Magnaporthe oryzae Induces the Expression of a MicroRNA to ... · The rice blast disease, caused by the fungus Magnaporthe oryzae, is the most important disease of rice (Oryza sativa)

Magnaporthe oryzae Induces the Expression of a MicroRNAto Suppress the Immune Response in Rice1[OPEN]

Xin Zhang,a,b,c Yalin Bao,a,b Deqi Shan,a,b Zhihui Wang,a,b Xiaoning Song,a,b Zhaoyun Wang,a,b

Jiansheng Wang,a,b Liqiang He,d Liang Wu,e Zhengguang Zhang,a,b Dongdong Niu,a,b Hailing Jin,f andHongwei Zhaoa,b,2

aCollege of Plant Protection, Nanjing Agricultural University, Nanjing 210095, ChinabKey Laboratory of Integrated Management of Crop Diseases and Pests, Nanjing Agricultural University,Ministry of Education, Nanjing 210095, ChinacInstitute of Industrial Crops, Shanxi Academy of Agricultural Sciences, Taiyuan 030000, Shanxi, ChinadState Key Laboratory of Crop Genetics and Germplasm Enhancement, Cytogenetics Institute, NanjingAgricultural University, Nanjing 210095, ChinaeDepartment of Agronomy, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058,ChinafDepartment of Plant Pathology and Microbiology, University of California, Riverside, California 92521

ORCID ID: 0000-0001-9107-765X (H.Z.).

MicroRNAs play crucial roles in plant responses to pathogen infections. The rice blast disease, caused by the fungusMagnaportheoryzae, is the most important disease of rice (Oryza sativa). To explore the microRNA species that participate in rice immunityagainst the rice blast disease, we compared the expression of small RNAs between mock- and M. oryzae-treated rice. We foundthat infection by M. oryzae strain Guy11 specifically induced the expression of rice miR319 and, consequently, suppressed itstarget gene TEOSINTE BRANCHED/CYCLOIDEA/PROLIFERATING CELL FACTOR1 (OsTCP21), which encodes a transcriptionfactor. Using transgenic rice that overexpresses miR319b (OE) or expresses OsTCP21-Res (which is resistant to miR319-mediatedsilencing), we found that OsTCP21 is a positive regulator of the rice defense response against the blast disease. When wild-typeand miR319b-OE rice were infected by Guy11, multiple jasmonic acid (JA) synthetic and signaling components were suppressed,indicating that Guy11 suppresses JA signaling through inducing miR319. In particular, we found that LIPOXYGENASE2 (LOX2)and LOX5 were specifically suppressed by miR319 overexpression or by Guy11 infection. LOXs are the key enzymes of JAsynthesis, which catalyze the conversion of a-linoleic acid to hydroperoxy-octadecadienoic acid. The application of a-linoleicacid rescued disease symptoms on the OsTCP21-Res rice but not wild-type rice, supporting our hypothesis that OsLOX2 andOsLOX5 are the key JA synthesis genes hijacked by Guy11 to subvert host immunity and facilitate pathogenicity. We proposethat induced expression of OsLOX2/5 may improve resistance to the rice blast disease.

Small RNAs (sRNAs) are a novel group of short,noncoding RNA molecules ranging from 20 to morethan 40 nucleotides in length (Yu et al., 2017; Niu et al.,2018). sRNAs act as master modulators that negativelyregulate gene expression at either the transcription orposttranscription level (known as gene silencing;

Iwakawa and Tomari, 2015; Jonas and Izaurralde, 2015).By recognizing sequence reverse complementary tosRNA, genes containing conserved sequences are silencedcoordinately, thus offering sRNA a tremendous regulat-ing capacity on genes with conserved structures (and re-lated functions derived from conserved structures). Forexample, in plants, such as Medicago spp., Nicotiana ben-thamiana, tomato (Solanum lycopersicum), and Arabidopsis(Arabidopsis thaliana), the sequences encoding the Toll/IL-1 receptor (TIR) domain of nucleotide-binding Leu-richrepeat resistance proteins are targeted by several groupsof sRNAs, which offers these sRNAs great regulatorypotentials on plant innate immunity (Zhai et al., 2011; Liet al., 2012; Shivaprasad et al., 2012; Boccara et al., 2014).

According to their biogenesis and mode of function,sRNA consists of small interfering RNA (siRNA) andmicroRNA (miRNA), both of which are capable of regu-latingnumerous biological processes associatedwith plantgrowth, development, and reproduction (Shivaprasadet al., 2012; Niu et al., 2015). sRNAs participating in plantresponses to biotic stresses have received more attention

1 This work was supported by a key project of the NationalNatural Science Foundation of China (31530063), the FundamentalResearch Funds for the Central Universities (KYTZ201403), and anInnovation Team Program for Jiangsu Universities (2017) to H.Z.

2 Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Hongwei Zhao ([email protected]).

H.J. and H.Z. designed the experiments; X.Z., Y.B., D.S., X.S., Z.W.,Z.W., J.W., L.H., L.W., and D.N. performed the experiments; X.Z.,Z.Z., and H.Z. analyzed the data; Z.X. and H.Z. wrote the article; allauthors reviewed the article.

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during the past decade (Navarro et al., 2006; Katiyar-Agarwal and Jin, 2007; Zhai et al., 2011; Khraiwesh et al.,2012; Li et al., 2012, 2017; Shivaprasad et al., 2012). Forexample,miR393was the first identifiedmiRNA involvedin plant innate immune regulation. miR393 suppressesthe expression of TRANSPORT INHIBITOR RESPONSE1(TIR1) that encodes the receptor of the auxin signalingpathway, which is a negative regulator of plant defenseresponse. By suppressingTIR1,miR393 enhances theplantdefense response against Pseudomonas syringae infection(Navarro et al., 2006). Later, more miRNA regulatorymodules associated with plant innate immunity wereidentified (Li et al., 2014; Jin andWu, 2015;Wu et al., 2015),highlighting the master role these sRNAs may play inplant innate immunity.Rice (O. sativa) endogenous miRNAs play important

roles between rice and associated pathogens. For ex-ample, miR160a and miR398b participate in modulat-ing rice innate immunity against the blast fungusM. oryzae. miR160 targets ARF16, whose Arabidopsishomolog is associated with immune responses againstbacterial pathogens (Zhang et al., 2011; Li et al., 2014).miR398 targets SUPEROXIDE DISMUTASE2 thatmaintains the dynamic balance of superoxide anionand hydrogen peroxide (H2O2) metabolism duringM. oryzae infection. Overexpression of miR160a andmiR398b enhances rice resistance against the blast dis-ease, confirming their contribution to rice defenseagainst M. oryzae infection (Li et al., 2014). miR168 is acritical player in the response against infectionwithRicestripe virus (RSV) and Rice dwarf virus, in which it mayfunction through incorporation into the Argonaute1(AGO1)-associated RNA-induced silencing complex.When miR168 is loaded into other AGOs, suchas AGO18, which is triggered by virus infection, thedefense responses are suppressed (Wu et al., 2015),manifesting a critical role of miR168 in regulatingbroad-spectrum resistance against viral diseases in rice.miR444 is a positive regulator of the RNA-DependentRNA Polymerase1-dependent RNA-silencing path-way. Upon induction byRSV infection, miR444 silencesspecific viral RNAs and host genes and contributes todefense against RSV (Wang et al., 2016). Also in riceinfected with RSV, it was found that the reducedmiR171b expression contributed to the viral stuntingand yellowing symptoms on RSV-infected rice plants(Tong et al., 2017). A different group showed thatmiR169 acted as a negative regulator in rice immunityagainst M. oryzae by repressing the expression of NU-CLEAR FACTOR Y-A genes (Li et al., 2017b). Takentogether, rice endogenous miRNAs are active immuneregulators against various pathogen infections.ThemiR319 family is an ancient miRNA group that is

widely conserved in plants (Talmor-Neiman et al.,2006; Fattash et al., 2007; Cuperus et al., 2011). The targetgenes of miR319 include TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1 (TCP),which encodes transcription factors regulating manyplant growth and developmental processes (Martín-Trilloand Cubas, 2010; Lopez et al., 2015). TCPs are important

growth regulators controlling leaf morphogenesis (Martín-Trillo and Cubas, 2010; Lopez et al., 2015). In addition, themiR319/TCP module also participates in plant responsesto biotic and abiotic stresses. For example, overexpressingricemiR319 or suppressing the corresponding target genes,such as OsPCF5, OsPCF6, OsPCF8, and OsTCP21, led toenhanced tolerance to cold stress (Yang et al., 2013; Wanget al., 2014). In tomato, sl-miR319 behaves both as a sys-temic signal and a regulatory factor of the defense responseto root-knot nematodes (Zhao et al., 2015).

