicon for infection and/or VIGS.

. Materials and methods

.1. Plant material, geminiviruses and growth conditions

The plant/geminivirus combinations tested in these experi-ents were Arabidopsis thaliana and Nicotiana benthamiana

noculated with Cabbage leaf curl virus (CaLCuV), N. ben-hamiana inoculated with Tomato golden mosaic virus (TGMV),r Nicotiana tabacum, Capsicum annuum and Nicandra physa-odes inoculated with Pepper yellow vein virus (PHYVV) orepper golden mosaic virus (PepGMV). Arabidopsis plantsere grown in short-day conditions (8-h light:16-h dark) at0 ◦C, while N. benthamiana, N. tabacum, C. annuum and N.hysalodes plants were grown in long-day conditions (16-hight:8-h dark) at 25 ◦C. N. benthamiana plants were inocu-ated at approximately the 6-leaf stage. Inoculated A. thalianalants had 16–18 true leaves. N. tabacum Xanthi, C. annuum cvonora Anaheim and N. physalodes were inoculated at the 5–7

eaf stage.

.2. Geminivirus hemidimers and silencing vectors

TGMV A and B hemidimers were described by Fontest al., 1994. TGMV-derived silencing vectors pTGA1.3 andTGB1.3Su were used to silence the N. benthamiana sulfur geneCHL1), encoding a subunit of magnesium chelatase (Kjemtrupt al., 1998; Peele et al., 2001). Bacterial plasmids coding fornfectious CaLCuV components A and B, pCPCLCVA.001 and

CPCbLCVB.001, were described by Turnage et al. (2002).noculation of PHYVV was performed with plasmids pIGV21nd pIGV22 (Garzon-Tiznado et al., 1993) or plasmids pTPVAnd pTPVB for PepGMV (Torres-Pacheco et al., 1996). The

dTaa

Virological Methods 142 (2007) 198–203 199

bilities of all of the hemidimers to cause infection have beenerified in bombardment experiments.

.3. Geminivirus infection

To infect plants by DNA abrasion, carborundum (glass pow-er has also been used successfully) was evenly dispersed ontoarget leaves from a 15 mL conical tube through 12 layers ofheesecloth (the abrasive should be almost invisible on the leaf)ollowed by the inoculation of 1–5 �g of plasmid DNA in 20 �Later or up to 20 �g of total DNA from infected plants. The0 �L DNA solution was placed at the leaf base and spread byubbing with 10 soft but firm strokes from the base to tip with aloved finger. Each plant was inoculated on two or three youngeaves larger than 1.5 cm in length. At the end of the manipula-ions, the leaves were gently washed with a stream of water toemove excess carborundum. Plants were covered with a plasticome for 24 h and placed in a growth chamber. Agroinocula-ion of TGMV onto N. benthamiana plants was performed asescribed by Egelkrout et al. (2001).

.4. Total DNA purification

Four discs (0.6 cm) or an equivalent amount of tissue∼100 mg) from symptomatic leaves were flash frozen in liq-id nitrogen and ground in a Retsch MM301 (Newtown, PA)rinder using two steel beads. Tissue was mixed with 750 �L ofNO buffer (7 M Urea, 0.35 M NaCl, 0.05 M Tris–HCl, 0.02 MDTA, 1% sarcosyl) and incubated at 65 ◦C for 10 min. Alter-atively, tissue was ground at room temperature with plasticestles in 250 �L of 3NO buffer. Five hundred microliters ofNO buffer was added, vortexed and incubated at room tem-erature for 30 min. Samples were centrifuged at 16,000 rcf formin, and the supernatant was transferred to a new tube. A

ingle volume of phenol:chloroform:isoamylalcohol (25:24:1,:v:v) was added, followed by vortexing and centrifugation asescribed above. The aqueous layer was transferred to a new tubend re-extracted with 1 volume of chloroform:isoamylalcohol24:1,v:v) followed by precipitation with 0.2 volumes of 10 Mmmonium acetate and one volume of isopropanol. Samplesere mixed and centrifuged for 20 min at 16,000 rcf. Tubesere decanted and washed with 500 �L of 70% ethanol and cen-

rifuged for 5 min at 16,000 rcf. The ethanol wash was decantednd the samples were air-dried. This procedure yields DNArom infected N. benthamiana that is brown in color, but readilyigested with restriction enzymes. Total DNA was resuspendedn 40 �L water and double-stranded DNA was quantified with aourometer using Hoechst H 33258 dye according to the man-facturers instructions (Hoefer DyNa Quant 200).

.5. DNA gel and squash blots

Total DNA (1 �g) from TGMV-infected plants was

igested with SacI and electrophoresed in a 1% TAE (40 mMris–acetate, 1 mM EDTA) agarose gel to resolve doublend single-stranded viral DNAs. The gel was transferred tonylon membrane and hybridized to a probe corresponding

2 al of Virological Methods 142 (2007) 198–203

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o the TGMV B component previously labeled with biotinsing a NEBlot Phototope kit (New England Biolabs, Bev-rly, MA). Hybridization was performed as recommendedy Licor (see http://biosupport.licor.com/docs/whatsnew/outhern-08331.pdf). Signals were detected using IRdye680

abeled streptavidin (Invitrogen) and quantified using theicor-Odyssey instrument.

For squash blots, symptomatic leaves were sampled byunching with the lid of a microcentrifuge tube (1.5 mL). Equiv-lent samples were taken from mock-inoculated plants. Leafamples were rubbed onto a nylon membrane using a plasticestle to push over the membrane, UV crosslinked, and probedith a [32P]-labeled TGMV B DNA using standard methods forNA blotting (Egelkrout et al., 2001). Radioactive signals were

maged and quantified using a Bio-Rad FX molecular imager.

. Results

.1. Efficiency of inoculation and silencing

The efficiency of using plasmid DNA directly as an inoculumor geminiviruses was tested by monitoring TGMV and CaLCuVnfection in N. benthamiana (Table 1). Symptoms were readilypparent when either TGMV (Fig. 1A) or CaLCuV (Fig. 2A)

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ig. 1. TGMV inoculated by DNA abrasion is infectious. (A) Nicotiana benthamianomponents by DNA abrasion (center) or agrobacterium (right). (B) Leaf inoculated ws indicated by the arrow. (C) Representative squash blot showing TGMV infection ovays post-inoculation (dpi). Signal intensities are shown below. (D) Southern blot shubbing of TGMV A and B component containing plasmids.

10/10 10/10 ND

noculation efficiencies were determined at 4 weeks post-inoculation.

ontaining plasmids were introduced into young leaves usingarborundum as an abrasive. Infection efficiency was propor-ional to inoculum amount. Using 5 �g of DNA to inoculate N.enthamiana on each of three leaves resulted in 100% infectionates for both TGMV and CaLCuV. Lower amounts of DNAesulted in more variable infection rates. One microgram ofNA resulted in infection rates that varied from 50% for the

ase of CaLCuV or 66% for TGMV. When 0.625 �g of DNAas used, efficiency was the same as the 1 �g inoculated plants

Table 1). Interestingly, silencing with 0.625 �g of DNA was

ighly efficient (higher than 80%, Table 1). Other viruses dis-layed different inoculation properties when 1 �g of DNA wasnoculated onto different plant species. PHYVV inoculated onto. tabacum, C. annuum and N. physalodes gave 60, 30 and 70%

a plants mock inoculated (left, uninfected) or inoculated with TGMV A and Bith TGMV by DNA abrasion showing adaxial browning and localized infectioner time in agrobacterium (A) or DNA abrasion (R) inoculated plants at various

owing replication of TGMV B in symptomatic leaves of a plant inoculated by

J.T. Ascencio-Ibanez, S.B. Settlage / Journal of Virological Methods 142 (2007) 198–203 201

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ig. 2. CaLCuV infection and silencing can be obtained by DNA abrasion. (Anoculation by DNA abrasion method. (B) Leaf depicting silencing of a magnesNA abrasion method. Silencing is manifested as white streaks and blotches in

noculation rates while PepGMV inoculated onto N. tabacumnd C. annuum gave 60 and 70% inoculation efficiencies, respec-ively (Table 2).

The timing and severity of TGMV infection in N. benthami-na was compared for the DNA abrasion and agroinoculationethods. With DNA abrasion, some abraded leaves showed

rowning on the adaxial surface 1 day post-inoculation (dpi),nd yellow spots were observed on inoculated leaves at 4–7 dpi,ost likely reflecting localized infection at inoculation sites,

Fig. 1B). The browning and yellow spots were not seen ongroinoculated plants. At 10–14 dpi, plants inoculated usingither protocol displayed similar symptoms with newly emerg-ng leaves showing curling and chlorosis typical of TGMVnfection. By 21 dpi, plants inoculated by both methods devel-ped severe symptoms characterized by overall stunting, leafurling and yellowing (Fig. 1A). For both treatments, newlymerged leaves showed less curling and yellowing after 21 dpi,ypical of N. benthamiana recovery from TGMV infection.

.2. DNA accumulation in inoculated plants

TGMV DNA accumulation was examined at 14, 18 and3 dpi on squash blots of symptomatic leaf tissue from N. ben-hamiana plants inoculated by DNA abrasion or agroinoculationFig. 1C). Similar levels of TGMV DNA were detected at 18 dpior both treatments, while lower levels of viral DNA wereetected at 14 and 23 dpi in plants infected by DNA abrasionhan agroinoculation (Fig. 1C). For both treatments, viral DNA

evels were highest at 18 dpi, a few days before peak symptomeverity. DNA gel blotting detected both double- and single-tranded forms of TGMV DNA in symptomatic leaves of plantsnoculated directly with DNA (Fig. 1D).

able 2noculation efficiencies of PHYVV and PepGMV by DNA abrasion

otal DNA (1 �g) Nicotianatabacum

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LCuV-infected plant showing severe leaf curling, chlorosis and stunting afterheletase gene after inoculation of a geminivirus derived silencing vector by the

areas of the foliage.

.3. Silencing of magnesium chelatase is possible throughhe DNA abrasion method

The use of geminivirus vectors to silence endogenous genes isecoming increasingly popular as a method to study gene func-ion in plants (Carrillo-Tripp et al., 2006). Therefore, a silencingector was also inoculated using the DNA abrasion method.or this experiment, a TGMV-based plasmid was used that hadreviously been shown to silence expression of a subunit ofagnesium cheletase in N. benthamiana via biolistic inocula-

ion (Kjemtrup et al., 1998; Peele et al., 2001). Like bombardedlants, N. benthamiana plants inoculated by DNA abrasion withhe silencing plasmid developed white patches on their leavesndicative of chlorophyll loss (Fig. 2B), establishing that DNAbrasion delivery can be used for silencing experiments.

.4. Other virus/host combinations are also susceptible toNA abrasion inoculation

Other geminivirus/host combinations were also tested. Inocu-ation of Capsicum annuum cv Sonora Anaheim and N. tabacumamsun by DNA abrasion onto leaves with PHYVV or PepGMVemidimers resulted in infection of at least half of the inoculatedlants (Table 2). Similar results were seen for Nicandra physa-odes and PHYVV. In contrast, A. thaliana plants inoculated withaLCuV by DNA abrasion only showed damage caused by thebrasive even though agroinfection and bombardment with theame hemidimers result in infection (Egelkrout et al., 2001).

. Discussion

Many different methods for viral inoculation have been devel-ped in the past (Dijkstra and de Jager, 1998). While most workor RNA viruses, inoculating plant DNA viruses has proven moreifficult. The use of whiteflies, agroinoculation, and biolisticsepresent the majority of inoculation methods currently usedith geminiviruses. Each of these methods requires particu-

ar expertise, specialized equipment, or a containment systemn the case of whiteflies. Whitefly inoculations can also beroblematic because of their preferential feeding habits on cer-ain plant species. Agroinoculations require a previous cloning

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tep into agrobacterium cloning vectors. Biolistic equipments expensive and bulky. However, geminiviruses can be easilyntroduced onto several solanaceous plant species using a DNAbrasion technique that is achievable in the absence of high-techquipment.

