metabolismo sec 2006 en hongos filamentosos.pdf

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Annu. Rev. Phytopathol. 2005. 43:43758doi: 10.1146/annurev.phyto.43.040204.140214

Copyright c 2005 by Annual Reviews. All rights reserved

REGULATION OF SECONDARY METABOLISMIN FILAMENTOUS FUNGI

Jae-Hyuk Yu1 and Nancy Keller1,21Department of Food Microbiology and Toxicology, 2Department of Plant Pathology,

University of Wisconsin, Madison, Wisconsin 53706;

email: [email protected], [email protected]

Key Words fungi, mycotoxins, transcriptional control, G proteins, RGS proteins

Abstract Fungal secondary metabolites are of intense interest to humankind dueto their pharmaceutical (antibiotics) and/or toxic (mycotoxins) properties. In the pastdecade, tremendous progress has been made in understanding the genes that are associ-ated with production of various fungal secondary metabolites. Moreover, the regulatorymechanisms controlling biosynthesis of diverse groups of secondary metabolites havebeen unveiled. In this review, we present thecurrent understanding of thegenetic regula-tion of secondary metabolism from clustering of biosynthetic genes to global regulators

balancing growth, sporulation, and secondary metabolite production in selected fungiwith emphasis on regulation of metabolites of agricultural concern. Particularly, theroles of G protein signaling components and developmental regulators in the mycotoxinsterigmatocystin biosynthesis in the model fungus Aspergillus nidulans are discussedin depth.

INTRODUCTION

Secondary metabolite production in fungi is a complex process coupled withmorphological development (reviewed in 27). Secondary metabolites often have

obscure or unknown functions in the producing organism but have tremendous

importance to humankind in that they display a broad range of useful antibiotic

and pharmaceutical activities as well as less desirable immunosuppressant and

toxic activities.

In most cases, the function of secondary metabolites for the producing fungus is

unknown but is inferred from a few studies using mutants or enzyme inhibitors. The

most obvious fungal natural products are the pigmentstypically brown and black

pigments referred to as melaninsgiving color to spores, appressoria, sclerotia,sexual bodies, and other developmental structures. Studies of pigment function

in these structures have shown that they act as plant (58, 71) and animal (114)

virulence factors or that they are required for general survival, presumably as UV

0066 4286/05/0908 0437$20 00 437

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protectants (72, 77), antigrowth deterrents (98), or ROS scavengers (37). Fungal

phytotoxins are proven pathogenicity or virulence factors that cause significant

disease on agricultural crops (5, 54, 127). Another notorious group of agriculturally

important secondary metabolites are the mycotoxins, which are excreted by fungias they grow in various commodities (8). The primary aim of this review is to

present our current understanding of how secondary metabolites are regulated in

fungi, with emphasis on regulation of metabolites of agricultural concern.

BIOSYNTHETIC GENE CLUSTERS

The inherent properties of secondary metabolites, both desirable and destructive,

spurred efforts toward identifying genes involved in their synthesis. Accumulatingdata from studies of known secondary metabolite biosynthetic genes dispelled an

original premise that fungal metabolic genes would be scattered throughout the

genome. Rather, the hallmark of secondary metabolite genesin contrast to genes

involved in primary metabolismis that they are clustered in fungal genomes

(reviewed in 68, 135). As described below, the contiguous clustering of metabolic

genes specific to one product has considerable bearing on the regulation of these

genes.

Aflatoxin and SterigmatocystinWith the possible exception of the penicillin metabolic cluster, the most thor-

oughly examined fungal secondary metabolite gene clusters are those involved

in mycotoxin biosynthesis, particularly the aflatoxin (AF) and sterigmatocystin

(ST) biosynthetic clusters found in several Aspergillus spp. (Figure 1). Both car-

cinogenic metabolites are products of the same lengthy pathway where ST is

the penultimate precursor of AF (16, 129). The AF cluster in A. parasiticus and

A. flavus contains genes that constitute a cluster spanning more than 70 kb. Among

these genes, 21 have been verified or predicted to encode biosynthetic enzymes, in-

cluding fatty acid synthases, a polyketide synthase, monooxygenases, reductases,dehydrogenases, methyltransferases, an esterase, a desaturase, and an oxidase (85,

129). One gene in the cluster, aflR, encodes a binuclear zinc cluster (Zn(II)2Cys6)

Figure 1 Order and direction of transcription of genes in the sterigmatocystin (ST)

and aflatoxin (AF) gene clusters. Orthologous genes in the two clusters are indicated

by the same bar pattern. Solid black bars represent ST and AF genes that have not been

characterized.

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REGULATION OF MYCOTOXIN PRODUCTION 439

transcription factor regulating transcription of the aflatoxin biosynthetic genes (33,

126). Another cluster gene, aflJ, also seems to have a role in regulating aflatoxin

production in A. flavus (83). In A. nidulans the 60-kb sterigmatocystin cluster con-

sists of circa 25 genes also regulated by aflR (16, 44, 130). The functions of mostof the sterigmatocystin cluster genes have been determined and are orthologs of

aflatoxin cluster genes (55).

Trichothecenes

Trichothecenes comprise a large family of sesquiterpenoid metabolites produced

by a number of fungal genera, including Fusarium, Myrothecium, Stachybotrys,

Cephalosporium, Trichoderma, and Trichothecium (62, 104, 120). These com-

pounds not only exhibit toxicity to vertebrates and plants, but also are associated

with virulence in specific plant-pathogen interactions (38, 53, 95).