The jasmonic acid (JA) signaling pathway is a keyregulator of plant defense responses against both hemi-biotrophic and necrotrophic pathogens (Thaler et al.,2012; Tamaoki et al., 2013; Zhang et al., 2016). Ath-miR319regulates the expression of a key JA synthetic component,LIPOXYGENASE2 (LOX2), through silencing TCP4 andTCP20 (Schommer et al., 2008; Danisman et al., 2012). Inrice infected byRice ragged stunt virus (RRSV), the inducedexpression of miR319 is associated with promoted viralinfection, suppression ofOsTCP21, down-regulation of JAsynthetic genes, and reduced JA accumulation, indicatingthat RRSV may manipulate endogenous JA levelsthrough miR319 (Zhang et al., 2016). Therefore, it is sug-gested that miR319/TCP may manipulate the plant in-nate response againstRRSV by affecting JA synthesis andsignaling.However,whether similar regulatorymachineryis conserved in the rice immune system againstM. oryzae is unknown.

Rice is the staple food crop worldwide, which makesthe rice-M. oryzae interaction of great significance atboth scientific and practical levels (Liu et al., 2013). Toidentify more miRNAs involved in the regulation of riceinnate immunity against the blast disease, we comparedand analyzed differentially expressed sRNAs uponM. oryzae infection. We show that miR319 is inducedspecifically by theM. oryzae strain Guy11, which leads tothe suppression of genes, such as OsTCP21, and subse-quent pathways regulated by this transcription factor.Our results show that M. oryzae infection blocks theconversion of a-linoleic acid (LnA) to hydroperoxy-octadecadienoic acid (HPODE), which is a critical stepof JA synthesis. We further confirm the involvement ofmiR319 in the key JA biosynthetic step by geneticapproaches and an external LnA application assay. Ourstudy indicates that the M. oryzae pathogen can inten-sify its pathogenicity by blocking the key steps of thehost JA biosynthetic and signaling pathway. Our studyidentifies critical molecular components affecting therice immune response against the blast disease, whichmay lead to engineered rice varieties with enhancedresistance.

RESULTS

M. oryzae Infection Induces the Expression of miR319

To identify endogenous blast disease-responsivemiRNAs that are potentially involved in rice immu-nity against M. oryzae infection, we infected rice (cvNipponbare) with the M. oryzae strain Guy11 for

Plant Physiol. Vol. 177, 2018 353

M. oryzae Induces miR319 to Overcome Rice Immunity

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different times (0, 24, 48, and 120 h). Total RNAfrom each sample was used for sRNA library con-struction as described previously (Zhang et al., 2011).As shown in Table I, 14,588,511, 15,931,384, 15,695,043,and 13,275,491 total reads were generated from eachlibrary, representing the different time points, respec-tively. By mapping to the rice genome (Rice GenomeAnnotation Project; http://rice.plantbiology.msu.edu/),10,638,183, 10,840,688, 11,977,454, and 8,505,278 ricesRNA reads were obtained, corresponding to 925,406,1,552,456, 977,547, and 805,238 unique reads, respec-tively. These reads were further searched against theRfam database (http://xfam.org/) to remove knownsRNAs, such as rRNAs, tRNAs, and small nucleolarRNAs. In the end, 7,376,173, 6,516,391, 6,815,060, and4,637,845 reads were retrieved from each library andwere considered as sRNAs originated from rice. Weclassified total rice sRNAs into three categoriesaccording to their origination and structural features:(1) conserved miRNAs that have a stem-loop precursorstructure and are listed in themiRNAdatabase (http://www.mirbase.org/); (2) novel rice miRNAs that have astem-loop precursor structure but are not listed in themiRNA database; and (3) rice siRNAs that do not havea stem-loop precursor structure. According to thesecriteria, we retrieved 3,948, 5,721, 3,598, and 3,467unique conserved rice miRNAs (osa-miRNAs) in eachlibrary, respectively (Table I; Supplemental Table S1).In addition, we obtained 2,155, 3,542, 2,080, and 1,820novel rice miRNAs and 271,594, 410,909, 312,402, and252,439 rice siRNAs, which will be discussed in sepa-rate articles.

To identify the osa-miRNAs responsive to blast in-fection, we compared miRNA expression betweenGuy11- and mock-treated rice. Osa-miRNAs both

abundantly and significantly differentially expressedwere identified by employing the following criteria:(1) total reads $ 150; and (2) [treated/mock] $ 2or [treated/mock] # 0.5 in each stage. At the end,we found 11 significantly differentially expressedconserved osa-miRNAs (Table II). By using reverse-cDNA fragments as probes, we validated our bio-informatic prediction by northern-blot assay (there isonly a one-nucleotide difference between miR169aand miR169h, between miR166k-3p and miR166j-3p,and between miR1317-3p, miR5150-3p, miR169a,miR319b, and their corresponding family members;therefore, detection using probes reverse comple-mentary to the aforementioned sRNAs representsthe expression of the family). In the six northern blotsthat were used to detect the up-regulated miRNAs,miR1317-3p, miR166k-3p, miR169 (Fig. 1A), andmiR319 (Fig. 1C) showed discernible up-regulation;miR162a showed weak expression with no notice-able expression variation. Surprisingly, miR5150-3pshowed a significant reduction on expression, whichis opposite to the bioinformatic prediction (Fig. 1A).For the three down-regulated miRNAs, miR156f-3pand miR435 were expressed under the detectablelevel. miR444d.3 showed an obvious reduction at24 and 120 hpi (Fig. 1B).

In these miRNAs that showed significant up- ordown-regulated expression, miR162a, miR156f-3p, andmiR435 were expressed at relatively low levels andwere not studied further. miR166 is known for itsdevelopment-related function (Fujioka et al., 2008).miR169a was identified as a rice blast-responsive sRNAin a previous study (Li et al., 2014). miR1317 was pre-dicted to target a gene encoding a transposon protein(LOC_Os10g20480), which is less likely to be involved

Table I. Distribution of sRNAs among different categories in each library

A rice sRNA deep-sequencing summary is shown. The abundance in different libraries was normalized to library 0 hpi, and sRNAs were calculatedas reads per million.

