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. 2019 Jan 7;179(4):1330–1342. doi: 10.1104/pp.18.01013

OsSYP121 Accumulates at Fungal Penetration Sites and Mediates Host Resistance to Rice Blast1

Wen-Lei Cao 1,2,3, Yao Yu 1,2, Meng-Ya Li 1, Jia Luo 1,4, Rui-Sen Wang 1, Hai-Juan Tang 1, Ji Huang 1, Jian-Fei Wang 1, Hong-Sheng Zhang 1,5, Yong-Mei Bao 1,5,6
PMCID: PMC6446747  PMID: 30617050

OsSYP121 accumulates at fungal penetration sites and plays an important role in rice blast resistance.

Abstract

Magnaporthe oryzae is a fungal pathogen that causes rice (Oryza sativa) blast. SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are key components in vesicle trafficking in eukaryotic cells and are known to contribute to fungal pathogen resistance. Syntaxin of Plants121 (SYP121), a Qa-SNARE, has been reported to function in nonhost resistance in Arabidopsis (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 resistance to rice blast. OsSYP121 is plasma membrane localized and its expression was obviously induced by the rice blast in both the blast-resistant rice landrace Heikezijing and the blast-susceptible landrace Suyunuo (Su). Overexpression of OsSYP121 in Su resulted 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) and Vesicle-associated membrane protein714/724. The interaction between OsSYP121 and OsSNAP32 may contribute to host resistance to rice blast. Our study reveals that OsSYP121 plays an important role in rice blast resistance as it is a key component in vesicle trafficking.

Vesicle trafficking plays crucial roles in plant development and immune responses ( Somerville et al., 2004; Lipka et al., 2007; Kwon et al., 2008a; Van Damme and Geelen, 2008; Meyer et al., 2009). SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are key components in vesicle trafficking in eukaryotic cells (Heese et al., 2001; Wick et al., 2003) and play a universal role in diverse biological processes including cytokinesis, defense response, pollen tube and root hair tip growth, root formation, and hormone response in plants (Dacks and Doolittle, 2002; Lipka et al., 2007; Enami et al., 2009). Four different types of SNAREs form a SNARE complex through their R-, Qa-, Qb-, and Qc-SNARE domains to determine the specificity of intracellular fusion (Antonin et al., 2000; Fukuda et al., 2000). Syntaxins (Qa-SNAREs) and interacting SNARE proteins (R-, Qb-, and Qc-SNAREs) contribute to the fusion of intracellular transport vesicles with acceptor membranes in diverse trafficking pathways (Pajonk et al., 2008; Reichardt et al., 2011).The SYP1 (Syntaxin of Plants1) subfamily is a plant-specific syntaxin family that belongs to the Qa-SNARE family. Nine SYP1 genes, SYP111, SYP112, SYP121, SYP122, SYP123, SYP124, SYP125, SYP131, and SYP132, are found in Arabidopsis (Arabidopsis thaliana; divided into three groups) and all localized on the plasma membrane (Uemura et al., 2004). The expression of SYP1s is tissue specific, only SYP132 ubiquitously expressed in various tissues throughout plant development (Enami et al., 2009). SYP111/KNOLLE is well known as a cytokinesis-specific syntaxin that is specifically expressed during mitosis and localizes to the forming cell plate (Lukowitz et al., 1996; Heese et al., 2001). SYP112 can functionally replace the cell cycle-regulated KNOLLE protein (Sanderfoot et al., 2000; Müller et al., 2003). As a calcium-dependent phosphorylation protein in Arabidopsis, SYP122 has redundant functions with its closest homolog 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 in root hairs and localizes to the tip region of root hairs, can function with SYP132 to mediate tip-focused membrane trafficking for root hair tip growth (Ichikawa et al., 2014). SYP124, SYP125, and SYP131 are pollen-specific syntaxins involved in pollen tube growth (Kato et al., 2010; Silva et al., 2010; Ul-Rehman et al., 2011). NbSYP132 in Nicotiana benthamiana acts as the cognate target-SNARE for the exocytosis of vesicles containing PR proteins 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; Kwon et al., 2008b). SYP121/SYR1 was originally identified in tabacco (Nicotiana tabacum), and it can prevent the potassium and chloride ion channel response to abscisic acid in stomatal guard cells (Leyman et al., 1999, 2000). SYP121/PEN1 in Arabidopsis was also shown to directly interact with the potassium and chloride ion channel through an FxRF motif to facilitate solute uptake 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 penetration resistance in Arabidopsis (Collins et al., 2003; Kwon et al., 2008a, 2008b, 2008c). SYP121/ROR2 in barley (Hordeum vulgare) was localized at the plasma membrane in nonpathogen challenged epidermal cells but accumulated focally near the papilla structure below 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 vesicle fusion events in an extracellular defense pathway by specifically forming a ternary SNARE complex with SNAP33 (Synaptosome-associated protein of 33 kD) and the VAMP721/722 (Vesicle-associated membrane protein721/722) to deliver defense components to the space between the plasma membrane and the plant cell wall 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 them has been well characterized. In our previous work, we cloned five SNARE genes, including OsSNAP32 (Bao et 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 gene OsSNAP32 was induced by H2O2, PEG6000, low temperature, and rice blast fungus inoculation treatments in rice seedlings (Bao et al., 2008b). The overexpression of OsSNAP32 and OsSYP71 in rice showed enhanced tolerance to oxidative stress and rice blast (Bao et al., 2012; Luo et al., 2016).