Biochemical and genetic analyses of the T-2 toxin producer F. sporotrichioides

ledto the identification of the first trichothecene biosynthetic gene cluster. The gene

cluster for deoxynivalenol production has also been identified in F. graminearum.

The two clusters contain 10 to 12 ORFs and span circa 29 kb (17, 18, 57). The

functions of ten genes have been determined. Seven of them encode biosynthetic

enzymes (6, 18, 19, 81). Tri6 and Tri10 are regulatory proteins and Tri12 is the

efflux pump that is implicated to play a self-protection role (7, 110). Recently, a

second mini-cluster has been found in F. graminearum that contains two more

enzymatic genes required for deoxynivalenol production (17). A trichothecene

cluster has also been described in Myrothecium roridum (113).

Fumonisins

Fumonisins are a group of polyketide mycotoxins that are produced primarily

by the economically important maize and sorghum pathogens Fusarium verticil-

lioides and Fusarium proliferatum (87). Fumonisin B1, the most toxic fumonisin,

promotes cancer and causes equine leukoencephalomalacia. The genes involved

in fumonisin biosynthesis are clustered in a 45-kb stretch of DNA. Expressionanalysis of F. verticillioides indicated that 15 genes (ORF1 and ORF6-19) are

coregulated and exhibited patterns of expression that were correlated with fumon-

isin production. These ORFs are designated as FUM genes (92). FUM5 encodes

the polyketide synthase gene that was shown to be required for fumonisin biosyn-

thesis (93). Disruption of FUM6 and FUM8 blocked production but did not lead

to accumulation of detectable intermediates (103); FUM9 and FUM13 both are

involved in side chain decoration of the carbon backbone (22, 23). Most recently,

a transcription factor, ZFR1, important in fumonisin regulation has been identified

(45). However, in contrast to the regulatory genes of the AF, ST, and trichothecenepathways, ZFR1 is not located in the fumonisin cluster.

Other identified gene clusters include those involved in production of other

mycotoxins and phytotoxins (HC toxin, 4; dothiostromin, 13; sirodesmin, 47;

gibberellin, 118; ergot alkaloids, 119; paxillin, 128; aflatrem, 135), antibiotics

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(cephalosporin, 48; penicillin, 79), melanins (71, 115), and pharmaceuticals (com-

pactin, 2; lovastatin, 69).

TRANSCRIPTIONAL REGULATION

The coregulation of the cluster genes can be in part explained by transcriptional

control of structural genes by two classes of transcription factors, one class briefly

mentioned in the previous section that are specific to a particular metabolic path-

way (i.e., aflR) and a second class that mediate environmental signals including

pH, carbon, and nitrogen sources. This multilevel regulation by both specific and

broad-domain transcription factors ensures that secondary metabolite pathways

can respond to the demands of general cellular metabolism and the presence ofspecific pathway inducers.

Pathway-Specific Transcription Factors

Many, but not all, clusters contain genes encoding transcription factors that pos-

itively regulate gene expression. Perhaps the archetypal protein in this group is

AflR, the Zn(II)2Cys6 domain protein required for AF and ST biosynthetic gene

activation (33, 44, 126). Typical for this group of DNA binding proteins, AflR

recognizes and binds to a palindromic sequence, 5-TCG(N5)GCA, found in the

promoters of the AF/ST biosynthetic genes (41, 44, 90). A second binding site,5-TTAGGCCTAA, has also been reported for A. flavus and A. parasiticus and

is considered important in autoregulation of aflR transcript in these spp. (34, 35,

40, 90). Disruption ofaflR eliminates the expression of structural genes (130) and

modifications of its promoter region alter not only its own but subsequent clus-

ter gene expression (40). How AflR is negatively regulated by protein kinase A

signaling is described below.

Othercluster transcription factors include additionalZn(II)2Cys6 proteins (MlcR

for compactin biosynthesis, 1), Cys2His2 zinc finger proteins (Tri6 and MRTRI6

for trichothecene production, 94), an ankyrin repeat protein (ToxE for HC-toxinproduction, 91), a two-peptide forkhead complex (AcFKH1 and CPCR1 for cepha-

losporin production, 99, 100), and a HAP-like transcriptional complex (PENR1

for penicillin, 78). Additionally, PENR1 has also been shown to be important in

taka-amylase, xylanase, and cellobiohydrolase production (14).

Pathway-specific regulatory genes not obviously encoding transcription factors

include aflJrequired for AF/ST biosynthesis and Tri10 involved in trichothecene

biosynthesis (83, 110). Inactivation or mutation ofaflJgives a phenotype similar to

an aflR deletion, i.e., a great reduction in AF or ST production (83; R.A. Butchko &

N.P Keller, unpublished data). Although AflJ has not been studied in A. nidulans,several studies have partially defined its function inA. parasiticus (32, 83). Despite

lack of AF production in aflJ deletion strains, structural genes are still expressed

at a reduced level. This complex phenotype suggests that AflJ is not directly

responsible for AF/ST gene transcription or for any particular enzymatic step in

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the pathway, but is in some way enhancing transcription. Chang (32) demonstrated

an interaction of AflJ with AflR using a yeast two-hybrid system. Two regions of

AflR are required for this interaction, one located between aa 230238, the other

at the C terminus. However, it appears that the full-length AflJ protein is likelyrequired for activity as deletion of the first 9 amino acids reduces activity of the

protein by 85%90%, anddeletion of the final 11 amino acids eliminates its activity.

