OsSYP121 Accumulates at Fungal Penetration Sites and ... · OsSYP121 Accumulates at Fungal...

OsSYP121 Accumulates at Fungal Penetration Sites andMediates Host Resistance to Rice Blast1

Wen-Lei Cao,2,3 Yao Yu,2 Meng-Ya Li, Jia Luo,4 Rui-Sen Wang, Hai-Juan Tang, Ji Huang, Jian-Fei Wang,Hong-Sheng Zhang,5 and Yong-Mei Bao5,6

State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Agriculture, JiangsuCollaborative Innovation Center for Modern Crop Production, Nanjing Agricultural University, Nanjing210095, China

ORCID IDs: 0000-0002-7262-3640 (M.L.); 0000-0002-9057-4299 (J.H.); 0000-0002-1410-915X (Y.B.).

Magnaporthe oryzae is a fungal pathogen that causes rice (Oryza sativa) blast. SNAREs (soluble N-ethylmaleimide-sensitive factorattachment protein receptors) are key components in vesicle trafficking in eukaryotic cells and are known to contribute to fungalpathogen resistance. Syntaxin of Plants121 (SYP121), a Qa-SNARE, has been reported to function in nonhost resistance inArabidopsis (Arabidopsis thaliana). However, the functions of SYP121 in host resistance to rice blast are largely unknown.Here, we report that the rice SYP121 protein, OsSYP121, accumulates at fungal penetration sites and mediates host resistanceto rice blast. OsSYP121 is plasma membrane localized and its expression was obviously induced by the rice blast in both theblast-resistant rice landrace Heikezijing and the blast-susceptible landrace Suyunuo (Su). Overexpression of OsSYP121 in Suresulted in enhanced resistance to blast. Knockdown of OsSYP121 expression in Su resulted in a more susceptible phenotype.However, knockdown of OsSYP121 expression in the resistant landrace Heikezijing resulted in susceptibility to the blast fungus.The POsSYP121::GFP-OsSYP121 accumulated at rice blast penetration sites in transgenic rice, as observed by confocal microscopy.Yeast two-hybrid results showed that OsSYP121 can interact with OsSNAP32 (Synaptosome-associated protein of 32 kD) andVesicle-associated membrane protein714/724. The interaction between OsSYP121 and OsSNAP32 may contribute to hostresistance to rice blast. Our study reveals that OsSYP121 plays an important role in rice blast resistance as it is a keycomponent in vesicle trafficking.

Vesicle trafficking plays crucial roles in plant devel-opment and immune responses ( Somerville et al., 2004;

Lipka et al., 2007; Kwon et al., 2008a; Van Damme andGeelen, 2008; Meyer et al., 2009). SNAREs (solubleN-ethylmaleimide-sensitive factor attachment proteinreceptors) are key components in vesicle trafficking ineukaryotic cells (Heese et al., 2001;Wick et al., 2003) andplay a universal role in diverse biological processesincluding cytokinesis, defense response, pollen tubeand root hair tip growth, root formation, and hormoneresponse in plants (Dacks and Doolittle, 2002; Lipkaet al., 2007; Enami et al., 2009). Four different types ofSNAREs form a SNARE complex through their R-, Qa-,Qb-, and Qc-SNARE domains to determine the speci-ficity of intracellular fusion (Antonin et al., 2000; Fukudaet al., 2000). Syntaxins (Qa-SNAREs) and interactingSNARE proteins (R-, Qb-, and Qc-SNAREs) contributeto the fusion of intracellular transport vesicles withacceptor membranes in diverse trafficking pathways(Pajonk et al., 2008; Reichardt et al., 2011).The SYP1(Syntaxin of Plants1) subfamily is a plant-specific syn-taxin family that belongs to the Qa-SNARE family.Nine SYP1 genes, SYP111, SYP112, SYP121, SYP122,SYP123, SYP124, SYP125, SYP131, and SYP132, arefound in Arabidopsis (Arabidopsis thaliana; divided intothree groups) and all localized on the plasma mem-brane (Uemura et al., 2004). The expression of SYP1s istissue specific, only SYP132 ubiquitously expressed invarious tissues throughout plant development (Enamiet al., 2009). SYP111/KNOLLE is well known as acytokinesis-specific syntaxin that is specifically expressed

1This work was supported by grants from the National Key Projectfor Transgenic Crops (2016ZX08009-003-001), the Fundamental Re-search Funds for the Central Universities (KYZ201704), the NationalNatural Science Foundation of China (31871602, 31171516, and30900888), the Jiangsu Agriculture Science and Technology Innova-tion Fund (CX151054), and the Open Fund of State Key Laboratory ofRice Biology (160101).

2These authors contributed equally to the article.3Present address: College of Agriculture, Yangzhou University,

225009 Yangzhou, China.4Present address: Chongqing Academy of Agricultural Sciences,

401329 Chongqing, China.5Senior authors.6Author for contact: [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:Yong-Mei Bao ([email protected]).

Y.B., H.Z., and W.C. designed the research; Y.B. cloned the geneOsSYP121; W.C., Y.Y., and Y.B. performed most experiments; J.L.provided technical assistance to W.C.; R.W. performed the real-timePCR; W.C., Y.Y., and M.L. performed the blast fungus inoculation;H.T. performed rice transforming experiments; J.H. and J.W. pro-vided assistance in data analysis; Y.B. andW.C. conceived the projectand wrote the article; Y.B. and H.Z. supervised and complementedthe writing.