In this article, we isolated and analyzed the expression of OsSYP111, OsSYP121, and OsSYP132 distributed in three SYP1 subgroups from rice, and only the expression of OsSYP121 was induced by the blast fungus. To elucidate the function of OsSYP121 in rice resistance to blast, we overexpressed and knocked down the expression of OsSYP121 in transgenic rice and observed the location of PSYP121:GFP-SYP121 in transgenic rice inoculated by blast fungus by microscopy.

RESULTS

Expression of OsSYP121 Is Induced by the Blast Fungus

The expression profiles of OsSYP111, OsSYP121, and OsSYP132 in rice landrace Heikezijing (Hei) were detected in various tissues. OsSYP121 and OsSYP132 were predominantly detected in leaf blades and leaf sheaths (Fig. 1A). In order to determine three genes’ expression in Hei and Suyunuo (Su) after blast fungus inoculation, the expression of SYP121 at 48 h in Hei and 8 h in Su with same expression level was normalized as 1 and relative expression of these genes was detected. It was found that OsSYP121 in Hei was continually increased after the blast fungus inoculation until 48 h, while the expression of OsSYP121 was increased to the peak at 8 h in Su and dropped back to a lower level at 24 h (Fig. 1B). The expression of OsSYP132 was induced at 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 Arabidopsis and rice revealed that all of these proteins were clustered into three subgroups: SYP11s, SYP12s, and SYP13s (Supplemental Fig. S1A; Uemura et al., 2004). Three genes OsSYP111, OsSYP121, and OsSYP132 distributed in three subgroups were cloned from rice (Supplemental Table S1), and protoplast subcellular localization results showed that GFP-OsSYP111 was mainly localized in the plasma membrane and cytoplasm, while GFP-OsSYP121 and GFP-OsSYP132 were localized at the plasma membrane comparing with GFP control that was globally localized in the cytoplasm and the nucleus (Supplemental Fig. S1, B–I). The syntaxin domain of SYP121 proteins in different organisms contains three α-helix domains: Ha, Hb, and Hc at the N terminus (Supplemental Fig. S2).

Figure 1.

Figure 1.

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Expression of OsSYP121 is induced by blast fungus inoculation. A, Tissue-specific expression assays of SYP1s in rice landrace Hei. The expressions of OsSYP111, OsSYP121, and OsSYP132 were detected by reverse transcription quantitative PCR (RT-qPCR). Rice Actin was used as an internal control. B, The expression patterns of four OsSYP1 genes in landraces Hei and Su inoculated with the M. oryzae strain Hoku1 were investigated by qPCR. The seedlings of Hei and Su were collected after inoculation for 0, 8, 24, 48, and 72 h. The expressions of OsSYP121 at 48 h after inoculation in Hei and at 8 h after inoculation in Su were the same and defined as 1. The amplification of the rice 18s-rRNA was used as an internal control. Error bars represent sd of three technical replicates.