Global Regulatory Factors

CreA, AreA, PacC Secondary metabolite biosynthesis is responsive to environmen-

tal cues including carbon and nitrogen source, ambient temperature, light, and

pH. Several studies (42, 84) indicate that these environmental signals are mediated

through Cys2His2 zinc finger global transcription factors conveying carbon (CreA,

39), nitrogen (AreA, 59), and pH (PacC, 79, 112) signaling. Gene expression inseveral gene clusters including AF, ST, penicillin, and gibberellin clusters is reg-

ulated, either positively or negatively, by these zinc finger proteins. For example,

disruption of the positive-acting nitrogen regulatory areA-GF gene in Gibberella

fujikuroi led to a 10%20% reduction of gibberellin production in gibberellin

induction medium. In addition, the loss-of-function areA-GFstrains were insensi-

tive to ammonium-mediated gibberellin repression, supporting the conclusion that

gibberellin biosynthesis is under the control of AreA-GF (84).

The involvement of CreA in secondary metabolism may reflect the differences

seen in metabolite production when fungi are grown in different carbon sources(43). Another related factor in metabolite production may be the availability of

precursor units. For example, each secondary metabolite ultimately depends on

the available pools of a limited number of primary precursors for peak produc-

tion. Many secondary metabolites are classified as polyketides (ST, AF, fumonisin,

lovastatin, compactins, and melanins). This chemical class is derived from reit-

erative condensations of acetyl-CoA and malonyl-CoA moieties, malonyl-CoA

itself being derived from acetyl-CoA. Availability of acetyl-CoA might be ex-

pected to impact polyketide formation. Such a prediction has been recently borne

out in genetic and biochemical studies of A. nidulans where mutants in path-ways affecting either acetyl-CoA concentration (-oxidation mutants; 78a) or

availability via altered acyl-CoA ratios (methyl citrate mutants; 133, 134) can

reduce or even eliminate polyketide production despite an otherwise wild-type

phenotype.

LaeA A novel mechanism of gene cluster regulation was uncovered by com-

plementation of an A. nidulans ST mutant that was unable to express aflR. The

complementing gene, termed laeA for loss ofaflR expression, encodes a nuclear

protein with closest identity to arginine and histone methyltransferases (11). Lossof LaeA function silences not only ST cluster expression but also a multitude of

other metabolites including penicillin and numerous mycelial pigments inA. nidu-

lans and gliotoxin in A. fumigatus, whereas overexpression of laeA upregulates

cluster gene expression. Furthermore, microarray examination of the A. nidulans

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laeA deletion and laeA overexpression strains clearly shows that LaeA transcrip-

tionally regulates multiple novel secondary metabolite clusters, several of which

are currently being examined in our lab (J.W. Bok, L. Maggio-Hall, D. Hoffmeister

& N.P Keller, unpublished data). The findings that LaeA regulates multiple clus-ters may support a coregulation model for clustering (135), possibly via chromatin

remodeling of cluster loci. Putative LaeA orthologs are found in all filamentous

and dimorphic fungi examined to date, and it will be interesting to see if these

LaeA homologs play a role in secondary metabolism in other genera.

UPSTREAM SIGNALING MECHANISMS

All cells have the capacity to sense and respond to various external signals, such

as nutrients, hormones, as well as physical and chemical stimuli including envi-ronmental stress. Among various signaling elements, heterotrimeric G proteins

(G proteins) are conserved in all eukaryotes and play a central role in relaying

external cues into the cells to elicit appropriate physiological and biochemical

responses (reviewed in 86). In the past decade, G proteinmediated signaling has

been intensively studied in various filamentous fungal species, and outcomes of the

studies provided an important clue to understand the upstream regulation of fungal

secondary metabolite biosynthesis, which was hypothesized to be intimately asso-

ciated with sporulation (reviewed in 27). Progress has been made with the model

fungus A. nidulans, and this section primarily discusses signaling mechanismsgoverning development and ST production in this fungus with a few additional

examples.

The basic unit of heterotrimeric G protein is comprised of a seven-transmem-

brane-spanning domain G proteincoupled receptor (GPCR), a G protein consist-

ing of, , and subunits, and an intracellular effector that produces a second

messenger. G protein signaling is activated when ligand-bound GPCRs catalyze

GDP/GTP exchange of the G subunit, which provokes subsequent dissociation

of G-GTP and G. It is turned off when the intrinsic GTPase activity of the

G subunit hydrolyzes GTP to GDP, causing the formation of the inactive het-erotrimer GGDP:G. Dissociated (activated) G-GTP and/or Gcan trigger

the production or release of a large variety of second messengers including cAMP,

inositol 1,4,5-trisphosphate (IP3), diacylglycerol, cGMP, Ca2+, and nitric oxide.

These second messengers in turn initiate amplified cellular responses (reviewed in

86). In fungi, GPCR-G proteininitiated signaling is primarily transmitted to two

downstream signaling branches defined by adenylyl cyclase cAMP protein

kinase (PKA) and/or mitogen-activated protein kinase (MAPKKKMPAKK

MAPK) cascades, which eventually elicit cellular responses such as growth, mat-

ing, cell division, cell-cell fusion, morphogenesis, toxicogenesis, chemotaxis, andpathogenic development (12, 76, 122).