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during mitosis and localizes to the forming cell plate(Lukowitz et al., 1996; Heese et al., 2001). SYP112 canfunctionally replace the cell cycle-regulated KNOLLEprotein (Sanderfoot et al., 2000; Müller et al., 2003). As acalcium-dependent phosphorylation protein in Arabi-dopsis, SYP122 has redundant functions with its closesthomolog SYP121 in the secretion of cell wall deposits(Nühse et al., 2003; Assaad et al., 2004; Zhang et al.,2007). SYP123, which is predominantly expressed inroot hairs and localizes to the tip region of root hairs,can function with SYP132 to mediate tip-focusedmembrane trafficking for root hair tip growth (Ichikawaet al., 2014). SYP124, SYP125, and SYP131 are pollen-specific syntaxins involved in pollen tube growth (Katoet al., 2010; Silva et al., 2010; Ul-Rehman et al., 2011).NbSYP132 in Nicotiana benthamiana acts as the cognatetarget-SNARE for the exocytosis of vesicles containing PRproteins in plant basal and salicylate-associated defense(Kalde et al., 2007).SYP121 is the most intensively studied and well-

characterized syntaxin (Collins et al., 2007; Kwonet al., 2008b). SYP121/SYR1 was originally identifiedin tabacco (Nicotiana tabacum), and it can prevent thepotassium and chloride ion channel response to abscisicacid in stomatal guard cells (Leyman et al., 1999, 2000).SYP121/PEN1 in Arabidopsis was also shown to di-rectly interact with the potassium and chloride ionchannel through an FxRF motif to facilitate solute up-take for cell expansion and plant growth (Sutter et al.,2006; Honsbein et al., 2009, 2011; Grefen et al., 2010).SYP121/PEN1 has been verified to contribute to pene-tration resistance in Arabidopsis (Collins et al., 2003;Kwon et al., 2008a, 2008b, 2008c). SYP121/ROR2 inbarley (Hordeum vulgare) was localized at the plasmamembrane in nonpathogen challenged epidermal cellsbut accumulated focally near the papilla structure be-low the penetration sites infected by powdery mildew(Assaad et al., 2004; Bhat et al., 2005; Collins et al.,2007). SYP121 is believed to act in mediating vesiclefusion events in an extracellular defense pathway byspecifically forming a ternary SNARE complex withSNAP33 (Synaptosome-associated protein of 33 kD)and the VAMP721/722 (Vesicle-associated membraneprotein721/722) to deliver defense components to thespace between the plasma membrane and the plant cellwall where fungus is attacking (Collins et al., 2003;Kwon et al., 2008b).As a major food crop, rice has a genome encoding 57

SNARE proteins (Sanderfoot, 2007), but none of themhas been well characterized. In our previous work, wecloned five SNARE genes, including OsSNAP32 (Baoet al., 2008b; Luo et al., 2016), OsSYP71 (Bao et al.,2012), and OsNPSN11 to OsNPSN13 (Bao et al.,2008a). The expression of the SNAP25-type geneOsSNAP32 was induced by H2O2, PEG6000, lowtemperature, and rice blast fungus inoculation treat-ments in rice seedlings (Bao et al., 2008b). The over-expression ofOsSNAP32 andOsSYP71 in rice showedenhanced tolerance to oxidative stress and rice blast(Bao et al., 2012; Luo et al., 2016).

In this article, we isolated and analyzed the expres-sion of OsSYP111, OsSYP121, and OsSYP132 distrib-uted in three SYP1 subgroups from rice, and only theexpression of OsSYP121 was induced by the blast fun-gus. To elucidate the function of OsSYP121 in rice re-sistance to blast, we overexpressed and knocked downthe expression of OsSYP121 in transgenic rice and ob-served the location of PSYP121:GFP-SYP121 in transgenicrice inoculated by blast fungus by microscopy.

RESULTS

Expression of OsSYP121 Is Induced by the Blast Fungus

The expression profiles ofOsSYP111,OsSYP121, andOsSYP132 in rice landrace Heikezijing (Hei) weredetected in various tissues. OsSYP121 and OsSYP132were predominantly detected in leaf blades and leafsheaths (Fig. 1A). In order to determine three genes’expression in Hei and Suyunuo (Su) after blast fungusinoculation, the expression of SYP121 at 48 h in Hei and8 h in Su with same expression level was normalized as1 and relative expression of these genes was detected. Itwas found that OsSYP121 in Hei was continually in-creased after the blast fungus inoculation until 48 h,while the expression of OsSYP121 was increased to thepeak at 8 h in Su and dropped back to a lower level at24 h (Fig. 1B). The expression ofOsSYP132was inducedat 8 h with lower expression level both in Hei and Su,while the expression of OsSYP111 was rarely detected.A phylogenetic analysis of SYP1 proteins from Arabi-dopsis and rice revealed that all of these proteins wereclustered into three subgroups: SYP11s, SYP12s, andSYP13s (Supplemental Fig. S1A; Uemura et al., 2004).Three genes OsSYP111, OsSYP121, and OsSYP132 dis-tributed in three subgroups were cloned from rice(Supplemental Table S1), and protoplast subcellularlocalization results showed that GFP-OsSYP111 wasmainly localized in the plasma membrane and cyto-plasm, while GFP-OsSYP121 and GFP-OsSYP132 werelocalized at the plasmamembrane comparingwith GFPcontrol that was globally localized in the cytoplasm andthe nucleus (Supplemental Fig. S1, B–I). The syntaxindomain of SYP121 proteins in different organismscontains three a-helix domains: Ha, Hb, andHc at theNterminus (Supplemental Fig. S2).

