Binding of the Magnaporthe oryzae Chitinase MoChia1 bya Rice Tetratricopeptide Repeat Protein Allows Free Chitin toTrigger Immune Responses

Chao Yang,a,1 Yongqi Yu,a,1 Junkai Huang,a,b Fanwei Meng,a,b Jinhuan Pang,a Qiqi Zhao,a,c Md. Azizul Islam,a,b

Ning Xu,a Yun Tian,d and Jun Liua,b,2

a State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, ChinabCollege of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, Chinac School of Life Sciences, University of Inner Mongolia, Hohhot, Inner Mongolia 010021, ChinadCollege of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China

ORCID IDs: 0000-0003-1714-3959 (C.Y.); 0000-0001-9846-4126 (Y.Y.); 0000-0002-6107-6899 (J.H.); 0000-0003-2193-7815(F.M.); 0000-0002-6569-028X (J.P.); 0000-0001-6264-0554 (Q.Z.); 0000-0001-5116-6738 (M.A.I.); 0000-0002-2621-5603 (N.X.);0000-0002-0906-3919 (Y.T.); 0000-0002-3255-269X (J.L.)

To defend against pathogens, plants have developed complex immune systems, including plasma membrane receptors thatrecognize pathogen-associated molecular patterns, such as chitin from fungal cell walls, and mount a defense response.Here, we identify a chitinase, MoChia1 (Magnaporthe oryzae chitinase 1), secreted by M. oryzae, a fungal pathogen of rice(Oryza sativa). MoChia1 can trigger plant defense responses, and expression of MoChia1 under an inducible promoter in riceenhances its resistance to M. oryzae. MoChia1 is a functional chitinase required for M. oryzae growth and development;knocking out MoChia1 significantly reduced the virulence of the fungus, and we found that MoChia1 binds chitin to suppressthe chitin-triggered plant immune response. However, the rice tetratricopeptide repeat protein OsTPR1 interacts withMoChia1 in the rice apoplast. OsTPR1 competitively binds MoChia1, thereby allowing the accumulation of free chitin and re-establishing the immune response. Overexpressing OsTPR1 in rice plants resulted in elevated levels of reactive oxygenspecies during M. oryzae infection. Our data demonstrate that rice plants not only recognize MoChia1, but also use OsTPR tocounteract the function of this fungal chitinase and regain immunity.

INTRODUCTION

Plants are under constant attack from various microbial patho-gens. Many phytopathogens colonize the plant apoplast, par-ticularly the biotrophic pathogens and hemibiotrophic pathogensduring their biotrophic stage (Jashni et al., 2015; Cao et al., 2016).Plants have developed a sophisticated immune system thatrecognizes the presence of pathogens by their conserved fea-tures. The plasma membrane-located pathogen recognition re-ceptors (PRRs) contain extracellular domains that can bindand recognize various pathogen-associated molecular pat-terns (PAMPs). Upon activation, the PRRs transduce signals todownstream components to initiate the immune responses(Zipfel, 2014; Boutrot and Zipfel, 2017), which typically involvesmitogen-activated protein kinase (MAPK) signaling, callose de-position, a reactive oxygen species (ROS) burst, and pathogen-related gene expression, referred to as PAMP-triggered immunity(PTI; Jones and Dangl, 2006; Zipfel, 2014).

Two common PAMPs are bacterial flagellin and the elongationfactor-Tu (EF-Tu), perceived in Arabidopsis (Arabidopsis thaliana)

by FLAGELLIN-SENSITIVE2 (FLS2) and EF-Tu receptor, re-spectively (Jones and Dangl, 2006; Zipfel, 2014). The recognitionof these PAMPs can trigger PTI. During infection by a filamentousfungus, chitin, the key constituent of the fungal cell wall, is rec-ognizedbyplant receptors categorizedas lysinmotif receptor-likekinases. Chitin is the most abundant amino polysaccharide in thebiosphere and is composed of polymeric N-acetylglucosamine(GlcNAc; Langner andGöhre, 2016). InArabidopsis, the lysinmotifreceptor-like kinases chitin-elicitor receptor kinase 1 (AtCERK1)and lysin motif receptor kinase 5 form a receptor complex thatrecognizes chitin (Cao et al., 2014); however, in rice (Oryza sativa),the LysM domain of chitin-elicitor binding protein (CEBiP) bindschitin and associates with OsCERK1, which phosphorylatesdownstream receptor-like cytoplasmic kinase 185 (OsRLCK185)to activate intracellular immune pathways (Yamaguchi et al.,2013).Successful pathogens must overcome host immunity to col-

onize the apoplast, either by avoiding the activation of, or directlysuppressing,PTI. Pep1 (Protein essential duringpenetration 1), aneffector of the maize smut fungus (Ustilago maydis), directly in-terferes with the generation of ROS in the host apoplast by in-hibiting peroxidase activity (Hemetsberger et al., 2012). InCladosporium fulvum, the effector protein Ecp6 (extracellularprotein 6) possesses LysM domains and sequesters fungal chitinoligomers to avoid chitin triggering immunity in the host plant (deJonge et al., 2010). Similarly, Magnaporthe oryzae secretes Se-creted LysM Protein 1 (Slp1), a LysM-containing effector protein,

1 These authors contributed equally to this work.2 Address correspondence to Junliu@im.ac.cn.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Jun Liu (Junliu@im.ac.cn).www.plantcell.org/cgi/doi/10.1105/tpc.18.00382

The Plant Cell, Vol. 31: 172–188, January 2019, www.plantcell.org ã 2019 ASPB.

to compete with OsCEBiP for chitin binding, thereby preventingthe activation of PTI in rice (Mentlak et al., 2012). In addition tocircumventing the extracellular perception by the PRRs, manypathogens deliver effectors into the plant cells, which perturbmultiple signaling pathways in the host, including subvertinghost apoplastic immunity by reducing the ROS burst, callosedeposition, and other PTI responses. The bacterial pathogenPseudomonas syringae secretes an effector protein AvrPto toblock FLS2 phosphorylation (Xiang et al., 2008), and the othereffector, AvrPtoB, targets FLS2 and CERK1 for degradation(Göhre et al., 2008; Gimenez-Ibanez et al., 2009).

In addition to the well-characterized PAMPs flagellin, EF-Tu,and chitin, many pathogen-secreted metabolites or virulenceproteins can also activate the plant immune system; these includelipopolysaccharides, peptidoglycan volatiles, glycoproteins, cellwall degradation enzymes (CWDE), and other pathogen-secretedproteins (Liu et al., 2012; Ranf et al., 2015). Proteins that are se-creted into the plant apoplast by pathogens are believed to playa fundamental role in plant apoplastic immunity (Jashni et al.,2015); for example, the pathogen-secreted CWDEs primarilytarget andmodulate plant cell walls to damage the cell’s integrity.Plants, on the other hand, secrete diverse proteases or proteaseinhibitors into the apoplast, presumably suppressing the activitiesof pathogen-derived CWDEs or other virulence proteins (Rooneyet al., 2005; Jashni et al., 2015).

Host–pathogen interactions in the plant apoplast may be ele-gantly regulated. One recent example is the interaction of theoomycete pathogen Phytophthora sojae and its host plant soy-bean (Glycine max; Ma et al., 2017). The pathogen secretes anapoplastic xyloglucan-specific endoglucanase (PsXEG1) thatfacilitates infection, but its activity couldpotentially be inhibitedbythe soybean-derived apoplastic glucanase inhibitor protein,GmGIP1; however, P. sojae secretes a paralogous nonfunctionalPsXEG1-like protein, PsXLP1, that acts as a decoy, binding toGmGIP1 with higher affinity than PsXEG1, thereby freeing theendoglucanase to support the infection (Ma et al., 2017). This

finding suggests the importance of apoplast decoys in the soy-bean response to pathogen invasion; however, this strategy hasnot yet been observed in other plant–pathogen interactions.Fungal pathogens harbor multiple chitinases that continuously

remodel their polymeric chitin and ensure the plasticity of their cellwalls during growth and infection (Langner and Göhre, 2016).Chitinases are essential for cell separation in U. maydis (Langneret al., 2015). However, the endochitinase of Verticillium dahliainhibits early fungal spore germination and triggers immune re-sponses in Arabidopsis and cotton (Gossypium hirsutum; Chenget al., 2017). In this study, we report that MoChia1 (M. oryzaechitinase1), achitinase fromthe riceblastpathogenM.oryzae, canactivate the immune response in the rice apoplast.MoChia1bindschitin and suppresses the chitin-triggered ROS burst in rice;however, the rice plants secrete a tetratricopeptide-repeat pro-tein, OsTPR1, which prevents MoChia1 from binding chitin in theapoplast, thereby rescuing the chitin-triggered ROS burst andregaining immunity.

RESULTS

Identification of MoChia1

Wepreviously predicted thatM. oryzae secretes over 150 effectorproteins into the rice apoplast, many of which target plant cell wallcomponents and are therefore unlikely to be delivered into theintracellular space during infection (Cao et al., 2017). Kim et al.identified over 400 proteins secreted by M. oryzae into the riceapoplast during infection, which could potentially be perceived bythe rice cells as “non-self” fungal proteins (Kim et al., 2013). Toscreen the fungus-secreted proteins for new elicitors/PAMPs thattrigger the plant immune response, we used fast protein liquidchromatography (LC) to isolate and purify the proteins that in-duced a ROS burst in rice suspension cells. After molecular ex-clusion and purification using anion exchange, we isolated

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a fraction that could significantly activate the ROS burst(Supplemental Figures 1A to 1D). Further protein identificationusing LC-MS/MS led to the discovery of five proteins in total(Supplemental Table 1).

We next expressed these proteins and tested whether theycould induce the ROSburst in rice cells. The sequences encodingthese proteinswere fusedwith theEscherichia coli gene encodingthe maltose binding protein (MBP). Four recombinant proteinswere produced in E. coli (the recombinant MGG-04436 could notbe expressed). The proteins were purified and then tested for theirability to cause a ROS burst in a suspension of rice cells. Only oneof the proteins, MGG_08054, activated a strong ROS burst(Supplemental Figures 1E and 1F). MGG_08054 is a putativechitinase, and was named MoChia1. Notably, the MoChia1-triggered ROS burst is much slower than that caused by pre-viously identified PAMPs, such as chitin or flg22 (Figure 1A).MoChia1 is phylogenetically distant from its closest orthologs inM. oryzae (Supplemental Figure 2; Supplemental Data Set).