OsSYP121 Is Associated with Penetration Resistance to Rice Blast Fungus

Three OsSYP121 overexpression transgenic lines (OE5-Su, OE8-Su, and OE11-Su) and two knockdown lines (RI3-Su and RI7-Su) in Su and two OsSYP121 knockdown transgenic lines (RI1-Hei and RI57-Hei) in Hei were obtained using an Agrobacterium tumefaciens-mediated method (driven by 35S promoter; Supplemental Figs. S3–S5). The OE5-Su, OE8-Su, and OE11-Su transgenic lines showed significantly dwarf phenotype compared with wild-type Su, while other agronomic traits showed no difference (Supplemental Fig. S6). All the knockdown transgenic lines (RI3-Su, RI7-Su, RI1-Hei, and RI57-Hei) showed the same agronomic traits as their wild-type controls (Supplemental Fig. S6). After inoculation with rice blast fungus (strain Hoku1) at the three- to four-leaf stage, OE5-Su, OE8-Su, and OE11-Su showed more resistance than wild-type Su, with less lesions, whereas RI3-Su, RI7-Su, RI1-Hei, and RI57-Hei were more susceptible to blast than their wild-type controls (Fig. 2, A and B). The lesion length of all transgenic plants showed no differences (Fig. 2B).

Figure 2.

Figure 2.

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OsSYP121 was associated with penetration resistance to rice blast fungus. A, The rice blast-resistant phenotypes of OsSYP121-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 and wild-type plants. Lesion number and lesion length were measured at 7 days post inoculation. Each bar indicates the average and sd of at least 30 seedlings. Significantly different values compared with wild-type plants are denoted by double asterisks (**, P < 0.01 by Dunnett’s test). C, Four types of individual 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 = 20 μm. D, The percentages of rice-M. oryzae interactions in each of the four types were detected in transgenic plants. At least 150 penetration sites were observed in each sample. Error bars represent sd of three technical replicates.

To gain a mechanistic insight into the enhanced blast resistance in the OsSYP121-OE lines and the susceptible phenotype in the OsSYP121-RI lines, we observed the penetration process of blast fungus to classify rice defense responses through a quantitative microscopic assessment of the interaction of rice and Magnaporthe oryzae (Nakao et al., 2011). In wild-type Su, 48.09% of the penetrated cells were in type IV stage, 58.08% to 64.1% of penetrated cells in RI3-Su and RI7-Su were in the type IV stage, and 54.2% to 76.74% of penetrated cells in OE5-Su, OE8-Su, and OE11-Su were unable to develop into differentiated appressoria (type II; Fig. 2, C and D). In wild-type Hei, more than 90% of the cells were in the type I stage, and 19.3% to 25.9% of the penetrated cells in RI1-Hei and RI57-Hei were in the type III and type IV stages. Thus, overexpression of OsSYP121 in transgenic plants more frequently prevented the penetration of rice blast fungus and the establishment of infection hyphae.

OsSYP121 Accumulates at Pathogen Penetration Sites

In noninoculated leaf sheaths, either GFP-OsSYP121 or GFP-OsSYP132 was exclusively distributed in the plasma membrane (Fig. 3, A and E), in agreement with the results in rice protoplasts (Supplemental Fig. S1, C and E). After inoculation with the compatible strain Hoku1 for 30 h, the accumulation of GFP-OsSYP121 as cup-shaped structures was observed beneath the appressoria of M. oryzae (Fig. 3, B–D), while no difference in GFP-OsSYP132 distribution was observed between noninoculated and inoculated leaf sheaths (Fig. 3F). The observed cup-shaped structures were specifically caused by the accumulation of GFP-OsSYP121 but not autofluorescence because the fluorescence was not observed in nontransgenic Su plants (Fig. 3, F–H).

Figure 3.

Figure 3.

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OsSYP121 accumulated at rice blast fungus penetration sites. Microscopy analysis of GFP-OsSYP121 and GFP-OsSYP132 localization in transgenic plants inoculated with compatible M. oryzae strain Hoku1 is shown. A, GFP-OsSYP121 was localized at the plasma membrane before inoculation. B to D, GFP-OsSYP121 accumulated at rice blast fungus penetration sites in PSYP121::GFP-SYP121 transgenic plants. E and F, GFP-OsSYP132 was localized at the plasma membrane in PSYP132::GFP-SYP132 transgenic plants before (E) or after inoculation (F). G and H, No autofluorescence was detected in wild-type plant Su before (G) or after inoculation (H). Arrowheads mark the appressorium of M. oryzae. BF, Bright field. Bars = 10 μm.

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

To explore the ternary SNARE complexes composed of OsSYP121, seven genes OsVAMP711, OsVAMP714, OsVAMP721, OsVAMP722, OsVAMP724, OsVAMP727, and OsSNAP32 were cloned from rice as candidates to identify any interactions (Fig. 4A). The yeast two-hybrid results showed that OsSYP121 could interact with OsSNAP32, OsVAMP714, and OsVAMP724 (Fig. 4A). The interaction of OsSYP121 and OsSNAP32 was also confirmed using a bimolecular fluorescence complementation (BiFC) assay in the N. benthamiana transient expression system (Fig. 4B).