OsSYP121 Is Associated with Penetration Resistance toRice Blast Fungus

ThreeOsSYP121 overexpression transgenic lines (OE5-Su, OE8-Su, and OE11-Su) and two knockdown lines(RI3-Su andRI7-Su) in Su and twoOsSYP121 knockdowntransgenic lines (RI1-Hei and RI57-Hei) in Hei wereobtained using an Agrobacterium tumefaciens-mediatedmethod (driven by 35S promoter; Supplemental Figs.S3–S5). The OE5-Su, OE8-Su, and OE11-Su transgeniclines showed significantly dwarf phenotype comparedwith wild-type Su, while other agronomic traits showed

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OsSYP121 Mediates Host Resistance to Rice Blast


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no difference (Supplemental Fig. S6). All the knockdowntransgenic lines (RI3-Su, RI7-Su, RI1-Hei, and RI57-Hei)showed the same agronomic traits as their wild-typecontrols (Supplemental Fig. S6). After inoculation withrice blast fungus (strain Hoku1) at the three- to four-leafstage, OE5-Su, OE8-Su, and OE11-Su showed more re-sistance than wild-type Su, with less lesions, whereasRI3-Su, RI7-Su, RI1-Hei, and RI57-Hei were more sus-ceptible to blast than their wild-type controls (Fig. 2, Aand B). The lesion length of all transgenic plants showedno differences (Fig. 2B).

To gain a mechanistic insight into the enhanced blastresistance in the OsSYP121-OE lines and the susceptiblephenotype in the OsSYP121-RI lines, we observed thepenetration process of blast fungus to classify rice defenseresponses through a quantitative microscopic assessmentof the interaction of rice and Magnaporthe oryzae (Nakaoet al., 2011). Inwild-type Su, 48.09%of the penetrated cellswere in type IV stage, 58.08% to 64.1% of penetrated cellsin RI3-Su and RI7-Suwere in the type IV stage, and 54.2%to 76.74% of penetrated cells in OE5-Su, OE8-Su, andOE11-Su were unable to develop into differentiated ap-pressoria (type II; Fig. 2, C andD). In wild-typeHei, morethan 90% of the cells were in the type I stage, and 19.3% to25.9% of the penetrated cells in RI1-Hei and RI57-Heiwere in the type III and type IV stages. Thus, over-expression of OsSYP121 in transgenic plants more fre-quently prevented the penetration of rice blast fungus andthe establishment of infection hyphae.

OsSYP121 Accumulates at Pathogen Penetration Sites

In noninoculated leaf sheaths, either GFP-OsSYP121or GFP-OsSYP132 was exclusively distributed in the

plasma membrane (Fig. 3, A and E), in agreement withthe results in rice protoplasts (Supplemental Fig. S1, Cand E). After inoculation with the compatible strainHoku1 for 30 h, the accumulation of GFP-OsSYP121 ascup-shaped structures was observed beneath the ap-pressoria of M. oryzae (Fig. 3, B–D), while no differencein GFP-OsSYP132 distribution was observed betweennoninoculated and inoculated leaf sheaths (Fig. 3F). Theobserved cup-shaped structures were specificallycaused by the accumulation of GFP-OsSYP121 but notautofluorescence because the fluorescence was not ob-served in nontransgenic Su plants (Fig. 3, F–H).

OsSYP121 Can Interact with OsSNAP32 and Mediates theHost Resistance to Rice Blast Fungus

To explore the ternary SNARE complexes composedof OsSYP121, seven genes OsVAMP711, OsVAMP714,OsVAMP721, OsVAMP722, OsVAMP724, OsVAMP727,and OsSNAP32 were cloned from rice as candidates toidentify any interactions (Fig. 4A). The yeast two-hybrid results showed that OsSYP121 could interactwith OsSNAP32, OsVAMP714, and OsVAMP724(Fig. 4A). The interaction of OsSYP121 and OsSNAP32was also confirmed using a bimolecular fluorescencecomplementation (BiFC) assay in the N. benthamianatransient expression system (Fig. 4B).

In this study, OsSNAP32 RNA interference trans-genic line OsSNAP32RI in Su showed more susceptiblephenotype (Fig. 5), which is consistent with our previousresults that OsSNAP32 RNA interference transgenic linesin Hei decreased resistance to blast (Luo et al., 2016). Inorder to study the genetic interaction between OsSYP121and OsSNAP32, OsSYP121RI transgenic plants in Su

Figure 1. Expression ofOsSYP121 is induced by blast fungus inoculation. A, Tissue-specific expression assays of SYP1s in rice landraceHei. The expressions ofOsSYP111,OsSYP121, andOsSYP132were detected by reverse transcription quantitative PCR (RT-qPCR). RiceActin was used as an internal control. B, The expression patterns of four OsSYP1 genes in landraces Hei and Su inoculated with theM. oryzae strain Hoku1 were investigated by qPCR. The seedlings of Hei and Su were collected after inoculation for 0, 8, 24, 48, and72 h. The expressions ofOsSYP121 at 48 h after inoculation inHei and at 8 h after inoculation in Suwere the same and defined as 1. Theamplification of the rice 18s-rRNA was used as an internal control. Error bars represent SD of three technical replicates.

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were used to cross with OsSNAP32RI transgenicplants in Su to generate OsSYP121RIOsSNAP32RIdouble knockdown transgenic plants. The rice blastdisease assay showed that the susceptibilities of OsSY-P121RI, OsSNAP32RI, and OsSYP121RIOsSNAP32RI

were similar, with more lesions and higher percent-age of type IV infected cells than wild-type Su (Fig. 5).These results indicate OsSYP121 may genetically in-teract with OsSNAP32 and mediate host resistancein rice.

Figure 2. OsSYP121 was associated with penetration resistance to rice blast fungus. A, The rice blast-resistant phenotypes ofOsSYP121-OE lines (OE5, OE8, and OE11), OsSYP121-RI lines (RI3 and RI7), and their wild-type plant Su, OsSYP121-RI lines(RI1 and RI57), and their wild-type plant Hei inoculated by M. oryzae strain Hoku1. The leaves with lesions are shown here.Bar = 1 cm. B, The lesion number per leaf and the lesion length of transgenic lines andwild-type plants. Lesion number and lesionlength were measured at 7 days post inoculation. Each bar indicates the average and SD of at least 30 seedlings. Significantlydifferent values compared with wild-type plants are denoted by double asterisks (**, P, 0.01 by Dunnett’s test). C, Four types ofindividual conidia were classified by microscopy using Uvitex-2B staining: type I,M. oryzae conidium (CO) without germ tubes;type II, differentiated appressorium (APP) formation; type III, establishment of infection hypha (primary hypha [PHY]); type IV,branch formation on infection hypha (secondary hypha [SHY]). Bars = 20mm.D, The percentages of rice-M. oryzae interactions ineach of the four types were detected in transgenic plants. At least 150 penetration sites were observed in each sample. Error barsrepresent SD of three technical replicates.