MoChia1 Functions as a PAMP

Because MoChia1 can activate a ROS burst in rice cells, wespeculated that this protein acts as an elicitor/PAMP during riceblast infection.MoChia1was found tobe secretedduringmycelialgrowth, and the secretion depends on secretion signal peptide(SP), because removal of SP (MoChia1NSP) abolishes MoChia1secretion to the supernatant (Supplemental Figure 3A). ClassicalPAMPs induceMAPK signaling and callose deposition (Luo et al.,2017). When MoChia1 was exogenously applied to rice cells,we found that the MAPK pathway was significantly activated,whereas the mock treatment did not activate this pathway(Figure 1B). In addition,MoChia1 inducedcallosedeposition in therice cell walls, similar to the chitin treatment (Figure 1C). Wetherefore concluded that MoChia1 is an active PAMP for riceplants.

To further study its role in planta,we expressedMoChia1 in rice;however, the constitutive expression of MoChia1 driven by theUbiquitinpromoter resulted in adwarf phenotypeand the eventualdeathof the transgenicplants (Figure1D), indicating thatMoChia1may cause an autoimmune response in transgenic plants. Wetherefore used a dexamethasone (DEX)-inducible promoter (Luoet al., 2017) andgenerated theDEX:MoChia1plants. As expected,MoChia1 was mainly localized in the apoplasts of the transgenicrice plants; however, the rice transcription factor RERJ1, whichhas been reported to be localized in the nucleus (Miyamoto et al.,2013), cannot be detected in the rice apoplasts (SupplementalFigure 3B). To investigate whether MoChia1 triggers ROS pro-duction in vivo, we examined the ROS levels of the transgenicplants using 3,3-diaminobenzidine (DAB) staining, revealing thatmore H2O2 accumulated in the DEX:MoChia1 plants followingtreatmentwith30mMDEX (Figure1E). This result is consistentwiththe in vitro assays using rice suspension cells (Figure 1A) anddemonstrates that MoChia1 can activate ROS accumulation inplanta. Notably, the major PRR genes, except for OsFLS2, werenot significantly induced in Dex-treated DEX:MoChia1 plants(Supplemental Figure 4A), suggesting that theMoChia1-triggeredimmune response is likely independent of known PRRs.

MoChia1 Chitinase Activity is Dispensable for the ROS Burstin Rice

Because MoChia1 is phylogenetically distant from other putativechitinases in the M. oryzae genome (Supplemental Figure 2), weinvestigated whether this protein is a functional chitinase by ex-amining its enzymatic activity using colloidal chitin as the sub-strate (Niu et al., 2016). The recombinant MoChia1 couldeffectively hydrolyze chitin into monomeric GlcNAc (Figure 1F),indicating that this protein is a functional chitinase. Sequencealignmentof thechitinase familyproteinssuggested thatGlu137 isa conserved amino acid residue among chitinases, which waspreviously reported to be essential for chitinase activity(Supplemental Figure 4B; Lienemann et al., 2009). We thereforemutated the Glu137 (E) of MoChia1 to Gln (Q; MoChia1E137Q); thismutation abolished its chitinase activity (Figure 1F). Nevertheless,we found that chitinase activity is not required for MoChia1 toinduce the ROS burst in rice cells, because MoChia1E137Q couldstill activate the ROS burst (Figure 1G).Furthermore, we generated the MoChia1E137Q transgenic rice

plants with both Ubiquitin and Dex-inducible promoters. Theconstitutive expression of MoChia1E137Q led to an autoimmuneresponse in rice, as the transgenic plants showed a dwarfphenotype and eventual death (Supplemental Figure 4C). TheDAB staining assay also revealed that 30 mM DEX-treatedDEX:MoChia1E137Q plants accumulated more H2O2 in compar-ison with the untreated plants (Supplemental Figure 4D). Theseresults demonstrate that the chitinase activity is not required forMoChia1-activated ROS accumulation in planta. By contrast,rice chitinases could not activate the ROS burst in rice cells(Supplemental Figures 5A and 5B), implying that plants haveevolved amechanism to avoid activation of the immune responseby their own chitinases.

MoChia1 Plays a Fundamental Role in Fungal Growth

BecauseMoChia1 is a functional chitinase,wecheckedwhether itis indeed required for fungal growth. MoChia1 was found to bedifferentially expressed in various fungal tissues (Figure 2A); forexample, theexpressionofMoChia1washigher in themycelia andappressoria. Interestingly, during infection, MoChia1 was notexpressedat high levels until 48hpost inoculation (hpi; Figure 2B).Next, we knocked out MoChia1 in M. oryzae using the re-

combinantexchangemethod (SupplementalFigures6Aand6B;Liet al., 2017). When grown on complete minimal (CM) media, theOMoChia1-2 mutant displayed a higher growth rate than thewild type (Figures 2C and 2D), whereas the growth rate of thefungal strain overexpressing MoChia1 was significantly reduced(Supplemental Figures 7A and 7B). The OMoChia1-2 mutantsproduced many fewer conidia than the wild type; however, thisphenotypic defect could be rescued by complementation withwild-type MoChia1 (Figure 2E), indicating that MoChia1 plays anessential role in conidia production.Because chitin is a major constituent of the fungal cell wall, we

questioned whether M. oryzae could respond to cell wall stresstreatments in the absence of MoChia1. We found that theOMoChia1-2 strain was resistant to treatment with 0.1% CongoRed, a cell wall stressor (Figure 2F), indicated by the larger

174 The Plant Cell

diameter of its colony in comparison with the wild type afterculturing for 7d.Thesedatasuggest thatMoChia1maymodify thefungal cell wall to regulate the response to cell wall stress.We thenmeasured the chitin contents in themycelia andconidia of thewildtype and the OMoChia1 mutants; the two OMoChia1 mutantstrains exhibitedmuch higher chitin contents than the wild type in

both the mycelia and conidia, indicating that MoChia1 has chi-tinase activity in vivo (Figure 2G). MoChia1 is also required forproper chitin deposition in the cell walls. As shown in Figures 2Hand 2I, abnormal chitin deposition was observed. The fluores-cence signal was found primarily in the tip of the mycelium of wildtype, but was dispersed in the OMoChia1 mutants (Figure 2H),

Figure 1. MoChia1 Activates Pathogen-Triggered Immune Responses in Rice.

(A)MoChia1 activates the ROS burst in rice suspension cells. Recombinant MBP-MoChia1 protein was purified from E. coli, and 2mg/ml protein was usedfor the luminol-based ROS burst assay. Flg22 (100 nM) and chitin (0.1 mg/ml) served as positive controls. MBP served as a negative control. Values aremeans 6 SD (n = 4). RLU, relative light units.(B)MoChia1 protein can activate MAP kinase signaling in rice. MoChia1 protein (1 mg/ml) was incubated with rice suspension cells. MBP protein (1 mg/ml)was used as the negative control. Activated MAPKs were detected by immunoblotting with the phospho-p38 MAPK antibody at the indicated times. Thecorresponding bands are represented for the phosphorylation of mitogen-activated protein kinase 3 and mitogen-activated protein kinase 6. Coomassiebrilliant blue (CBB) staining of ribulose-1,5-bis-phosphate carboxylase/oxygenase was used to ensure equal loading in each lane. The experiment wasrepeated three times with similar results.(C)MoChia1 inducescallosedeposition in rice. The leavesof 2-week-old rice seedlingswere treatedwith0.6mg/mlMoChia1or 8nMchitin for 16h, and thenstained using aniline blue. MBP served as the mock treatment.(D) The overexpression ofMoChia1 leads to an autoimmune response in rice.MoChia1, driven by the Ubiquitin promoter, was ligated into the pC1390Ubinary vector and transformed into the rice variety Nipponbare (wild type). Lines 5 and 8 are two representative independent lines. Bar = 1 cm.(E)MoChia1 overexpression leads to ROS accumulation in rice. Two-week-oldDEX:MoChia1 plants were treated with 30mMDEX for 24 h; then the leaveswere stained with DAB. Lines 4 and 6 are two independent transgenic lines. Bar = 1 cm.(F) MoChia1 is a functional chitinase, and the Glu137 mutation abolishes its enzymatic activity. The chitinase enzyme activity of the recombinant MBP-MoChia1 protein was analyzed, using colloid chitin as substrate. The reaction of DNS (3,5-dinitrosalicylic acid) and GlcNAc wasmonitored at OD 565 nm.One unit of chitinase activity was defined as the amount of enzyme required to release 1 mmol ofN-acetyl-D-glucosamine per hour. Values are means6 SD

(n = 3). **Significant differences from MBP at P < 0.01 (Student’s t test).(G) The enzyme activity of MoChia1 is not required for the ROS burst. Rice suspension cells were used to measure ROS production in a luminol-basedassay, using 1 mg/ml protein. MBP was used as a negative control. Values are means 6 SD (n = 4).

OsTPR Binds Fungal Chitinase for Defense 175

Figure 2. MoChia1 is Required for the Growth and Development of M. oryzae.

(A)MoChia1 expression levels in different tissue or developmental stages.MoChia1 expressionwas investigated using RT-qPCR. The fungi were grown inCM media. Values are means 6 SD (n = 3).(B)MoChia1 expression levels duringM. oryzae infection in rice. The rice leaveswere inoculatedwithM. oryzae spores at a concentration of 13 105 permL.The leaves were sampled at the indicated time points for RT-qPCR assays. Values are means 6 SD (n = 3).(C) MoChia1 affects mycelial growth. The images were taken from the wild-type Guy11 and DMoChia1 mutants grown on the CM medium for 2 weeks.Bar = 1 cm.(D)Statistical analysis of the growth rate ofmycelia in (C). Values aremeans6 SD (n= 8). **Significant differences fromwild type at P<0.01 (Student’s t test).(E) DMoChia1mutants produce fewer conidia. Conidia were observed under a light microscope after illumination for 24 h. The experiments were repeatedthree times with similar results.(F)DMoChia1mutants are less sensitive to cell wall stress. TheGuy11,DMoChia1mutants, and complementation strainswere grownon completemediumcontaining 0.1% Congo Red. Photographs were taken after 7 d in culture at 28°C. Bar = 1 cm.(G)DMoChia1mutantsdisplay chitin accumulation in themycelia andconidia. Thechitin contents of theGuy11andDMoChia1myceliaweremeasuredaftera 2-d culture at 28°C. Fresh conidia after light induction were used for chitin content measurements. DW, dry weight. Values are means 6 SD (n = 3).**Significant differences from Guy11 at P < 0.01 (Student’s t test).(H) and (I) Knocking outMoChia1 leads to abnormal chitin deposition in the mycelia, conidia, and germination tubes. Mycelia were cultured for 14 days at28°C. Mycelia, fresh conidia, and germinated conidia were used for cell wall staining with calcofluor. The fluorescence was observed under a fluorescencemicroscope using the DAPI channel. Bar = 10 mm.