Figure 4.

Figure 4.

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Characterization of OsSYP121 interaction with OsSNAP32 protein. A, Yeast two-hybrid assays indicate interactions of OsSYP121 with OsSNAP32 and OsVAMP714/724. 3-AT, 3-Amino-1,2,4-triazole; SD(-LW), synthetic dextrose (-Leu,-Trp) medium; SD(-LWAH), synthetic dextrose (-Trp,-Leu,-His,-Ade) medium. B, BiFC assay for OsSYP121 and 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 μm.

In this study, OsSNAP32 RNA interference transgenic line OsSNAP32RI in Su showed more susceptible phenotype (Fig. 5), which is consistent with our previous results that OsSNAP32 RNA interference transgenic lines in Hei decreased resistance to blast (Luo et al., 2016). In order to study the genetic interaction between OsSYP121 and OsSNAP32, OsSYP121RI transgenic plants in Su were used to cross with OsSNAP32RI transgenic plants in Su to generate OsSYP121RIOsSNAP32RI double knockdown transgenic plants. The rice blast disease assay showed that the susceptibilities of OsSYP121RI, OsSNAP32RI, and OsSYP121RIOsSNAP32RI were similar, with more lesions and higher percentage of type IV infected cells than wild-type Su (Fig. 5). These results indicate OsSYP121 may genetically interact with OsSNAP32 and mediate host resistance in rice.

Figure 5.

Figure 5.

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OsSYP121 interacts with OsSNAP32 to mediate penetration resistance to rice blast fungus. A, The phenotypes of OsSYP121RI, OsSNAP32RI, and OsSYP121RIOsSNAP32RI lines and wild-type plant Su infected by M. oryzae. The leaves with lesions are shown here. Bar = 1 cm. B, The lesion number per leaf of transgenic lines and wild-type plants. Lesion numbers were measured at 7 days post inocluation . Each bar indicates the average and sd of at least 30 seedlings. Significantly 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. At least 150 penetration sites were observed and categorized into the four types. D, The lesion lengths of transgenic lines and wild-type plants were measured. Lesion lengths were measured at 7 days post inoculation . Each bar indicates the average and sd of at least 30 seedlings.

OsSYP121 Promotes Rice Defense Response to Blast Fungus

To identify the genes probably affected by OsSYP121, we compared the transcriptomes of R1-Hei, R57-Hei, and wild-type Hei through microarray analysis. Compared with the Hei background, 51 genes were down-regulated by less than 0.66-fold changes both in RI1-Hei and RI57-Hei, and 89 genes were up-regulated by greater than 1.5-fold both in RI1-Hei and RI57-Hei (Fig. 6A; Supplemental Tables S2 and S3). To identify genes related to metabolic reconfiguration in the different combinations, the AGRIGO and MapMan tools were used to conduct the GO enrichment and display the significantly regulated pathways. By AGRIGO GO enrichment analysis, only the GO term cellular component was identified with default significance levels (FDR < 0.05), and 20% of down-regulated and up-regulated DEGs were associated with cytoplasmic membrane-bound vesicles, membrane-bound vesicles, cytoplasmic vesicles, and vesicles (Fig. 6, B and C). By MapMan analysis, we found that two down-regulated genes were associated with vesicle trafficking (OsSNAP32) and auxin trafficking (OsPILS7a) in the transport overview (Supplemental Fig. S7). One down-regulated gene and three up-regulated genes were associated with biotic stress, and one up-regulated gene was associated with development and two genes were associated with abiotic stress in the cellular response pathway (Supplemental Fig. S7B). Twelve down-regulated genes and 26 up-regulated genes were related to pathogen/pest attack pathways (Supplemental Fig. S7C). We further investigated the expression of six down-regulated genes OsSNAP32 (Os02g0437200), OsPILS7a (Os09g38130), OsMYB20 (Os02g49986), OsWRKY21 (Os01g60640), OsRbohF (Os08g35210), and OsHSP90 (Os09g0482610) as well as OsSGT1 (Os01g0624500) in OsSYP121 overexpression and knockdown expression transgenic plants. These results suggest that OsSYP121 can affect the expression of OsSNAP32, OsPILS7a, OsMYB20, OsWRKY21, OsRbohF, and OsHSP90 to trigger plant immunity responses (Fig. 7).