OsSYP121 Promotes Rice Defense Response toBlast Fungus

To identify the genes probably affected byOsSYP121,we compared the transcriptomes of R1-Hei, R57-Hei,and wild-type Hei through microarray analysis. Com-pared with the Hei background, 51 genes were down-regulated by less than 0.66-fold changes both in RI1-Heiand RI57-Hei, and 89 genes were up-regulated bygreater than 1.5-fold both in RI1-Hei and RI57-Hei(Fig. 6A; Supplemental Tables S2 and S3). To identifygenes related to metabolic reconfiguration in the dif-ferent combinations, the AGRIGO and MapMan toolswere used to conduct the GO enrichment and displaythe significantly regulated pathways. By AGRIGO GOenrichment analysis, only the GO term cellular componentwas identified with default significance levels (FDR ,0.05), and 20% of down-regulated and up-regulated DEGswere associated with cytoplasmic membrane-boundvesicles, membrane-bound vesicles, cytoplasmic vesi-cles, and vesicles (Fig. 6, B and C). ByMapMan analysis,we found that two down-regulated genes were associ-ated with vesicle trafficking (OsSNAP32) and auxintrafficking (OsPILS7a) in the transport overview(Supplemental Fig. S7). One down-regulated gene andthree up-regulated genes were associated with bioticstress, and one up-regulated gene was associated withdevelopment and two genes were associated with abi-otic stress in the cellular response pathway (SupplementalFig. S7B). Twelve down-regulated genes and 26 up-regulated genes were related to pathogen/pest attackpathways (Supplemental Fig. S7C). We further in-vestigated the expression of six down-regulated genesOsSNAP32 (Os02g0437200), OsPILS7a (Os09g38130),

OsMYB20 (Os02g49986), OsWRKY21 (Os01g60640),OsRbohF (Os08g35210), and OsHSP90 (Os09g0482610)as well as OsSGT1 (Os01g0624500) in OsSYP121 over-expression and knockdown expression transgenicplants. These results suggest that OsSYP121 can affectthe expression of OsSNAP32, OsPILS7a, OsMYB20,OsWRKY21, OsRbohF, and OsHSP90 to trigger plantimmunity responses (Fig. 7).

DISCUSSION

Compared with yeast and mammals, which onlyhave two and four syntaxins, there are 18 syntaxins inArabidopsis and 14 syntaxins in rice (Uemura et al.,2004; Lipka et al., 2007; Sanderfoot, 2007; Reichardtet al., 2011). In SYP1 subgroup of syntaxin, there arenine AtSYP1s in Arabidopsis and six OsSYP1s in rice. Incontrast to Arabidopsis, less OsSYP1s were detected inrice and the roles of OsSYP1 proteins in rice host re-sistancewere largely unknown. Subcellular localizationanalysis of OsSYP111, OsSYP121, and OsSYP132 dis-tributed in three OsSYP1 subgroups showed thatOsSYP121 and OsSYP132 were localized to plasmamembrane, while OsSYP111 was localized to plasmamembrane and cytoplasm. The subcellular localizationof OsSYP111, OsSYP121, and OsSYP132 is similar totheir homologs in Arabidopsis (Uemura et al., 2004).The expression of these three genes in response toM. oryzae showed that only OsSYP121 was signifi-cantly induced by M. oryzae. In resistant landraceHei, the expression of OsSYP121 was obviously andstably induced until 48 h upon blast fungus inocu-lation. In susceptible landrace Su, the expression of

Figure 3. OsSYP121 accumulated at rice blastfungus penetration sites. Microscopy analysis ofGFP-OsSYP121 and GFP-OsSYP132 localizationin transgenic plants inoculated with compatibleM. oryzae strain Hoku1 is shown. A, GFP-OsSYP121 was localized at the plasma mem-brane before inoculation. B to D, GFP-OsSYP121accumulated at rice blast fungus penetration sitesin PSYP121::GFP-SYP121 transgenic plants. E and F,GFP-OsSYP132 was localized at the plasmamembrane in PSYP132::GFP-SYP132 transgenicplants before (E) or after inoculation (F). G and H,No autofluorescence was detected in wild-typeplant Su before (G) or after inoculation (H). Ar-rowheadsmark the appressorium ofM. oryzae. BF,Bright field. Bars = 10 mm.


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OsSYP121 was induced at 8 h and then declined.Overexpression of OsSYP121 in Su leads to enhancedresistance and knockdown expression of OsSYP121in Hei and Su showedmore susceptibility. These datasuggest that the expression level of OsSYP121 iscorrelated with the susceptible and resistant pheno-type and OsSYP121 might play an important role inthe rice defense response to M. oryzae attack.Overexpression of OsSYP121 in Su significantly de-

creased the number of lesions but not lesion lengthin transgenic rice, indicating that pathogen penetra-tion was prevented in the early stages. Furthermore,microscopic observation of the blast fungus infec-tion process in the transgenic plants revealed thatpenetration-stage defense was induced in OsSYP121-OE rice, which indicates that OsSYP121 may functionduring M. oryzae penetration into rice epidermal cells.