The DNA abrasion technique is amenable to multiple plantpecies and geminivirus combinations. Infection efficiencyncreases with increasing amounts of inoculated DNA but asittle as 625 ng of pure plasmid DNA is effective. Optimalmounts of DNA produced 100% infection rates. N. benthami-na, C. annuum, N. physalodes and N. tabacum were also readilynfected by the technique and it is likely that optimization ofnoculum amount will result in 100% infection rates in thesepecies. In contrast, the DNA abrasion technique was not suc-essful when used with A. thaliana, and it is possible that itspplication is limited to solanaceous species. However, it is alsoossible that a systematic study of inoculation conditions mayesult in a wider range of infected species. For example, it may beeasible to optimize the technique to include A. thaliana throughhe use of different buffers and abrasive compounds, which aremportant for mechanical transmission of a tospovirus in peanutMandal et al., 2001). The use of celite instead of carborundumas also been proposed (Gilbertson et al., 1993; Sunter et al.,001).

Some geminiviruses are found only in cells associated withhe plant phloem tissue but others are distributed among manyells throughout the leaf and stems. There is not a clear rela-ion between the ability to mechanically inoculate a geminivirusnd its preferential location. The geminiviruses (TGMV, CaL-uV and ACMV) that are readily inoculated using the DNAbrasion method are not phloem-limited. Squash leaf curl virus,phloem limited virus, is mechanically inoculated with very

ow efficiency. It is likely that the primary cell targets of theNA abrasion protocol are mesophyll cells. Hence, a prerequi-

ite for success of the method may be the ability of the inoculatedirus to infect mesophyll cells and move to the vascular cellsor translocation through the phloem and establishment of sys-emic infection. Phloem-limited viruses may be precluded by theature of the inoculation to reach susceptible cells and to developymptoms. However, the lack of success of the DNA abrasionechnique in A. thaliana is not due to tissue tropism becauseaLCuV is not limited to the phloem in A. thaliana (Ascencio-

banez et al., in preparation). In addition, Maize Streak Virus ispread throughout mesophyl cells but cannot be mechanicallyransmitted. It is more likely that a combination of host/virusactors influence the success of this technique.

An especially interesting application of DNA abrasion inoc-lation technology is the silencing of genes using geminiviralectors (Muangsan and Robertson, 2004). This technologyllows the study of gene function by inhibition of transcrip-ion and/or translation in vivo and the testing of genes that aressential for plant survival without having to generate geneticnock-outs, which are often not viable. Silencing is effective

n even highly attenuated virus infections (Peele et al., 2001).herefore, it is likely that the requirements to induce silenc-

ng will be less stringent than the requirements for full-blownymptom development and may allow the use of DNA abra-

K

Virological Methods 142 (2007) 198–203

ion for silencing in host/virus combinations that have not beenreviously successfully used for wild-type infections.

cknowledgments

This work was supported by National Science Foundationrant MCB0110536 to Linda Hanley-Bowdoin. JTA-I thanksr. Rafael Rivera-Bustamante for his support in performing theNA abrasion inoculations on C. annuum, N. physalodes and. tabacum at Cinvestav’s facilities, Irapuato, Mexico. We arelso grateful to Dr. Niki Roberston for providing the silencingectors and Dr. Linda Hanley-Bowdoin for providing laboratoryacilities, guidance and for the critical reading of this manuscript.

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Global Analysis of Arabidopsis Gene ExpressionUncovers a Complex Array of Changes ImpactingPathogen Response and Cell Cycle duringGeminivirus Infection1[W][OA]

Jose Trinidad Ascencio-Ibanez*, Rosangela Sozzani2, Tae-Jin Lee, Tzu-Ming Chu, Russell D. Wolfinger,Rino Cella, and Linda Hanley-Bowdoin

Departments of Molecular and Structural Biochemistry (J.T.A.-I., L.H.-B.) and Horticultural Science (T.-J.L.),North Carolina State University, Raleigh, North Carolina 27695; Department of Genetics and Microbiology,University of Pavia, 27100 Pavia, Italy (R.S., R.C.); and SAS Institute Inc., Cary, North Carolina 27513–2414(T.-M.C., R.D.W.)

Geminiviruses are small DNA viruses that use plant replication machinery to amplify their genomes. Microarray analysis ofthe Arabidopsis (Arabidopsis thaliana) transcriptome in response to cabbage leaf curl virus (CaLCuV) infection uncovered 5,365genes (false discovery rate ,0.005) differentially expressed in infected rosette leaves at 12 d postinoculation. Data miningrevealed that CaLCuV triggers a pathogen response via the salicylic acid pathway and induces expression of genes involved inprogrammed cell death, genotoxic stress, and DNA repair. CaLCuV also altered expression of cell cycle-associated genes,preferentially activating genes expressed during S and G2 and inhibiting genes active in G1 and M. A limited set of core cellcycle genes associated with cell cycle reentry, late G1, S, and early G2 had increased RNA levels, while core cell cycle geneslinked to early G1 and late G2 had reduced transcripts. Fluorescence-activated cell sorting of nuclei from infected leavesrevealed a depletion of the 4C population and an increase in 8C, 16C, and 32C nuclei. Infectivity studies of transgenicArabidopsis showed that overexpression of CYCD3;1 or E2FB, both of which promote the mitotic cell cycle, strongly impairedCaLCuV infection. In contrast, overexpression of E2FA or E2FC, which can facilitate the endocycle, had no apparent effect.These results showed that geminiviruses and RNA viruses interface with the host pathogen response via a commonmechanism, and that geminiviruses modulate plant cell cycle status by differentially impacting the CYCD/retinoblastoma-related protein/E2F regulatory network and facilitating progression into the endocycle.

Geminiviruses are a large, diverse family of plant-infecting viruses that cause serious crop losses world-wide (Rojas et al., 2005). They have singled-strandedDNA genomes that are transcribed, replicated, andencapsidated in the nuclei of infected cells. They alsotraffic within and between cells and move systemicallythrough infected plants. Geminiviruses usurp plantenzymes and metabolites for these processes, whiletheir hosts mount defense responses to limit infection.Studies of plant RNAviruses showed that viral infectionis accompanied by many changes in the plant tran-

scriptome (Whitham et al., 2003; Marathe et al., 2004;Yang et al., 2007). Earlier studies showed that gemini-viruses induce the accumulation of a host replicationprotein (Nagar et al., 1995) and a protein kinase (Kongand Hanley-Bowdoin, 2002), indicating that plant DNAviruses also alter host gene expression. However, unlikeRNA viruses that encode replicases to amplify theirgenomes, geminiviruses represent a unique opportunityto study changes in the expression of host genes in-volved in cell cycle regulation and DNA replication aswell as the induction of the pathogen response.

The Geminiviridae is classified into four genera based ongenome structure, insect vector, and host range (Fauquetet al., 2003). Cabbage leaf curl virus (CaLCuV), a member ofthe Begomovirus genus (Hill et al., 1998), encodes sevenproteins, including two viral replication proteins desig-nated as AL1 (AC1, C1, or Rep) and AL3 (AC3, C3, orREn), and does not specify a DNA polymerase. Instead, itdepends on host DNA replication machinery to amplifyits small, circular genome via a combination of rollingcircle replication (RCR) and recombination-mediatedreplication (RDR). This dependence on host machineryconstitutes a barrier to infection of mature plant cells,which have exited the cell cycle and no longer supportDNA replication (for review, see Hanley-Bowdoin et al.,

1 This work was supported by the National Science Foundation(grant nos. MCB–0110536 and DBI20421651 to L.H.-B.) and byMiUR (grant no. 2006057940 to R.S.).

2 Present address: Department of Biology, Duke University, Dur-ham, NC 27708.

* Corresponding author; e-mail jtascenc@ncsu.edu.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Jose Trinidad Ascencio-Ibanez (jtascenc@ncsu.edu).

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

scription.www.plantphysiol.org/cgi/doi/10.1104/pp.108.121038

436 Plant Physiology, September 2008, Vol. 148, pp. 436–454, www.plantphysiol.org � 2008 American Society of Plant Biologists

2004). To overcome this constraint, the geminivirus AL1protein binds to the host retinoblastoma-related protein(RBR) and relieves repression of E2F transcription factors.This, in turn, allows activation of genes required fortransition into S phase and establishment of a DNAreplication-competent environment (Egelkrout et al.,2001, 2002; Desvoyes et al., 2006).

Interactions between geminivirus proteins andother host factors are also likely to impact plant geneexpression mechanisms. The binding of AL3 to a NACtranscription factor enhances viral DNA replication(Selth et al., 2005), while interactions between AL1 anda putative mitotic kinesin or histone H3 might con-tribute to the altered chromosomal structure charac-teristic of infected cells and indirectly influence hostgene expression (Bass et al., 2000; Kong and Hanley-Bowdoin, 2002). The viral AL2 protein binds to adeno-sine kinase to suppress host gene silencing (Wanget al., 2005), while interactions between the viralnuclear shuttle protein BR1 and the nuclear acetyl-transferase AtNSI may prevent DNA modificationsthat interfere with replication and transcription(Carvalho et al., 2006). In addition, both AL1 and AL2function as transcriptional regulators of viral genes andmight impact the activities of yet-to-be-identified hostgenes (Eagle et al., 1994; Sunter and Bisaro, 1997).

Geminiviruses could also influence host gene ex-pression by altering signal transduction pathwaysthrough interactions with host protein kinases. Re-duced activity of a SNF1-related kinase (SnRK1) inresponse to AL2 binding has been implicated in hostsusceptibility to infection (Hao et al., 2003). The BR1protein binds to NIK1, NIK2, and NIK3, members ofthe Leu-rich repeat-receptor-like kinase (RLK) family,and inhibits their phosphorylation and antiviral activ-ities (Fontes et al., 2004; for a list of gene acronyms anddescriptions, see Supplemental Table S1). In contrast,interaction and phosphorylation of BR1 by a PERK-like RLK, NsAK, is necessary for efficient infection andfull symptom development (Florentino et al., 2006).AL1 binding to GRIK1 and GRIK2, which accumulatein infected cells (Kong and Hanley-Bowdoin, 2002),may modulate their proposed dual roles in controllingprecursor and energy resources needed for DNAreplication and activation of SnRK1 and the pathogenresponse (Shen and Hanley-Bowdoin, 2006). The di-vergent AL4 and C4 proteins may alter cell signalingthrough their interactions with two members of theshaggy protein kinase-related family involved in bras-sinosteroid signaling (Piroux et al., 2007).

The diverse interactions and activities of the viralproteins suggest that geminiviruses modulate a vari-ety of plant processes by altering host gene expression.Serial analysis of gene expression of cassava mosaicdisease annotated 30 differentially expressed genesencoding proteins associated with systemic acquiredresistance, a b-tubulin, and a WRKY transcriptionfactor among 12,786 sequenced tags (Fregene et al.,2004). Differential display analysis of Capsicum an-nuum response to Pepper huasteco yellow vein virus iden-

tified two clones encoding a methyltransferase and anNADP-malic enzyme (Anaya-Lopez et al., 2005). Amicroarray study of Arabidopsis (Arabidopsis thaliana)cultured cells expressing the AC2 proteins of Mung-bean yellow mosaic virus (MYMV) or African cassavamosaic virus (ACMV) identified 139 genes that wereelevated by both viral proteins (Trinks et al., 2005). Allof these studies used resistant virus/host combina-tions or focused on differences between resistant andsusceptible infections. The best-characterized exampleof host gene expression change during a compatiblegeminivirus infection is activation of the Nicotianabenthamiana PCNA gene in response to CaLCuV ortomato golden mosaic virus infection (Egelkrout et al.,2001; Egelkrout et al., 2002).