Figure 6.

Figure 6.

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Gene Ontology (GO) enrichment analysis of microarrays showed that OsSYP121 can trigger vesicle trafficking response. A, Venn diagrams of the genes from different comparisons. Three biological replicates and two transgenic lines were used for microarray analysis. The genes with 1.5-fold changes compared with control were considered as differentially expressed genes (DEGs). B, GO enrichment analysis was carried by AGRIGO. GO terms, such as biological process, molecular function, and cellular component, were identified using AGRIGO (http://bioinfo.cau.edu.cn/agriGO/ndex.php) with default significance levels (false discovery rate [FDR] < 0.05).

Figure 7.

Figure 7.

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Expression patterns of DEGs in microarray- and reported plant immunity pathway-associated genes in transgenic lines OE8-Su, OE11-Su, RI1-Hei, and RI57-Hei and wild types Su and Hei. The expressions of all genes in the microarray (transgenic line RI57-Hei and Hei) are also shown. Three biological replicates were performed both in microarray and RT-PCR experiments. Significantly different expressions compared with those of the wild-type controls are denoted by asterisks (*, P < 0.05 and **, P < 0.01 by Dunnett’s test).

DISCUSSION

Compared with yeast and mammals, which only have two and four syntaxins, there are 18 syntaxins in Arabidopsis and 14 syntaxins in rice (Uemura et al., 2004; Lipka et al., 2007; Sanderfoot, 2007; Reichardt et al., 2011). In SYP1 subgroup of syntaxin, there are nine AtSYP1s in Arabidopsis and six OsSYP1s in rice. In contrast to Arabidopsis, less OsSYP1s were detected in rice and the roles of OsSYP1 proteins in rice host resistance were largely unknown. Subcellular localization analysis of OsSYP111, OsSYP121, and OsSYP132 distributed in three OsSYP1 subgroups showed that OsSYP121 and OsSYP132 were localized to plasma membrane, while OsSYP111 was localized to plasma membrane and cytoplasm. The subcellular localization of OsSYP111, OsSYP121, and OsSYP132 is similar to their homologs in Arabidopsis (Uemura et al., 2004). The expression of these three genes in response to M. oryzae showed that only OsSYP121 was significantly induced by M. oryzae. In resistant landrace Hei, the expression of OsSYP121 was obviously and stably induced until 48 h upon blast fungus inoculation. In susceptible landrace Su, the expression of OsSYP121 was induced at 8 h and then declined. Overexpression of OsSYP121 in Su leads to enhanced resistance and knockdown expression of OsSYP121 in Hei and Su showed more susceptibility. These data suggest that the expression level of OsSYP121 is correlated with the susceptible and resistant phenotype and OsSYP121 might play an important role in the rice defense response to M. oryzae attack.

Overexpression of OsSYP121 in Su significantly decreased the number of lesions but not lesion length in transgenic rice, indicating that pathogen penetration was prevented in the early stages. Furthermore, microscopic observation of the blast fungus infection process in the transgenic plants revealed that penetration-stage defense was induced in OsSYP121-OE rice, which indicates that OsSYP121 may function during M. oryzae penetration into rice epidermal cells. In Arabidopsis, knockout PEN1 leads to enhanced penetration of nonhost powdery mildew pathogen but results in enhanced resistance to adapted powdery mildew (Zhang et al., 2007; Kwon et al., 2008b). Silencing of MdSYP121 increased resistance to Botryosphaeria dothidea (He et al., 2018). In our study, it is interesting that knocking down of OsSYP121 in the resistant landrace Hei and susceptible landrace Su leads to susceptibility. This indicates that SYP121 may play different roles among phytopathosystems of biotrophs, necrotrophs, and seminecotrophs. While SYP121 plays a positive role in penetration resistance, it also plays a negative role in salicylic acid (SA) signaling that is required for resistance against biotrophic pathogens. However, SA signaling is generally antagonistic to jasmonate and ethylene signals that are required for resistance against necrotrophic pathogens. Powdery mildew fungi are biotrophs, B. dothidea is a necrotroph, whereas M. oryzae is a seminecrotroph. All these fungi have to penetrate the host cell wall, but postpenetration resistance in the host requires different hormone signaling. Both jamsonate and ethylene signals play positive roles in blast-disease resistance. Therefore, SYP121 shows conserved penetration resistance but differences in postpenetration resistance. Overexpression of OsSYP121 showed enhanced resistance and dwarfism phenotype. It is not clear that there is a relationship between resistance and dwarfism phenotype and whether OsSYP121 can induce a constitutive defense response. Loss of PEN genes in Arabidopsis affects not only penetration resistance against nonadapted powdery mildew but also hypersensitive response induced after recognition of pathogenic effectors (Johansson et al., 2014). In further research, we would identify the SA concentration and hypersenstive response phenotype to learn more about the functions of OsSYP121 in the defense response.