Types

Normalized Reads

0 hpi 24 hpi 48 hpi 120 hpi

reads % reads % reads % reads %

Total sRNA 14,588,511 100.00 15,931,384 100.00 15,695,043 100.00 13,275,491 100.00Mapped sRNA 10,638,183 72.92 10,840,688 68.05 11,977,454 76.31 8,505,278 64.07Unique reads 925,406 1,552,456 977,547 805,238

Rfam 3,262,010 22.69 4,324,297 27.44 5,162,394 33.07 3,867,433 29.48rRNA 2,523,283 17.30 34,90,529 21.91 4,144,215 26.40 2,859,734 21.54tRNA 713,233 4.89 789,733 4.96 955,329 6.09 974,162 7.34snRNA 2,498 0.35 2,487 0.31 1,792 0.19 3,666 0.38Small nucleolar RNA 22,996 0.16 41,548 0.26 61,058 0.39 29,871 0.23

Rice sRNA 7,376,173 50.23 6,516,391 40.60 6,815,060 43.25 4,637,845 34.59Conserved miRNA (total) 131,480 0.90 247,126 1.55 81,347 0.52 84,275 0.63Conserved miRNA (unique) 3,948 5,721 3,598 3,467Novel miRNA (total) 11,179 0.08 22,391 0.14 10,398 0.07 9,198 0.07Novel miRNA (unique) 2,155 3,542 2,080 1,820trans-acting siRNA (total) 6,512 0.04 16,197 0.10 6,864 0.04 6,438 0.05trans-acting siRNA (unique) 401 643 312 354siRNA (total) 7,227,002 49.54 6,230,677 39.11 6,716,451 42.79 4,537,934 34.18siRNA (unique) 271,594 410,909 312,402 252,439

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Zhang et al.


http://rice.plantbiology.msu.edu/

http://xfam.org/

http://www.mirbase.org/

http://www.mirbase.org/

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directly in the plant defense response. miR5150-3pwas predicted to target a receptor-like proteinkinase (LOC_Os01g70260); miR444d.3 could targetOsMADS57 (LOC_Os02g49840), which is a MADS-boxfamily gene. Investigation of these two positive im-mune regulators will be discussed in a separate article.The induced expression of miR319 also was observed

at the transcriptional level, which was confirmed bythe examination of the miR319b precursor using real-time PCR (Fig. 1C). Interestingly, when inoculated withthe avirulent strain 2539, the expression of miR319 wasnot obviously affected (Supplemental Fig. S1). Strain2539 is an avirulent strain with a moderately patho-genic phenotype to many rice genotypes, which ismediated by an avirulence gene, AVR1-CO39 (Smithand Leong, 1994; Zheng et al., 2011). Therefore, ourobservation indicates that the elicitation of miR319 ex-pression is Guy11 specific. miR319 is predicted to targetOsTCP21 (LOC_Os07g05720) and a GA MYB gene(OsGAmyb [LOC_Os01g59660]; Fig. 2A; SupplementalTable S2), both of which are transcription factors.Transcription factors are key regulators of gene expres-sion in response to various developmental and environ-mental cues (Danisman, 2016; Samad et al., 2017).Therefore, we focused on the functional study of miR319in rice immune responses against the blast disease.To validate whether OsTCP21 and OsGAmyb are

authentic target genes regulated by miR319, both themiRNA and the Flag tag-fused OsTCP21 (35S::Flag:OsTCP21) and OsGAmyb (35S::Flag:OsGAmyb)were transiently coexpressed in N. benthamiana. Whendetected by an anti-Flag antibody, both OsTCP21 andOsGAmyb were obviously suppressed by coexpressionwith miR319, but not in the control assay or by an ir-relevant miRNA (such as miR169a, a miRNA withoutreverse complementarity to both target genes; Fig. 2A).This result suggests that miR319 silences the expressionof bothOsTCP21 andOsGAmyb in vivo. The expressionof OsTCP21 and OsGAmyb also was checked in rice

infected by Guy11 (Fig. 2B). The transcription of bothOsTCP21 and OsGAmyb declined obviously after48 hpi, which corresponded to the induced expres-sion of miR319 by Guy11 infection (Fig. 1C). Takentogether, we concluded that OsTCP21 and OsGAmybare authentic target genes of miR319. OsGAmyb is atranscription factor involved in many developmen-tal processes, such as regulating the expression ofa-amylase in the aleurone layer of rice seed; it alsoplays a role in pollen development (Liu et al., 2010;Sutoh et al., 2015). By contrast, OsTCP21 function isrelated to rice responses to biotic and abioticstresses. For example, OsTCP21 is involved in thesynthesis and signaling of JA in response to coldstress and virus infection (Wang et al., 2014; Zhanget al., 2016). Therefore, we concentrated our study onthe miR319/OsTCP21 regulatory module in rice de-fense responses against the blast disease.

miR319b Overexpression Causes Rice Susceptibility to theBlast Disease

To investigate the involvement of miR319 in rice im-munity against the blast disease, we employed a miR319boverexpression line (miR319b-OE) and an OsTCP21 mu-tant line (OsTCP21-Res) in which the OsTCP21 gene is re-lieved from regulation by miR319b due to engineeredsynonymous mismatches (Zhang et al., 2016). When dis-ease responses against Guy11 infection were comparedamong wild-type, miR319b-OE, and OsTCP21-Res rice,miR319b-OE showed obviously stronger disease symp-toms than the wild-type rice, such as heavily developedlesions, obvious chlorosis (Fig. 3A), and significantly morehyphae accumulation (biomass; Fig. 3B). In contrast,OsTCP21-Res plants showed relatively resistant pheno-types against Guy11 infection, manifested by fewer visiblelesions, less severe chlorosis (Fig. 3A), and reduced hyphaeaccumulation (Fig. 3B).We further checked lesionnumbersand quantitatively analyzed the composition of lesion

Table II. List of some candidate miRNAs involved in rice immunity against M. oryzae in each library

Expression variations were analyzed. Osa-miRNAs with significant changes (greater than 2-fold) and more than 150 reads in four libraries wereselected.

miRNA Identifier Normalized Reads Fold Change

0 hpi 24 hpi 48 hpi 120 hpi 24 hpi/0 hpi 48 hpi/0 hpi 120 hpi/0 hpi

reads mg21

Up-regulatedosa-miR169a 63.22 418.67 879.59 532.46 6.62 13.91 8.42osa-miR1317-3p 12,512.79 29,328.42 139,549.83 62,645.91 2.34 11.15 5.01osa-miR169h 109.21 231.20 338.30 348.15 2.12 3.10 3.19osa-miR162a 5,402.86 10,966.52 51,230.29 15,308.21 2.03 9.48 2.83osa-miR5150-3p 557.53 1,212.25 3,473.24 1,546.18 2.17 6.23 2.77osa-miR166j-3p 425.33 915.44 2,954.51 983.00 2.15 6.95 2.31osa-miR319b 137.95 306.19 304.47 286.71 2.22 2.21 2.08osa-miR166k-3p 7,443.30 18,580.60 34,619.64 14,898.63 2.50 4.65 2.00

Down-regulatedosa-miR444d.3 959.87 484.28 112.77 399.34 0.50 0.12 0.42osa-miR435 488.56 240.58 135.32 204.79 0.49 0.28 0.42osa-miR156f-3p 827.67 187.46 112.77 215.03 0.23 0.14 0.26








types in bothwild-type and transgenic plants (Fig. 3, C andD). In miR319b-OE plants, more lesions were visible(8.7 cm22), and more type 4 and type 5 lesions (26.8% intotal)were observed than in thewild-type plants (5.1 cm22;13.1% in total). In contrast, OsTCP21-Res plants exhibitedfewer lesions on the leaf surface (2 cm22), and no type 4 ortype 5 lesions formed.

Notably, in both the wild-type and the miR319b-OEplants, the OsTCP21 expression level was reduced sig-nificantly by Guy11 infection, but not in the OsTCP21-Res mutant (Fig. 3E). This is an indication that anintegralmiR319/OsTCP21 regulatorymodule is functionalin thewild-type and themiR319b-OE plants. Furthermore,the reduced expression of OsTCP21 in miR319b-OE andthe elevated expression in OsTCP21-Res plants appearedcoincidentallywith the observeddisease symptoms. These

results implicate a tight association between theOsTCP21 expression level and rice resistance againstthe blast disease.