An important aspect of achieving pertinent cellular response to a (or multiple)

signal is to properly control the intensity of G protein signaling. Among various

controlling elements, regulators of G protein signaling (RGS proteins) play a key

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role in tightly controlling G protein signaling upstream or at the same level of

G proteins. RGS proteins contain a conserved 130 amino acid core domain

(RGS box) that functions in enhancing the intrinsic GTPase activity of the G

subunit, which results in increased GTP to GDP hydrolysis (inactivation) ratesof G subunits (Figure 2). Through activities of multiple RGS proteins, cells

can coordinate diverse incoming signals and fine-tune their cellular responses

(reviewed in 36). G proteins and RGS proteins function in global coordination of

fundamental biological processesin filamentous fungi including vegetative growth,

sporulation, mycotoxin/pigment production, pathogenicity, and mating.

AspergillusSpecies

ST/AF PRODUCTION REGULATION BY FadA (G) AND FlbA (RGS PROTEIN) G pro-

tein and its proper regulation play a central role coordinating hyphal growth,asexual/sexual development, and secondary metabolite production in A. nidulans.

Hyphal growth signaling is mediated by both the (FadA) and (SfaD) subunits of

a heterotrimeric G protein (96, 132). When FadA (G) is in its active-GTP bound-

state, it is dissociated with its cognate Gdimer (SfaD:GpgA) and the free FadA-

GTP and SfaD:GpgA can both activate downstream effectors for proliferation

(Figure 2), which inhibits both sexual and asexual development (96, 132; J.-A. Seo

& J.-H. Yu, unpublished data). In addition, activated FadA signaling also blocks

production of the mycotoxin sterigmatocystin (ST) and mRNA expression ofaflR,

indicating that FadA-GTP signaling negatively controls ST biosynthesis (56, 131,132) (Figure 3). For asexual/sexual development as well as ST biosynthesis to oc-

cur, FadA-GTP/SfaD:GpgA signaling needs to be at least partially inactivated. In

this attenuation of growth signaling, FlbA (an RGS protein) plays a key role and is

presumed to rapidly convert FadA-GTP to FadA-GDP by increasing the intrinsic

GTPase activity of FadA (Figure 3). Loss of flbA function results in the fluffy-

autolytic colony (Figure 2) that lacks sexual/asexual sporulation and ST production

due to the absence of proper down-regulation of FadA signaling (56, 73, 132).

While the primary role of FlbA in development and ST production is inactivat-

ing FadA, FlbA is found to have additional roles in conidiation and ST biosynthesis(56, 132). This was further supported by the observation that whereas overexpres-

sion of the conidiation activator brlA rescued conidiation in loss-of-function flbA

mutants (J. Hicks & N.P. Keller, unpublished data), forced expression of aflR or

site-directed mutagenized hyperactive AflR could not restore ST production in the

absence of flbA function (106). These results suggest that FlbA is necessary for

activity of the AflR protein via unknown mechanisms. While loss of SfaD (G)

or GpgA (G) function has clear effects on vegetative growth, conidiation, and

sexual development, their roles in ST production remain to be examined (96; J.-A.

Seo & J.-H. Yu, unpublished data).Importantly, G proteins are found to be functionally conserved in Aspergillus

species, in that the FadA-homologous pathway negatively controls AF biosynthesis

in both A. parasiticus and A. flavus. A study by Hicks et al. (56) showed that intro-

duction of the A. nidulans constitutively active FadA allele into an A. parasiticus

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Figure 3 Antagonistic pathways coordinate vegetative growth, conidiation, and pro-

duction of secondary metabolites in A. nidulans. Vegetative growth signaling by

(FadA) and (SfaD with a presumed G, GpgA) subunits of a heterotrimeric G pro-tein inhibits asexual/sexual sporulation and ST biosynthesis, but stimulates penicillin

production (56, 96, 111, 132). Activation of asexual development requires at least

partial inhibition of FadA-mediated signaling, which requires two genes, fluG and

flbA (7375). FluG is proposed to activate conidiation via removing repressive effects

imposed by multiple negative regulators (101). FlbA is an RGS protein that inhibits

FadA-GTP/SfaD::GpgA-mediated vegetative growth signaling (96, 132; J.-A. Seo &

J.-H. Yu, unpublished data).

strain inhibited production of AF (and/or intermediate products) as well as coni-diation in a dominant manner. Moreover, the similar results were observed in

A. flavus (82).

ProteinkinaseAandAflR FadA growth signaling is transduced in part via protein

kinase A (PKA; 107). Deletion of the pkaA gene encoding the primary PKA

catalytic subunit inA. nidulans resulted in elevated conidiationand highlyrestricted

vegetative growth. Analyses of epistatic interactions between fadA-flbA and pkaA

revealed that PkaA functions downstream of FlbA/FadA, i.e., deletion of pkaA

suppressed both developmental and ST biosynthesis defects caused by the absenceofflbA function. Conversely, overexpression ofpkaA caused reduced conidiation,

increased vegetative growth as well as inhibition ofaflR expression necessary for

ST biosynthesis (107). This study clarified that FadA-mediated signaling is (at

least in part) transmitted to a cAMP PKA signaling cascade and PkaA plays

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a major role in activation of vegetative growth and repression of both conidiation

and ST production (Figure 3).