Microscopic observation of GFP-OsSYP121 transgenic plants clearly showed the accumulation of OsSYP121 in penetration sites at 24 to 48 h after inoculation, while OsSYP132 remained localized in the plasma membrane after inoculation. It provides evidence that OsSYP121 contributes to penetration resistance in rice-M. oryzae interaction. As the first line of plant defense against fungi, penetration resistance is achieved by localized cell wall appositions or papillae at fungal penetration sites and functions as physical and chemical barriers to cell penetration (Aist, 1976; Schmelzer, 2002; Hardham et al., 2007; Yang et al., 2014 ). Penetration resistance of Arabidopsis against powdery mildew fungi relies on PEN1 as well as PEN2/PEN3, which can contribute to the synthesis and secretion of antimicrobial proteins and metabolites (Collins et al., 2003; Lipka et al., 2005; Stein et al., 2006; Bednarek et al., 2009). The syntaxin PEN1 in Arabidopsis has been identified as an important molecular component in nonhost resistance to Bgh (Collins et al., 2003; Thordal-Christensen, 2003; Zhang et al., 2007). We found that OsSYP121 plays a critical role in rice penetration resistance against M. oryzae and that the OsSYP121 accumulated at rice blast fungi penetration sites and mediates host resistance in rice. Some clues showed that the rice-M. oryzae system is a good system for the study of fungus penetration and preinvasion resistance (Robatzek, 2007; Faivre-Rampant et al., 2008; Ribot et al., 2008). Although the relocalization and concentration of SYP121 proteins at penetrations sites to powdery mildew in Arabidopsis and barley are well studied, the SYP121 proteins appeared to be actively recruited to papillae at the penetration sites of powdery mildew fungus (Assaad et al., 2004; Bhat et al., 2005). However, the function of OsSYP121 in the rice-blast fungus interaction system is still not well known. It is well known that there are no papillae in the blast fungi penetration sites. It is worth studying the function and location of OsSYP121 in rice, a staple food crop.

In this study, we cloned the candidate Qb-SNAREs and OsVAMPs and used yeast two-hybrid systems to check the interactions between OsSYP121 and the protein candidates. It was found the OsSNAP32 and OsVAMP714/724 can interact with OsSYP121, whereas the AtSYP121 in Arabidopsis can interact with AtSNAP33 and AtVAMP721/722 (Kwon et al., 2008b). This suggests that there may be different elements in the OsSYP121 SNARE complex in rice and Arabidopsis. Sugano et al. (2016) reported that the OsVAMP714-mediated trafficking pathway plays an important role in rice blast resistance. Overexpression of OsVAMP714 in rice leads to enhanced resistance, while knockdown expression of OsVAMP714 in rice showed serious susceptibility. In our previous study, OsSNAP32 has been proven to function in rice blast resistance (Luo et al., 2016). In this study, the working model for OsSYP121 could be speculated as follows: OsSYP121 can interact with OsSNAP32 and VAMP714/724 to form the SNARE complex; in the blast fungi invasion phase, OsSYP121 can accumulate at fungi penetration sites; the vesicle trafficking- and defense-associated genes OsMYB20, OsWRKY21, OsRbohF, and OsHSP90 could be affected by knockdown expression of OsSYP121 (Fig. 8).

Figure 8.

Figure 8.

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Working model for the roles of OsSYP121 in rice-blast fungus interaction. In rice cells, OsSYP121 can interact with OsSNAP32 and VAMP714/724 to form the SNARE complex. In the blast fungi invasion phase, OsSYP121 can accumulate at blast fungi penetration sites. The vesicle trafficking- and defense-associated genes could be affected by knockdown expression of OsSYP121.