To further confirm that miR319 has a negative regu-latory role in rice resistance against the blast disease,miR319b-OE andOsTCP21 silencingmutant (OsTCP21-RNAi) lines from a different background (cv Kongyu131) also were tested (Wang et al., 2014). The wild-typecv Kongyu 131 is almost completely immune to Guy11infection, which developed nearly no visible lesions at120 hpi (Fig. 4A); on miR319b-OE plants, prominentlesion formation and hyphae accumulation were ob-served (Fig. 4B). Quantitative examination of lesionnumbers and the composition of lesion types revealedthat both miR319b-OE and OsTCP21-RNAi plants de-veloped significantly more lesions (6.5 and 4.5 cm22,

Figure 1. Expression patterns of miRNAs uponM. oryzae infection. Two-week-old seedlings were inoculated with Guy11 spores(1 3 105 mL21), and total RNA was extracted from leaves at the indicated time points. A, Detection of the up-regulated osa-miRNAs using RNA blotting. A total of 100 mg of total RNAwas loaded. RNA blots were hybridized with DNA oligonucleotide probescomplementary to the indicated miRNAs. U6 was used as a loading control. Values below each section represent the relative abundance ofmiRNA normalized to U6. B, Detection of the down-regulated osa-miRNAs using RNA blotting. C, RNA-blotting detection of mR319b andquantitative reverse transcription-PCR (RT-qPCR) analyses of themR319b precursor at the indicated time points uponGuy11 infection. Valuesrepresent means6 SD of three independent samples. Student’s t test was used to determine the significance of differences between 0 h postinfection (hpi) and the indicated time points. Asterisks indicate significant differences (**, P, 0.01). The RT-qPCR experimentswere repeatedthree times with similar results.


Zhang et al.



respectively) than the wild-type plants (1.4 cm22) aswell as more prominent type 4 and 5 lesions (22.7% and11.4%, respectively; Fig. 4, C and D), indicating that theelevated miR319 expression corresponds to the de-creased resistance. OsTCP21-RNAi plants also showedreduced resistance to Guy11 infection, but to a lesserdegree in comparisonwith themiR319b-OE plants (Fig.4, A–D), which may be due to partial suppression in theRNAi plants (Wang et al., 2014). A similar observationwas made in the cv Kongyu 131 background plants,whereby OsTCP21 expression was significantly re-duced upon Guy11 infection in the wild-type or mutantplants (Fig. 4E). Therefore, we conclude that theOsTCP21 expression is positively associated with riceresistance against the blast disease resistance.Taken together, our results confirmed the association

between the expression level of OsTCP21 and enhancedresistance against M. oryzae infection in different rice va-rieties. Our results suggest thatOsTCP21may play a rolein the rice defense response against the blast disease,which is regulated by miR319-mediated gene silencing.

OsTCP21 Overexpression Leads to Early Activation ofDefense Responses

To validate the involvement of miR319/OsTCP21 inrice resistance against the blast disease, we examinedreactive oxygen species (ROS) accumulation in bothwild-type and mutant plants. ROS is produced whenplants are under stress conditions and is one of the earlyresponses against pathogen infection (Lamb andDixon,1997; Thaler et al., 2012). A recent report showed that

OsTCP21 could enhance cellular ROS levels and con-tribute to plant tolerance against cold stress (Yang et al.,2013; Wang et al., 2014). In mock-treated wild-type riceplants (cv Nipponbare), H2O2 accumulation was neg-ligible, indicating that a low ROS level was produced inricewithoutM. oryzae infection (Fig. 5A).When infectedby Guy11, wild-type plants accumulated measurablelevels of H2O2, which could be detected by dia-minobenzidine (DAB) staining. In miR319b-OE plants,where the expression of OsTCP21 is suppressed, no mea-surable H2O2 accumulation was detected in either mock-or Guy11-infected rice; in contrast, in the OsTCP21-Resplants, H2O2 accumulated to a significantly higher levelthan in the mock-treated plants (Fig. 5A). We furthertested H2O2 accumulation in wild-type and OsTCP21-RNAiplants in the cvKongyu131 background.Consistentwith its resistance to the blast disease, wild-type cv Kon-gyu 131 accumulated high levels of H2O2 when infectedwith Guy11. However, in either the miR319b-OE or theOsTCP21-RNAi plants, significantly less H2O2 accumula-tion than that in the wild-type cv Kongyu 131 was ob-served (Fig. 5B). Taken together, the co-occurrencebetween cellular H2O2 levels and the in vivo OsTCP21expression levels suggests an association of this gene withthe early immune response against the blast disease in rice.

miR319 Impedes JA Synthesis and Signaling Elicited byM. oryzae Infection

JA is a phytohormone that plays a critical role indefense responses against necrotrophic and hemi-biotrophic pathogens (Thaler et al., 2012; Tamaoki al.,

Figure 2. miR319b targets OsTCP21and OsGAmyb. A, Western-blot anal-ysis of OsTCP21 and OsGAmyb ex-pression by the transient expressionsystem. Agrobacterium tumefaciens-mediated transient coexpression ofOsTCP21orOsGAmybwithmiR319bor an irrelevant miRNA (miR169a) inNicotiana benthamiana for 48 h isshown. The expression levels ofOsTCP21 and OsGAmyb were nor-malized to b-actin. B, RT-qPCR anal-yses of OsTCP21 and OsGAmybexpression levels at the indicated timepoints upon Guy11 infection. Valuesrepresent means 6 SD of three inde-pendent samples. Student’s t test wasused to determine the significance ofdifferences between 0 hpi and the in-dicated time points. Asterisks indicatesignificant differences (**, P , 0.01).Similar results were obtained fromthree biological replicates.





2013; Zhang et al., 2016). It has been reported that, inboth Arabidopsis and rice, some TCP transcriptionfactors are involved in the JA signaling pathway(Schommer et al., 2008; Zhang et al., 2016). Therefore,we hypothesized that the miR319-mediated regulationofOsTCP21might affect rice resistance against the blastdisease by manipulating JA signaling.

To validate our hypothesis, we examined the expres-sion levels of JA signaling pathway reporter genes, suchas PATHOGENESIS-RELATED PROTEIN1B (OsPR1b),JA synthetic component genes, such as PHOSPHOLI-PASE D a1 (OsPLDa1), OsLOX2, OsLOX5, and ALLENEOXIDE SYNTHASE2 (OsAOS2), and JA signaling com-ponent genes, such as CORONATINE INSENSITIVE1B(OsCOI1b), OsCOI2, and JASMONATE ZIM-DOMAINPROTEIN8 (OsJAZ8; Chini et al., 2007; Thines et al.,2007;Katsir et al., 2008;Yamadaet al., 2012; Lee et al., 2013).

In wild-type rice, both the JA signaling reporter gene,OsPR1b, and JA synthesis and signaling componentswere induced by Guy11 infection at the early stage butdeclined at the late stage (Supplemental Fig. S2). Thisunique expression pattern suggests a close associationbetween the up-regulated JA signaling pathway and theprogress of pathogen infection. Moreover, in plants inwhich the miR319/OsTCP21 regulatory module wasperturbed, the expression of JA signaling pathway com-ponents changed. For example, after Guy11 infection, theexpression of OsPR1b was suppressed in miR319b-OErice but was highly induced in theOsTCP21-Res rice (Fig.6A), indicating that the JA signaling pathway is affectedby the miR319/OsTCP21 regulatory module. Addition-ally, the expression of components involved in JA syn-thesis alsowas affected. Specifically, genes involved in theconversion frommembrane lipids to LnA (OsPLDa1) and

Figure 3. Overexpressing miR319b results in elevated susceptibility toM. oryzae in the cv Nipponbare (NPB) background. Two-week-old seedlings of the wild type (cv Nipponbare) and transgenic lines (miR319b-OE andOsTCP21-Res) were inoculated withGuy11 spores (13 105 mL21). A, Rice pathogenicity assays. Leaf phenotypes were observed at 120 hpi. B, RT-qPCR analyses ofM. oryzae biomass (expression level of MGG03982.6) on the indicated rice at 120 hpi. Values represent means 6 SD of threeindependent samples. Student’s t test was used to determine the significance of differences between the wild type and the in-dicated lines. C and D, Count of lesion numbers (per 1 cm2) and composition of lesion types of wild-type and transgenic rice.Lesions in eight diseased leaves were analyzed at 120 hpi. Values in C represent means 6 SD of three biological replicates.Student’s t test was used to determine the significance of differences between wild-type cv Nipponbare and the indicated rice lines. E,RT-qPCRanalyses ofOsTCP21 expression levels inmock- orGuy11-infectedwild-type and transgenic rice. Values representmeans6 SD

of three independent samples. Student’s t test was used to determine the significance of differences between the mock- and Guy11-infected rice. Asterisks indicate significant differences (**, P, 0.01). Similar results were obtained in three independent experiments.