Our limited knowledge of host gene expression dur-ing geminivirus infection in planta is due in part to theabsence of a well-characterized, compatible virus/hostsystem suitable for transcriptome profiling studies. Thislimitation is compounded by the technical challenge ofdetecting changes that occur in only a small fraction ofvirus-positive cells. To address these constraints and togain new insight into geminivirus/host interactions, weestablished a carefully controlled experimental systembased on CaLCuV and its susceptible host Arabidopsisto examine global changes in host gene expressionduring infection. Using this system, we showed thatCaLCuV infection alters the expression of a large num-ber of plant genes involved in diverse processes rang-ing from the pathogen response and programmed celldeath to DNA replication and cell cycle control.

RESULTS

CaLCuV Infection of Arabidopsis Ecotype Columbia

For the gene profiling studies, we implemented aninfection protocol for CaLCuV in its compatible hostArabidopsis ecotype Columbia (Col-0) that minimizedsecondary effects due to development and environ-ment. Plants were grown under short-day conditions(8-h/16-h photoperiod) to produce a large number ofrosette leaves and prevent flowering during the courseof the experiment. This approach ensured adequate leafmaterial for RNA extraction and minimized any devel-opmental effects associated with the vegetative to floraltransition that would have complicated the analysis.Previous experiments showed that geminivirus-mediatedactivation of PCNA expression was readily detected inplants inoculated by agroinfection but not by bom-bardment (Egelkrout et al., 2001). Hence, Arabidopsisplants at the 16- to 18-leaf stage were agroinoculatedwith partial tandem dimers of CaLCuV and monitoredfor symptom development over time.

CaLCuV symptoms began to appear at 9 to 10 dpostinoculation (dpi) with all plants showing symp-toms by 12 dpi (Fig. 1A). A general yellowing associ-ated with agroinfection disappeared at least 3 d beforethe onset of viral symptoms, which were characterized

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by overall stunting and chlorosis. Upper leaves weresmaller, narrower, and curled and displayed a reticu-lated chlorosis beginning at the base and expandingthrough the blade as infection progressed. Lowerleaves were thicker and darker and showed a moder-ate upward curvature and helical twisting. Mock-inoculated plants showed normal leaf developmentand no visible effect of agroinoculation at 12 dpi (Fig. 1B).Viral DNA accumulation was examined in leaves 5 to 20and in a pool comprised of the four uppermost visibleleaves (1–4) from a single infected Arabidopsis plant at12 dpi. DNA gel blotting detected double- and single-stranded forms of CaLCuV DNA in all the leaves (Fig.1E, lanes 1–11), with the highest amounts in leaves 7, 8,and 9 (lanes 4, 5, and 6). Leaves 6 and 10 containedintermediate amounts of viral DNA (Fig. 1E, lanes 3 and

7), while leaves 1 to 4, 5, 12, and 14 had lower levels(lanes 1, 2, 8, and 9). Leaves 16 and 20 had trace amountsof CaLCuV DNA (Fig. 1E, lanes 10 and 11). Similardistributions of viral DNA were seen in two other plants(data not shown).

Expression Profiling of CaLCuV-InfectedArabidopsis Leaves

We used the Affymetrix ATH1 GeneChip to com-pare the expression profiles of mock-inoculated andCaLCuV-infected plants at 12 dpi, the earliest timeafter inoculation at which we could reliably identifyinfected leaves. At this time, leaves 7, 8, and 9contained the highest amounts of viral DNA andwere larger than 1 cm in length (Fig. 1E). Leaves

Figure 1. CaLCuV infection differentially impacts Arabidopsis gene expression across functional categories. CaLCuV symptomsin Arabidopsis Col-0 rosette leaves at 12 dpi are shown in A. The arrows mark the size of leaves harvested for RNA extraction. Themock-inoculated plant at 12 dpi is shown in B. Immunolocalization of the AL1 protein in leaf 9 of a CaLCuV-infected plant at 12dpi can be observed in C. D, Control section from an equivalent, mock-inoculated leaf. Endogenous peroxidase activity is seen invascular tissue (rectangle). Total DNA (0.5 mg) from a CaLCuV-infected plant at 12 dpi was analyzed by DNA gel blotting using a32P-labeled probe corresponding to full-length CaLCuV A DNA. In E, the DNA samples were from a pool of leaves 1 to 4 (lane 1)or individual leaves (lanes 2–11). The leaves are numbered according to their positions relative to the shoot apex from theyoungest (1–4, lane 1) to the oldest (20, lane 11). The bands corresponding to double-stranded (ds) and single-stranded (ss) formsof CaLCuV A are identified on the left. The leaves used for the microarray experiments are marked at the top (sample). A pie chartshowing the distribution of the differentially expressed genes across functional categories defined by the TAIR7 GO is depicted inF. The numbers are the percentage of genes in each category differentially expressed during infection. Interval analysis of selectedfunctional groups can be seen in G. Genes were ordered according to their q values and grouped into intervals of 1,000. Thefrequencies of selected functional classes normalized to their overall representation in the GO database were plotted for fiveintervals corresponding to the 5,000 genes with the lowest P values. The abiotic/biotic stimulus is represented by triangles, thestress response by diamonds, DNA/RNA metabolism by circles, and transcription by squares. The colors of the lines are the sameas in F. The black line corresponds to the mean representation of all GO categories for each interval.

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greater than 1 cm in length contain few cells under-going DNA replication, while leaves smaller than1 cm contain significant numbers of replicating cells(Donnelly et al., 1999). Based on these observations,we hypothesized that viral effects on host gene ex-pression would be maximal in leaves 7, 8, and 9,while leaf developmental effects related to DNAreplication and the cell division cycle would be min-imal. We also took into account that at a given thermaltime, the expanded leaves are equivalent to the leavesdescribed by Donnelly et al. (1999); therefore, leavesbigger than 1 cm will be suitable for sampling(Granier et al., 2002).

Arabidopsis plants were agroinoculated withCaLCuV or mock inoculated with a control strain toeliminate effects due to Agrobacterium infection. Foreach sample, leaves 7, 8, and 9 from at least six plantsat 12 dpi were pooled to minimize plant-to-plantvariation. Three independent sets of plants were sam-pled to generate three biological replicas. Total RNAfrom the leaf samples was converted to cRNA for useas target in microarray experiments. The six targetsamples were hybridized, processed, and scanned inparallel, and the consistency of the perfect match datawas inspected by MA plots (data not shown). LOESSnormalization using the mean across the chips as thebaseline (Dudoit and Fridlyand, 2002) was applieddirectly to the probe data because of its higher repro-ducibility (Chu et al., 2006). After normalization, thecorrelation coefficients of perfect match probes be-tween chips ranged from 0.89 to 0.98, with a meanvalue of 0.93 (data not shown). The expression datawas analyzed by an ANOVA model (Chu et al., 2002)using JMP Genomics (http://www.jmp.com/software/genomics/index.shtml), and the resulting P values wereused to determine a false discovery rate (Storey andTibshirani, 2003). Using a q value ,0.005 (P value,0.002), we identified 5,365 RNAs that are differentiallyexpressed in CaLCuV-infected versus mock-inoculatedArabidopsis in our experimental system. Of these, 3,004RNAs were elevated and 2,361 were reduced in infectedleaves (Supplemental Table S2). The microarray resultswere validated for 30 of the differentially expressedgenes (q values from 0.0031–4.0 3 10229) and for sixgenes with q values .0.005. The ATH1 results wereconfirmed for 34 of the 36 genes examined (Supplemen-tal Fig. S1).

Functional Categorization of DifferentiallyExpressed Genes

As a first step toward characterization of the 5,365differentially expressed genes, we used the TAIR7Gene Ontology (GO) descriptions to categorize thegenes by biological process. All GO biological processcategories were represented among the significantgenes, with the number ranging from 66 to 2,552across the various processes (Fig. 1F). Categoriescorresponding to abiotic/biotic stimuli (33%) andstress response (31%) were overrepresented, while

the categories for DNA/RNA metabolism (16%) andtranscription (15%) were underrepresented.

To gain insight into the differences between cate-gories, the genes in order of their q values weregrouped into bins of 1,000. The bins were classifiedby GO biological processes and normalized relative tothe total number of annotations in each category. Theresults for the bins with the 5,000 lowest q values areshown for selected categories in Figure 1G. This anal-ysis revealed that genes annotated in the abiotic/bioticstimulus (dark red) and stress response (orange) cat-egories are highly represented in the first three bins,while genes annotated in the DNA/RNA metabolism(blue) and transcription (green) categories are poorlyrepresented in the same bins. In both cases, the repre-sentations diverge from the overall mean of all cate-gories in each bin (black line). The normalizedfrequencies for the four categories are more similar inbins 4 and 5 and converge on the overall mean (blackline). Our analysis is consistent with more robust and,hence, more readily detected expression changes forgenes in the overrepresented versus the underrepre-sented categories. The uneven distribution of the differ-ent categories may reflect different aspects of theinfection process, with the overrepresented categoriesincluding a large fraction of systemic events and theunderrepresented categories containing a large propor-tion of events that are cell autonomous with CaLCuVpresence. This idea is supported by studies showing thatthe host factors PCNA and GRIK, which are expressed inyoung tissues of healthy plants, accumulate specificallyin virus-positive cells of mature leaves during infection(Nagar et al., 1995; Kong and Hanley-Bowdoin, 2002).

Earlier studies showed that the AL1 protein is anearly marker for geminivirus infection and can bedetected prior to viral DNA at the cellular level (Nagaret al., 2002). To determine the relative fraction ofCaLCuV-positive cells under our infection condition,we used a polyclonal antiserum to immunolocalizeAL1 in infected Arabidopsis leaves at 12 dpi. The AL1protein was readily detected in cross sections of leaf 9(Fig. 1C), consistent with high viral DNA level in thisleaf (Fig. 1E). The viral replication protein was seenmost often in cells adjacent to vascular tissue but wasalso found in the occasional mesophyll cell and therare epidermal cell. In all cases, AL1 was detected innuclei, which showed quenching of 4#,6-diamidino-2-phenylinodole (DAPI) staining by the peroxidase pre-cipitate (data not shown). Mock-inoculated leaves hadbackground staining in the vasculature, but no nuclearsignals were seen in any leaf cell types (Fig. 1D). Thenumber of infected cells varied across sections but wasno more than 10% of the total number of cells. Similarresults were obtained when in situ hybridization wasused to detect viral DNA (data not shown). Based onthese results, we reasoned that changes in gene ex-pression associated with cell-autonomous eventswould be diluted at least 10-fold, making them moredifficult to detect than systemic changes that are notconfined to virus-positive cells.

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CaLCuV and RNA Viruses Alter the Expression of a

Common Set of Genes

Although plant DNA and RNA viruses use differentreplication and expression strategies, many aspects oftheir infection cycles are related and may lead tosimilar changes in host gene expression. We comparedthe CaLCuV infection profile with the profiles of plantsinoculated with Turnip mosaic virus (TuMV; Yang et al.,2007) or Cucumber mosaic virus-Y (CMV-Y; Maratheet al., 2004) in Figure 2A. The TuMV study was in thesusceptible Arabidopsis Col-0 ecotype, while theCMV-Y study was in the resistant C24 ecotype. Com-parison of the CaLCuV profile uncovered greateroverlap with the TuMV infection profile (267/556)than the CMV-Y resistome (107/444), but both com-parisons also identified many genes with oppositeexpression (TuMV, 72/556; CMV-Y, 77/444). In con-trast, comparison to a common set of genes differen-tially expressed in response to five different RNAviruses (including TuMV) representing four families(Whitham et al., 2003) uncovered 63 of 102 similarlyexpressed genes and only three with opposite expres-sion during CaLCuV infection (Supplemental TableS3). Together, these comparisons indicated that a sub-set of the host genes differentially regulated duringvirus infection is common to both DNA and RNAviruses (Fig. 2A).