176 The Plant Cell

revealing that MoChia1 affects chitin distribution. In addition, thefluorescence signal was also found at the germination point of thegermination tube (Figure 2H); however, in theOMoChia1 conidiacells it was found at the septal area. These data demonstrate thatMoChia1 is a critical gene for modulating fungal growth and theresponse to cell wall stresses.

MoChia1 Contributes to M. oryzae Virulence

In general, developmental defects in fungi affect their virulence.We therefore evaluated the pathogenicity ofOMoChia1mutantsas well as the MoChia1-OE strains on rice. On a hydrophobicglass surface, the OMoChia1 mutants exhibited much slower

germination tube development and appressoria formation thanthewild type (Figure 3A). At 3 hpi, thegermination tubewasalmostcompletely developed in the wild type, and at 6 hpi, appressoriahad formed; however, the development of these features wasdelayedbyabout 3 h in theOMoChia1mutants. Accordingly, only58.3%of theOMoChia1mutantshad formedappressoriaat6hpi,but 91.2%of thewild-type fungi had done so (Figures 3B and 3C).Nevertheless, we found that the fungal hyphae of theOMoChia1mutants had developed normally when observed at 24 and 48 hpi;however, the mutant hyphae grew more slowly, with a delayof ;12 h compared with the wild type (Figure 3D). This slowdevelopment also largely reduced the pathogenicity of theOMoChia1 mutants on rice leaves following spray inoculation

Figure 3. MoChia1 is Required for M. oryzae Virulence.

(A) Germination of conidial spores of the wild type and DMoChia1 mutants. Conidia were germinated on glass cover slips. The germination tubes andappressorium development were observed at the indicated time points.DMoChia1-2 andDMoChia1-11 are two independent mutant strains. Bar = 10mm.(B)Appressorium formation is largely delayed in theDMoChia1mutants. Appressoriumdevelopmentwasobserved after a 6-h incubationon ahydrophobicglass surface. Bar = 100 mm.(C) Statistical analysis of appressorium formation in the wild type and DMoChia1 mutants observed in (B). Values are means 6 SD (n = 3). **Significantdifferences from the wild type at P < 0.01 (Student’s t test).(D)DMoChia1display reducedvirulenceonrice.The rice leafsheathwas inoculatedwithaconidial suspensionataconcentrationof13105conidiapermL in0.2% Tween 20. The hyphae images were taken at 24 and 48 hpi. Bar = 10 mm.(E) Disease symptoms of rice leaves infected with the wild-type M. oryzae, DMoChia1 mutants, and the complementation strains. Conidial suspensions(13 105 conidia permL in 0.2%Tween 20)were sprayed onto the leaf surfaces of 2-week-old seedlings. Imageswere taken at 7 dpi (days post inoculation).Bar = 1 cm.(F) Relative fungal biomass for (E). The fungal biomass was determined using qPCR of theM. oryzae Pot2 gene against the rice OsUbi1 gene. Values aremeans 6 SD (n = 4). **Significant differences from the wild type at P < 0.01 (Student’s t test).

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(Figures3Eand3F), indicatedby fewer lesions thanwereobservedfollowing inoculation with the wild-type strain. By contrast, theMoChia1 overexpression strains showed lower chitin levelsthan the control and the MoChia1E137Q overexpression strains(Supplemental Figure 7C). Interestingly, the MoChia1 over-expression strains also displayed lower virulence than the wild-type Guy11 (Supplemental Figures 7D and 7E).

Melanin is known to contribute to fungal pathogenicity (Soaneset al., 2012). Knocking outMoChia1 remarkably reduced melaninaccumulation in the fungus (Supplemental Figure 7F), and de-creased the expression levels of themelanin biosynthesis-relatedgenes RSY1 and 4HNR; however, the expression of AIB1 andBUF1wasnot affected (Supplemental Figure7G;Kawamuraet al.,1997). In addition, the infection of rice cells with the OMoChia1mutant fungus activated stronger expression of RbohB andRbohD, the defense-related marker genes, than infection with thewild-type strain (Supplemental Figures 8A and 8B). The abovedata imply that the loss of MoChia1 function reduces pathogenvirulencebecauseof theslowerdevelopmentof thehyphae,whichis at least partially mediated by the enhanced immune responsein rice.

Ectopic Expression of MoChia1 Activates theImmune Response

The data described above show that transiently inducedMoChia1expression leads to ROS accumulation in rice plants (Figures 1Dand1E). Inaddition,MoChia1actsasa typicalPAMPfor riceplants(Figures 1B and 1C). We therefore investigated whether the ec-topic expression of MoChia1 in rice could enhance the diseaseresistance of these plants. Because the constitutive expression ofMoChia1 in rice caused an autoimmune response, we tested thedisease response of the DEX:MoChia1. The DEX:MoChia1 plantsshowed enhanced resistance to M. oryzae when pretreated with30 mM DEX (Figures 4A and 4B).

We next examined the infection process at the cellular levelusing the rice leaf sheath. The inoculation of the leaf sheathrevealed that the induced ectopic expression of MoChia1 in ricecells restricted the mycelial growth of the pathogen (Figure 4C).Using DAB staining, we found elevated H2O2 levels in the infectedcells of the DEX:MoChia1 plants compared with the wild type,which may result in the enhanced disease resistance of theseplants (Figure 4D). We also found that the expression of OsPR10and OsRbohA was significantly upregulated in the DEX-treatedDEX:MoChia1 plants (Figures 4E and 4F). These genes are in-dicators of the activation of the immune response in rice plants(Yang et al., 2017), suggesting that MoChia1 leads to the acti-vation of the immune response. These data demonstrate that theectopic expression of MoChia1 can enhance blast disease re-sistance in rice.

MoChia1 Interacts with OsTPR1 in the Rice Apoplast

We attempted to identify the proteins that mediate MoChia1recognition in the rice apoplast, or proteases in rice that couldpotentially degrade MoChia1. To this end, we conducted a yeasttwo-hybrid screen against a rice cDNA library using MoChia1 as

the bait. This approach did not lead to the identification of theMoChia1-interacting protease, nor PRRs; however, we dididentify one of proteins that can interact with MoChia1, OsTPR1.This protein is a member of the tetratricopeptide-repeat (TPR)family, which often function as scaffold proteins in protein–proteininteractions (Cerveny et al., 2013).We confirmed the interaction ofMoChia1 and OsTPR1 by expressing their full-length cDNA se-quences in yeast (Figure 5A). Notably, the MoChia1E137 mutationdid not affect the interaction (Figure 5A). The OsTPR1 proteincontains three TPR motifs from amino acids 64 to 172(Supplemental Figure 9A). We further identified the interactionregion of OsTPR1 and found that the C-terminal of OsTPR1 waslikely responsible for the interaction (Supplemental Figures9Aand9B). GST pull-down assays using the recombinant GST-MoChia1and MBP-OsTPR1 showed that the two proteins interactedwith each other in vitro (Figure 5B). Because the sequence ofMoChia128-370 is predicted to be a carbohydrate binding domain(CBD), we then tested the interaction betweenMoChia1 CBD andOsTPR1 by pull-down assays. We confirmed that MoChia1 CBDinteracted with OsTPR1 (Figure 5B). We also tested their in-teraction in planta.MoChia1was found to interactwithOsTPR1 inNicotiana benthamiana leaves using both split-luciferase andbimolecular fluorescence complementation (BiFC) assays (Fig-ures 5C to 5E). The above results confirm the interaction ofMoChia1 and OsTPR1 in vitro and in vivo.It is worth noting thatMoChia1 apoplast localization is essential

for the interaction of MoChia1 and OsTPR1, because removal ofMoChia1 SP (MoChia1NSP) abolished the interaction (Figures 5Dand 5E). In addition, it is necessary to check the subcellular lo-cation of OsTPR1. OsTPR1 was found to colocalize with theplasmamembranemarker PCD-1002 in rice protoplasts aswell asinN. benthamiana leaves (Figures 5F and 5G; Du et al., 2013). Theplasmolysis assay further supports that OsTPR1 localizes to thericeplasmamembrane (Figure 5G). To investigatewhether there isany extracellular domain/region(s), we used protease protectionassays to explore the region. Both trypsin and protease K coulddegradeOsTPR1butnotgreenfluorescentprotein (GFP)orACTINin rice protoplasts and N. benthamiana leaves (SupplementalFigures 9C to 9E), indicating that OsTPR1 carries extracellulardomain/region(s).With useof theplasmolysis assay, theMoChia1subcellular localization was determined, which was found in theplantapoplast following its transientexpression inN.benthamianaleaves, similar to the reported apoplastic anionic gaiacol perox-idase of G. hirsutum (GhPOD10; Figure 5H; Li et al., 2016). Theresults also showed that, before plasmolysis, the MoChia1 andGhPOD10 colocalized with PCD-1002; however, after plasmol-ysis, they were separated from PCD-1002 and were localized inthe plant apoplast (Figure 5H). The evidence suggests thatMoChia1 likely interacts with OsTPR1 in the plant apoplast.

MoChia1 Suppresses the Chitin-Triggered ROS Burst inRice, Whereas OsTPR1 Removes this Suppression

As described above, MoChia1 likely interacts with OsTPR1 in therice apoplastduringM.oryzae infection.We therefore investigatedthe biological significance of this interaction. MoChia1 is a chiti-nase and uses chitin as its substrate; MoChia1 as well asMoChia1E137Qwere able to bind chitin in vitro in a chitin pull-down

178 The Plant Cell

assay (Figure 6A), indicating that the chitinase activity is dis-pensable for binding chitin. OsTPR1 can also bind MoChia1;therefore, we investigated whether it competes with chitin to bindMoChia1.The result showsthat lessMoChia1 ispulleddown in thechitin pull-down assay following the addition of increasingamounts of OsTPR1 (Figure 6A), indicating that OsTPR1 can in-terfere with the interaction between MoChia1 and chitin.