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from LnA to HPODE (OsLOX2/5) were both suppressed,while the gene involved in the conversion of HPODE to12-oxo-phytodienoic acid (OsAOS2) was induced (Fig.6B). These genes showed reversed expression patterns inthe OsTCP21-Res rice. Genes involved in JA signaling(OsCOI1b,OsCOI2, andOsJAZ8) also varied in expressionwhen miR319b was overexpressed or suppressed. Inparticular, the expression of OsCOI1 and OsCOI2 wassuppressed in the miR319b-OE rice but induced in theOsTCP21-Res rice, while OsJAZ8 showed a reversedpattern (Fig. 6B). These results demonstrated that both JAsynthesis and signaling are affected by the miR319/OsTCP21 regulatory module.Similarly, we also checked the expression of key

genes involved in the salicylic acid (SA) signalingpathway. The results showed that neither synthesis(OsICS1 and OsPAL1) nor signaling (OsPAD4 andOsEDS1) of the SA pathway was affected noticeably by

miR319/OsTCP21 (Fig. 6C). Taken together, our resultsdemonstrated that the miR319/OsTCP21 module reg-ulates multiple key factors in the JA signaling pathway,among whichOsLOX genes (OsLOX2 andOsLOX5) areseverely affected. We speculated that, in Guy11-infected rice, JA synthesis is weakened due to thejeopardized LOX activities.

Guy11 Infection Induces miR319b and InhibitsJA-Mediated Defense Responses

To validate our hypothesis, we applied both methyljasmonate (MeJA) and LnA to wild-type and mutantrice. We speculated that MeJA would demonstrate adisease-rescue effect on both wild-type and transgenicrice, while LnA could only complement the diseasesymptoms in plants in which the LOX activities are notinhibited.

Figure 4. Overexpressing miR319b results in elevated susceptibility to M. oryzae in the cv Kongyu 131 background. Two-week-oldseedlings of wild-type (cv Kongyu 131) and transgenic (miR319b-OE and OsTCP21-RNAi) rice were inoculated with Guy11 spores(1 3 105 mL21). A, Rice pathogenicity assays. Leaf phenotypes were observed at 120 hpi. B, RT-qPCR analyses ofM. oryzae biomass(expression level of MGG03982.6) on the indicated lines at 120 hpi. Values represent means 6 SD of three independent samples.Student’s t testwas used to determine the significance of differences between thewild type and the indicated lines. C andD,Comparisonof disease lesion number (per 1 cm2) and composition of disease lesion types of wild-type and transgenic rice. Lesions in eight diseasedleaves were analyzed at 120 hpi. Values in C represent means6 SD of three biological replicates. Student’s t test was used to determinethe significance of differences between the wild type and the indicated lines. E, RT-qPCR analyses of OsTCP21 expression levels inmock- or Guy11-infectedwild-type and transgenic rice. Values represent means6 SD of three independent samples. Student’s t test wasused todetermine the significanceof differences between themock- andGuy11-infected plants. Asterisks indicate significant differences(**, P , 0.01). Similar results were obtained in three independent experiments.





On bothwild-type (cvNipponbare) andmiR319b-OErice, but not OsTCP21-Res rice, Guy11 infection causedapparent disease symptoms at 120 hpi. Application ofMeJA on both wild-type and miR319b-OE rice obvi-ously rescued the disease susceptibility (Fig. 7A). Spe-cifically, MeJA-treated plants displayed much lesssevere disease symptoms, demonstrated by fewer le-sions (about 60% less), less hyphae accumulation (about50% less), and about 70% fewer type 4 and 5 lesionsthan the mock-treated rice (Fig. 7, B and C). In contrast,LnA treatment displayed no discernible effects on ei-ther wild-type or miR319b-OE rice, suggesting thatMeJA treatment, but not LnA, could rescue the reducedresistance on these plants (Fig. 7A). Similarly, the effectsof MeJA and LnA were tested on OsTCP21-Res rice. InOsTCP21-Res rice, which was relatively resistant toGuy11 infection, the application of MeJA and LnAshowed no visible difference at 120 hpi. However,when quantitative examinations were employed, bothMeJA-treated and LnA-treated rice showed signifi-cantly less Guy11 infection than the mock-treatedplants (Fig. 7B). Specifically, the MeJA-treated riceformed 50% fewer lesions and accumulated 75% fewerhyphae than the mock-treated rice; the LnA-treated rice

formed 50% fewer lesions and accumulated 85% fewerhyphae than the mock-treated rice. Neither MeJA-treatednor LnA-treated rice formed any type 4 or 5 lesions, while6.7% of the lesions formed on the mock-treated rice weretype 4 or 5 (Fig. 7, C and D). We also measured theexpression levels ofmiR319b andOsTCP21 in bothMeJA-and LnA-treated rice. In both miR319-OE and OsTCP21-Res plants, the expression of miR319b and OsTCP21wasnot affected by the application of hormones, indicatingthat the effects of MeJA or LnA are genetically down-stream of OsTCP21 (Supplemental Fig. S3). These resultsconfirmed our hypothesis that the elevated disease sus-ceptibility in wild-type or miR319b-OE rice is due to theblocking of JA synthesis at the LnA step, which conse-quently disrupted JA-mediated disease resistance againstthe blast disease. Our results demonstrated that the LnA-to-HPODE conversion is the key JA synthesis step that ismanipulated by the blast fungus.

M. oryzae Induces miR319 Expression throughcis-Elements in Its Promoter Region

To investigate how the infection signals are perceivedby potential cis-elements in the miR319 promoter, we

Figure 5. The expression ofOsTCP21results in the activation of early defenseresponses. Two-week-old leaves ofwild-type and transgenic lines weredipped in a Guy11 spore suspension(1 3 105 mL21). A, Sustained expres-sion of OsTCP21 results in the accu-mulationofH2O2 in the cvNipponbare(NPB) background. B, Silencing ofOsTCP21 blocks H2O2 accumulationin the cv Kongyu 131 background. Todetect the accumulation of H2O2, rep-resentative rice leaves from the indi-cated lines (48 hpi) were stained withDAB to show the accumulation ofH2O2; mock-treated wild-type cv Nip-ponbare and cv Kongyu 131were usedas controls. Expression levels werequantified using ImageJ as instructed.Similar results were obtained fromthree biological replicates. Values rep-resent means 6 SD of three biologicalreplicates. Student’s t test was used todetermine the significance of differ-ences between the indicated lines andtreatments.


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analyzed the 1,452-bp sequence located immediatelyupstream of the miR319 stem-loop region by usingPlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/; Lescot et al., 2002). Somestress/hormone-responding cis-elements, such as the fun-gal elicitor-responsive element (Box-W1), defense/stress-responding element (TC-rich repeats), abscisic acid-responding element (ABRE), MeJA-responsive element(CGTCA motif), and SA-responsive element (TCA ele-ment)were identified (Supplemental Fig. S4; SupplementalTable S3). We fused a GFP reporter gene to either the wildtype or different deletion mutants of the miR319 promoter(Fig. 8A). When transiently expressed in rice protoplasts

with and without elicitor (Guy11 cell wall extraction)treatment (Forlani et al., 2011), GFP fluorescence wasunder the detectable level, indicating that the miR319promoter is not a strong promoter. When driven by thewild-type promoter, the GFP gene was expressed sig-nificantly higher in the elicitor-treated samples than inthe mock-treated samples, indicating that the proto-plast transient expression assay is a good system for ourpromoter analysis (Fig. 8B). Under the mock-treatedcondition, the reporter driven by either the wild-typeor themutant promoterwas expressed at a similar level,indicating that none of the cis-elements tested (namelyTC-rich repeats, Box-W1, ABRE, CGTCA motif, and