In Arabidopsis, knockout PEN1 leads to enhancedpenetration of nonhost powdery mildew pathogen butresults in enhanced resistance to adapted powderymildew (Zhang et al., 2007; Kwon et al., 2008b). Si-lencing of MdSYP121 increased resistance to Botryos-phaeria dothidea (He et al., 2018). In our study, it isinteresting that knocking down of OsSYP121 in theresistant landrace Hei and susceptible landrace Su leadsto susceptibility. This indicates that SYP121 may playdifferent roles among phytopathosystems of biotrophs,necrotrophs, and seminecotrophs. While SYP121 playsa positive role in penetration resistance, it also plays anegative role in salicylic acid (SA) signaling that is re-quired for resistance against biotrophic pathogens.However, SA signaling is generally antagonistic tojasmonate and ethylene signals that are required forresistance against necrotrophic pathogens. Powdery

Figure 4. Characterization of OsSYP121 interaction with OsSNAP32 protein. A, Yeast two-hybrid assays indicateinteractions of OsSYP121 with OsSNAP32 and OsVAMP714/724. 3-AT, 3-Amino-1,2,4-triazole; SD(-LW), syntheticdextrose (-Leu,-Trp) medium; SD(-LWAH), synthetic dextrose (-Trp,-Leu,-His,-Ade) medium. B, BiFC assay for OsSYP121and OsSNAP32 interaction in N. benthamiana leaves. The chlorophyll autofluorescence (red), YFP fluorescence(yellow), bright-field, and combined images were taken with a confocal microscope 2 to 4 d after transfection. PM,Plasma membrane; YFPC, Yellow Fluorescent Protein C terminus; YFPN, Yellow Fluorescent Protein N terminus.Bars = 20 mm.





mildew fungi are biotrophs, B. dothidea is a necrotr-oph, whereas M. oryzae is a seminecrotroph. All thesefungi have to penetrate the host cell wall, but post-penetration resistance in the host requires differenthormone signaling. Both jamsonate and ethylenesignals play positive roles in blast-disease resistance.Therefore, SYP121 shows conserved penetration re-sistance but differences in postpenetration resistance.Overexpression of OsSYP121 showed enhanced re-sistance and dwarfism phenotype. It is not clear thatthere is a relationship between resistance and dwarf-ism phenotype and whether OsSYP121 can induce aconstitutive defense response. Loss of PEN genes inArabidopsis affects not only penetration resistanceagainst nonadapted powdery mildew but also hy-persensitive response induced after recognition ofpathogenic effectors (Johansson et al., 2014). In furtherresearch, we would identify the SA concentration andhypersenstive response phenotype to learn moreabout the functions of OsSYP121 in the defenseresponse.

Microscopic observation of GFP-OsSYP121 trans-genic plants clearly showed the accumulation ofOsSYP121 in penetration sites at 24 to 48 h after inoc-ulation, while OsSYP132 remained localized in the

plasma membrane after inoculation. It provides evi-dence that OsSYP121 contributes to penetration resis-tance in rice-M. oryzae interaction. As the first line ofplant defense against fungi, penetration resistance isachieved by localized cell wall appositions or papillaeat fungal penetration sites and functions as physicaland chemical barriers to cell penetration (Aist, 1976;Schmelzer, 2002; Hardham et al., 2007; Yang et al., 2014 ).Penetration resistance of Arabidopsis against powderymildew fungi relies on PEN1 as well as PEN2/PEN3,which can contribute to the synthesis and secretion ofantimicrobial proteins and metabolites (Collins et al.,2003; Lipka et al., 2005; Stein et al., 2006; Bednareket al., 2009). The syntaxin PEN1 in Arabidopsis hasbeen identified as an important molecular componentin nonhost resistance to Bgh (Collins et al., 2003;Thordal-Christensen, 2003; Zhang et al., 2007). Wefound that OsSYP121 plays a critical role in rice pene-tration resistance against M. oryzae and that theOsSYP121 accumulated at rice blast fungi penetrationsites and mediates host resistance in rice. Some cluesshowed that the rice-M. oryzae system is a good systemfor the study of fungus penetration and preinvasionresistance (Robatzek, 2007; Faivre-Rampant et al., 2008;Ribot et al., 2008). Although the relocalization and

Figure 5. OsSYP121 interacts with OsSNAP32 to mediate penetration resistance to rice blast fungus. A, The phenotypes ofOsSYP121RI, OsSNAP32RI, and OsSYP121RIOsSNAP32RI lines and wild-type plant Su infected by M. oryzae. The leaveswith lesions are shown here. Bar = 1 cm. B, The lesion number per leaf of transgenic lines and wild-type plants. Lesionnumbers were measured at 7 days post inocluation . Each bar indicates the average and SD of at least 30 seedlings. Signif-icantly different values compared with wild-type plants are denoted by double asterisks (**, P , 0.01 by Dunnett’s test). C,Histograms show the percentages of rice-M. oryzae interactions in each of the four types represented in transgenic plants. Atleast 150 penetration sites were observed and categorized into the four types. D, The lesion lengths of transgenic lines andwild-type plants were measured. Lesion lengths were measured at 7 days post inoculation . Each bar indicates the averageand SD of at least 30 seedlings.


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concentration of SYP121 proteins at penetrations sitesto powdery mildew in Arabidopsis and barley are wellstudied, the SYP121 proteins appeared to be activelyrecruited to papillae at the penetration sites of powderymildew fungus (Assaad et al., 2004; Bhat et al., 2005).However, the function of OsSYP121 in the rice-blastfungus interaction system is still not well known. It iswell known that there are no papillae in the blast fungipenetration sites. It is worth studying the function andlocation of OsSYP121 in rice, a staple food crop.