The only published microarray study for geminivi-ruses examined the impact of expressing individualviral proteins in suspension cells (Trinks et al., 2005).The RNA profiles of Arabidopsis cells transientlyexpressing MYMV or ACMV AC2 at 8 h posttransfec-tion (Trinks et al., 2005) did not show a strong rela-tionship to the expression profile of CaLCuV-infectedleaves, with only 18% to 20% of the RNAs showingsimilar trends and 21% to 24% showing oppositetrends. It was also reported that overexpression ofMYMV AC1 altered expression of 259 RNAs, but wecould not compare these to our results, because theelevated mRNAs were not identified. However, thereported lack of overlap between the AC1 and AC2datasets is consistent with the observation that manymore RNAs are differentially expressed in infectedleaves than in cells expressing individual viral pro-teins. The low correlation between the two experimen-tal systems may also reflect the use of different virus/host combinations (natural host versus nonhost), tim-ing of analysis (12 dpi versus 8 h), and greater dilutionof cell-autonomous changes in intact plants versuscultured cells.

Pathogen Response Genes

Plants respond to pathogens via the salicylic acid(SA), jasmonic acid (JA), and ethylene (ET) pathways.RNA viruses typically activate the SA pathway(Whitham et al., 2006), but equivalent information isnot available for geminiviruses. The gene profilingdata showed that PR1, PR2, and PR5 transcripts,

which are markers for the SA response (Pieterse andVan Loon, 2004), were elevated during CaLCuV infec-tion (Fig. 2B). Genes specifying SA biosynthetic en-zymes and signaling components (EDS1, PAD4,SAG101, FMO1, ALD1, SID2, EDS5, NPR1, NPR2,NPR3, and NPR4) as well as downstream transcriptionfactors (WRKY70, TGA1, TGA3, and TGA5) also hadincreased RNA levels (Pieterse and Van Loon, 2004;Wiermer et al., 2005). In contrast, transcripts for someJA/ET markers (JR1, VSP1, PDF1.2, and CLH1) werereduced, whereas others (GST1, b-CHI, and HEL) wereup in infected leaves (Lorenzo and Solano, 2005).RNAs encoding components of the JA pathway weredown (FAD3, FAD7, AOS, ACO1, OPR3, SSI2, andMYC2) or showed no change on the arrays (COI1 andJAR1, although JAR1 transcripts were reduced in re-verse transcription [RT]-PCR reactions; Lorenzo andSolano, 2005; Beckers and Spoel, 2006). The mRNAsfor EIN2 and EIN5, which are associated with the ETpathway (Etheridge et al., 2006), were up, possiblyleading to the downstream activation of JA late re-sponse genes.

We used semiquantitative RT-PCR to verify induc-tion of the SA pathway and down-regulation of theET/JA pathways. These experiments confirmed themicroarray data for selected RNAs (Supplemental Fig.S1A; EDS1, PAD4, NPR2, FAD3, PR1, and PDF1.2). Themicroarray experiments failed to detect a significantchange in JAR1 transcripts even though RT-PCR indi-cated that they were reduced in four independentRNA samples from infected leaves. Based on theseresults, we included JAR1 among the down-regulatedgenes. FAD8, which was not represented on the array,was also down in infected versus mock-inoculatedtissues (Supplemental Fig. S1A), and COI1-inducedgenes were reduced in CaLCuV microarray data (datanot shown), lending further support for repression ofthe JA pathway. In addition, we examined the expres-sion of a general set of defense response genes in-volved in oxidative stress and cell wall metabolism byquantitative-RT PCR using a commercial kit (Supple-mental Fig. S1C). These results paralleled the micro-array data but showed a greater dynamic range.Among the analyzed RNAs, only PDF1.2 transcriptswere reduced during CaLCuV infection. Transcriptsspecifying thioredoxins, glutathione S-transferases,and b-glucanases were represented among the ele-vated transcripts in CaLCuV-infected leaves. The cat-alase and invertase RNAs, which showed the greatestchanges in the microarray experiments, also displayedstrong differences in mock and infected leaves bysemiquantitative RT-PCR.

We tested a number of Arabidopsis lines carryingmutations in various pathogen response genes inCaLCuV infectivity assays. These experiments didnot uncover any differences with respect to timing orseverity of symptoms between SA (pad4, mpk4, sid2-2,npr1, and wrky70), JA (coi1-16), or ET (ein2) pathwaymutants and wild-type Col-0 plants (data not shown).Although the aggressive nature of CaLCuV infection

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Figure 2. Pathogen response during CaLCuV infection. Genes differentially expressed during CaLCuV infection were comparedto the expression profiles of Arabidopsis genes that change in response to other plant pathogens or pathogen proteins. A, Thecomparisons include: (1) genes differentially expressed during the compatible TuMV infection (Yang et al., 2007); (2) genesdifferentially expressed during the resistance response to CMV-Y (Marathe et al., 2004); (3) transcripts elevated by five RNAviruses in planta (Whitham et al., 2003); and (4) transcripts increased in cultured cells expressing MYMV or ACMV AC2 protein(Trinks et al., 2005). The numbers of shared genes that are up- or down-regulated in CaLCuV-infected leaves are indicated foreach comparison. The totals for each treatment were adjusted to include only genes represented on the ATH1 array. B, A model ofthe interactions between key genes involved in the SA (orange), JA (blue), and ET (yellow) pathogen response pathways. Thegenes are shown in their proposed order in the regulatory network or order of expression. Genes with elevated mRNA levels arein red typeface, while genes with reduced mRNA are in green typeface. The genes with red or black backgrounds are in F. Thecorresponding Arabidopsis gene numbers are in Supplemental Table S1. C, Infected Col-0 plant showing signs of senescence andcell death at 25 dpi is depicted. CaLCuV-inoculated cpr1 mutant (Bowling et al., 1994) at 25 dpi is shown in D, followed by amock-inoculated cpr1 mutant at 25 dpi (E). Graph in F shows the percentage of overlap of mRNAs that are increased (red arrow)or decreased (green arrow) during CaLCuV infection to genes with reduced (MPK4 1) or elevated (MPK4 2) transcripts in anmpk4 mutant (Andreasson et al., 2005), with reduced RNA in an eds1/pad4 mutant (EDS1/PAD4 1; Bartsch et al., 2006), or withincreased RNA in MKK3 (1)- or MKK5 (1)-overexpressing lines (Takahashi et al., 2007). The number of genes is indicated foreach bar.

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may have masked subtle differences, similar resultshave also been reported for RNA viruses, suggestingthat individual knockout mutations do not dramati-cally impact plant virus infection (Huang et al., 2005).In contrast, Arabidopsis cpr1 plants, which constitu-tively express PR1 (Bowling et al., 1994), were lesssusceptible to CaLCuV infection. CaLCuV-inoculatedcpr1 plants did not display symptoms at 25 dpi (Fig.2D), while Col-0 plants were highly symptomatic atthis time (Fig. 2C). The cpr1 plants eventually devel-oped weak symptoms (data not shown), indicatingthat up-regulation of the downstream components ofthe SA pathway impair but do not prevent CaLCuVinfection. A recent study also reported a delayedsymptom phenotype for cpr1 plants infected withCauliflower mosaic virus (CaMV), which has a double-stranded DNA genome (Love et al., 2007). Interest-ingly, CaMV induces Arabidopsis PR1, PR2, andPR5 expression during infection (Love et al., 2005)like CaLCuV. However, CaMV up-regulates PDF1;2expression while CaLCuV down-regulates its expres-sion, suggesting that are differences as well as paral-lels in the host responses to these two DNA virusfamilies.

Signaling and the Pathogen Response

EDS1 and PAD4 are key activators of the SA path-way (Wiermer et al., 2005), while the mitogen-activatedprotein kinase MPK4 is a negative regulator of theEDS1/PAD4 pathway (Brodersen et al., 2006). DuringCaLCuV infection, EDS1 and PAD4 transcripts wereelevated in both the microarray and RT-PCR analyses(Supplemental Fig. S1A). MPK4 RNA levels were alsoelevated in the microarray experiment, but this wasnot confirmed by semiquantitative RT-PCR of fourindependent RNA samples (Supplemental Fig. S1A).Comparison of our microarray data to expressionprofiles of Arabidopsis lines carrying knockout muta-tions in these regulators revealed that 87% of the genesdisplaying EDS1/PAD4-dependent expression (Bartschet al., 2006) and 67% of MPK4-repressed genes(Andreasson et al., 2005) showed enhanced expressionin CaLCuV-infected leaves (Fig. 2F). In contrast, 84%of the genes showing MPK4-dependent expression(Andreasson et al., 2005) were down. For both the eds1/pad4 and mpk4 mutant studies, many of the affectedgenes are known components of the SA pathway andamong the most significant genes differentially ex-pressed during CaLCuV infection (SupplementalTable S4). Together, these results showed that CaLCuVinfection activates the SA pathway through inductionof EDS1 and PAD4 and that MPK4 activity is sup-pressed posttranscriptionally during infection.

The mitogen-activated protein kinases MKK4,MKK5, and MPK6 are activated in response to path-ogen infection as part of the ET pathway, and MKK3and MPK6 regulate transcription in the JA pathway(Takahashi et al., 2007). MKK4, MKK5, and MPK6transcripts were elevated in CaLCuV-infected leaves,

while MKK3 RNA levels did not change. Comparisonof our microarray data to expression profiles of Arabi-dopsis lines that inducibly express MKK3 or MKK5(Takahashi et al., 2007) revealed that 55% of the genesactivated by MKK5 overexpression were differentiallyexpressed in CaLCuV-infected leaves, with the major-ity showing up-regulation (Fig. 2F). There was lessoverlap between the CaLCuV profile and MKK3-induced genes and the trend was generally opposite.These results are consistent with the activation of theMKK5 cascade and not the MKK3 cascade duringCaLCuV infection.

Senescence

Arabidopsis plants infected with CaLCuV at the 16-to 18-leaf stage display strong chlorosis and signs ofdesiccation at 25 dpi (Fig. 2C) and die approximately 6weeks postinoculation. Mock-inoculated plants do notshow signs of tissue death (data not shown) andflower during the same time frame. Because of itsextent and delay, the cell death cannot be attributed toinduction of a hypersensitive response and, instead,may reflect premature senescence or some other formof programmed cell death. To address this possibility,we identified several genes associated with senescencethat were differentially expressed during CaLCuVinfection (see Supplemental Table S5). We also com-pared the expression profiles of CaLCuV infection,natural leaf senescence (Buchanan-Wollaston et al.,2005), and Glc-induced senescence (Pourtau et al.,2006). The Venn diagram in Figure 3A shows that theprofiles of the three treatments overlap and share 77common genes. However, 82% (349/424) of the genesshared between CaLCuV and natural senescence wereabsent in the Glc treatment, while only 36% (42/117) incommon between CaLCuV and Glc-induced senes-cence were not included in the natural senescenceprofile. There was little overlap between the expressionprofiles during infection and nitrogen starvation-induced senescence (Pourtau et al., 2006; data not shown).

Hallmarks of leaf senescence include a decline inphotosynthetic capacity, nitrogen recycling, and pro-tein degradation (Lim and Nam, 2005). DuringCaLCuV infection, there was a general reduction inmRNAs encoding parts of the photosynthetic appara-tus, including both photosystems, light-harvestingcomplexes, and dark reaction enzymes, as well asenzymes involved in chlorophyll synthesis (see Sup-plemental Table S6). Genes (GDH2, GAD1, GLN1;3,and GLN1;4) specifying enzymes involved in nitrogenrecycling via Gln were more highly expressed ininfected tissues. Genes encoding components of theubiquitin-proteasome pathway also showed increasedtranscript accumulation (Smalle and Vierstra, 2004). Atotal of 32 genes specifying 11 core and 13 regulatorysubunits of the 26S proteasome complex had elevatedtranscripts. There were also increases in the mRNAsspecifying two of two E1 ubiquitin-activating en-zymes, eight of 37 E2 ubiquitin-conjugating enzymes,

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and 149 of 1,570 E3 ubiquitin ligases. The elevated E3transcripts represented eight of the 10 classes, withthe majority in the F-box, RING, and PHD groups(http://plantsubq.genomics.purdue.edu/plantsubq/html/master.html). Only 23 E3 ligase genes showedreduced mRNA levels.