To further verify the competition betweenOsTPR1andchitin forMoChia1 binding, we used microscale thermophoresis (MST)assays to study their interactions. The results show that theOsTPR1 and MoChia1 interaction has a smaller Kd (dissociationconstant; Kd = 0.052) than that of MoChia1 and chitin (Kd = 0.31),suggesting that OsTPR1 has a higher binding affinity for MoChia1

than does chitin (Figures 6B and 6C). By contrast, OsTPR1 wasunable to bind with chitin (Figure 6C). In the presence of OsTPR1,chitin cannot efficiently bind to MoChia1; we found that 20 mMrecombinant OsTPR1 caused the disassociation of chitin andMoChia1 (Kd=1.34),whereas 100mMOsTPR1almost completelyprevented the interactionofchitin andMoChia1 (Figure6C). Thesedata confirm the competition between OsTPR1 and chitin forMoChia1 binding, which essentially releases chitin to activate theimmune response.Because chitin binds to MoChia1, we therefore checked

whether MoChia1 suppresses the chitin-triggered ROS burst asSlp1 does (Mentlak et al., 2012). Purified MoChia1 protein wasfound to substantially suppress the chitin-triggered ROS burst in

Figure 4. Ectopic Expression of MoChia1 Enhances Rice Resistance to M. oryzae.

(A)DEX:MoChia1plantsexhibit enhanceddisease resistance toM.oryzaeafterDEX treatment. Two-week-oldDEX:MoChia1plantswere treatedwith30mMDEX applied to the roots. After 24 h, the seedlingswere spray-inoculatedwith conidial suspensions (13 105 conidia permL in 0.2%Tween 20). The imageswere taken at 5 dpi. Bar = 1 cm.(B) Relative fungal biomass in (A). Values are means 6 SD (n = 4). **Significant differences from wild type at P < 0.01 (Student’s t test).(C) The DEX:MoChia1 plants displayed enhanced disease resistance to M. oryzae after DEX treatment. Rice leaf sheaths were inoculated with conidialsuspension at a concentration of 13 105 conidia per mL in 0.2% Tween 20. The hyphae images were taken at 24 and 48 hpi, respectively. Bar = 10 mm.(D) DEX:MoChia1 plants exhibit enhanced ROS accumulation during M. oryzae infection after DEX treatment. The rice leaf sheath was inoculated witha conidial suspension at a concentration of 1 3 105 conidia per mL in 0.2% Tween 20. The DAB staining was performed at 24 and 48 hpi, respectively.Bar = 10 mm.(E) and (F)OsPR10 andOsRbohA expressionwas elevated inDEX:MoChia1plants. Two-week-oldwild type andDEX:MoChia1plantswere pretreatedwith30mMDEX.After24h,RT-qPCRwasused toexamine thegene transcription levels inplants.Valuesaremeans6 SD (n=4). **Significantdifferences fromwildtype at P < 0.01 (Student’s t test).

OsTPR Binds Fungal Chitinase for Defense 179

Figure 5. MoChia1 Interacts with OsTPR1 In Vitro and In Vivo.

(A)MoChia1 andMoChia1E137Q interact withOsTPR1 in yeast. AD-MoChia1, AD-MoChia1E137Q, andBD-OsTPR1 plasmidswere cotransformed into yeastcells andscreenedonsyntheticdextrosemedia lackingLeuandTrp (SD-2). Thesingle colonieswere seriallydilutedontoSD-2andSD-3 (synthetic dextrosemedia lacking Leu, Trp, and His) to observe the yeast cell growth. Yeast cotransformed with pGADT7-T+pGBKT7-53 served as a positive control. Yeastcotransformed with pGADT7-T+pGBKT7-lam served as a negative control. EV, empty vector.(B) MoChia1 and its CBD interact with OsTPR1, revealed using GST pull-down assays. The recombinant MBP-OsTPR1, GST-MoChia1, and GST-MoChia1CBD proteins purified from E. coli were subjected to a GST pull-down analysis. Interacting proteins were visualized with immunoblotting.(C)MoChia1 interactswithOsTPR1 inN. benthamiana, revealed using split luciferase assays.N.benthamiana leaveswere co-infiltratedwith35S:MoChia1-nLUC and 35S:cLUC-OsTPR1. Luciferase complementation imaging assays were performed 2 d later. The gels at the right show the expression of therespective proteins. This experiment was repeated three times with similar results.(D)MoChia1but notMoChia1NSP interactswithOsTPR1 inN.benthamiana, revealedusing split YFPassays. Theexperimental procedurewassimilar to thatused in (C).MoChia1orMoChia1NSPwas fusedwithcYFPat theC terminus, andOsTPR1was fusedwithnYFPat theN terminus. The imageswereobservedunder a confocal microscope 2 d later. Bar = 50 mm.(E) Immunoblotting showed the expression of respective proteins in (D).(F)OsTPR1 is localized to theplasmamembrane in riceprotoplasts.OsTPR1-GFPand theplasmamembranemarkerPCD-1002-CFPwereco-expressed inrice protoplasts and visualized by confocal microscopy. GFP co-expressed with PCD-1002-CFP was the negative control. PCD-1002-CFP was assignedthe pseudocolor red. Bar = 10 mm.(G)OsTPR1 is localized to the plasmamembrane inN. benthamiana.Proteins fusedwith respective fluorescence proteins were transiently expressed inN.benthamiana leaves following Agrobacterium-mediated transformation. Plasmolysis was performed by treatments with 10 mM NaCl. The images werecaptured using a confocal microscope. PCD-1002-CFP was assigned the pseudocolor red. Bar = 25 mm.(H)MoChia1 is located in the plant apoplast.Gossypium hirsutum apoplastic peroxidase GhPOD10 andMoChia1-GFPwere expressed inN. benthamianaleaves. The protein subcellular localization was observed using a confocal microscope. Top panel, protein localization before plasmolysis; bottom panel,protein localization after plasmolysis. The fluorescence curves were obtained following the direction of the white arrows. Bar = 25 mm.

180 The Plant Cell

rice cells (Figure 6D). MoChia1 is a PAMP, but it did not trigger anenhanced immune response in the presence of chitin revealed byeither MAPK activation or callose deposition on the rice cell walls(Supplemental Figure 10). This result suggested that MoChia1suppressed the chitin-triggered immune response in planta(Supplemental Figure 10). OsTPR1 significantly reverses thechitin-triggered ROS burst suppressed by MoChia1; however,OsTPR1 alone cannot induce a ROS burst (Figure 6D), implyingthatOsTPR1maycompetewithchitin tobind to thesame regionofMoChia1. This competition should lead to increased levels of freechitin in the apoplast, which is perceived by the plant PRRs. Toconfirm this observation, we also tested the ROS burst in a sus-pension of rice cells overexpressing OsTPR1. MoChia1 sup-presses the chitin-inducedROSburst in wild-type cells, but couldnot suppress the ROS burst in OsTPR1-overexpressing cells,

demonstrating that OsTPR1 prevents the MoChia1-mediatedsuppression of the chitin-induced ROS burst in vivo (Figure 6E).Notably, OsTPR1 does not interfere with the MoChia1-activatedimmune response (Supplemental Figure 11).

OsTPR1 Promotes Blast Disease Resistance

TPR proteins have previously been reported to be recruited byvarious pathogens to enhance their virulence; however, thefunction of these proteins in higher plants remains elusive(Cerveny et al., 2013). Because OsTPR1 disarms the MoChia1-mediated suppression of the chitin-triggered immune response(Figures 6D and 6E), it is speculated that OsTPR1 may contributeto disease resistance in plants.

Figure 6. OsTPR1 Removes the MoChia1-mediated Suppression of the Chitin-Triggered ROS Burst.

(A) OsTPR1 interferes with the ability of MoChia1 to bind chitin. Left: Chitinase activity is dispensable for binding chitin. Right: Colloid chitin and purifiedrecombinant proteins were used for chitin pull-down assays. Each 50-mL reaction mixture contained 50 mg colloid chitin and 2 mg MBP-MoChia1. GST-OsTPR1protein (0, 40, and80mg)wasadded to themixtureand incubated for1hwithconstant shaking. Thechitin-associatedMBP-MoChia1wasdetectedusing immunoblotting. The experiment was repeated three times with similar results.(B)MST assays show thatMoChia1 interacts withOsTPR1. The recombinant proteinswere contained in NT standard capillaries. The solid curve is the fit ofthe data points to the standard Kd-fit function. The experiment was repeated at least three times with similar results. Kd, dissociation constant. Bars6 SD

(n = 3).(C) OsTPR1 competes with chitin for MoChia1 binding, as revealed using MST assays. The experimental procedure was the same as in (B).(D)MoChia1 suppresses the chitin-triggered ROS burst in rice cells, whereas OsTPR1 removes this suppression. A 1 mg/ml MoChia1 or 1 mg/ml OsTPR1aliquotwas incubatedwith 0.2mg/ml chitin in the buffer [50mMTris-HCl (pH7.0), 100mMNaCl] for 10minwith constant shaking at 4°C. The reactionswerethen suppliedwith aROS reactionmixture to detect their luminescence. Values aremeans6 SD (n=6). **Significant differences at P< 0.01 (Student’s t test).RLU, relative light units.(E)Rice suspension cells overexpressingOsTPR1 display enhanced chitin-triggeredROSbursts in the presence ofMoChia1. Rice cells sub-culturedmorethan 20 times were used for the assays. Values are means 6 SD (n = 6). **Significant differences at P < 0.01 (Student’s t test). RLU, relative light units.

OsTPR Binds Fungal Chitinase for Defense 181

We examined OsTPR1 expression in rice leaves during blastinfection, and found that the expressionwas significantly elevatedfollowing their inoculation with M. oryzae, implying that OsTPR1expression is induced by infection (Figure 7A). Interestingly, wealso found that MoChia1 could slightly activate OsTPR1 tran-scription (Figure 7B). We then generatedOsTPR1 overexpression(OsTPR1-OE) and RNA interference (RNAi) lines (OsTPR1-RNAi)and confirmed the OsTPR1 expression levels using reversetranscription-quantitative PCR (RT-qPCR; Figures 7C and 7D).Then, the homozygousOsTPR1-OEplants and silencedOsTPR1-RNAi lines were used for the pathogen inoculation assays.OsTPR1-OE plants exhibited enhanced disease resistance to M.oryzae, whereas the OsTPR1-RNAi plants were more suscepti-ble than the wild type (Figures 7E and 7F). This result is fur-ther supported by the presence of higher levels of H2O2 in the

OsTPR1-OE plants following pathogen infection as revealed byDAB staining, but very low levels in the OsTPR1-RNAi plants(Figure 7G). These data indicate that OsTPR1 positively con-tributes to disease resistance to M. oryzae by enhancing riceimmunity.

DISCUSSION

Plant apoplast immunity is emerging as an important branch ofplant innate immunity (Doehlemann and Hemetsberger, 2013).Many phytopathogens secrete virulence factors into the plantapoplast, to either suppress theperceptionofPAMPsby thePRRsor to directly damage the host cell structure for their own use(Jashni et al., 2015; Langner and Göhre, 2016; Ma et al., 2017).Plants are also known to secrete proteases or protease inhibitors

Figure 7. OsTPR1 Positively Contributes to Blast Disease Resistance.