In this study, we cloned the candidate Qb-SNAREsand OsVAMPs and used yeast two-hybrid systems tocheck the interactions between OsSYP121 and the pro-tein candidates. It was found the OsSNAP32 andOsVAMP714/724 can interact withOsSYP121, whereasthe AtSYP121 in Arabidopsis can interact with AtS-NAP33 andAtVAMP721/722 (Kwon et al., 2008b). Thissuggests that there may be different elements in theOsSYP121 SNARE complex in rice and Arabidopsis.Sugano et al. (2016) reported that the OsVAMP714-

Figure 6. Gene Ontology (GO) enrich-ment analysis of microarrays showed thatOsSYP121 can trigger vesicle traffickingresponse. A, Venn diagrams of the genesfrom different comparisons. Three bio-logical replicates and two transgeniclines were used for microarray analysis.The genes with 1.5-fold changes com-pared with control were considered asdifferentially expressed genes (DEGs).B, GO enrichment analysis was carriedby AGRIGO. GO terms, such as bio-logical process, molecular function,and cellular component, were identi-fied using AGRIGO (http://bioinfo.cau.edu.cn/agriGO/ndex.php) with defaultsignificance levels (false discovery rate[FDR] , 0.05).




http://bioinfo.cau.edu.cn/agriGO/ndex.php



mediated trafficking pathway plays an important rolein rice blast resistance. Overexpression of OsVAMP714in rice leads to enhanced resistance, while knockdownexpression of OsVAMP714 in rice showed serious sus-ceptibility. In our previous study, OsSNAP32 has beenproven to function in rice blast resistance (Luo et al.,2016). In this study, the working model for OsSYP121could be speculated as follows: OsSYP121 can inter-act with OsSNAP32 and VAMP714/724 to form theSNARE complex; in the blast fungi invasion phase,OsSYP121 can accumulate at fungi penetration sites;the vesicle trafficking- and defense-associated genesOsMYB20, OsWRKY21, OsRbohF, and OsHSP90 couldbe affected by knockdown expression of OsSYP121(Fig. 8).

In summary, our study demonstrates that OsSYP121functions in fungi penetration, andOsSYP121 can interactwith OsSNAP32 and mediate host resistance to rice blast.This indicates OsSYP121 might play an important role inthe rice defense response to M. oryzae attack.

MATERIALS AND METHODS

Plant Materials and Growth

Two rice (Oryza sativa subsp. japonica) landraces, Hei and Su, with resistanceand susceptibility to the blast fungus (Magnaporthe oryzae) strain Hoku1, re-spectively (Wang et al., 2002), and seven OsSYP121 overexpression andknockdown expression transgenic lines (T2) generated including OE5-Su, OE8-Su, OE11-Su, RI3-Su, RI7-Su, RI1-Hei, and RI57-Hei were used in this study.

Rice seeds of two landraces and transgenic lines were sown in plastic pots(diameter = 10 cm and height = 10 cm) containing garden soil (75% ordinary

garden soil and 25%nutrient soil) and grown in a greenhouse (16-h-light/8-h-dark period at 25°C 6 3°C) 3 weeks for the blast fungus inoculation andinduction expression analysis of target genes. Some landrace seedlingswere transplanted in the fields in Nanjing. At the flowering stage of Hei,root, stem, leaf blade, leaf sheath, immature panicle (5–6 cm), and flow-ering panicle samples were collected for tissue-specific expression analysisof target genes.Nicotiana benthamiana plants were grown in the greenhouseat 24°C for 4 to 5 weeks for BiFC transient expression assay (Waadt andKudla, 2008).

Figure 7. Expression patterns of DEGsin microarray- and reported plant im-munity pathway-associated genes intransgenic lines OE8-Su, OE11-Su, RI1-Hei, and RI57-Hei and wild types Suand Hei. The expressions of all genes inthe microarray (transgenic line RI57-Hei and Hei) are also shown. Threebiological replicates were performedboth in microarray and RT-PCR experi-ments. Significantly different expres-sions compared with those of the wild-type controls are denoted by asterisks(*, P , 0.05 and **, P , 0.01 by Dun-nett’s test).

Figure 8. Workingmodel for the roles of OsSYP121 in rice-blast fungusinteraction. In rice cells, OsSYP121 can interact with OsSNAP32 andVAMP714/724 to form the SNARE complex. In the blast fungi invasionphase, OsSYP121 can accumulate at blast fungi penetration sites. Thevesicle trafficking- and defense-associated genes could be affected byknockdown expression of OsSYP121.


Cao et al.



Pathogen Inoculation and Disease Evaluation

The blast strain Hoku1 (provided by Zhiyi Chen, Jiangsu Academy ofAgricultural Science) was used for blast fungus inoculation in this study.Three-week-old rice seedlings were inoculated by spraying with sporesuspension (13 105 spores mL21 in 0.025% [w/v] Tween 20) as previouslyreported (Wang et al., 2002). The inoculated seedlings were kept in a darkincubation room with 100% relative humidity and 26°C for 24 h, thenmoved to the greenhouse for the disease inducing. Seven days after in-oculation, OsSYP121 transgenic plants and OsSYP121RIOsSNAP32RIcrossed plants were assessed for lesion number on each inoculated leaf andlesion length according to the methods of Shi et al. (2010) and Mackill andBonman (1992).

qPCR and RT-qPCR Analysis

Total RNA was extracted from various rice tissues using the Trizol reagent(Invitrogen), according to the manufacturer’s instructions. First-strand cDNAwas synthesized with 2 mg of purified total RNA using the RT-PCR system(Promega). Leaves of Hei and Su were sampled at 0, 8, 24, 48, and 72 h afterinoculation, frozen in liquid nitrogen immediately, and then stored at 280°C.The leaves of transgenic lines were collected and stored at 280°C for RNAextraction and qPCR analysis and RT-qPCR analysis. All the primers are shownin Supplemental Table S4.

qPCR was performed using FastStart Universal SYBR Green Mastermix (ROX;Roche) anda7500FastReal-TimePCRSystem(AppliedBiosystems).Reactionswereset up with the following program: 1 min at 95°C, followed by 40 cycles of 95°C for10 s, 60°C to 62°C for 15 s, and 72°C for 40 s. The relative expression level of eachgenewas calculated using the 2–△△CTmethod (Livak and Schmittgen, 2001). Threebiological replicates were performed for each qPCR. The expression level of 18S-rRNAwasused as an internal control (Jain et al., 2006). RT-qPCRwas set upwith thefollowing program: 1min at 95°C, followed by 27 to 36 cycles of 95°C for 30 s, 58°Cto 62°C for 30 s, and 72°C for 45 s. The expression level ofActin gene in ricewas usedas an internal control (Martin, 1999).