The role of PkaA in negatively controlling ST production was further sup-

ported by a recent study by Shimizu et al. (106), where it was demonstrated thatAflR is phosphorylated by PkaA in vitro. Furthermore, probable posttranscrip-

tional negative regulation of AflR activity by PkaA-dependent phosphorylation

in vivo was also shown by substitution of the putative phosphorylation target

amino acid Ser to Ala in AflR. Such site-directed mutations abolished inhibitory

effects of overexpression ofpkaA on AflR activity. Moreover, the authors showed

that the requirement of FlbA for AflR-mediated ST production is PkaA indepen-

dent. This is consistent with the previous hypothesis that, in addition to inhibiting

FadA signaling, FlbA has an additional role in activating conidiation as well as

ST production (56, 132). Later, Bok & Keller (11) showed that PkaA negativelycontrols LaeA, which is required for the expression of clustered genes for ST,

penicillin, and lovastatin biosynthesis, respectively (see section on LaeA).

Recently, the roles of FadA, cAMP and PKA in regulation of AF biosynthesis

and conidiation in A. parasiticus were examined by Roze et al. (97). Interestingly,

while introduction of cAMP or dibutyryl-cAMP (DcAMP) onto solid medium

resulted in a 100-fold increase in intracellular cAMP/DcAMP, total cellular PKA

activity was lowered by 40- to 80-fold in the tested strain. These findings explained

why cAMP or DcAMP stimulated AF synthesis and conidiation in A. parasiticus

despite the functionally conserved FadA-mediated signaling mechanisms. Theauthors concluded: (a) the FadA/PKA signaling cascade negatively regulates AF

biosynthesis and conidiation via similar mechanisms in Aspergillus species; and

(b) intracellular cAMP levels, at least in part, mediate a PKA-dependent regulatory

influence on conidiation and AF synthesis.

GanBandRgsA,thesecondG-RGSpair As in most filamentous fungal genomes

(12), the A. nidulans genome contains three G subunits, FadA, GanA, and GanB.

Recent studies identified three additional RGS proteins (RgsA, RgsB, and RgsC)

in A. nidulans (52). Han et al. (52) revealed that RgsA down-regulates pigmentproduction and conidial germination, but stimulates conidiation (and ST produc-

tion) via inhibiting GanB signaling. This study showed that deletion ofrgsA caused

reduced colony size with increased aerial hyphae, elevated accumulation of brown

(mycelial) pigments, but reduced ST production (Figure 4). The fact that dele-

tion of both flbA and rgsA resulted in an additive phenotype led the authors to

speculate that the G protein pathways controlled by FlbA and RgsA are differ-

ent. Morphological alterations, increased pigment but reduced ST production, as

well as restricted colony growth caused by deletion of rgsA were suppressed by

deletion of ganB, indicating that the primary role of RgsA is to negatively con-trol GanB-mediated signaling (Figure 4). The observations that overexpression

ofrgsA as well as deletion or dominant interfering mutations of ganB caused in-

appropriate hyperactive conidiation in liquid-submerged culture further support

that RgsA and GanB function in opposite manner and GanB-mediated signaling

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represses conidiation (and ST production; 31, 52). This second RGS-G pair in

A. nidulans may govern upstream regulation of fungal cellular responses to envi-

ronmental changes such as carbon sources and stresses. While the precise mech-

anism of GanB-mediated reduction of ST production remains to be determined,it was speculated that elevated accumulation of brown pigment(s) might partially

contribute to this phenotype (52). Taken together, a genetic model incorporating

the activities of two G protein signaling pathways and the cognate RGS proteins

in governing growth, development and ST production is presented (Figure 5).

Conidiation-specific functions and ST production A close relationship between

fungal development and secondary metabolite production has been observed in

Aspergillus species. Bennett and colleagues (9, 10) first observed that A. para-

siticus morphological mutants also lost the ability to produce wild-type levels of

Figure 5 G protein-RGS mediated regulation of development and ST production in

A. nidulans (adapted from Reference 52). Two independent G-RGS signaling path-

ways coordinately control cellular responses to various signals. FlbA-FadA primarily

governs vegetative growth versus development and ST production, and RgsA-GanB

controls stress response (pigmentation), carbon sensing, and germination. Conidiationoccurs through activation of brlA, which requires multiple upstream genes including

fluG (74). GanB and presumed SfaD:GpgA-mediated signaling is proposed to repress

asexual sporulation (31, 96). Possible direct activation of conidiation (and ST produc-

tion) by FlbA and RgsA is presented as dotted arrows.

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AF. Later, these sporulation defective mutants were found to fail to accumulate

mRNA ofaflR and genes encoding enzymes for AF biosynthesis (64, 65). Genetic

mechanisms interconnecting conidiation and AF/ST production were uncovered

by a series of studies that identified and characterized genes (fluG, flbE, flbD,flbB, flbC, and brlA) required for conidiation in A. nidulans (reviewed in 3, 7375,

124). BrlA is a key transcription factor that activates conidiophore (asexual spore-

bearing structure) formation, andfluG, flbE, flbD, flbB, andflbCare required for the

expression of brlA. Mutations in these upstream regulatory genes resulted in the

absence or delay of conidiation and overproliferation of hyphae, termed fluffy

phenotypes. Accordingly, a genetic model proposed that conidiation occurs via

activities of multiple positive regulators (reviewed in 3). Among these, the fluG

gene that functions at the most upstream of this genetic cascade was also found to

be necessary for ST production.Hicks et al. (56) demonstrated that loss of fluG function resulted in lack of ST

production. Importantly, the role of FluG in ST biosynthesis was found to be indi-

rectly inhibiting FadA-mediated vegetative growth signaling. They demonstrated

that although mutational inactivation offadA did not overcome the sporulation de-

fects caused by deletion offluG, it restored ST biosynthesis in the absence offluG

function (56, 132). This finding and discovery of the interdependent relationship

offluG and flbA in conidiation (75) strongly suggested that the role of fluG in ST

production is indirect, via activating FlbA, which in turn inhibits FadA signaling

(Figure 3). Later, Seo et al. (101) isolated and characterized suppressors offluG lossof function and proposed that the primary role of FluG in activating conidiation and

ST production is to remove repressive effects imposed by multiple negative regu-

lators of conidiation. These studies provided partial understanding of the genetic

mechanism for intimate correlation between conidiation and ST production as well

as upstream regulation of secondary metabolite biosynthesis in A. nidulans (see

Figure 3). Although a probable FluG homolog has been found in all Aspergilli ex-

amined (J.-H. Yu, unpublished data), precise mechanisms coordinating conidiation

and toxin biosynthesis in individual Aspergillus species remain to be uncovered.