Figure 6. miR319b impedes JA synthesis and signaling. Two-week-old seedlings of the wild type (WT; cv Nipponbare [NPB]) andtransgenic lines (miR319b-OE andOsTCP21-Res) were used for the assays. A, RT-qPCR analyses of the JA reporter geneOsPR1bin the indicated lines upon Guy11 infection at 24 hpi. B, RT-qPCR analyses of the key JA synthesis and signaling components inthe indicated lines. C, RT-qPCR analyses of the key SA synthesis and signaling components in the indicated lines. Values representmeans6 SD of three independent samples. Student’s t test was used to determine the significance of differences between the wildtype and the indicated lines. Asterisks indicate significant differences (**, P , 0.01). The RT-qPCR experiments were repeatedthree times with similar results. OPDA, 12-Oxo-phytodienoic acid.




http://bioinformatics.psb.ugent.be/webtools/plantcare/html/






TCA element) affect the expression of miR319 withoutelicitor treatment. However, when treated with elici-tors, deletion of either the TC-rich repeats or the Box-W1motif caused significantly reduced expression of the

GFP gene, while mutation in other motifs (ABRE,CGTCA motif, and TCA element) did not cause mea-surable change (Fig. 8B). When both the TC-rich repeatsand the Box-W1 motif were deleted, the expression of

Figure 7. Overexpressing miR319b inhibitsJA-mediated defense responses. Two-week-oldseedlings of wild-type (cv Nipponbare [NPB]) andtransgenic (miR319b-OE and OsTCP21-Res) ricewere treated with mock, MeJA, or LnA. Two dayslater, the plants were inoculated with Guy11spores (1 3 105 mL21). A, Rice pathogenicityassays. Leaf phenotypes in the indicated rice uponmock, JA, or LnA treatment were observed at120 hpi. B, RT-qPCR analyses of M. oryzae bio-mass (expression level of MGG03982.6) on theindicated lines upon the different treatments at120 hpi. C and D, Count of lesion number (per1 cm2) and composition of lesion types on theindicated lines upon the different treatments. Le-sions in eight diseased leaves were analyzed at120 hpi. Values represent means 6 SD of threebiological replications. Student’s t test was used todetermine the significance of differences in themock and the indicated treatments. Asterisks in-dicate significant differences (**, P , 0.01). Sim-ilar results were obtained in three independentexperiments.


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the reporter gene was similar to that of the single mu-tation, indicating that these two cis-elements do notfunction redundantly. Therefore, our results indicatedthat the TC-rich repeats and the Box-W1 motif arecritical for the Guy11-induced expression of the miR319precursor gene.In summary, we found that rice endogenous

miR319b is induced specifically by M. oryzae strainGuy11 infection via recognizing conserved motifs, suchas the TC-rich repeats or Box-W1, in the promoter re-gion. miR319b suppresses OsTCP21 and some keyJA synthetic component genes, such as OsPLDa1,OsLOX2, and OsLOX5, which specifically interruptJA synthesis at two early steps (from membrane lipidsto LnA, then to HPODE). Stalled JA synthesis, aswell as signaling transduction, led to reduced de-fense responses, including ROS accumulation and theproduction of pathogenesis-related proteins. The in-volvement of miR319a/b in cold tolerance also wasreported, which involves OsPCF5, OsPCF6, OsPCF8,and OsTCP21, and ROS elimination in rice cells (Yanget al., 2013; Wang et al., 2014). Therefore, the miR319/OsTCP21 regulatory module may serve as a signalinghub mediating rice responses to both biotic and abioticstresses (Fig. 9). Whether there is any interplay between

resistance to the blast disease and tolerance to coldneeds further investigation.

DISCUSSION

As a master modulator of gene expression, miRNA-mediated gene silencing is a potent regulatory mecha-nism involved in plant responses against pathogeninfection (Padmanabhan et al., 2009; Ding, 2010). In therice-M. oryzae interaction, both compatible and incom-patible infections trigger specific gene expression vari-ation on endogenous sRNAs, either initiated bypathogen infection or by the host innate immunity (Liet al., 2014, 2017). As a consequence, gene expressionregulated by these sRNAs may facilitate disease de-velopment (Li et al., 2017a) or contribute to disease re-sistance (Li et al., 2014). Therefore, identification ofsRNAs involved in the interaction between M. oryzaeand rice and investigation of the target gene functionsare crucial in discovering critical rice immune compo-nents against the blast disease. Previous studies haveshown that miR160a and miR398b participate in riceinnate immunity against the blast disease, over-expression of which significantly enhances disease re-sistance (Li et al., 2014, 2017). In this study, we found

Figure 8. Identification of M. ory-zae-responsive cis-elements. A,Schematic maps of the cis-elementsin the miR319b promoter sequenceand corresponding deletion muta-tions. B, RT-qPCR analyses of thereporter gene in the wild type andthe indicated mutants, which weretransiently expressed in rice proto-plasts with or without elicitor treat-ment. Values represent means 6 SD

of three independent samples. Stu-dent’s t test was used to determinethe significance between the wild-type and mutant promoter sequences.Different letters at the top of eachcolumn indicate significant differences(P , 0.01). The RT-qPCR experimentswere repeated three times with similarresults.





that miR319 is a novel factor that determines the rela-tionship between rice and M. oryzae. Moreover, theblast pathogen can manipulate rice immune responsesby blocking JA signaling. This blockage is accom-plished by the miR319-mediated silencing of a tran-scription factor gene (OsTCP21) and its target genes(such as OsLOX2 and OsLOX5 that encode key syn-thetic components of JA). By repressing the rice JAsignaling pathway, the blast pathogen facilitates itsinfection, growth, and propagation in the host plant.Our discovery identified a potential focal point thatcould be used for efficient control of the blast disease.

Researchers have shown that members of the miR319family are involved in plant responses against a varietyof biotic stresses (Zhang et al., 2011; Shen et al., 2014).Although a common resistant mechanism has not beenidentified, a lot of evidence points to a potential

association with the modulation of JA signaling. The JAsignaling pathway is a potent mechanism employed byplants that can defend against necrotrophic and hemi-biotrophic pathogens, including the blast pathogen(Thaler et al., 2012; Tamaoki et al., 2013; Zhang et al.,2016). In tomato, miR319 behaves as a systemic signaland a negative regulator of defense responses againstroot-knot nematodes through decreasing the JA level(Zhao et al., 2015). In rice, RRSV infection inducesmiR319 expression. The resultant suppressed expres-sion of TCP inhibits plant defense responses and pro-motes RRSV infection and disease symptomdevelopment (Zhang et al., 2016). In our study, JA sig-naling was induced at the early stage but declinedat the late stage (Supplemental Fig. S2), which matchedthe expression pattern of miR319 very well (Fig. 1C).The fluctuating JA signaling is a good indicator of an

Figure 9. A model of miR319/OsTCP21modulates the rice and M. oryzae interaction through affecting the JA pathway. Guy11infection induces miR319b expression, which diminishes the stimulative roles of OsTCP21 on key JA synthesis and signalingcomponents (such as OsPLDa, OsLOXs, and OsCOL). Disruption of JA signaling leads to reduced defense responses, includingreduced ROS accumulation and PR1 expression. Transgenic rice expressing LOXs in an induced manner (miR319::LOXs) couldbe a potential resistance mechanism for the blast disease. Cold stress suppresses miR319 expression in rice, which results ininduced expression of TCPs, weakened ROS elimination, and, consequently, reduced tolerance to cold stress. A potential crosstalk between rice resistance to biotic stresses and tolerance to abiotic stresses is suggested. OPDA, 12-Oxo-phytodienoic acid.