Bioinformatics Analysis of OsSYP1s

The phylogenetic analysis of SYP1s in rice and Arabidopsis (Arabidopsisthaliana) was performed using MEGA6 software (Tamura et al., 2013). Full-length amino acid sequences of 15 SYP1 proteins, AtSYP111, AtSYP112,AtSYP121, AtSYP122, AtSYP123, AtSYP124, AtSYP125, AtSYP131, AtSYP132,OsSYP111, OsSYP121, OsSYP124, OsSYP125, OsSYP131, and OsSYP132, wereused to generate a bootstrap neighbor-joining phylogenetic tree. Bootstrapprobabilities were obtained from 1,000 replicates. Multiple sequence alignmentof SYP1 proteins was carried out by ClustalX 1.8 (Thompson et al., 1997), andthe results were edited by GENEDOC (https://www.softpedia.com/get/Science-CAD/GeneDoc.shtml). Pfam (http://pfam.xfam.org/) and TMHMM(http://www.cbs.dtu.dk/services/TMHMM/) were used to annotate theprotein domain of SYP1 proteins.

Subcellular Localization of OsSYP1s in Protoplasts

Full-length cDNA fragments of OsSYP111, OsSYP121, and OsSYP132 wereamplified from Hei cDNA and cloned into the pGEM-T vector (Takara). Toconstruct the transient expression plasmids, the full-length cDNA fragmentswere inserted into the pUC18 vector, N terminal of the fragments framedwith GFP.

Protoplast extraction of young rice seedlings (landrace Hei) and plasmidtransient transformationwere performed as described (Chen et al., 2006). A totalof 10 mg of plasmid DNA for each construct was mixed with 200 mL of sus-pended protoplasts (13 106 cells mL21) and then incubated in the dark at 28°C.The transformed cells were observed by a Zeiss 710 laser confocal microscopeafter 12 and 16 h.

Generation and Identification of Transgenic Plants

Full-length OsSYP121 was inserted into the pCAMBIA1300S vector togenerate the overexpression transgenic vector pCAMBIA1300S-OsSYP121. A246-bp OsSYP121-specific fragment was used to generate the knockdown ex-pression transgenic vector pTCK303-OsSYP121, as described by Wang et al.(2004). The fragments with native promoter and coding regions of OsSYP121

or OsSYP132 were inserted into pCAMBIA1304 and framed with GFP to gen-erate the final vectors POsSYP121::GFP-OsSYP121 or POsSYP132::GFP-OsSYP132(Supplemental Fig. S8). These vectors were transformed into rice plants usingAgrobacterium tumefaciens-mediated methods (Toki et al., 2006).

Southern blotting was conducted to identify the transgenic plants using DIGHigh Prime DNA Labeling and Detection Starter Kit I (Version 10.0; Roche)according to the manufacturer’s instructions. Twenty micrograms of EcoRI-digested genomic DNA was hybridized to the hygromycin phosphotransferase-specific fragment probe.

Microscopy Observation of Inoculated Leaves

As previously described (Chen et al., 2010), the inoculated leaves weresampled at 24 h after inoculation and submerged in lactophenol:ethanol(1:2, v/v) solution for 1 to 2 d. The samples were treated with Uvitex-2Bstaining. According to Nakao et al. (2011) methods, the fungal growth wasobserved under a fluorescence microscope (Nikon Eclipse 80i). Four types offungal growth stage: type I, M. oryzae conidium without germ tubes; type II,differentiated appressorium formation; type III, establishment of infection hy-pha (primary hypha); type IV, branch formation on infection hypha (secondaryhypha) were identified, and the percentage of each type in the total observedcells was calculated. At least nine leaves from three plants of each transgenicline or landrace were sampled.

Localization of OsSYP121-GFP and OsSYP132-GFPin Cells

To identify the localization of OsSYP121-GFP and OsSYP132-GFP intransgenic plants, the sixth leaf sheaths were placed in blast fungus conidialsuspension (1 3 105 conidia mL21) and incubated for 30 h at 25°C in the dark,then the epidermal cells of leaf sheaths were sampled for microscopy as de-scribed by Tanabe et al. (2009).

Yeast Two-Hybrid Assay

Based on a report in Arabidopsis that the AtSYP121 interacting proteins areAtSNAP33 andAtVAMP721/722, seven SNAREmember homologous proteinsin rice were selected for yeast two-hybrid assay. Full-length cDNAofOsSYP121was inserted into the pBT3-N vector (bait), and full-length cDNAs of OsS-NAP32, OsVAMP711, OsVAMP714, OsVAMP721, OsVAMP722, OsVAMP724,and OsVAMP727 were inserted into pPR3-N (prey; Dualsystems Biotech). Theconstructs were transformed into yeast strain NMY51 according to the protocolfor the DUALMembrane Kit 1. The positive clones on synthetic dextrose (SD; -Leu,-Trp) medium were transferred to SD (-Trp,-Leu,-His,-Ade) medium con-taining 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside acid (20 mg mL21)and 3-amino-1,2,4-triazole (5 mM) to identify protein-protein interactions. Theinteraction between Cub-OsSYP121 and NubI served as a positive control,whereas coexpression of Cub-OsSYP121 and NubG served as a negative con-trol. Yeast NMY51 cells harbored the C-terminal half of ubiquitin (Cub) and anartificial transcription factor (LexA-VP16) fusion construct and the mutatedN-terminal half of ubiquitin (NubG) fusion constructs. The yeast cells werespotted on SD (-Leu,-Trp) medium (selection for positive transformants), and10-fold dilutions of the yeast cells were spotted on SD (-Trp,-Leu,-His,-Ade)medium and 5 mM 3-amino-1,2,4-triazole (selection for interaction) and incu-bated for 5 d at 30°C.