Sexual developmental genes and ST/AF production An important characteristic

ofA. nidulans distinguishing it from many Aspergilli is that A. nidulans has both

sexual and asexual reproductive cycles (see 29, 63). Recent studies identified

a number of genes required for and/or associated with sexual development in

A. nidulans such as GPCRs (51, 102), G proteins (96), MAPKKK (123), various

transcription factors (50, 121), and novel genes such as veA with unclear functions

(70). Among these, the veA gene encoding a novel protein coordinating balanced

sexual/asexual development, particularly in response to light, is also found to

be required for ST production as well as aflR expression in A. nidulans (67).Furthermore,Calvoetal.(24)showedthata veA ortholog in the aflatoxin-producing

fungus A. parasiticus is essential for formation of sclerotia (protective spherical

structures) as well as production of AF (and expression of necessary genes). It

appears that deletion ofveA resulted in pleiotrophic effects in Aspergilli.

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Role of small GTP-binding protein RasA in ST production Recently, Shimizu

et al. (106) reported that RasA (a small GTP-binding protein), a homolog of the

yeast Ras proteins, is negatively associated with ST production in A. nidulans.

High levels of activated RasA also inhibited production of ST via repression ofaflR expression (106). While this RasA-mediated transcriptional control of aflR

was independent of PkaA, posttranscriptional regulation of AflR by RasA was

found to be partially mediated by PkaA (106).

G PROTEIN SIGNALING AND PENICILLIN/CYCLOPIAZONIC ACID PRODUCTION Tag

et al. (111) showed that while the constitutively active FadA allele (G42R) in-

hibited conidiation and production of ST, it also caused elevated mRNA levels of

the isopenicillin synthetase gene (ipnA) in the penicillin biosynthetic gene cluster

as well as enhanced production of the well-known antibiotic penicillin in A. nidu-lans. This result implies that FadA-mediated activation of vegetative growth has

opposite roles in regulating the biosynthesis of two major secondary metabolites,

penicillin and ST, in A. nidulans (see Figure 3). This same allele, when expressed

in A. flavus, repressed both AF biosynthesis as well as cyclopiazonic acid produc-

tion (82). The veA gene was also shown to be necessary for penicillin production.

Kato et al. (67) demonstrated that although VeA repressed transcription of ipnA,

it was found to be required for expression of acvA, a gene encoding the delta-

(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase that acts at the first step of

penicillin biosynthesis.

FusariumSpecies

TRICHOTHECENES Tag et al. (111) also examined the morphological and physi-

ological consequences of a constitutively active FadA allele in Fusarium sporotri-

chioides. They demonstrated that the introduced mutant FadA allele reduced

Fusarium spore production by 50% to 95%, restricted colony growth, but ele-

vated production of the mycotoxin trichothecene. This result indicates that the

cellular responses to a given G protein signal can be different between fungalgenera. Jain et al. (60, 61) identified and characterized G protein and sub-

units in Fusarium oxysporum and found that these G proteins are necessary for

normal development and pathogenicity. However, roles of these G proteins in

controlling production of secondary metabolites in F. oxysporum remain to be

examined.

FUMONISINS Fumonisins are a group of mycotoxins produced by the maize

pathogen Fusarium verticillioides. Shim & Woloshuck (105) isolated a mutant

that was unable to produce fumonisins on cracked corn and identified the mu-tant locus named FCC1 for Fusarium cyclin C1. The fcc1 mutant produced a

dark purple substance when grown on cracked-corn medium. This study sug-

gests a possible role of cell-cycle regulators in coordinately controlling biosynthe-

sis of various metabolites including fumonisins. However, detailed mechanisms

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of FCC1-mediated regulation of secondary metabolite production remain to be

studied.

Pigment Production in Other FungiZuber et al. (136) identified the gasCgene encoding a G protein subunit (GanB-

homolog) in the opportunistic human pathogen Penicillium marneffei. Analyses

of the deletion, dominant-interfering, and constitutively active GasC mutations re-

vealed that GasC-mediated signaling is positively associated with germination as

well as production of an unknown secondary metabolite. In appropriate medium,

the gasCdeletion mutant and strains carrying a dominant-interfering GasC allele

exhibited reduced production of red pigment, whereas strains carrying a constitu-

tively active GasC allele overproduced red pigment and appeared dark red. This is

somewhat consistent with physiological outcomes (i.e., increased brown pigment

production) of GanB-mediated signaling in A. nidulans (52; see above).

In Cryphonectria parasitica, the chestnut-blight fungus, deletion of cpg1 and

cpgb-1 encoding a G protein subunit and subunit, respectively, resulted in

reduced hyphal growth, lowered spore formation, a loss of virulence as well as de-

creased pigment production (46, 66), indicating that these G proteins are necessary

for normal level pigment production.