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enhanced immune effort at the early stage of M. oryzaeinfection, which was later suppressed by the pathogenas the infection proceeded.Target genes of miR319 include OsTCPs, which en-

code transcription factors that participate in JA syn-thesis and homeostasis (Palatnik et al., 2003; Schommeret al., 2008). In Arabidopsis, OsTCP promotes OsLOX2expression at the transcriptional level (Schommer et al.,2008; Hao et al., 2012). By usingmultiple transgenic andmutant rice lines, our results built a strong connectionbetween miR319 expression and JA signaling. For in-stance, the miR319b-OE and OsTCP21-RNAi plantswere more susceptible to Guy11 infection, while theOsTCP21-Res plants weremore resistant (Figs. 3 and 4).Both theOsTCP21-OE and theOsTCP21-Res lines showedsimilar resistant phenotypes to Guy11 infection, suggest-ing that the function of miR319 is through manipulatingthe expression of OsTCP21 (Supplemental Fig. S5). Fur-ther observations showed that enhanced H2O2 accumu-lation was observed in OsTCP21-Res but not inmiR319b-OE rice (Fig. 5), indicating an enhanced earlydefense response when OsTCP21 expression is elevated.Many JA synthetic component genes possess OsTCP21-binding sites (GTGGNCCC, TGGGCY, andGGACCA) inthe promoter regions, through which the in vivo JA levelis tightly controlled (Zhang et al., 2016). Therefore, thesuppression of miR319-mediated JA signaling may be acounter defense mechanism that is conserved amongmany plant/pathogen systems, which is worthy of moreattention.Despite multiple observations suggesting that the JA

signaling pathway is a potent defense machineryagainst many pathogens (includingM. oryzae; Mei et al.,2006; Thaler et al., 2012; Tamaoki et al., 2013; Zhanget al., 2016), the detailed molecular mechanism re-garding how this regulation is carried out, and how JAsignaling is maneuvered in the interaction between riceand the blast pathogen, is not clear. Our results showedthat, in miR319b-OE rice, key JA synthesis components,such as OsPLDa1, OsLOX2, and OsLOX5, were specif-ically suppressed (Fig. 6). This observation suggeststhat Guy11 infection can suppress the expression ofthese genes and evade the JA-mediated rice defenseresponse. This is manifested by the MeJA and LnAspray-application assay on both wild-type and trans-genic plants. MeJA boosted the rice defense responseagainst Guy11 in both wild-type and transgenic plants(miR319b-OE and OsTCP21-Res), while LnA was onlyeffective on OsTCP21-Res rice (Fig. 7). Therefore, wepropose that, although the JA signaling pathway is aneffective defense mechanism against the M. oryzae path-ogen, some strains have evolved successful machinery toward off the host defense and invade its host. In the caseof Guy11, through the miR319-mediated silencing ofOsTCP21, key JA synthetic components are suppressed,which sabotages host immunity by stalling JA signaling(Fig. 9). Puccinia striiformis uses a similar strategy to breakdown wheat (Triticum aestivum) resistance (Wang et al.,2017). Therefore, our discovery may reveal a commonhost resistance-suppressing strategy employed byvarious

pathogens, which is of great significance in understand-ing the pathogenicity mechanism. Our discovery poten-tializes engineered rice harboring a chimeric genewith theLnA-encoding gene driven by the miR319b promoter.This engineered rice should express LnA upon blastpathogen infection (and maybe other pathogens as well)and could restore the stalled JA signaling caused byinfection.

Intrigued by the inducing mechanism of Guy11 in-fection, we analyzed the miR319b promoter sequenceand identified several cis-elements in the miR319bpromoter region, including potential fungal elicitor-responding elements, defense-responding elements,and hormone-responding elements (Supplemental Fig.S4; Supplemental Table S3). Deletion analysis indicatedthat the Box-W1 and TC-rich repeats regions are re-quired for response to Guy11 infection (Fig. 8). Box-W1has been identified as an elicitor-responsive elementthat is critical for the elicitor-induced expression of theparsley (Petroselinum crispum) PR1 genes and the Ara-bidopsis cytosolic thioredoxin h5 gene (Rushton et al.,1996, 2002; Laloi et al., 2004). The TC-rich repeatsregions are conserved defense-responsivemotifs that playan important role in the response toErysiphe necator (Diaz-De-Leon et al., 1993; Wen et al., 2017). Interestingly, theexpression of miR319b was not affected by an avirulentM. oryzae strain (strain 2539), either at the precursortranscript or at the mature miRNA level (SupplementalFig. S1), indicating that the induced expression ofmiR319b is Guy11 specific. Thus, our results favor ascenario that some unidentified Guy11 effector(s) maybe secreted to the rice cells and bind directly or indi-rectly to these cis-elements, which triggers the inducedexpression of themiR319b precursor. By using these cis-elements as bait molecules, potential Guy11 effectorsmay be identified in future studies.

Our results further suggest a potential cross talk be-tween rice resistance against biotic and abiotic stresses.Overexpression of miR319a/b or reduced expression ofendogenous target genes (such as OsPCF5, OsPCF6,OsPCF8, and OsTCP21) could enhance plant cold tol-erance through adjusting the in vivo ROS level (Yanget al., 2013; Wang et al., 2014). This suggests thatmiR319 is responsive to both cold stress and fungalinfection. Cold stress suppresses miR319 expression inrice, which results in induced expression ofOsTCPs andweakened ROS elimination. The correlation betweenOsTCPs and ROS also is observed in rice infected byGuy11. It would be interesting to investigate how tol-erance to cold and resistance toM. oryzae are reconciledby miR319.

MATERIALS AND METHODS

Pathogen Infection and sRNA Library Construction

Rice (O. sativa japonica ‘Nipponbare’) was grown in a growth roommaintained at 26°C and 70% relative humidity with a 12-h/12-h light/darkphotoperiod. M. oryzae strain Guy11 was used for rice infection. Briefly, three-leaf-stage plants were spray inoculated with gelatin or the indicated conidialsuspensions (1 3 105 spores mL21 in 0.2% gelatin). The inoculated plants were











kept in darkness at 80% relative humidity for 24 h before they were transferredto a growth chamber at 25°C, 80% relative humidity, and a 12-h/12-h light/dark photoperiod. Seedlings from at least 15 to 20 rice plants from the sametreatment were pooled for RNA extraction, library construction, and RT-qPCRanalysis. Plants that received the same treatment and were maintained underthe same conditions were used as biological replicates.

sRNA library construction and Illumina sequencing were performed asdescribed (Mi et al., 2008). The sRNA reads with length over 16 nucleotideswere mapped to the rice nuclear, chloroplast, and mitochondrial genomes(http://rice.plantbiology.msu.edu/; version 6.0) and M. oryzae genomes(http://www.broadinstitute.org). The perfect genome-matched sRNAs wereanalyzed following a previous report (Wu et al., 2009). The normalized abun-dance of sRNAs was calculated as reads per million.

Northern-Blot Analysis

RNA-blot analyses for miRNAs from total extracts were performed as de-scribed previously (Katiyar-Agarwal and Jin, 2007). The inoculated plants wereused for RNA extraction at 0, 24, 48, and 120 hpi. Total RNA was extractedusing TRIzol reagent (Takara) following the manufacturer’s instructions. RNAwas resolved on a 14% denaturing 8 M urea-PAGE gel and then transferred andchemically cross-linked onto a Hybond N+ membrane (GE Healthcare LifeSciences) using N-(3-dimethylaminopropyl)-N9-ethylcarbodiimide hydro-chloride. miRNA probes were end labeled with [g-32P]ATP by T4 polynu-cleotide kinase (New England Biolabs). Expression levels were quantifiedusing ImageJ as instructed. Probes used for RNA blotting are listed inSupplemental Table S4.

RT-qPCR

Total RNA was extracted from rice leaves or rice protoplasts using TRIzolreagent (Takara). ANanoDrop-1000was used to detect the quality and quantityof RNA (NanoDrop). The RNA was then reverse transcribed into cDNA usingthe PrimeScript RT Reagent Kit (Takara). RT-qPCR was performed in 15 mL ofreaction mixture consisting of 1.5 mL of 13 SYBR Green (Invitrogen), 1.5 mL ofPCR buffer, 0.3mL of 10mMdeoxyribonucleotide triphosphates (Takara), 0.3mLof Taq, 0.3 mL of ROX DYE2 (Vazyme), 1.5 mL of 2 mM each primer, and 2 mL ofappropriate diluted cDNA. The conditions for RT-qPCR were as follows: 94°Cfor 3 min, then 40 cycles at 94°C for 30 s and 58°C for 30 s, followed by 72°C for35 s for PCR amplification. Transcript levels of each gene were measured by theApplied Biosystems 7500 system according to the manufacturer’s instructions.18s rRNA was used as a quantitative control in the RT-qPCR analysis. Primersused in this study are listed in Supplemental Table S4.