(A) The OsTPR1 expression is induced by M. oryzae infection. Two-week-old rice seedlings were spray-inoculated with a conidial suspension ata concentration of 13 105 conidia per mL in 0.2% Tween 20. The expression ofOsTPR1was examined using RT-qPCR at the indicated times. Values aremeans 6 SD (n = 4). **Significant differences from 0 hpi at P < 0.01 (Student’s t test).(B) TheOsTPR1 expression in DEX:MoChia1 transgenic plants. Two-week-old DEX:MoChia1 plants were treated with 30 mMDEX applied to the roots. At24 h, the leaves were sampled and the expression ofOsTPR1was examined using RT-qPCR. Values are means6 SD (n = 4). **Significant differences fromwild type at P < 0.01 (Student’s t test).(C) and (D) OsTPR1 expression levels in the OsTPR1 transgenic plants. The leaves were sampled from 2-week-old rice seedlings of the OsTPR1-overexpressing lines and the RNAi-silenced lines. OsTPR1-OE-3 and OsTPR1-OE-11 are two independent overexpression lines. OsTPR1-RNAi-5 andOsTPR1-RNAi-7 are two independent silenced lines. Others are as in (B).(E)OsTPR1positively contributes toblast disease resistance in rice. TheOsTPR1overexpression (OE) lines3and11and theOsTPR1-silencing lines5and7were spray-inoculated with M. oryzae spores at a concentration of 1 3 105 conidia per mL in 0.2% Tween 20. At 5 dpi, the disease lesions were pho-tographed. The experiment was repeated at least three times.(F) Relative fungal biomass in (E). Values are means 6 SD (n = 4). **Significant differences from wild type at P < 0.01 (Student’s t test).(G) DAB staining of H2O2 production inOsTPR1 transgenic plants after pathogen infection. Two-week-old rice seedling leaf sheaths were inoculated withconidial spores at a concentration of 13 105 conidia per mL. The infected leaf tissue was stained with DAB, and the images were taken at 24 and 48 hpi.

182 The Plant Cell

into the apoplast during pathogen invasion, presumably targetingthe virulence factors to repress infection (Doehlemann andHemetsberger, 2013), demonstrating the importance of apoplastimmunity.

As a key constituent of the fungal cell wall, chitin is a majorimmune activator in plant–fungi interactions. Pathogens havedeveloped multiple strategies to sequester chitin oligomers andprevent their recognition by plant PRRs; for example, the effectorprotein Ecp6 secretedbyC. fulvum canbind chitin and hide it fromplant receptors, preventing the activation of the immune response(de Jonge et al., 2010). Another C. fulvum effector, Avr4, directlyprotects fungal chitin from degradation by plant chitinases (vanden Burg et al., 2006). Similarly, M. oryzae secretes the effectorSlp1,whichcompeteswithOsCEBiP tobindchitinoligomers, thusblocking the OsCEBiP-OsCERK1 activated immune response inrice (Mentlak et al., 2012). It is noteworthy that chitin is dynamicallymodifiedbychitinase(s)during fungigrowthand infection,but theirroles in plant immune responses are largely unknown. In an at-tempt to screen new elicitors/PAMPs during blast infection, weidentifiedaM.oryzaechitinase,MoChia1,whichplaysdual roles inrice immunity (Figure 8).

Chitinases are widely distributed in prokaryotic and eukaryoticcells, and have diverse biological functions (Langner and Göhre,2016); for example, the bacterial pathogen Xylella fastidiosa se-cretes chitinase that hydrolyzes the chitin of its host fungi andinsects for use as a carbon source (Labroussaa et al., 2017).Fungal pathogens use chitinases to continuously remodel theircellwall plasticityduringgrowthand infection (LangnerandGöhre,2016). Langner et al. (2015) also showed that chitinases wereessential for cell separation inU.maydis. Because chitin is the keycomponent of fungal cell walls, plants secrete chitinases to

directly degrade the cell walls of invading fungal pathogens andhalt their infection (Cletus et al., 2013). Overexpressing plantchitinases has been shown to effectively enhance the resistanceof wheat (Triticum aestivum) to Fusarium pathogens and tomato(Solanum lycopersicum) to Botrytis cinerea (Cletus et al., 2013).We show here that MoChia1 is indispensable for the growth andvirulence of this fungus; knocking outMoChia1 results in a fastergrowth rate, theproductionof fewer conidia, and the redistributionofchitin in thecellwalls (Figure2).MoChia1alsomodulates thecellwall composition and the cell wall stress response in M. oryzae(Figure 2). In addition, knockingoutMoChia1 significantly reducesthe virulence of M. oryzae, because the mutant formed its ap-pressoria more slowly than the wild-type strain (Figure 3). Thehigher chitin content in the fungal cell wall could therefore beresponsible for the activation of the immune response and thecompromised virulence of the DMoChia1 mutant.Rice plants can recognize MoChia1 and mount an immune

response; we demonstrate that MoChia1 exhibits the typicalfeatures of a PAMP, activating the ROS burst, causing callosedeposition, and activating MAPK signaling (Figure 1). Notably,plant chitinases putatively orthologous to MoChia1 cannot acti-vateROSbursts in rice cells, although theypossess theenzymaticactivity to hydrolyze chitin (Supplemental Figure 5). Our phylo-genetic analysis shows the evolutionary distance betweenMoChia1 and the rice chitinases; the plant chitinases are distantlyrelated to the microbial chitinases (Supplemental Figure 2). Be-cause rice chitinases cannot activate the ROS burst but possesschitinase activity (Supplemental Figure 5),wehypothesize that theevolution of the plant chitinases followed a different path to thefungi following the divergence of these species. The evolutionaryconsequence is that plants are able to carefully avoid activatingtheir own immune systems. It is noteworthy that the MoChia1-triggered ROS burst is slower than thosemediated by chitin orother PAMPs in rice (Figure 1A), implying the existence ofan unknown PRR that specifically recognizes fungus-derivedchitinases.Unexpectedly, we also found that MoChia1 suppressed the

chitin-triggered ROS burst in rice cells (Figure 6D). MoChia1 likelybinds tochitin toprevent it frombeing recognizedbyOsCEBiPandOsCERK1, functioning inasimilarmanner toSlp1duringM.oryzaeinfection (Mentlak et al., 2012); however, we found that the riceprotein OsTPR1 could disarm the MoChia1-mediated suppres-sion of the ROS burst. The TPR family proteins contain three ormore TPR structural motifs, originally identified in yeast (Sikorskiet al., 1990; Cerveny et al., 2013). They usually mediate pro-tein–protein interactions or the assembly of multi-protein com-plexes (Cerveny et al., 2013). These TPR proteins thereforefunction in a variety of cell processes, including being directlyinvolved in virulence-associated functions in bacteria (Cervenyet al., 2013). The influenza virus was reported to recruit a 58-kDTPR protein to downregulate the interferon-induced double-stranded RNA-activated protein kinase in bovine kidney cells toavoid the kinase’s deleterious effects on viral protein synthesisand replication (Lee et al., 1994). However, the functions of theTPR proteins are not yet fully understood in higher plants. Similarto OsTPR1, several TPR family proteins were found to be locatedto the plasma membrane, although they have no predictedtransmembrane domain (Lin et al., 2008, 2009). Our data indicate

Figure 8. Proposed Working Model.

During M. oryzae infection, the rice PRR receptor OsCERK1 recognizesfungal chitin and mounts an immune response; however, the fungus-secreted chitinase MoChia1 is able to bind chitin and repress the chitin-mediated activation of the immune response. MoChia1 is perceived by anunknownPRR,which also triggers theROSburst. In addition, the rice plantdeploysOsTPR1 tocompetewithchitin andbind toMoChia1, freeingchitinand thereby re-establishing the activation of the immune response.

OsTPR Binds Fungal Chitinase for Defense 183

that OsTPR1 may carry atypical transmembrane domain/region(Supplemental Figure 9). In addition, the TPR domain has beenfound to function in protein translocation across membranes(Schlegel et al., 2007). Lin et al. reported that the TPR1 proteins inArabidopsis and tomato interact with ethylene receptors to in-fluence the ethylene signaling pathway, and that overexpressionof the TPR1 proteins resulted in dwarf phenotypes in plants (Linet al., 2008, 2009). However, the molecular mechanism behindthese responses remains unclear. In our study, we speculate thatthe site at which OsTPR1 interacts with MoChia1 is probably alsothe MoChia1 binding site for chitin (Figure 6). The competition ofOsTPR1 for MoChia1 binding essentially exposes chitin to Os-CEBiP and OsCERK1, which subsequently re-activates rice im-munity. Asa result, transgenic riceplants overexpressingOsTPR1display enhancedblast disease resistance (Figure7). Although theroles of OsTPR1 need further investigation, we conclude that itfunctionally acts as a “decoy” chitin in this case (for model, seeFigure 8); therefore, our study demonstrates that the apoplastdecoy strategy also exists in higher plant.

In summary, we have identified a chitinase in M. oryzae,MoChia1, which contributes to fungal virulence by modulatingfungal growth and suppressing the chitin-mediatedROSburst inthe host plant. In addition, MoChia1 also acts as a PAMP. Wepropose a scenario that during M. oryzae infection, MoChia1 issecreted, which modulates the fungal cell wall and also bindschitin to suppress rice immune response; rice plants, however,not only deploy OsTPR1 to counteract the immune suppressioncaused by MoChia1, but also use the unknown PRR to perceiveMoChia1 (Figure 8). This work unravels a mechanism involvingthe chitin-related immune response during rice blast infection.Nevertheless, the ultimate immune output should depend on theOsTPR1 and MoChia1 recognition receptor in different ricespecies. Our work demonstrates the complex arms race thatoccurs in plant apoplast immunity. Although the importance ofMoChia1 in triggering the immune response of different ricevarieties requires further investigation, the discovery of chitinasein the plant immune response may be useful for rice breeding.Future work should focus on the signaling components involvedin MoChia1 recognition in rice.

METHODS

Plant and Fungal Strain Growth Conditions

The Magnaporthe oryzae strain Guy11 was cultured at 28°C on oatmealagar medium (oatmeal 40 g/L, calcium carbonate 0.6 g/L, agar 30 g/L) for2weeks.Conidial formationswere inducedunderwhite light (20,000 lux) for2 to;3 days after removing the surface mycelium. Then, the spores werecollected in 10 mL sterile water. Rice (Oryza sativa, subsp. japonica cvNipponbare) plants were grown at 28°C under 16-h light and 8-h darkcondition.