LigandsThe demonstration of how important G protein signaling is in secondary meta-

bolism, sporulation, and virulence indicated that various environmental ligands

must also be important in initiating these cascades, presumably through GPCRs or

similar transmembrane proteins. One of the first extracellular signals described to

regulate both asexual and sexual spore development is psi factor, the collective term

for a series of oleic, linoleic, and linolenic acid-derived oxylipins, produced by

A. nidulans (25, 28, 30, 80) and other fungal genera (15, 49, 109). The proportion

of these three compounds to each other was reported to regulate asexual to sexual

spore development in A. nidulans (28).These fungal sporogenic lipids bear structural and biosynthetic similarities to

plant defense oxylipins, particularly the lipoxygenase products 9S-HPODE (9S-

hydroperoxy-10E,12Z-octadecadienoic acid) and 13S-HPODE (13S-hydroperoxy-

9Z,11E-octadecdienoic acid). Detailed studies of Aspergillus spp. showed that

purified linoleic acid and hydroperoxy linoleic acids derived from seed exhibit

sporogenic activities towards several Aspergillus spp. including A. nidulans,

A. flavus, and A. parasiticus (26) and, furthermore, that Aspergillus infection of

seed induces expression of seed lipoxygenases responsible for synthesis of 9S-

HPODE (20, 125). Additionally, the seed oxylipins also had a profound effect onAF and ST production in these species where 9S-HPODE had a stimulatory ef-

fect and 13S-HPODE an inhibitory effect on toxin biosynthesis (21). These results,

coupled with the studies from other fungal research groups (88, 89, 108), suggested

that linoleic acid and its derivatives are conserved signal molecules modulating

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450 YU KELLER

mycotoxin biosynthesis, fungal sporulation, and other aspects of fungal differen-

tiation processes.

The fungal oxylipin biosynthetic pathway has been partially elucidated in A.

nidulans. Two oxygenases, bearing similarity to mammalian prostaglandin syn-thetases and, to a lesser degree, plant lipoxygenases have been recently charac-

terized (116, 117). Eliminination of ppoA (psi-producing oxygenase) from the

genome results in a strain with an increased asexual to sexual spore ratio and re-

duced levels of the linoleic acid-derived psiB (117), whereas the deletion ofppoC

reduces levels of oleic acid-derived psiB and decreases the asexual to sexual spore

ratio (116). Moreover, the PpoA::gfp fusion protein located the oxygenase to both

asexual and sexual spore-bearing structures (117). These results complemented

previous physiological and biochemical studies that pointed out an important role

for oxylipins in integrating mitotic and meiotic spore development (30). Currentstudies have revealed a third oxygenase, ppoB, deletion of which greatly increases

asexual spore production (116a).

ST biosynthesis is also affected in ppo mutants. Deletion of both ppoA and

ppoC in the same strain eliminated ST production, whereas deletion of ppoB

greatly stimulated its synthesis (D.I. Tsitsigiannis & N.P. Keller, unpublished

data). These opposite effects on ST are reminiscent of the differential effects

of 9S- and 13S-HPODE on toxin biosynthesis (21). The myriad effects of Ppo

activity were reflected at a transcriptional level where expression of transcription

factors required for ST (aflR), asexual sporulation (brlA), and sexual sporulation(nsdA) were upregulated or down-regulated, respectively, with concomitant toxin

and spore production (116, 116a, 117; D.I. Tsitsigiannis & N.P. Keller, unpub-

lished data). Ppo orthologs have been found in all filamentous fungi examined by

genome database, and deletion of a ppo gene in F. sporotrichiodes generated a

strain impaired in both conidiation and T toxin production (82). Current studies

in our laboratories suggest a model where the different oxylipin products gener-

ated by Ppo oxygenases are secreted and function as ligands activating specific

GPCR signaling cascades inAspergillus and other fungi (Figure 5). The conserved

presence of ppo genes in fungal genomes coupled with conserved lipid stimula-tion of sporulation in several filamentous fungi suggests a putative global oxylipin

signaling cascade in the fungal kingdom.

CONCLUSION

Although still not complete, our knowledge of the molecular genetics of fungal

secondary metabolism has soared in the past decade. The establishment of the

secondary metabolite cluster motif and identification of both pathway-specific

and global regulators of these clusters lend themselves well to identification and

manipulation of additional clusters. Signaling cascades link sporulation processes

with metabolite synthesis. Coupling of secondary metabolism with morphologi-

cal development of the fungus appears to be a universal constant in filamentous

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fungi and may indicate an underlying evolutionary mechanism important in fungal

survival and possibly aspects of pathogenesis.

ACKNOWLEDGMENTSThe authors are thankful to those who made contributions to the subject areas.

This work was supported by National Science Foundation grant MCB-04,21863

to J.H.Y. and MCB-02,36393 to N.P.K.

The Annual Review of Phytopathology is online at

http://phyto.annualreviews.org

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REGULATION OF MYCOTOXIN PRODUCTION C-1

Figure 2 RGS proteins enhance the intrinsic GTPase activity of G subunits.