We examined the involvement of the SA, JA, and ETsignaling pathways in induction of senescence-associatedgene expression during CaLCuV infection. The Venndiagram in Figure 3B compares senescence-associatedgenes that are not activated in NahG-expressing plantsor in ein2 or coi1 mutants (Buchanan-Wollaston et al.,2005). Of the 122 genes that are specifically activatedthrough the SA pathway (down in NahG plants), 72%had elevated transcripts in CaLCuV-infected leaves.The correlation was less for genes specifically activatedthrough the ET (ein2 mutant) and JA (coi1 mutant)pathways, which display 54% and 40% overlap, re-spectively, with the CaLCuV profile. When the inter-secting regions of the Venn diagram were included inthe comparisons, the SA pathway included 73% of thetotal number of senescence-associated genes induced inCaLCuV plants, and the SA and ET pathways together

comprised 90%. Transcripts for several senescencemarkers (PAP1, STP13, GLN1;4, and GPT2) not associ-ated with the SA or ET pathways were also enhancedduring CaLCuV infection, indicating that induction ofprogrammed cell death was not simply a consequenceof activating the pathogen response. In addition,mRNAs for some SA-responsive genes specificallyassociated with senescence like WRKY53 were in-creased during CaLCuV infection. In contrast, theWRKY53 repressor ESR, which is regulated throughthe JA pathway, was down during infection.

Genotoxic Stress and DNA Repair

The single-stranded and nicked viral DNA formsthat accumulate in infected nuclei may be perceived asdamaged DNA and induce a genotoxic stress response(Weitzman et al., 2004). To address this possibility, wecompared the CaLCuV infection profile with theprofiles induced by four treatments known to causegenotoxic stress (Chen et al., 2003; Molinier et al.,2005). UV-C radiation and mitomycin C induce theformation of nucleotide dimers, bleomycin (Blm) causesdouble-stranded DNA breaks (DSBs), and xylanase(Xyl) is a general fungal elicitor that induces an oxi-dative burst and the defense response. Comparison ofgenes represented on the two array platforms used forCaLCuV and genotoxic stress profiling revealed lim-ited overlap (UV-C, 201/732; Blm, 54/584; Xyl, 210/758 genes) as well as opposite effects on expression(UV-C, 51/732; Blm, 79/584; Xyl, 71/758). In contrast,comparison to a common set of genes differentiallyexpressed in response to all three treatments uncov-ered 105 of 198 similarly expressed genes and onlythree with opposite expression. A higher correlationwas also seen with a combined Blm/mitomycin Ctreatment, with 68 of 130 genes showing similar ex-pression and seven displaying opposite expression.Thus, CaLCuV infection alters the expression of a coreset of genes involved in response to genotoxic stress.However, the CaLCuV profile only shows weak over-lap (13 up and eight down) with a set of 67 transcriptsthat are increased by nine different types of stress(Swindell, 2006), indicating that infection does notactivate a general stress response.

Like all eukaryotes, plants have evolved multipleDNA repair pathways (Kimura and Sakaguchi, 2006).Base excision repair (BER), nucleotide excision repair(NER), and photoreactivation repair DNA cross-links,while nonhomologous end-joining and homologousrecombination repair DSBs. To gain insight into theimpact of geminivirus infection on the various repairpathways and DNA damage tolerance, we analyzedthe expression profiles of Arabidopsis genes involvedin these processes (Table I). CaLCuV infection andgenotoxic stress altered the expression of componentsof BER, NER, and DSB pathways but did not alterexpression of genes encoding the replication-associatedmismatch repair machinery or the photolyases in-volved in photoreactivation.

Figure 3. CaLCuV infection induces senescence. A, Genes differen-tially expressed during CaLCuV infection at 12 dpi compared to genesthat are up-regulated during natural senescence (Buchanan-Wollastonet al., 2005) and Glc-induced senescence (Pourtau et al., 2006). B,Genes differentially expressed during infection compared to genes (firstnumber in each section) that are expressed in response to SA (NahG),ET (ein2), or JA (coi1) during senescence (Buchanan-Wollaston et al.,2005). The numbers of mRNAs elevated during infection are given atthe top of the bracket, while the numbers of depressed transcripts aregiven below.

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Expression of genes involved in DSB repair duringCaLCuV infection is of particular interest because ofthe role of recombination in the geminivirus DNAreplication process (Jeske et al., 2001). Increased

RAD50 and KU80 mRNA levels in infected leaves(Table I) suggested that both the homologous recom-bination and nonhomologous end-joining pathwaysare activated during infection (Schuermann et al.,

Table I. Differentially expressed DNA repair genes

Bold type designates similar regulation during CaLCuV infection and genotoxic stress. No results areshown for genes not on the array. *, Plastid protein. nc, No change.

Gene Identifier Gene Name CaLCuV Genotoxic Stressa

Damage toleranceAt3g04880 DRT102 up ncAt1g23260 MMS1 upAt1g70660 MMS2 upAt2g39100 RAD18 nc up/downAt5g44740 RAD30 nc downAt4g35740 RECQ3 nc upAt1g10930 RECQ4A nc downAt1g67500 REV3 nc upAt1g52410 TSA1 downAt5g13930 CHS (TT4) down downAt3g55120 CFI (TT5) down up/downAt3g51240 F3H (TT6) downAt3g12610 *DRT100 downAt1g30480 *DRT111 nc upAt1g20340 *DRT112 down nc

BERAt4t02390 PARP1 up up/downAt2g31320 PARP2 up upAt1g80420 XRCC1 upAt2g41460 ARP nc upAt3g10010 DML2 (DNA glycosylase) upAt1g75090 DNA glycosylase downAt3g12710 DNA glycosylase downAt4g12740 DNA glycosylase downAt5g44680 DNA glycosylase downAt3g50880 *Base excision upAt1g21710 *OGG1 up nc

NERAt3g50360 CEN2 upAt4g37010 CEN2-like up upAt1g03750 CSB-like nc upAt2g18760 ERCC6/CSB up upAt1g79650 RAD23 upAt3g02540 RAD23-3 downAt5g38470 RAD23-4 upAt1g55750 TFB1-1 nc upAt1g55680 TFB1-2 up ncAt1g12400 TFB5 up

DSB repairAt4g21070 BRCA1 nc upAt3g22880 DMC1 up ncAt1g64750 DSS1(1) up ncAt5g45010 DSS1(V) upAt3g57300 INO80 upAt1g48050 KU80 upAt5g61460 MIM nc up/downAt5g66130 RAD17 upAt2g31970 RAD50 up ncAt2g28460 RAD51B nc upAt2g45280 RAD51C nc upAt1g10930 REC4QA down ncAt3g26680 SMN1 nc up

aMolinier et al. (2005).

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2005). Increased expression of DMC1, DSS1(1), andDSS1(V), which encode proteins that interact with theRAD51 recombinase, further supported activation ofrecombination during infection. None of the tran-scripts for the four Arabidopsis RAD51 genes waselevated during infection, even though RAD51B andRAD51C are activated by genotoxic stress. However,activation of DNA repair pathways may only occur incells that replicate the viral DNA and, as such, may bedifficult to detect.

DNA repair and replication depend on adequatedNTP pools and share many enzymes and factors(Kimura and Sakaguchi, 2006). During CaLCuV infec-tion, nine Arabidopsis genes encoding dNTP biosyn-thetic enzymes or DNA replication machinery (Shultzet al., 2007) were differentially expressed, while 10 weredifferentially expressed during genotoxic stress (TableII). The datasets share four genes: one encoding thePOLD3 subunit, two encoding RPA1 subunits, and oneencoding the TSO small subunit of ribonucleotide re-ductase. RPA, a trimeric single-stranded DNA bindingcomplex, has been implicated in all four eukaryoticrepair pathways as well as in DNA damage checkpointcontrol (Zou et al., 2006). POLD, the four-subunit repli-cative DNA polymerase, is involved in NER and mis-match repair (Hubscher et al., 2002). POLD expressionduring infection was also supported by up-regulation ofthe POLD4 gene specifying the smallest subunit of thepolymerase complex. Based on earlier studies showingthat CaLCuV infection induces transcription of thePCNA gene in N. benthamiana (Egelkrout et al., 2002),we anticipated that expression of the POLD4 clampwould also be elevated during infection. However, thegene profiling results indicated that transcripts for thePCNA (At1g07370) gene were reduced, while RNAcorresponding to the PCNA-like gene (At2g29570) didnot change in infected leaves. Semiquantitative RT-PCRconfirmed these results (Supplemental Fig. S1B). Down-regulation of the PCNA gene is consistent with itsexpression only in embryonic tissues (https://www.genevestigator.ethz.ch/). The lack of detectable up-regulation of the PCNA-likegene may reflect the difficultyin detecting cell autonomous events during infection,especially if such genes are expressed in a limited timeperiod during the process.

Because many of the plant genes encoding dNTPbiosynthetic enzymes and replication proteins are un-der cell cycle control, we compared the expression pat-terns of Arabidopsis core cell cycle genes (Vandepoeleet al., 2002; Menges et al., 2005) during infection andgenotoxic stress (Table II). This comparison uncoveredsimilar expression patterns for four core cell cyclegenes among the 12 and 18 genes that showed differ-ential expression during infection and DNA damage,respectively. CKL6 and CYCB1;1 mRNAs were up,while CYCD1;1 and CYL1 transcripts were down inboth cases. Up-regulation of CYCB1;1 expression dur-ing infection is associated with a q value of 7.5 3 1028,making it the most significant core cell cycle gene inour dataset. CYCB1;1 RNA levels are highly induced

by a variety of genotoxic stresses, suggesting thatactivation of CYCB1;1 expression is a general feature ofgenotoxic stress (De Veylder et al., 2007). The CaLCuVprofile did not show a change in expression for WEE1or its upstream activators ATR and ATM, which havebeen implicated in activation of Arabidopsis DNAcheckpoints in response to DNA damage (De Schutteret al., 2007).

Table II. Differentially expressed DNA replication and core cellcycle genes

Bold type indicates similar regulation during CaLCuV infection andgenotoxic stress. No results are shown for genes not on the array. nc, Nochange.

Gene Identifier Gene Name CaLCuV Genotoxic Stressa

DNA replicationAt1g44900 MCM2 nc up/downAt5g46280 MCM3 nc upAt5g44635 MCM5 downAt4g02060 MCM7 down ncAt2g37560 ORC2 nc upAt4g29910 ORC5 nc upAt1g07370 PCNA downAt1g78650 POLD3 up upAt1g09815 POLD4 upAt2g27120 POLE1 nc upAt1g19080 PSF3 upAt2g06510 RPA1 up upAt4g19130 RPA1 up upAt5g61000 RPA1 downAt3g07800 TK upAt3g27060 TSO2 up up

Core cell cycleAt1g76540 CDKB2;1 downAt1g66750 CDKD;2 upAt5g63370 CDKG:1 upAt5g44290 CKL5 up ncAt1g03740 CKL6 up upAt1g70210 CYCD1;1 down downAt4g34160 CYCD3;1 nc downAt5g67260 CYCD3;2 downAt5g65420 CYCD4;1 nc upAt5g10440 CYCD4;2 upAt1g77390 CYCA1;2 nc upAt5g25380 CYCA2;1 nc downAt4g37490 CYCB1;1 up upAt5g06150 CYCB1;2 downAt2g26760 CYCB1;4 nc downAt2g17620 CYCB2;1 nc downAt4g35620 CYCB2;2 nc upAt1g16330 CYCB3;1 nc downAt1g27630 CYCT;1 nc downAt4g34090 CYL1 down up/downAt3g12280 RBR ncAt2g36010 E2Fa nc upAt1g47870 E2Fc upAt3g48160 E2Fe downAt5g03455 CDC25 upAt1g02970 WEE1 nc upAt2g26760 KRP1 nc down

aCompiled from Chen et al. (2003) and Molinier et al. (2005).