In summary, our study demonstrates that OsSYP121 functions in fungi penetration, and OsSYP121 can interact with OsSNAP32 and mediate host resistance to rice blast. This indicates OsSYP121 might play an important role in the 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 resistance and susceptibility to the blast fungus (Magnaporthe oryzae) strain Hoku1, respectively (Wang et al., 2002), and seven OsSYP121 overexpression and knockdown 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 ± 3°C) 3 weeks for the blast fungus inoculation and induction expression analysis of target genes. Some landrace seedlings were transplanted in the fields in Nanjing. At the flowering stage of Hei, root, stem, leaf blade, leaf sheath, immature panicle (5–6 cm), and flowering panicle samples were collected for tissue-specific expression analysis of target genes. Nicotiana benthamiana plants were grown in the greenhouse at 24°C for 4 to 5 weeks for BiFC transient expression assay (Waadt and Kudla, 2008).

Pathogen Inoculation and Disease Evaluation

The blast strain Hoku1 (provided by Zhiyi Chen, Jiangsu Academy of Agricultural Science) was used for blast fungus inoculation in this study. Three-week-old rice seedlings were inoculated by spraying with spore suspension (1 × 105 spores mL−1 in 0.025% [w/v] Tween 20) as previously reported (Wang et al., 2002). The inoculated seedlings were kept in a dark incubation room with 100% relative humidity and 26°C for 24 h, then moved to the greenhouse for the disease inducing. Seven days after inoculation, OsSYP121 transgenic plants and OsSYP121RIOsSNAP32RI crossed plants were assessed for lesion number on each inoculated leaf and lesion length according to the methods of Shi et al. (2010) and Mackill and Bonman (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 cDNA was synthesized with 2 μg 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 after inoculation, frozen in liquid nitrogen immediately, and then stored at −80°C. The leaves of transgenic lines were collected and stored at −80°C for RNA extraction and qPCR analysis and RT-qPCR analysis. All the primers are shown in Supplemental Table S4.

qPCR was performed using FastStart Universal SYBR Green Mastermix (ROX; Roche) and a 7500 Fast Real-Time PCR System (Applied Biosystems). Reactions were set up with the following program: 1 min at 95°C, followed by 40 cycles of 95°C for 10 s, 60°C to 62°C for 15 s, and 72°C for 40 s. The relative expression level of each gene was calculated using the 2–△△CT method (Livak and Schmittgen, 2001). Three biological replicates were performed for each qPCR. The expression level of 18S-rRNA was used as an internal control (Jain et al., 2006). RT-qPCR was set up with the following program: 1 min at 95°C, followed by 27 to 36 cycles of 95°C for 30 s, 58°C to 62°C for 30 s, and 72°C for 45 s. The expression level of Actin gene in rice was used as an internal control (Martin, 1999).

Bioinformatics Analysis of OsSYP1s

The phylogenetic analysis of SYP1s in rice and Arabidopsis (Arabidopsis thaliana) 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, were used to generate a bootstrap neighbor-joining phylogenetic tree. Bootstrap probabilities were obtained from 1,000 replicates. Multiple sequence alignment of SYP1 proteins was carried out by ClustalX 1.8 (Thompson et al., 1997), and the 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 the protein domain of SYP1 proteins.

Subcellular Localization of OsSYP1s in Protoplasts

Full-length cDNA fragments of OsSYP111, OsSYP121, and OsSYP132 were amplified from Hei cDNA and cloned into the pGEM-T vector (Takara). To construct the transient expression plasmids, the full-length cDNA fragments were inserted into the pUC18 vector, N terminal of the fragments framed with GFP.

Protoplast extraction of young rice seedlings (landrace Hei) and plasmid transient transformation were performed as described (Chen et al., 2006). A total of 10 μg of plasmid DNA for each construct was mixed with 200 μL of suspended protoplasts (1 × 106 cells mL−1) and then incubated in the dark at 28°C. The transformed cells were observed by a Zeiss 710 laser confocal microscope after 12 and 16 h.

Generation and Identification of Transgenic Plants

Full-length OsSYP121 was inserted into the pCAMBIA1300S vector to generate the overexpression transgenic vector pCAMBIA1300S-OsSYP121. A 246-bp OsSYP121-specific fragment was used to generate the knockdown expression 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 generate the final vectors POsSYP121::GFP-OsSYP121 or POsSYP132::GFP-OsSYP132 (Supplemental Fig. S8). These vectors were transformed into rice plants using Agrobacterium tumefaciens-mediated methods (Toki et al., 2006).