When a GPCR is sensitized by ligand binding, it catalyzes GDP to GTP exchange of

a G subunit, which subsequently provokes dissociation of G-GTP and G. Once

freed, either G-GTP or G, or both, can mediate signaling. Hydrolysis of G-

GTP to G-GDP causes the formation of the inactive heterotrimer G-GDP:G,

thereby turning off the signal. InA. nidulans, FadA-GTP and the cognate G het-

erodimer mediate signaling for vegetative growth, which involves PkaA (107) and

presumed transcription factors (TFs). FlbA is proposed to rapidly turn off FadA-

mediated vegetative growth signaling by acting as a GTPase Activating Protein

(132). Colony photographs are wild type (WT) and thef lbA deletion mutant (f lbA).Note that the f lbA mutant does not produce asexual spores and center of the f lbA

colony autolyzed (arrow).



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C-2 YU KELLER

Figure 4 GanB-RgsA signaling and production of pigment and ST (modified from

Reference 52) The ganBrgsA mutant exhibits colony growth and pigmentation

phenotypes identical to the ganB mutant. A TLC chromatograph shows that the

rgsA mutant produced a high level of fast migrating brown pigments (top two

arrows) while accumulating hardly detectable level of ST. Note that ST is readily

detectable in wild-type or ganB strains, and that the ganBrgsA mutant restored

the production of ST to the level of the ganB mutant.



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Annual Review of Phytopathology

Volume 43, 2005

CONTENTS

FRONTISPIECE, Robert K. Webster xii

BEING AT THE RIGHT PLACE, AT THE RIGHT TIME, FOR THE RIGHT

REASONSPLANT PATHOLOGY, Robert K. Webster 1

FRONTISPIECE, Kenneth Frank Baker

KENNETH FRANK BAKERPIONEER LEADER IN PLANT PATHOLOGY,

R. James Cook 25

REPLICATION OF ALFAMO- AND ILARVIRUSES: ROLE OF THE COAT PROTEIN,

John F. Bol 39

RESISTANCE OF COTTON TOWARDS XANTHOMONAS CAMPESTRISpv.

MALVACEARUM, E. Delannoy, B.R. Lyon, P. Marmey, A. Jalloul, J.F. Daniel,

J.L. Montillet, M. Essenberg, and M. Nicole 63

PLANT DISEASE: A THREAT TO GLOBAL FOOD SECURITY, Richard N. Strange

and Peter R. Scott 83

VIROIDS AND VIROID-HOST INTERACTIONS, Ricardo Flores,Carmen Hernandez, A. Emilio Mart nez de Alba, Jose-Antonio Daros,

and Francesco Di Serio 117

PRINCIPLES OF PLANT HEALTH MANAGEMENT FOR ORNAMENTAL PLANTS,

Margery L. Daughtrey and D. Michael Benson 141

THE BIOLOGY OF PHYTOPHTHORA INFESTANS AT ITS CENTER OF ORIGIN,

Niklaus J. Gr unwald and Wilbert G. Flier 171

PLANT PATHOLOGY AND RNAi: A BRIEF HISTORY, John A. Lindbo

and William G. Doughtery 191

CONTRASTING MECHANISMS OF DEFENSE AGAINST BIOTROPHIC AND

NECROTROPHIC PATHOGENS, Jane Glazebrook 205

LIPIDS, LIPASES, AND LIPID-MODIFYING ENZYMES IN PLANT DISEASE

RESISTANCE, Jyoti Shah 229

PATHOGEN TESTING AND CERTIFICATION OF VITIS AND PRUNUSSPECIES,

Adib Rowhani, Jerry K. Uyemoto, Deborah A. Golino,

and Giovanni P. Martelli 261

MECHANISMS OF FUNGAL SPECIATION, Linda M. Kohn 279

vii

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viii CONTENTS

PHYTOPHTHORA RAMORUM: INTEGRATIVE RESEARCH AND MANAGEMENT

OF AN EMERGING PATHOGEN IN CALIFORNIA AND OREGON FORESTS,

David M. Rizzo, Matteo Garbelotto, and Everett M. Hansen 309

COMMERCIALIZATION AND IMPLEMENTATION OF BIOCONTROL, D.R. Fravel 337

EXPLOITING CHINKS IN THE PLANTS ARMOR: EVOLUTION AND EMERGENCE

OF GEMINIVIRUSES, Maria R. Rojas, Charles Hagen, William J. Lucas,

and Robert L. Gilbertson 361

MOLECULAR INTERACTIONS BETWEEN TOMATO AND THE LEAF MOLD

PATHOGEN CLADOSPORIUM FULVUM, Susana Rivas

and Colwyn M. Thomas 395

REGULATION OF SECONDARY METABOLISM IN FILAMENTOUS FUNGI,

Jae-Hyuk Yu and Nancy Keller 437

TOSPOVIRUS-THRIPS INTERACTIONS, Anna E. Whitfield, Diane E. Ullman,and Thomas L. German 459

HEMIPTERANS AS PLANT PATHOGENS, Isgouhi Kaloshian

and Linda L. Walling 491

RNA SILENCING IN PRODUCTIVE VIRUS INFECTIONS, Robin MacDiarmid 523

SIGNAL CROSSTALK AND INDUCED RESISTANCE: STRADDLING THE LINE

BETWEEN COST AND BENEFIT, Richard M. Bostock 545

GENETICS OF PLANT VIRUS RESISTANCE, Byoung-Cheorl Kang, Inhwa Yeam,

and Molly M. Jahn 581

BIOLOGY OF PLANT RHABDOVIRUSES, Andrew O. Jackson, Ralf G. Dietzgen,

Michael M. Goodin, Jennifer N. Bragg, and Min Deng 623

INDEX

Subject Index 661

ERRATA

An online log of corrections to Annual Review of Phytopathology chapters

may be found at http://phyto.annualreviews.org/

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