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A limitation of the above comparisons is that thegenotoxic stress studies used array formats with fewerfeatures and, in some instances, different genes thanthose represented on the ATH1 array. As a conse-quence, it was only possible to compare expressionchanges for a subset of the DNA repair, DNA replica-tion, and core cell cycle genes shared by the arrayformats. Even with this constraint, the results indi-cated that while there was overlap between the ex-pression profiles, not all of the changes in the repairand cell cycle pathways during infection can be attrib-uted to genotoxic stress.

Cell Cycle Regulation

To gain further insight into modulation of host geneexpression leading to viral DNA replication, we com-pared the CaLCuV profile to those generated fromcultured Arabidopsis cells synchronized with aphidi-colin (Menges et al., 2003). Of the 1,081 genes thatshowed cell cycle regulation, 120 were up and 214were down during CaLCuV infection. Classification ofthe oscillating cell cycle-associated genes by peakexpression phase revealed that a higher proportionof S phase (35%) than M phase (26%) genes weredifferentially expressed during infection, while thepercentages of G1 (32%) and G2 (31%) phase geneswere intermediate (Fig. 4A). Genes with peak expres-sion in G1 and M were primarily down, while S andG2 phase genes tended to be up. The highest fractionof elevated transcripts was associated with S phase,while the greatest proportion of reduced RNAs wasassociated with M phase. These distributions are notcompatible with a general activation of the cell divi-sion cycle by geminivirus infection and, instead, indi-cated that infection specifically activates genes neededto establish a replication-competent environment andprevents expression of genes necessary for mitosis. Thisidea is supported by an earlier study showing that bothviral and plant chromosomal DNA replicate in infectedcells in the absence of proliferation (Nagar et al., 2002).

We identified transcripts for 12 core cell cycle genesthat differentially accumulate during infection in themicroarray experiments and confirmed their expres-sion patterns by semiquantitative RT-PCR (Supple-mental Fig. S1B). The same expression pattern wasalso detected at 9 dpi, which is prior to CaLCuVsymptom appearance, by quantitative RT-PCR (Sup-plemental Table S8). Ten of the differentially expressedcore cell cycle genes were grouped in Figure 4Baccording to when they are thought to act or aremaximally expressed during the cell cycle or reentry(Menges et al., 2005; De Veylder et al., 2007). For thisanalysis, we did not consider CDC25, which has anuncertain role in the plant cell division cycle, orCDKD;2, which functions primarily during transcrip-tion. It is also not known if CYL1 is involved in cellcycle control, although it is most highly expressedduring S phase (Menges et al., 2005). All three mRNAswere increased during infection.

Three core cell cycle genes that are enhanced uponcell cycle reentry (CDKG;1, CKL5, and CKL6) hadelevated transcripts in infected tissue (Fig. 4B), sug-gesting that CaLCuV induces quiescent cells to reenterthe cell division cycle. Because there is a generaldown-regulation of G1-associated genes during infec-tion (Fig. 4A), it is likely that infected cells only transitthrough late G1. This idea is supported by down-regulation of CYCD1;1 and CYCD3;2, which encodeearly activators of G1 (Masubelele et al., 2005; Dewitteet al., 2007). In contrast, transcripts corresponding tothe late G1 cyclin CYCD4;2 (Menges et al., 2005) wereincreased by infection. E2FC mRNA was also elevatedin infected leaves. We did not detect changes in theexpression of other components of the RBR/E2F net-work, which regulates transcription at the G1/Sboundary (Gutierrez et al., 2002).

CaLCuV infection has opposite effects on the ex-pression of CYCB1;1 and CDKB2;1, both of which

Figure 4. CaLCuV infection alters expression of cell cycle genes. Cellcycle-associated genes that are up-regulated (red arrow) or down-regulated (green arrow) in CaLCuV-infected leaves are grouped accord-ing to their peak expression phase (top) during the cell division cycle(Menges et al., 2003) can be seen in A. The total number of genesassociated with each phase that are differentially expressed duringinfection and the ratio of increased versus reduced transcripts areshown at the bottom. B, A model showing the core cell cycle transcriptsthat are higher (red) or lower (green) during CaLCuV infection isproposed. Each gene is positioned where it is thought to act or showspeak expression during the cell division cycle or during reentry(Menges et al., 2005). The stars mark three genes with reducedtranscripts in infected leaves and function at the indicated cell cyclestage. The Arabidopsis gene numbers for the genes in A and C are inSupplemental Table S1.

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promote mitosis and growth (De Veylder et al., 2007).Expression of CYCB1;1, which is induced upon cellcycle reentry and remains high throughout the cellcycle, is increased by infection. It has been proposedthat elevated CYCB1;1 expression leads to sequester-ing factors necessary for M phase and G2 arrest (DeVeylder et al., 2007). Consistent with this idea,CDKB2;1 expression, which peaks at the G2/M bound-ary, is reduced by infection (Fig. 4B). The CYCB1;1 andCDKB2;1 expression profiles resulting in G2 blockcorrelate with the absence of dividing cells and ena-tions and shut down of the meristem during infection(Fig. 1; J.T. Ascencio-Ibanez and L. Hanley-Bowdoin,unpublished data).

CaLCuV Infection and Endoreduplication

The asymmetric expression patterns observed forcell cycle-associated and core cell cycle genes sug-gested that CaLCuV infection specifically activates Sphase and inhibits M phase. This could be accom-plished by blocking transit into M phase or by by-passing M phase as part of an endocycle. Todistinguish between these possibilities, we asked ifinfected leaves are enriched for 4C cells indicative of aG2 block or contain a larger fraction of $8C cellsindicative of the endocycle. Efforts to isolate intactnuclei from plants inoculated at the 16- to18-leaf stagewere not successful, most likely because of the strongcell death phenotype. To minimize this effect, weinoculated older plants, which are less severely im-pacted by CaLCuV. These plants produced an inflo-rescence stem, allowing us to compare rosette andcauline leaves. FACS analysis of nuclei isolated fromboth leaf types of infected and mock-inoculated plantsat 28 dpi showed a small increase in ploidy levels ininfected versus control nuclei, with a 7% increase in8C, 16C, and 32C nuclei in rosette leaves and a 10%increase in cauline leaves (Fig. 5A). In both cases, theincrease in 8C, 16C, and 32C nuclei was accompaniedby a reduction in 4C nuclei. In two independentreplicates, rosette and cauline leaves showed 9% and12% increases in 8C, 16C, and 32C nuclei, respectively,and a concomitant reduction in 4C nuclei (data notshown). The numbers of 4C (P value 5 0.024) and $8C(P value 5 0.0041) nuclei in mock and infected leaveswere significantly different in a two-tailed Student’s ttest, but there was no significant difference for the 2Cnuclei (P value 5 0.68; Supplemental Table S8). The 7%to 12% increase in 8C, 16C, and 32C nuclei in infectedleaves is reminiscent of the small proportion of virus-positive cells in CaLCuV infected leaves (Fig. 1C),suggesting that the increase in ploidy is cell autono-mous with viral presence.

Several plant genes, including CDKB1;1, CDKA;1,CYCD2;1, CYCD3;1, CYCD3;2, CYCD3;3, CYCA2;3,E2FC, E2FE, KRP2, SIM, ILP1, FAS1, and CCS52A,influence the balance of mitotic and endoreduplicat-ing cells or the number of endocycles (Boudolf et al.,2004; Verkest et al., 2005; Churchman et al., 2006; Qi

and John, 2007; Ramırez-Parra and Gutierrez, 2007). Ofthose represented on the ATH1 array, E2FC and ILP1were elevated in infected leaves and CYCD3;2 wasreduced. ILP1 negatively regulates CYCA2 transcrip-tion (Yoshizumi et al., 2006), but we did not detectexpression changes for any of the four CYCA2 genesencoded by the Arabidopsis genome. Interestingly,E2FC and CYCD3;2 are part of the RBR/E2F regulatorynetwork, which is modulated by geminivirus infection(Hanley-Bowdoin et al., 2004).

CaLCuV Infection Is Differentially Impacted by E2FTranscription Factors

To gain insight into the impact of geminivirus in-fection on the RBR/E2F network, we compared genesup- or down-regulated by ectopic expression of E2FAand its DPA partner in Arabidopsis (Vlieghe et al.,2003). This comparison uncovered 107 genes (28%)that showed similar trends and 39 (6%) that showedopposite trends in infected leaves (Fig. 5B). We thenexamined the expression of Arabidopsis genes withpredicted E2F sites in their promoters (Vandepoeleet al., 2005). This analysis revealed that 24% of the 5,861putative E2F target genes were differentially expressedin CaLCuV-infected leaves (data not shown). Together,these results suggested that geminivirus infection im-pacts the expression of a subset of E2F-regulated genes,including those controlled by E2FA/DPA.

We also asked if overexpression of E2FA, E2FB, orE2FC, the three Arabidopsis E2F family members thatbind to RBR (Mariconti et al., 2002), impacts CaLCuVinfection. Plants ectopically expressing E2FA or E2FCdeveloped severe symptoms at the same time as wild-type plants and did not flower (data not shown). Incontrast, E2FB-overexpressing plants showed verymild symptoms in floral tissues after a significantdelay, while none of the E2FA and E2FC plants un-derwent the vegetative to floral transition. Viral DNAaccumulation was readily detected by tissue printingof rosette leaves of E2FA and E2FC plants but not in theleaves of E2FB plants (Fig. 5C). The E2FB resistancephenotype was observed when plants were inoculatedby bombardment or agroinfection, ruling out anyeffect due to the inoculation procedure.

We examined the ploidy distributions of uninfectedrosette leaves from the E2FA-, E2FB-, and E2FC-overexpressing lines. FACS analysis was performedon nuclei from mature leaves of plants with 16 to 18true leaves. The plants were at the same developmen-tal stage and grown under the same conditions asthose used for the microarray experiments. The FACSprofiles detected similar numbers of 8C, 16C, and 32Cnuclei in wild type (27%), E2FA (26%), and E2FC (24%)leaves (Fig. 5D). In contrast, only 12% of the nucleiisolated from E2FB leaves had ploidy values $8C.Based on these results, one possibility is that E2FBplants are resistant to CaLCuV infection because of areduced capacity to undergo endocycling and supportviral replication.

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The cyclin D family plays a key role in regulating theE2F/RBR pathway by activating cyclin-dependentkinase (CDK)-mediated phosphorylation of RBR anddisrupting its interactions with E2F (De Veylder et al.,2007). Hence, we asked if overexpression of CYCD3;1(Dewitte et al., 2003) or loss of CYCD3 function(DeWitte et al., 2007) impacts CaLCuV infection. Plantscarrying mutations in CYCD3;1, CYCD3;2, andCYCD3;3 genes developed severe symptoms by 12dpi, while plants overexpressing CYCD3;1 showed noevidence of disease even at later times (data notshown). High levels of viral DNA were detected inrosette leaves of cycd3 mutants at 22 dpi by tissueprinting (Fig. 5E). In contrast, only trace amounts ofviral DNA were observed in leaves from CYCD3;1-overexpressing plants. Quantitative RT-PCR showedthat the ratios of PR1 transcripts in E2FB- andCYCD3;1-overexpressing plants to wild-type plantswere 0.93 and 0.76, respectively, ruling out that the

resistance phenotypes reflected induction of pathogenresponse genes (Supplemental Table S8). Together,these results are consistent with CaLCuV replicatingin endocycling cells and the proposed role of theCYCD3 subfamily in promoting the mitotic cycle andinhibiting the endocycle (Dewitte et al., 2007).