Southern blotting was conducted to identify the transgenic plants using DIG High 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 were sampled 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-2B staining. According to Nakao et al. (2011) methods, the fungal growth was observed under a fluorescence microscope (Nikon Eclipse 80i). Four types of fungal growth stage: type I, M. oryzae conidium without germ tubes; type II, differentiated appressorium formation; type III, establishment of infection hypha (primary hypha); type IV, branch formation on infection hypha (secondary hypha) were identified, and the percentage of each type in the total observed cells was calculated. At least nine leaves from three plants of each transgenic line or landrace were sampled.

Localization of OsSYP121-GFP and OsSYP132-GFP in Cells

To identify the localization of OsSYP121-GFP and OsSYP132-GFP in transgenic plants, the sixth leaf sheaths were placed in blast fungus conidial suspension (1 × 105 conidia mL−1) and incubated for 30 h at 25°C in the dark, then the epidermal cells of leaf sheaths were sampled for microscopy as described by Tanabe et al. (2009).

Yeast Two-Hybrid Assay

Based on a report in Arabidopsis that the AtSYP121 interacting proteins are AtSNAP33 and AtVAMP721/722, seven SNARE member homologous proteins in rice were selected for yeast two-hybrid assay. Full-length cDNA of OsSYP121 was inserted into the pBT3-N vector (bait), and full-length cDNAs of OsSNAP32, OsVAMP711, OsVAMP714, OsVAMP721, OsVAMP722, OsVAMP724, and OsVAMP727 were inserted into pPR3-N (prey; Dualsystems Biotech). The constructs were transformed into yeast strain NMY51 according to the protocol for the DUAL Membrane Kit 1. The positive clones on synthetic dextrose (SD; -Leu,-Trp) medium were transferred to SD (-Trp,-Leu,-His,-Ade) medium containing 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside acid (20 μg mL−1) and 3-amino-1,2,4-triazole (5 mm) to identify protein-protein interactions. The interaction between Cub-OsSYP121 and NubI served as a positive control, whereas coexpression of Cub-OsSYP121 and NubG served as a negative control. Yeast NMY51 cells harbored the C-terminal half of ubiquitin (Cub) and an artificial transcription factor (LexA-VP16) fusion construct and the mutated N-terminal half of ubiquitin (NubG) fusion constructs. The yeast cells were spotted on SD (-Leu,-Trp) medium (selection for positive transformants), and 10-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 incubated for 5 d at 30°C.

BiFC Assay

A previously described protocol (Waadt and Kudla, 2008) was followed to observe BiFC signals with some modification. The full-length cDNA of OsSYP121 was 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 μL of 3 m MgCl2, and 100 μL of 150 mm acetosyringone) to OD600 at 0.7 to 0.8. The working suspensions were prepared by mixing at a 1:1:1 ratio with three A. tumefaciens strains carrying the YFPN fusion construct, the YFPC fusion construct, and the gene-silencing inhibitor p19 strain, respectively. The mixture was standing for 2 to 4 h. The A. tumefaciens suspensions were then coinfiltrated onto the abaxial surface of 4- to 5-week-old N. benthamiana plant leaves. Fluorescence of the epidermal cell layer of the lower leaf surface was examined at 2 to 4 d after infiltration. Images were captured with a Zeiss 710 laser scanning confocal microscope, with excitation wavelengths of 488 and 496 nm and an emission wavelength between 520 and 535 nm for YFP signals.

Microarray and Pathway Analyses

Three-week-old seedlings of OsSYP121-RI lines R1 and R57 and Hei were sampled, and three biological replicates were used for the microarray assay. RNA isolation, purification, and hybridization of Affymetrix microarrays were conducted by the Biotechnology Group (Biotechnology Corporation). We used the ordinary Student’s t test (P < 0.05) to identify significantly differentially expressed genes. Probe sets showing more than 1.5-fold changes for up-regulation and less than 0.66-fold changes for down-regulation in expression were considered to be DEGs. Functional enrichment analysis of DEGs using the GO domains molecular function, biological process, and cellular component was 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 the microarray data based on the expression value of each DEG. A metabolic pathway overview was produced by loading the DEGs with their expression values into the locally installed MapMan program and shown using color intensity.

Accession Numbers

Sequence data from this article can be found in the GenBank data libraries under 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.

Footnotes

1

This work was supported by grants from the National Key Project for Transgenic Crops (2016ZX08009-003-001), the Fundamental Research Funds for the Central Universities (KYZ201704), the National Natural Science Foundation of China (31871602, 31171516, and 30900888), the Jiangsu Agriculture Science and Technology Innovation Fund (CX151054), and the Open Fund of State Key Laboratory of Rice Biology (160101).

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