Transient Expression Analysis in Nicotiana benthamiana

To generate the construct for the miR319b lines, the primary amiR319bconstruct was engineered from pRS300 (miR319a) Arabidopsis (Arabidopsisthaliana) with the WMD3 online tool (http://wmd3.weigelworld.org) and wascloned into a pEarleyGate (pEG202) destination vector using LR clonase II(Invitrogen). To generate the overexpression lines, the OsTCP21 and OsGAmybcoding sequences were cloned into the pEG202 vector.

Transient coexpression assays in N. benthamiana were performed by infil-trating 3-week-old N. benthamiana plants with Agrobacterium tumefaciensGV3101 (OD600 = 1) harboring constructs containing miR319b (pEG202) withA. tumefaciens GV3101 (OD600 = 1) containing target gene coding sequences(pEG202), or with miR169a (pEG202) as a control. Leaf tissue was collected48 hpi, and protein expression was detected by western blot.

Western Blot

The tissue was ground in liquid nitrogen, and total proteins were extractedusing 23 SDS loading buffer. The samples were resolved on a 12% SDS-PAGEgel and transferred onto an Amersham Hybond-P PVDF membrane (GEHealthcare) using a Tris-Gly transfer buffer. Membranes were blocked with 5%milk in 0.05% TBS-plus Tween 20 (TBST) for 40 min and incubated overnight at4°C with 1:5,000 dilutions of primary antibodies of mouse anti-Flag conjugatedto horseradish peroxidase (Abmart), washed three times with TBST, then in-cubated overnight at 4°C with 1:1,000 dilutions of secondary antibodies (Bey-time). Membranes were washed three times with TBST. Detection wasperformed using the ECL Plus Western Blotting Detection Reagents (GE

Healthcare) and ChemiDoc Touch Imager (Bio-Rad). Image data were analyzedwith Image Lab Software (Bio-Rad) and assembled in Adobe Photoshop CS6.

Pathogenicity Assays and Chemical Treatments

The pathogenicity test was performed by spraying a conidial suspension at aconcentration of 1 3 105 spores mL21 onto the leaves of 2-week-old rice. Theinoculated plants were kept in darkness at 80% relative humidity for 24 h beforethey were transferred to a growth chamber at 25°C, 80% relative humidity, anda 12-h/12-h light/dark photoperiod. Leaf phenotypes were observed at 120 hafter fungal inoculation. The blast disease lesions were examined and classifiedinto six types. In brief, 0 = no disease lesions observed; 1 = small pinpoint-likedisease lesions between two small vascular bundles; 2 = lesions with diameter0.5 to 1 mm that developed over the two small vascular bundles but did notreach the big vascular bundles; 3 = disease lesions with diameter about 1 to3 mm that developed between the two big vascular bundles; 4 = disease lesionswith diameter about 3 to 4 mm that developed over the two big vascularbundles; and 5 = disease lesions with diameter over 4 mm that developed overthe main vein.

Chemical treatments of 2-week-old rice plants with 100 mM MeJA (in 10%ethanol) or LnA (in 1% methanol; Sigma) were sprayed on the leaves. Distilledwater containing 10% ethanol and 1% methanol was used as a control treat-ment. After 24 h, control-treated, JA-treated, and LnA-treated plants weresubjected to pathogenicity assays. Leaf phenotypes were observed at 120 h afterfungal inoculation. Image data were assembled in Adobe Photoshop CS6.

Detection of H2O2 Accumulation

To detect the accumulation of H2O2, rice leaves from at least three differentplants were stained with 1 mg of DAB (pH 3.8) per 1 mL for 8 h in the dark at25°C to 28°C. The DAB solution was then supplemented with 50 mM ascorbicacid, a scavenger of H2O2, to ensure the specificity of staining. Subsequently,rice leaves were cleared with 96% (v/v) ethanol and were preserved in 50% (v/v)ethanol. DAB staining visualizes H2O2 as red-brown precipitate using the lightmicroscope. Image data were assembled in Adobe Photoshop CS6. Expressionlevels were quantified using ImageJ as instructed.

Protoplast Isolation and Transformation

Rice protoplasts were isolated as described previously (Bart et al., 2006). Ineach tube, 1 mL of protoplasts was gently mixed with 10 mg of plasmid DNAand 1 mL of 40% PEG. After incubation at 28°C for 15 to 20 min, each tube wasadded with 4 mL of W5 solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, and2 mM MES [pH 5.7]) and gently mixed. The tubes were spun at 200 rpm for3 min, and the pellet was resuspended with 200 mL of W5 solution and thenincubated at 28°C in the dark overnight. The pXZP008 vector was used tooverexpress miR319b and OsTCP21 in rice protoplasts. The amiR319b and full-length cDNA fragment were cloned into the pXZP008 vector with the Ubiquitinpromoterwith the homologous recombinationmethod using the ClonExpress IIOne Step Cloning Kit (Vazyme). Primers used in this study are listed inSupplemental Table S4.

Promoter Cloning and Analysis

The 1,452-bp region upstream of miR319b was cloned by PCR and thencloned into the pXZP008 vector with the homologous recombination methodusing the ClonExpress II One Step Cloning Kit (Vazyme). The putative regu-latory elements were predicted using the PlantCARE program (Lescot et al.,2002; http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). Weobtained a single-site mutant of the putative regulatory elements (Box-W1 andTC-rich repeats) by overlap PCR and then cloned them into a pXZP008 vectorwith the homologous recombination method. All three constructs were trans-formed into rice protoplasts as described previously. Primers used in this studyare listed in Supplemental Table S3.

Accession Numbers

Sequence data from this study can be found in the Rice Genome Database(http://rice.plantbiology.msu.edu) and GenBank (https://www.ncbi.nlm.nih.gov/genbank) under the following accession numbers: OsTCP21(LOC_Os07g05720), OsGAmyb (LOC_Os01g59660), OsPLDa1 (LOC_Os01g07760),


Zhang et al.


http://rice.plantbiology.msu.edu/

http://www.broadinstitute.org



http://wmd3.weigelworld.org




http://rice.plantbiology.msu.edu

https://www.ncbi.nlm.nih.gov/genbank

https://www.ncbi.nlm.nih.gov/genbank


OsLOX5 (LOC_Os02g10120), OsLOX2 (LOC_Os03g08220), OsAOS2(LOC_Os03g12500), OsCOI1b (LOC_Os05g37690), OsCOI2 (LOC_Os03g15880),OsJAZ8 (LOC_Os09g23650), OsPR1b (LOC_Os01g28450), OsICS1(LOC_Os09g19734),OsPAD4 (LOC_Os11g09010),OsPAL1 (LOC_Os02g41630), andOsEDS1 (AK100117).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Expression of miR319b is not induced by theavirulent strain 2539.

Supplemental Figure S2. There is a close association between the JA path-way and the progress of pathogen infection.

Supplemental Figure S3. The expression levels of miR319b and OsTCP21in MeJA- and LnA-treated rice lines.

Supplemental Figure S4. Schematic location maps of the cis-elements inthe miR319b promoter sequence.

Supplemental Figure S5. The phenotype of different transgenic lines uponthe Guy11 infection.

Supplemental Table S1. List of identified miRNAs differentially expressedin M. oryzae-infected rice.

Supplemental Table S2. miRNA target genes.

Supplemental Table S3. Identified M. oryzae-responding cis-elements.

Supplemental Table S4. Primer information.

ACKNOWLEDGMENTS

We thank Dr. Yanming Zhu and Dr. Xiaoli Sun for kindly providing theKongyu 131 seeds. We also thank Tanzeela Zia for critical proofreading of thearticle.

Received November 27, 2017; accepted February 16, 2018; published March 16,2018.

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