DISCUSSION

Host-pathogen interactions involve a complex set ofevents that depend on the nature of the interactingpartners, developmental stage, and environmentalsignals. In plants, pathosystems share a common setof responses that are intertwined with unique re-sponses characteristic of the specific host/pathogencombination. Analysis of the Arabidopsis transcrip-tome in response to CaLCuV infection showed thatlike RNA viruses (Whitham et al., 2006), plant DNA

Figure 5. Ploidy and infectivity studies of wild-type Arabidopsis and plants altered in the CYCD/RBR/E2F pathway. A, Ploidydistribution of nuclei from rosette and cauline leaves of mock-inoculated and infected Arabidopsis plants. The percentage of 2C,4C, 8C, 16C, or 32C nuclei in each FACS profile is indicated. The experiment was repeated twice with similar results (data notshown). B, A comparison of CaLCuV expression profile to the RNA profile of transgenic Arabidopsis overexpressing E2FA and itsDPA partner is presented (Vandepoele et al., 2005). The red and green arrows indicate increased and reduced transcripts,respectively. The number of genes represented by each arrow is indicated. C, Viral DNA in leaves from mock and CaLCuV-inoculated Col-0, E2FA-, E2FB-, or E2FC-overexpressing plants was detected by tissue printing at 12 d after bombardment. Theleaves were photographed prior to rubbing onto nylon membrane. D, Ploidy distribution of nuclei from untreated, mature rosetteleaves of Col-0, E2FA-, E2FB-, or E2FC-overexpressing plants is presented. The percentage of 2C, 4C, 8C, 16C, or 32C nuclei fromFACS profile is indicated. The experiment was repeated twice with similar results (data not shown). E, Viral DNA in leaves frommock and CaLCuV-inoculated plants carrying a triple knockout in CYCD3;1, 2, and 3 or overexpressing CYCD3;1 was detectedby tissue printing at 22 d after bombardment.

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448 Plant Physiol. Vol. 148, 2008

viruses activate the SA pathway leading to the expres-sion of PR genes and the induction of programmed celldeath. The general pathogen response is superimposedon expression changes in core cell cycle and DNAreplication/repair machinery due to the unique depen-dence of geminiviruses on host enzymes to amplifytheir genomes. The extent and complexity of the re-sponse also reflect the asynchronous nature of theCaLCuV infection in intact plants and contributionsby both systemic and cell-autonomous events.

A recurring theme in the CaLCuV expression data iscross talk between the various pathways. Increasedexpression of the EDS1/PAD4/SAG101 complex anddownstream genes encoding biosynthetic enzymes,transcriptional regulators, and PR proteins (Bartschet al., 2006) and suppression of the MPK4 signalingpathway (Andreasson et al., 2005) provided strongevidence for the activation of a pathogen responsethrough the SA pathway (Wiermer et al., 2005). Thisconclusion was further supported by the observationthat CaLCuV symptoms are strongly delayed andattenuated in cpr1 plants that constitutively expressPR1 (Bowling et al., 1994). The overlap between theCaLCuV expression profile and senescence-associatedgenes regulated by SA also implicated SA signalingin induction of programmed cell death (Buchanan-Wollaston et al., 2005). However, the CaLCuV expres-sion profile suggested that regulation of the pathogenresponse and senescence during infection is morecomplex. Although the expression of genes encodingJA biosynthetic enzymes was reduced, consistent withthe ability of the SA pathway to suppress JA signaling(Beckers and Spoel, 2006), selected JA marker tran-scripts were elevated. This observation is indicative ofcross talk between the JA and ET pathways, whichshare the ERF transcription factors, (Lorenzo andSolano, 2005), and is compatible with activation ofMKK5 but not the MKK3 cascade (Takahashi et al.,2007) during infection. As observed for the SA pathway,there was significant overlap between the CaLCuVexpression profile and senescence-associated genesregulated by EIN2 (Buchanan-Wollaston et al., 2005),implicating ET signaling in programmed cell deathas well as the pathogen response.

There was also cross talk between the genotoxicresponse, the cell cycle changes, and the programmedcell death phenotype associated with CaLCuV infec-tion. CYCB1;1 is expressed in proliferating cell popu-lations of developing leaves (Beemster et al., 2005), inresponse to DNA damage (Chen et al., 2003), and inCaLCuV-infected leaves. RAD17, which increasedtranscript levels during infection, has been implicatedin checkpoint control and shown to be necessary forDSB repair in Arabidopsis (Heitzeberg et al., 2004).Recent animal studies linked activation of the DNAdamage checkpoint, aberrant DNA replication, andinduction of senescence (Bartkova et al., 2006; DiMicco et al., 2006). A similar relationship in plants issupported by the observation that silencing of the corecell cycle gene encoding the RBR protein causes ne-

crosis in mature leaves (Jordan et al., 2007). Thus,inhibition of RBR by CaLCuV AL1 binding mightcontribute to the activation of programmed cell deathas well as to the induction of host genes encodingDNA replication factors (Egelkrout et al., 2002;Arguello-Astorga et al., 2004).

Activation of both core cell cycle and DNA repairgenes during CaLCuV infection indicated that gem-iniviruses might rely on both host DNA synthesispathways for their replication, consistent with theiruse of RCR and RDR mechanisms (Jeske et al., 2001).However, because of the low frequency of homologousrecombination, which is required for RDR, versusnonhomologous end joining in plants (Kimura andSakaguchi, 2006), the relative contributions of RCRand RDR to viral DNA accumulation may differsignificantly. A very early event in infection is likelyto be AL1/RBR binding, leading to reprogramming ofcell cycle controls, the accumulation of host replicationmachinery, and the onset of RCR. The accumulation ofviral DNA replication products and intermediatescould then trigger a genotoxic response and the syn-thesis of host repair proteins and potentially a switchto RDR. A model in which RCR precedes RDR iscongruent with the absolute dependence of geminivi-rus infection on a functional origin that is recognizedby its cognate AL1 protein (Fontes et al., 1994), anal-ogous to bacteriophage T4, which replicates by se-quential RCR to RDR (Cox, 2001).

A longstanding question is the extent to whichgeminivirus infection impacts host DNA replication(Accotto et al., 1993; Nagar et al., 2002). Up-regulationof S phase-associated genes during CaLCuV infectionis consistent with the establishment of a replication-competent environment, while increased ploidy ininfected leaves demonstrated that infection inducescells to endocycle and fully replicate their genomes.Increased ploidy levels have also been reported inArabidopsis cauline leaves infected with the nano-virus Fava bean necrotic yellow virus (Lageix et al., 2007).Like geminiviruses, nanoviruses rely on host DNAreplication enzymes to amplify their single-strandedDNA genomes and encode a protein (CLINK) thatbinds to RBR and induces the expression of hostreplication machinery. These parallels suggest thatgeminiviruses and nanoviruses interact with theirhosts through conserved mechanisms that ultimatelylead to the establishment of an endocycle. However,hyperplasia and enations have been observed for a fewgeminivirus/host combinations, indicating that someplant DNA viruses can induce mitosis (Esau andHoefert, 1978; Briddon, 2003). The capacity to inducecell division is associated with the viral C4 protein(Latham et al., 1997), but C4 is not essential for infection(Stanley and Latham, 1992). Hence, geminiviruses thatinduce cell division do not depend on the mitotic cellcycle to replicate their genomes, underscoring theuniversal involvement of the endocycle in plant DNAvirus replication. This idea is supported further by theability of maize streak virus RepA, the RBR-binding

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Plant Physiol. Vol. 148, 2008 449

protein of monocot-infecting geminiviruses, to induceendoreduplication (Desvoyes et al., 2006).

Mammalian DNA tumor viruses also induce chro-mosomal DNA replication, resulting in 2C, 4C, and.4C cell populations (Friedrich et al., 1992; Belyavskyiet al., 1996). Unlike plant DNA viruses, rereplication inanimal cells is not associated with discrete C valuescharacteristic of complete endocycles. This differencemay reflect the fundamentally different ways the cellcycle and DNA replication are integrated with devel-opmental processes in the two kingdoms. In mam-mals, growth generally corresponds to increased cellnumber, and the endocycle is confined to a few spe-cialized cell types. In contrast, in plants, the endocycleis commonplace and linked to growth. It often repre-sents the final stage of DNA replication during plantdevelopment and, as such, may be more readily in-duced by viral infection. The mechanisms leading torereplication in mammalian cells are not known, butthere is evidence that checkpoint controls are disruptedand that interactions between viral oncoproteins andretinoblastoma family members are dispensable (Wuet al., 2004; Cherubini et al., 2006). In contrast, theRBR/E2F pathway has been implicated in endoredu-plication in plants and insects, which also undergoextensive endocycling during development (De Veylderet al., 2007; Swanhart et al., 2007). However, a recentreport showed that mammalian primary cells depletedfor Rb can undergo full rounds of genome amplica-tion, suggesting that Rb suppresses the endocycle innontransformed cells (Srinivasan et al., 2007). In con-trast, another recent report indicated that depletion ofArabidopsis RBR is not sufficient to induce endore-duplication in cultured plant cells (Hirano et al., 2008),suggesting that the mechanisms that govern inductionof the endocycle may differ in plant and animal systems.

Like mammalian DNA viruses (Felsani et al., 2006),geminiviruses bypass G1 cell cycle controls by bindingto RBR and altering the expression E2F target genes.Several lines of evidence indicate that geminivirusesselectively impact E2F activity related to endocycling.First, E2FC expression was elevated during CaLCuVinfection. Arabidopsis plants that overexpress E2FCand its DPB partner have increased DNA content, andE2FC RNAi lines undergo hyperplasia (del Pozo et al.,2006). Both of these phenotypes are indicative of E2FCpromoting the endocycle. Second, although we did notobserve increased E2FA expression, many genes dif-ferentially expressed in plants ectopically expressingE2FA and DPA showed similar trends in CaLCuV-infected leaves (Vlieghe et al., 2003; Vandepoele et al.,2005), suggesting that geminivirus infection altersexpression of a subset of E2FA/DPA-regulated genes.Third, like CaLCuV infection, inactivation of RBR bybinding to maize streak virus RepA resulted in up-regulation of E2FA and E2FC expression and anincreased number of endocycling cells in older devel-oping leaves (Desvoyes et al., 2006). Strikingly, E2FAoverexpression and RBR inactivation enhances celldivision early in leaf development and the endocycle

later in development (De Veylder et al., 2002; Desvoyeset al., 2006), indicating that geminivirus infectiondistinguishes between the mitotic and endocyclingactivities of RBR and E2FA and selectively activatesendoreduplication.

During CaLCuV infection, the increase in ploidywas accompanied by a reduction in 4C but not 2C cells,indicating that the virus preferentially targets 4C cells.A recent study proposed that the CYCD3 family trig-gers a commitment to the mitotic cell cycle (Dewitteet al., 2007). This model predicts the existence oftwo 4C populations, one programmed to completemitosis and the other destined to enter the endocycle(Fig. 6). Several observations suggested that gemini-viruses specifically target the second population. First,CYCD3;2 RNA levels were reduced in infected leaves.Down-regulation of CYCD3 expression would favoraccumulation of the endocycle 4C population andfacilitate infection. A triple CYCD3 mutant line didnot show enhanced susceptibility to CaLCuV, mostlikely because of the strong infection phenotype ofthe virus. However, plants ectopically expressingCYCD3;1 were highly resistant to infection, indicatingthat although these plants contain a larger proportionof 4C cells (Dewitte et al., 2003), they are not suitable

Figure 6. Model for CaLCuV induction of the endocycle. Duringinfection, CaLCuV selectively infects the 4C cell population (markedby the asterisk) predisposed to undergo endoreduplication but not 2Cor 4C cells in the mitotic cycle. The viral AL1 protein, which isproduced very early in the infection process, binds to RBR and relievesrepression of E2FC and E2FA, leading to activation of S phase genes andan increase in endocycling cells. Up-regulation of E2FC expression alsopromotes the endocycle during infection, while down-regulation ofCYCD3 expression prevents the mitotic cycle possibly through sup-pression of E2FB activity. Genes with elevated transcripts in CaLCuV-infected leaves are in red, while genes with reduced mRNAs are ingreen.