Preparation and Purification of Secreted Proteins from M. oryzae

To screen the elicitors, 40 L ofM. oryzae growth culture was collected andspun down at 12,000 g for 10 min at 4°C. The supernatant was filteredthrough a 0.22 mm Millex Syringe Filter Unit (Millipore). Then, 70 g (NH4)2SO4 powder was added to 100 mL of supernatant to precipitate totalproteins at 4°C for 12h. Thesedimentwas collected through centrifugation

at 12,000 g for 10 min at 4°C. The protein pellet was resuspended anddissolved in TE buffer [10mMTris-HCl, 1mMEDTA (pH 7.5)]. The proteinswere loaded onto a Superdex200 10/300GL (GE Healthcare) column andsubjected to fast protein liquid chromatography separation. The fractionswere collectedaccording to theabsorbancepeaksat 280nm.Theselectedfractions were further purified by anion-exchange chromatography on HiTrap TM QXL (GE Healthcare). The fractions that can trigger ROS burst inrice suspension cells were analyzed by liquid chromatography tandemmass spectrometry (LC-MS/MS, Thermo Fisher Scientific).

Rice Leaf and Leaf Sheath Inoculation Assays

Rice leaf and leaf sheath inoculation assays followed the method used byYang et al. (2017). For leaf inoculation, 2-week-old rice seedlings weresprayed with M. oryzae conidial suspensions at a concentration of 105

conidia/ml in 0.2% Tween-20. Inoculated plants were placed in a growthchamber at 28°C for 24 h in the dark, and switched back to the normalgrowth condition of a photoperiod of 16-h light (20,000 lux white fluo-rescent light) and 8-h dark. Photographs were taken at 5 to7 d after in-oculation. The respective fungal biomass was examined by qPCR usingspecific primers forPot2ofM. oryzae and normalized to the reference geneOsUbi1. SYBR Premix Taq (Toyobo) was used for qPCR. Reactions weredetected by the Bio-Rad system. For rice sheath inoculation assays, theconidial suspensions were injected into the inner leaf sheaths with a co-nidial concentration of 105 mL by a syringe, and then the inoculated leafsheaths were incubated under the same conditions as those that werespray inoculated. At the indicated time points, the leaf sheaths were ex-amined under a microscope (Olympus BX51) to observe the fungal de-velopment in rice tissues.

Plant Transformation

A full-length OsTPR1 CDS was cloned from the rice variety Nipponbare,and the fragment was ligated to the binary vector pCAMBIA 1390U at sitesthat were digested by KpnI and BamHI to generate the overexpressionconstruct. For the RNAi vector construction, a unique length of 231 bp to428bpofOsTPR1wascloned into theRNAivectorpTCK303 (BamHI/KpnI-digested for the forward fragment andSpeI/SacI-digested for theconversefragment). The primers used are listed in Supplemental Table 2. Theconstructs were introduced into Nipponbare through an Agrobacterium-mediated transformationmethod described previously (Xiong et al., 2017).

Fungal Transformation

MoChia1, MoChia1NSP, MoChia1E137Q overexpression, and MoChia1knockout and complementation constructs were generated as de-scribed by Li et al. (2017). Briefly, the CDS of MoChia1, MoChia1NSP, andMoChia1E137Q amplified from M. oryzae were ligated with the over-expression vector pRTN-eGFP that was digested with EcoRI and BamHI.For the knockout construct, two 800-bp flanking sequences from theupstream and downstream of theMoChia1 gene were introduced into thevector pKOV21 (EcoRI/BamHI-digested for the upstream and HindIII-digested for the downstream). For the complementation construct, a 1.1kb fragment upstream of start codon ofMoChia1 and the CDS ofMoChia1was introduced into the pRTN vector (KpnI/NotI-digested). All the ligationswere performed using aOne-stepCloningKit (VazymeBiotech, C112). Theprimers used are listed in Supplemental Table 2. The respective restrictionenzyme digestion sites were highlighted in the primer sequences. All theconstructs were confirmed by sequencing, and subsequently weretransformed into protoplasts of the Guy11 or DMoChia1 mutant strain.

184 The Plant Cell

Callose Deposition and ROS Burst Assay

The leaves of 2-week-old rice seedlings that had been treated with MBP(Mock treatment), MoChia1, and Chitin (hexa-N-acetylchitohexaose,Seikagaku) were fixed in ethanol:acetic acid (3:1 [v/v]) solution for 5 hwith frequently changed fresh solution. Then rice leaves were rehy-drated in 70%ethanol for 2 h and 50%ethanol for 2 h, and thenwere keptinwater overnight. After beingwashedwithwater three times, the leaveswere treated with 10% sodium hydroxide (NaOH) for 1 h to make thetissues transparent. After beingwashedwithwater four times, the leaveswere incubated in staining solution [150 mM K2HPO4 (pH 9.5), 0.01%aniline blue] for 4 h on an end-over-end shaker. The leaves were thenobserved using a Leica microscope under UV light (340 to 380 nm;Olympus BX51).

ForROSburst assays, reactionmixtures (50mMK3PO4, 17mM luminol,10 mg/ml horseradish peroxidase, and 8 nM elicitor) were mixed with therice suspension cells. Luminescence was measured continuously overa120-minperiodwith1-min intervalsbyaCentroXS3LB960Luminometer(Berthold Technologies). At least three replications were performed for allthe experiments.

DAB and Mycelium Calcofluor White Staining

DAB (Sigma-Aldrich) was used for H2O2 staining according to a previouslydescribedmethod (Yangetal., 2017).The rice tissueswerestained in50mLDAB solution (50 mg DAB, 0.5mM NaH2PO4, 20 mL Tween 20) and vac-uumed for 5 to10min toeliminateair inplant tissues. Then, the tissueswerecontinuouslystainedwithDABsolution for8hona lowspeedshaker. Then,the tissues were de-stained in a solution containing ethanol: lactic acid:glycerol (3:1:1) for 8 h with frequent changes of fresh solution. The tissuewas observed under a light microscope (Zeiss).

Calcofluor White staining of mycelia cell wall components (chitin/cellulose)was performed using Fluorescent Brightener 28 according to themanufacturer’sprotocol (10mg/ml, Sigma-Aldrich). The fungal strainsweretransferred onto cover slips that contained a thin layer of complete agarmedium, andcultured for 24h.Agar pieceswithmyceliawere removedandstainedwith 10mg/ml CalcofluorWhite for 10min in darkness. Themyceliawere rinsed twice with 10 N NaOH, and then were further washed threetimes with distilled, deionized water and viewed under a fluorescencemicroscope (Nikon).

Measurement of Chitin Contents in M. oryzae

The chitin content was determined by measuring the amount of glucos-amine released by acid hydrolysis from fungal cell walls, according toa previously describedmethod (Guerriero et al., 2010). One gram of freshlyharvested mycelia or conidia was ground in liquid nitrogen and thenhomogenized in 5mL of deionizedwater. After centrifugation at 13,000 gfor 10 min at 4°C, the pellets were lyophilized overnight (Labconco). Foreach 5 mg of the dried pellets, 1 mL of 6 M HCl was added. After thesamples were hydrolyzed at 100°C for 4 h, the hydrolysis was adjustedwith 10NNaOH to pH7.0. An aliquot (0.2mL) of the resultingmixturewasadded to 0.25 mL of 4% acetyl acetone in 1.25 M sodium carbonate andheated for 30min at 100°C. After cooling down, 2mLethanol and 0.25mLof Ehrlich reagent (1.6 g of N,N-dimethyl-p-aminobenzaldehyde in 60mLof a 1:1 mixture of ethanol and concentrated HCl) was added to themixture, and the mixture was heated for 1 h at 60°C. The mixture wasfurther centrifuged at 13,000 g for 10 min at room temperature, and thesupernatant wasmeasured for absorbance at 530 nm. The chitin contentwas calculated based on the standard curve established by measuringthe absorbance of known amounts of glucosamine hydrochloride(Sigma, G4875).

Chitinase Activity Assay

Colloidal chitin was prepared according to a previously described method(Niu et al., 2016). Briefly, 10 g chitin power was ground in 40 mL acetone,and then 400 mL concentrated HCl was added in the homogenate. Themixturewaskeptat4°C for24hand thenfiltered throughglasswool to2Lof50% prechilled ethanol with constant mixing. After centrifugation at10,000 rpm for 20 min at 4°C, the white precipitate was washed in coldprechilled distilled water several times until the pH was close to 5.5. Thesupernatant was discarded, and 1 L of distilled water was added to form0.5%colloidal chitin. Chitinaseactivitywasmeasuredusingcolloidal chitinas the substrate. One microgram of recombinant protein was incubatedwith 0.25mgcolloidal chitin at 37°C for 1h in100mL50mMsodiumacetatebuffer (pH 7.0). The reaction was terminated by adding 100 mL 3,5-dinitrosalicylic acid (DNS) following 5 min heating at 100°C. After cooledin an ice bath, the mixture was diluted with 800 mL H2O. The undigestedchitin was removed by centrifugation. The absorbance of supernatant wasmonitored at OD 565 nm. The standard curve was generated by the re-action ofGlcNAcandDNS.Oneunit of chitinase activitywasdefinedas theamount of enzyme required to produce 1 mmol of GlcNAc/h under theabove conditions.

Rice Apoplastic Protein Extraction

The rice apoplast protein extraction was prepared as described (Kim et al.,2013) with slight modification. Briefly, the leaves were washed with dis-tilled, deionized water three times to remove any dust. Then, the leaveswere vacuum infiltrated with distilled, deionized H2O2 under 7.5 psi for5 min. Then, the leaves were loaded into 50 mL centrifuge tubes andcentrifuged at 1000 g for 5 min. The extracted apoplastic proteins werefurther concentrated in the Millipore tubes by the TCA-DOC method(Haslam et al., 2003; O’Leary et al., 2014), and the proteins were re-suspended in lamelli buffer. The proteins were separated on a SDS-PAGEgel and probed by the FLAG (Sigma, F3165) and ribulose-1,5-bis-phosphate carboxylase/oxygenase (Huaxingbio, HX1989) antibodies,respectively.

Split-Luciferase Complementation Assay

The split-luciferase complementation assay was performed as described(Luo et al., 2017). Agrobacterium tumefaciens (strain C58C1) carrying theindicated nLUC and cLUC constructs was mixed and infiltrated into theleaves of 4-week-old N. benthamiana plants using a 1-mL needlelesssyringe. Two days after infiltration, the leaves were rubbed with 0.5 mMluciferin and kept in thedark for 5min to quench the fluorescence. A cooledCCD imaging apparatus (Roper Scientific) was used to capture luciferaseimages.