BiFC Assay

A previously described protocol (Waadt and Kudla, 2008) was followed toobserve BiFC signals with some modification. The full-length cDNA ofOsSYP121was cloned into the pSPYNE173 vector to generate OsSYP121:YFPN,and OsSNAP32 was inserted into the pSPYCE vector to generate OsSNAP32:YFPC. The constructs were transformed into the A. tumefaciens strain EHA105.Overnight cell cultures were collected and resuspended in 1 mL of AS medium(1 mL of 1 M MES-KOH, pH 5.6, 333 mL of 3 M MgCl2, and 100 mL of 150 mM

acetosyringone) to OD600 at 0.7 to 0.8. The working suspensions were preparedby mixing at a 1:1:1 ratio with three A. tumefaciens strains carrying the YFPNfusion construct, the YFPC fusion construct, and the gene-silencing inhibitorp19 strain, respectively. The mixture was standing for 2 to 4 h. The A. tumefa-ciens suspensions were then coinfiltrated onto the abaxial surface of 4- to 5-week-old N. benthamiana plant leaves. Fluorescence of the epidermal cell layer





https://www.softpedia.com/get/Science-CAD/GeneDoc.shtml

https://www.softpedia.com/get/Science-CAD/GeneDoc.shtml

http://pfam.xfam.org/

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of the lower leaf surface was examined at 2 to 4 d after infiltration. Images werecaptured with a Zeiss 710 laser scanning confocal microscope, with excitationwavelengths of 488 and 496 nm and an emission wavelength between 520 and535 nm for YFP signals.

Microarray and Pathway Analyses

Three-week-old seedlings of OsSYP121-RI lines R1 and R57 and Hei weresampled, and three biological replicates were used for the microarray assay.RNA isolation, purification, and hybridization of Affymetrix microarrays wereconducted by the Biotechnology Group (Biotechnology Corporation). We usedthe ordinary Student’s t test (P , 0.05) to identify significantly differentiallyexpressed genes. Probe sets showing more than 1.5-fold changes for up-regulation and less than 0.66-fold changes for down-regulation in expressionwere considered to be DEGs. Functional enrichment analysis of DEGs using theGO domains molecular function, biological process, and cellular componentwas performed by AGRIGO (http://bioinfo.cau.edu.cn/agriGO/ndex.php)with default significance levels (FDR , 0.05). The MapMan tool (Thimm et al.,2004) was employed to analyze the metabolic and signaling changes in themicroarray data based on the expression value of each DEG. A metabolicpathway overview was produced by loading the DEGs with their expressionvalues into the locally installed MapMan program and shown using colorintensity.

Accession Numbers

Sequence data from this article can be found in the GenBank data librariesunder the following accession numbers: OsSY121 (BAS86738.1), OsSYP132(BAT00191.1), OsSYP111 (BAS86268.1), OsSYP124 (BAD32916.1), OsSYP125(BAD25019.1), OsSYP131 (BAS96357.1), AtSYP111 (AEE28306.1), AtSYP112(AEC06747.1), AtSYP121 (AAF23198.1), AtSYP122 (AEE78943.1), AtSYP123(AEE82307.1), AtSYP124 (AEE33817.1), AtSYP125 (AEE28704.1), AtSYP131(AEE73995.2), AtSYP132 (AED91242.1), NtSYP121 (AAD11808.1), HvSYP121(AAP75621.1), ZmSYP121 (ACG40338.1), OsVAMP727 (BAD13129.1), OsVAMP724(BAD30660.1), OsVAMP722 (BAD30158.1), OsVAMP721 (BAS86911.1),OsVAMP714 (BAT09923.1), and OsVAMP711 (BAA95814.1).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Phylogenetic analysis of SYP1 proteins and sub-cellular localization of OsSYP111, OsSYP121, and OsSYP132.

Supplemental Figure S2. Multiple sequence alignment of SYP121 proteinsin different organisms.

Supplemental Figure S3. Identification of OsSYP121 overexpression trans-genic plants in Su.

Supplemental Figure S4. Identification of OsSYP121 knockdown expres-sion transgenic plants in Su.

Supplemental Figure S5. Identification of OsSYP121 knockdown expres-sion transgenic plants in Hei.

Supplemental Figure S6. Agronomic traits of OsSYP121-OE andOsSYP121-RI transgenic plants.

Supplemental Figure S7. Microarray analysis showed that OsSYP121 cantrigger the plant immunity response.

Supplemental Figure S8. The transgenic vector constructs POsSYP121::GFP-OsSYP121 and POsSYP132::GFP-OsSYP132.

Supplemental Table S1. Sequence characteristics of OsSYP111, OsSYP121,and OsSYP132.

Supplemental Table S2. Up-regulated genes in RI57-Hei and RI1-Hei com-pared with wild-type Hei by microarray analysis.

Supplemental Table S3. Down-regulated genes in RI57-Hei and RI1-Heicompared with wild-type Hei by microarray analysis.

Supplemental Table S4. Primers used in this study.

Received August 15, 2018; accepted December 10, 2018; published January 7,2019.

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OsSYP121 Accumulates at Fungal Penetration Sites and ... · OsSYP121 Accumulates at Fungal...

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