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450 Plant Physiol. Vol. 148, 2008

targets for viral infection, because they are prepro-grammed to undergo mitosis and not to enter theendocycle. Similarly, the resistance phenotype of E2FB-overexpressing plants but not E2FA or E2FC lines indi-cated that reducing the number of endocycle-competentcells strongly impairs infection. Up-regulation of CYCD4;2in infected leaves was consistent with its expressionoutside of meristematic regions (Kono et al., 2007) anddemonstrated that viral infection does not result in ageneral reduction of CYCD expression.

At first glance, geminivirus targeting of 4C cells andtheir dependence on the RBR/E2F pathway may seemincompatible. However, the endocycle consists of onlytwo phases, S and a gap phase that prepares the cell toreplicate its genome. In the absence of CYCD3-CDKactivity, RBR and E2F may play a role in inducing 4Ccells to transition to the endocycle early in infection.Alternatively, if the 4C cells are already in the endo-cycle gap phase, the RBR/E2F pathway may facilitatetransition into S phase of the first endocycle. In bothcases, AL1 binding to RBR would modulate E2FA andE2FC activities, leading to changes in host gene ex-pression and induction of the endocycle (Fig. 6). Thesemechanisms are not mutually exclusive and mayinvolve atypical E2F family members that do notinteract with RBR (Vlieghe et al., 2005) as well as thecanonical E2Fs. Future studies that examine the tran-script and protein accumulation patterns of core cellcycle and DNA replication genes in infected cells, aswell as the efficiency and tissue specificity of infectionof various core cell cycle mutant and overexpressinglines, will provide insight into requirements for theestablishment of a DNA replication-competent envi-ronment during geminivirus infection.

MATERIALS AND METHODS

Plant Growth and Inoculation Conditions

Arabidopsis (Arabidopsis thaliana) Col-0 plants were grown and inoculated

with CaLCuV as described previously (Shen and Hanley-Bowdoin, 2006).

Control plants were mock inoculated using the same Agrobacterium strain

transformed with pNSB690, which contains a 35S promoter uidA expression

cassette. Typically, 12 plants were inoculated with CaLCuV, while six were

mock inoculated per tray. The tray was covered with a clear plastic lid for 4 d

and then uncovered for the remainder of the growth period. Symptoms were

monitored from the first dpi until the CaLCuV-infected plants were dead at 6

weeks postinfection.

Transgenic Arabidopsis plants carrying expression cassettes for E2FA,

E2FB, E2FC, or CYCD3;1, with mutations in CYCD3;1, CYCD3;2, and CYCD3;3

or carrying the cpr1 mutation (Bowling et al., 1994; del Pozo et al., 2006;

Sozzani et al., 2006; Dewitte et al., 2003, 2007), were grown under the same

conditions. Overexpression of the transgenes was verified for each homozy-

gous line by RT-PCR (data not shown). The plants were agroinoculated as

described above or inoculated by bombardment using 1-mm gold

microprojectiles coated with plasmid DNA (mock) or a mixture of CaLCuV

A (pCPCBLCVA.003) and B (pCPCBLCVB.002) replicon plasmids (Turnage

et al., 2002).

Viral DNA Hybridization

Visible rosette leaves were collected at 12 dpi. Leaves 1 to 4 (with leaf

1 being the youngest) were pooled, while leaves 5 to 20 were harvested

individually. DNA (0.5 mg) isolated from each leaf sample was digested with

SacI, resolved by agarose gel electrophoresis, and visualized by DNA gel

blotting using a 32P-labeled, unit-length CaLCuV A probe (Ascencio-Ibanez

and Settlage, 2007). Viral DNA accumulation was monitored independently in

three plants. Tissue printing of viral DNA was performed by rubbing leaves

onto nylon membranes followed by UV crosslinking and hybridization with a32P-labeled, unit-length CaLCuV A probe.

AL1 Immunolocalization

The coding region for CaLCuVAL1 was released from pNSB958 (Arguello-

Astorga et al., 2004) as a 1.2-kb NdeI/XhoI fragment and subcloned into the

Escherichia coli expression vector pET-16b (Novagen). The resulting pNSB1044

clone encodes an AL1 protein fused to a 31-amino acid N-terminal extension

with 11 His residues and designated as His-AL1. The expression cassette was

transformed into the E. coli BL21 strain and His-AL1 was produced by

isopropylthio-b-galactoside induction and purified by nickel-nitrilotriacetic

acid agarose agarose affinity chromatography according to the manufacturer’s

instructions (Qiagen) with the following modifications to the recommended

growth conditions. A 5-mL aliquot from a fresh overnight culture was diluted

into 100 mL LB supplemented with 100 mg/mL ampicillin in a 250-mL baffle

flask and grown at 28�C to an OD of 0.8. Recombinant protein expression was

induced by the addition of 1 mM isopropylthio-b-galactoside, followed by

incubation at 37�C for 2.5 h. Antibodies were produced by Cocalico by

immunizing a rabbit with 0.25 mg purified His-AL1 and boosting with 0.25

mg three times at 14, 21, and 49 d. The antiserum was verified by immuno-

blotting total protein extracts from CaLCuV-infected and mock inoculated

leaves (Shen and Hanley-Bowdoin, 2006).

Leaf 9 from CaLCuV-infected plants at 12 dpi and equivalent leaves from

mock-inoculated plants were fixed and processed as previously reported (Shen

and Hanley-Bowdoin, 2006) with the following modifications. After a 10-min

exposure to AEC reagents (Vector Laboratories), sections were washed for 5 s in

ethanol to reduce background. Sections were immediately washed with

phosphate-buffered saline, pH 7.4, counter-stained with 1 mg/mL DAPI for

10 min, and mounted onto glass slides in 90% (v/v) glycerol in phosphate-

buffered saline. Sections were observed with a Nikon Eclipse E800 microscope.

RNA Isolation, Labeling, and Hybridization

Leaves 7 to 9 were harvested, pooled, flash-frozen, ground in liquid

nitrogen, and stored at 280�C. Three biological replicas corresponding to

infected and mock-inoculated samples were collected and processed indepen-

dently as described below. Total RNA was extracted from each sample using

the Plant RNeasy Mini kit (Qiagen). Double-stranded cDNA was synthesized

using total RNA (5 mg) template and an oligo(dT)-T7 primer (Qiagen) in 20-mL

reactions using the Superscript II system (Invitrogen) according to Affymetrix

protocols. Biotinylated cRNA was synthesized using 10 mL of the resulting

cDNA using the BioArray High Yield Transcript Labeling kit (Enzo) and

cleaned with RNeasy columns (Qiagen) according to the manufacturer’s

protocol except for a double passage through the column to increase yield.

The cRNA (15 mg) was hybridized to an ATH1 GeneChip (Affymetrix P/N

510690) for 16 h according to the Affymetrix GeneChip protocol. The arrays

were washed and stained using the EukGe-Wsv4 protocol in a GeneChip

Fluidic Station 450 and scanned using a GeneChip Scanner (Affymetrix). Array

quality was assessed following the Affymetrix recommended parameters

(GeneChip Expression Analysis, Technical Manual, 701021 rev 1). The hybrid-

ization, washing, and scanning protocols were performed at the NCSU

Genome Research Laboratory. The microarray data have been submitted to

ArrayExpress (www.ebi.ac.uk/arrayexpress; accession no. E-ATMX-34).

Microarray Data Analysis

The GeneChip scanning data were applied at probe-level to directly extract

the intensities from.CEL files. The data were first transformed by logarithm

base 2 and normalized across the chips by LOESS normalization (Dudoit and

Fridlyand, 2002) with the probe-wise mean intensities using the mean across

the arrays for each probe as baseline. Normalization was performed using

SAS/Proc LOESS (SAS Institute).

The following mixed ANOVA model (Chu et al., 2002) was applied for

further statistical analysis on the normalized data:

Yijkg 5 Tig 1 Pjg 1 TPijg 1 Akg 1 eijkg

Akg ; Nð0;skg2Þ

eijkg ; Nð0;sg2Þ

Arabidopsis Infected with Geminivirus

Plant Physiol. Vol. 148, 2008 451

The indexes i, j, k, and g indicate the treatment group, probe, chip, and probe

set (gene), respectively. The response variable, Y, represents the normalized

intensity. T and P represent the treatment and probe main effects, respectively.

TP represents the interaction effect of the two main effects. A represents the

random chip effect assumed to be normal distributed, while e corresponds to

the stochastic error assumed to be normal distributed and independent to

random chip effect. The P values from the ANOVA were converted to q values

using the R program (version 2.5.1; Storey and Tibshirani, 2003).

Nuclei Isolation and Ploidy Analysis

Plants grown under the same conditions used for the microarray exper-

iments were allowed to bolt, and the first flower bolts were removed at the

base. The plants were inoculated with CaLCuV DNA by bombardment. For

each sample, rosette and cauline leaves were harvested and pooled from at

least six plants at 24 dpi and frozen in liquid nitrogen prior to nuclei isolation.

The frozen tissues were chopped on ice with a single-edge razor blade in a

cold glass petri dish containing lysis buffer (15 mM Tris-HCl, pH 7.5, 2 mM

Na2EDTA, 80 mM KCl, 20 mM NaCl, 15 mM b-mercaptoethanol, 0.1% Triton

X-100, and 2 mg/L DAPI). The chopped leaf suspension was incubated on ice

for 10 min and filtered through a three-tiered nylon mesh (100, 50, and 30 mm).

The nuclei suspension was filtered through a 20-mm nylon mesh and imme-

diately subjected to FACS analysis using an InFlux cell sorter (Cytopeia) with a

355-nm UV laser (20 mW) tuned to excitation at 460/50 nm. Debris and

aggregates were excluded from the populations by gating. FlowJo software

(version 6.4.7) was used for FACS analysis.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Validation of microarray results.

Supplemental Table S1. Genes cited in the text and corresponding

references.

Supplemental Table S2. Significant genes changing during CaLCuV

infection.

Supplemental Table S3. Genes with increased transcripts during RNA

virus infection and significantly altered by CaLCuV infection.

Supplemental Table S4. Comparisons to host pathogen pathway profiles.

Supplemental Table S5. Expression of senescence-associated genes.

Supplemental Table S6. Photosynthesis-related genes affected by

CaLCuV infection.

Supplemental Table S7. Primer sets used in this study.

Supplemental Table S8. Core cell cycle genes at 9 dpi, PR1 expression in

E2FB and CYCB3;1 overexpressors, and T-test for ploidy analysis of

infected leaves.

Supplemental Methods and Materials S1. RT-PCR methods.

ACKNOWLEDGMENTS

We thank Dr. Crisanto Gutierrez (Universidad Autonoma de Madrid) for

providing the Arabidopsis E2FC-overexpressing line, Dr. James Murray

(Cambridge University) for providing the CYCD3 lines, and Dr. Steven Spoel

(Duke University) for the cpr1 mutant. We also thank Drs. Jose Alonso (North

Carolina State University) and Dominique Robertson (North Carolina State

University) for their comments on the manuscript.

Received April 23, 2008; accepted July 21, 2008; published July 23, 2008.

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