BiFC Assay

The pSAT1-nYFP and pSAT1-cYFP plasmids were used for the BiFCassay. The CDS of MoChia1 and MoChia1NSP were cloned into pSAT1-cYFPatEcoRI/SmaI sites, and theCDSofOsTPR1wascloned intopSAT1-nYFP at SalI/BamHI sites. The Agrobacterium tumefaciens strain C58C1carrying the indicated constructs was mixed and infiltrated into the leavesof 4-week-old N. benthamiana plants. Infiltrated leaves were observed 36to 48 h later using a confocal laser scanning microscope (Leica ModelTCS SP8).

Subcellular Localization

MoChia1, OsTPR1, andGhPOD10 proteinswithGFP-tag at theC-terminalwere co-expressed with PCD1002-CFP, respectively, in N. benthamiana.Forty-eight hours later, the expressed proteins were observed using

OsTPR Binds Fungal Chitinase for Defense 185

a confocal laser microscope (Leica Model TCS SP8). Rice protoplastpreparation and plasmid transformation were performed according to themethods of Xiong et al. (2017). The excitation wavelengths and emissionfilters are as follows: 488 nm/band-pass 500 to 550 nm for GFP, and 433nm/band-pass 475 to 503 nm for CFP. Confocal images were analyzedusing Leica LAS AF software.

The OsTPR1 extracellular domain/region was identified by proteaseprotection assays using trypsin (Amresco) or protease K (Sigma). Theproteaseswere dissolved in 50mMTris-HCl, pH 8.0. The rice protoplast orN. benthamiana that expressed OsTPR1-GFP or GFP proteins were in-cubated with proteases at 28°C. The anti-GFP antibody (TransGen Bio-tech, HT801) was used to examine the protein degradation.

GST and Chitin Pull-Down Assays

The MoChia1, MoChia1E37Q, MoChia1CBD, and OsTPR1 were cloned intorespective pGEX-4T-1and/or pMAl-C4Xvectors usingaOne-stepCloningKit (VazymeBiotech, C112) and expressed inE. coli strainBL21 to producerecombinant proteins. The GST pull-down assay was performed using themethod described by Luo et al. (2017). The chitin pull-down assay wasperformedusing themethoddescribedbyLiuet al. (2012).Briefly, insolublechitin was incubated with purified recombinant MBP-OsTPR1 in bindingbuffer [20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 0.1 mMphenylmethylsulfonyl fluoride, and 0.1% Triton X-100] at 4°C for 2 h withconstant shaking. Theglycanwasspundownbycentrifugation at1000gat4°C for 3min. The pellet waswashed five timeswithwashing buffer [20mMTris-HCl (pH 7.5), 200 mM NaCl, 0.1 mM EDTA, 0.1 mM phenyl-methylsulfonyl fluoride, and0.2%Nonidet P-40] and thenboiledwithSDS-PAGE loading buffer. The chitin-associated MBP-MoChia1 was detectedby immunoblotting using anti-MBP antibody (TransGen Biotech, HT701).

MST Analysis

Binding reactionsof recombinantMBP-MoChia1 toMBP-OsTPR1orchitinwas measured by MST in a Monolith NT.Label Free (Nano TemperTechnologiesGMBH) instrument that detects changes in size, charge, andconformation induced by binding. Labeled MBP-MoChia1 (10 mM) wasdisplaced by a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl,10 mM MgCl2, and 0.05% (v/v) Tween 20. A range of concentrations ofMBP-OsTPR1 or chitin in the assay buffer [50 mM Tris-HCl (pH 7.8),150mMNaCl, 10mMMgCl2, 0.05%Tween20]was incubatedwith labeledprotein (1:1, v/v) for 10min. The sample was loaded into the NT.Label Freestandard capillaries and measured with 20% LED power and 80% MSTpower. The KD Fit function of the Nano Temper Analysis Software (Version1.5.41) was used to fit the curve and calculate the value of the dissociationconstant (Kd ).

Phylogenetic Analyses

Theaminoacid sequencesof thechitinaseproteinsweredownloaded fromthe UniProt website (https://www.uniprot.org/). Sequence alignment wasperformed with ClustalW (Supplemental Data Set). A neighbor-joiningmethod implemented in MEGA7.0 was used to generate the phyloge-netic tree. The bootstrap values indicated at the nodes in the phylogenetictree are based on 1000 replications.

Statistical Analysis

All the datawere analyzed using aone-wayANOVAor two-tailedStudent’st test with SPSS 18.0. The values represented as means 6 SD.

Accession Numbers

Sequence data from this article can be found in the GenBank databaselibraries under the following accession numbers: MoChia1, MGG_08054;4HNR, MGG_07216; AIB1, MGG_07219; BUF1, MGG_02252; RSY1,MGG_05059; MoActin, MGG_03982; OsTPR1, LOC_Os10g34540;two rice chitinase genes, LOC_Os04g30770 and LOC_Os05g33130;OsRbohA, LOC_Os01g53294; OsRbohB, LOC_Os09g26660; OsRbohD,LOC_Os05g38980; OsPR10, LOC_Os03g18850; OsActin, LOC_Os10g36650.

Supplemental Data

Supplemental Figure 1. The identification of MoChia1 in M. oryzae.

Supplemental Figure 2. Phylogenetic tree of chitinase gene families.

Supplemental Figure 3. MoChia1 is a secreted protein.

Supplemental Figure 4. MoChia1 chitinase activity is not essential forimmune activation.

Supplemental Figure 5. Rice chitinases possess enzymatic activitiesbut do not cause a ROS burst in rice suspension cells.

Supplemental Figure 6. Strategy for the targeted gene knockout ofMoChia1.

Supplemental Figure 7. The phenotype of MoChia1-overexpressingstrains.

Supplemental Figure 8. OMoChia1 activates stronger immuneresponses than the WT in rice.

Supplemental Figure 9. MoChia1 interacts with the C-terminal ofOsTPR1.

Supplemental Figure 10. Chitin and MoChia1 activated immuneresponses in rice.

Supplemental Figure 11. Comparison of ROS burst activated byMoChia1 in rice suspension cells of WT and OsTPR1-OE.

Supplemental Table 1. The proteins identified by LC-MS/MS.

Supplemental Table 2. Primers used in this study.

Supplemental Data Set. Text file of alignment corresponding to thephylogenetic analysis in Supplemental Figure 2.

ACKNOWLEDGMENTS

We thank Prof. Youliang Peng for kindly providing the pRTN-eGFP andpKOV21 plasmids and Prof. Junfeng Liu at Chinese Agricultural Univer-sity for bioinformatics assays. We also thank Yao Wu for technicalassistance on the use of MST. The work was supported by theChinese Academy of Sciences (CAS) (Strategic Priority Research Pro-gram Grant XDB11020300), the National Natural Science Foundationof China (NSFC) (31570252, 31601629), the start-up fund of ‘One Hun-dred Talents’ program of the Chinese Academy of Sciences, and bythe grants from the State Key Laboratory of Plant Genomics (GrantO8KF021011 to J.L.).

AUTHOR CONTRIBUTIONS

C.Y. and J.L. conceived and designed the experiments, and wrote thearticle; C.Y., Y.Y., J.H., and F.M. performed most of the experiments; J.P.and Y.T. generated the transgenic plants; Q.Z., A.I., and N.X. helped withthe data analysis.

186 The Plant Cell

ReceivedMay 14, 2018; revised November 19, 2018; accepted December31, 2018; published January 4, 2019.

REFERENCES

Boutrot, F., and Zipfel, C. (2017). Function, discovery, and exploi-tation of plant pattern recognition receptors for broad-spectrumdisease resistance. Annu. Rev. Phytopathol. 55: 257–286.

Cao, J., Yang, C., Li, L., Jiang, L., Wu, Y., Wu, C., Bu, Q., Xia, G., Liu,X., Luo, Y., and Liu, J. (2016). Rice Plasma membrane proteomicsreveals Magnaporthe oryzae promotes susceptibility by sequentialactivation of host hormone signaling pathways. Mol. Plant MicrobeInteract. 29: 902–913.

Cao, J., Yu, Y., Huang, J., Liu, R., Chen, Y., Li, S., and Liu, J. (2017).Genome re-sequencing analysis uncovers pathogenecity-relatedgenes undergoing positive selection in Magnaporthe oryzae. Sci.China Life Sci. 60: 880–890.

Cao, Y., Liang, Y., Tanaka, K., Nguyen, C.T., Jedrzejczak, R.P.,Joachimiak, A., and Stacey, G. (2014). The kinase LYK5 is a majorchitin receptor in Arabidopsis and forms a chitin-induced complexwith related kinase CERK1. eLife 3: e03766.

Cerveny, L., Straskova, A., Dankova, V., Hartlova, A., Ceckova, M.,Staud, F., and Stulik, J. (2013). Tetratricopeptide repeat motifs inthe world of bacterial pathogens: Role in virulence mechanisms.Infect. Immun. 81: 629–635.

Cheng, X.X., Zhao, L.H., Klosterman, S.J., Feng, H.J., Feng, Z.L.,Wei, F., Shi, Y.Q., Li, Z.F., and Zhu, H.Q. (2017). The endochitinaseVDECH from Verticillium dahliae inhibits spore germination andactivates plant defense responses. Plant Sci. 259: 12–23.

Cletus, J., Balasubramanian, V., Vashisht, D., and Sakthivel, N.(2013). Transgenic expression of plant chitinases to enhance dis-ease resistance. Biotechnol. Lett. 35: 1719–1732.

de Jonge, R., van Esse, H.P., Kombrink, A., Shinya, T., Desaki, Y.,Bours, R., van der Krol, S., Shibuya, N., Joosten, M.H.A.J., andThomma, B.P.H.J. (2010). Conserved fungal LysM effector Ecp6prevents chitin-triggered immunity in plants. Science 329: 953–955.

Doehlemann, G., and Hemetsberger, C. (2013). Apoplastic immunityand its suppression by filamentous plant pathogens. New Phytol.198: 1001–1016.

Du, Y., Zhao, J., Chen, T., Liu, Q., Zhang, H., Wang, Y., Hong, Y.,Xiao, F., Zhang, L., Shen, Q., and Liu, Y. (2013). Type I J-domainNbMIP1 proteins are required for both Tobacco mosaic virus in-fection and plant innate immunity. PLoS Pathog. 9: e1003659.

Gimenez-Ibanez, S., Hann, D.R., Ntoukakis, V., Petutschnig, E.,Lipka, V., and Rathjen, J.P. (2009). AvrPtoB targets the LysM re-ceptor kinase CERK1 to promote bacterial virulence on plants. Curr.Biol. 19: 423–429.

Göhre, V., Spallek, T., Häweker, H., Mersmann, S., Mentzel, T.,Boller, T., de Torres, M., Mansfield, J.W., and Robatzek, S.(2008). Plant pattern-recognition receptor FLS2 is directed fordegradation by the bacterial ubiquitin ligase AvrPtoB. Curr. Biol. 18:1824–1832.

Guerriero, G., Avino, M., Zhou, Q., Fugelstad, J., Clergeot, P.H.,and Bulone, V. (2010). Chitin synthases from Saprolegnia are in-volved in tip growth and represent a potential target for anti-oomycete drugs. PLoS Pathog. 6: e1001070.

Haslam, R.P., Downie, A.L., Raveton, M., Gallardo, K., Job, D.,Pallett, K.E., John, P., Parry, M.A.J., and Coleman, J.O.D. (2003).The assessment of enriched apoplastic extracts using proteomicapproaches. Ann. Appl. Biol. 143: 81–91.

Hemetsberger, C., Herrberger, C., Zechmann, B., Hillmer, M., andDoehlemann, G. (2012). The Ustilago maydis effector Pep1

suppresses plant immunity by inhibition of host peroxidase activity.PLoS Pathog. 8: e1002684.

Jashni, M.K., Mehrabi, R., Collemare, J., Mesarich, C.H., and de Wit,P.J.G.M. (2015). The battle in the apoplast: further insights into the rolesof proteases and their inhibitors in plant-pathogen interactions. Front.Plant Sci. 6: 584.

Jones, J.D.G., and Dangl, J.L. (2006). The plant immune system.Nature 444: 323–329.

Kawamura, C., Moriwaki, J., Kimura, N., Fujita, Y., Fuji, S., Hirano,T., Koizumi, S., and Tsuge, T. (1997). The melanin biosynthesisgenes of Alternaria alternata can restore pathogenicity of themelanin-deficient mutants of Magnaporthe grisea. Mol. Plant Mi-crobe Interact. 10: 446–453.

Kim, S.G., Wang, Y., Lee, K.H., Park, Z.Y., Park, J., Wu, J., Kwon,S.J., Lee, Y.H., Agrawal, G.K., Rakwal, R., Kim, S.T., and Kang,K.Y. (2013). In-depth insight into in vivo apoplastic secretome ofrice-Magnaporthe oryzae interaction. J. Proteomics 78: 58–71.

Labroussaa, F., Ionescu, M., Zeilinger, A.R., Lindow, S.E., andAlmeida, R.P.P. (2017). A chitinase is required for Xylella fastidiosacolonization of its insect and plant hosts. Microbiology 163:502–509.

Langner, T., and Göhre, V. (2016). Fungal chitinases: Function, reg-ulation, and potential roles in plant/pathogen interactions. Curr.Genet. 62: 243–254.

Langner, T., Öztürk, M., Hartmann, S., Cord-Landwehr, S.,Moerschbacher, B., Walton, J.D., and Göhre, V. (2015). Chiti-nases are essential for cell separation in Ustilago maydis. Eukaryot.Cell 14: 846–857.

Lee, T.G., Tang, N., Thompson, S., Miller, J., and Katze, M.G.(1994). The 58,000-dalton cellular inhibitor of the interferon-induceddouble-stranded RNA-activated protein kinase (PKR) is a memberof the tetratricopeptide repeat family of proteins. Mol. Cell. Biol. 14:2331–2342.

Li, X., Gao, C., Li, L., Liu, M., Yin, Z., Zhang, H., Zheng, X., Wang, P.,and Zhang, Z. (2017). MoEnd3 regulates appressorium formationand virulence through mediating endocytosis in rice blast fungusMagnaporthe oryzae. PLoS Pathog. 13: e1006449.

Li, Y.B., Han, L.B., Wang, H.Y., Zhang, J., Sun, S.T., Feng, D.Q.,Yang, C.L., Sun, Y.D., Zhong, N.Q., and Xia, G.X. (2016). Thethioredoxin GbNRX1 plays a crucial role in homeostasis of apo-plastic reactive oxygen species in response to Verticillium dahliaeinfection in cotton. Plant Physiol. 170: 2392–2406.

Lienemann, M., Boer, H., Paananen, A., Cottaz, S., and Koivula, A.(2009). Toward understanding of carbohydrate binding and sub-strate specificity of a glycosyl hydrolase 18 family (GH-18) chitinasefrom Trichoderma harzianum. Glycobiology 19: 1116–1126.

Lin, Z., Arciga-Reyes, L., Zhong, S., Alexander, L., Hackett, R.,Wilson, I., and Grierson, D. (2008). SlTPR1, a tomato tetra-tricopeptide repeat protein, interacts with the ethylene receptors NRand LeETR1, modulating ethylene and auxin responses and de-velopment. J. Exp. Bot. 59: 4271–4287.

Lin, Z., Ho, C.W., and Grierson, D. (2009). AtTRP1 encodes a novelTPR protein that interacts with the ethylene receptor ERS1 andmodulates development in Arabidopsis. J. Exp. Bot. 60: 3697–3714.

Liu, B., et al. (2012). Lysin motif-containing proteins LYP4 and LYP6play dual roles in peptidoglycan and chitin perception in rice innateimmunity. Plant Cell 24: 3406–3419.

Luo, X., Xu, N., Huang, J., Gao, F., Zou, H., Boudsocq, M., Coaker,G., and Liu, J. (2017). A lectin receptor-like kinase mediatespattern-triggered salicylic acid signaling. Plant Physiol. 174: 2501–2514.

Ma, Z., et al. (2017). A paralogous decoy protects Phytophthora sojaeapoplastic effector PsXEG1 from a host inhibitor. Science 355:710–714.

OsTPR Binds Fungal Chitinase for Defense 187

Mentlak, T.A., Kombrink, A., Shinya, T., Ryder, L.S., Otomo, I.,Saitoh, H., Terauchi, R., Nishizawa, Y., Shibuya, N., Thomma,B.P.H.J., and Talbot, N.J. (2012). Effector-mediated suppression ofchitin-triggered immunity by magnaporthe oryzae is necessary forrice blast disease. Plant Cell 24: 322–335.

Miyamoto, K., Shimizu, T., Mochizuki, S., Nishizawa, Y., Minami, E.,Nojiri, H., Yamane, H., and Okada, K. (2013). Stress-induced expres-sion of the transcription factor RERJ1 is tightly regulated in response tojasmonic acid accumulation in rice. Protoplasma 250: 241–249.

Niu, X., Liu, C.C., Xiong, Y.J., Yang, M.M., Ma, F., Liu, Z.H., andYuan, S. (2016). The modes of action of ChiIII, a chitinase frommushroom Coprinopsis cinerea, shift with changes in the length ofGlcNAc oligomers. J. Agric. Food Chem. 64: 6958–6968.

O’Leary, B.M., Rico, A., McCraw, S., Fones, H.N., and Preston,G.M. (2014). The infiltration-centrifugation technique for extractionof apoplastic fluid from plant leaves using Phaseolus vulgaris as anexample. J. Vis. Exp. 94: e52113.

Ranf, S., Gisch, N., Schäffer, M., Illig, T., Westphal, L., Knirel, Y.A.,Sánchez-Carballo, P.M., Zähringer, U., Hückelhoven, R., Lee, J.,and Scheel, D. (2015). A lectin S-domain receptor kinase mediateslipopolysaccharide sensing in Arabidopsis thaliana. Nat. Immunol.16: 426–433.

Rooney, H.C.E., Van’t Klooster, J.W., van der Hoorn, R.A.L.,Joosten, M.H.A.J., Jones, J.D.G., and de Wit, P.J.G.M. (2005).Cladosporium Avr2 inhibits tomato Rcr3 protease required for Cf-2-dependent disease resistance. Science 308: 1783–1786.

Schlegel, T., Mirus, O., von Haeseler, A., and Schleiff, E. (2007). Thetetratricopeptide repeats of receptors involved in protein trans-location across membranes. Mol. Biol. Evol. 24: 2763–2774.

Sikorski, R.S., Boguski, M.S., Goebl, M., and Hieter, P. (1990). Arepeating amino acid motif in CDC23 defines a family of proteinsand a new relationship among genes required for mitosis and RNAsynthesis. Cell 60: 307–317.

Soanes, D.M., Chakrabarti, A., Paszkiewicz, K.H., Dawe, A.L., andTalbot, N.J. (2012). Genome-wide transcriptional profiling of ap-pressorium development by the rice blast fungus Magnaporthe or-yzae. PLoS Pathog. 8: e1002514.

van den Burg, H.A., Harrison, S.J., Joosten, M.H.A.J., Vervoort,J., and de Wit, P.J.G.M. (2006). Cladosporium fulvum Avr4 pro-tects fungal cell walls against hydrolysis by plant chitinases ac-cumulating during infection. Mol. Plant Microbe Interact. 19:1420–1430.

Xiang, T., Zong, N., Zou, Y., Wu, Y., Zhang, J., Xing, W., Li, Y., Tang,X., Zhu, L., Chai, J., and Zhou, J.M. (2008). Pseudomonas syringaeeffector AvrPto blocks innate immunity by targeting receptor kina-ses. Curr. Biol. 18: 74–80.

Xiong, Q., et al. (2017). Ethylene-inhibited jasmonic acid biosynthesispromotes mesocotyl/coleoptile elongation of etiolated rice seed-lings. Plant Cell 29: 1053–1072.

Yamaguchi, K., et al. (2013). A receptor-like cytoplasmic kinase tar-geted by a plant pathogen effector is directly phosphorylated by thechitin receptor and mediates rice immunity. Cell Host Microbe 13:347–357.

Yang, C., et al. (2017). Activation of ethylene signaling pathwaysenhances disease resistance by regulating ROS and phytoalexinproduction in rice. Plant J. 89: 338–353.

Zipfel, C. (2014). Plant pattern-recognition receptors. Trends Im-munol. 35: 345–351.

188 The Plant Cell

DOI 10.1105/tpc.18.00382; originally published online January 4, 2019; 2019;31;172-188Plant Cell

Xu, Yun Tian and Jun LiuChao Yang, Yongqi Yu, Junkai Huang, Fanwei Meng, Jinhuan Pang, Qiqi Zhao, Md. Azizul Islam, Ning

Allows Free Chitin to Trigger Immune Responses Chitinase MoChia1 by a Rice Tetratricopeptide Repeat ProteinMagnaporthe oryzaeBinding of the

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