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OsSYP121 accumulates at fungal penetration sites and mediates host 1
resistance to rice blast 2
3
Wen-Lei Cao1,2,3, Yao Yu1,2, Meng-Ya Li1, Jia Luo1,4, Rui-Sen Wang1, Hai-Juan Tang1, Ji 4
Huang1, Jian-Fei Wang1, Hong-Sheng Zhang1,*, Yong-Mei Bao1,* 5
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1State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of 7
Agriculture, Jiangsu Collaborative Innovation Center for Modern Crop Production, Nanjing 8
Agricultural University, Nanjing 210095, China 9
2These authors contributed equally to this work. 10
3Present address: College of Agriculture, Yangzhou University, 225009, Yangzhou, China 11
4Present address: Chongqing Academy of Agricultural Sciences, 401329, Chongqing, China 12
*Address correspondence to [email protected] (Y.M. Bao) and [email protected] 13
(H.S. Zhang) 14
15
This work was supported by grants from the National Key Project for Transgenic Crops 16
(2016ZX08009-003-001), the Fundamental Research Funds for the Central Universities 17
(KYZ201704), the Natural Science Foundation of China (31871602, 31171516, 30900888), 18
Jiangsu Agriculture science and technology innovation fund (CX(15)1054) and the Open Fund of 19
State Key Laboratory of Rice Biology (160101). 20
The authors responsible for distribution of materials integral to the findings presented in this 21
article in accordance with the policy described in the Instructions for Authors 22
(www.plantphysiol.org) are: Yongmei Bao ([email protected]) and Hongsheng Zhang 23
([email protected]) 24
Author Contributions 25
Y. B., H. Z. and W. C. designed the research; Y. B. cloned the gene OsSYP121; W. C., Y. Y. 26
and Y. B. performed the most experiments; J. L. provided technical assistance to W. C.; R. W. 27
performed the real-time PCR; W. C., Y. Y. and M. L. performed the blast fungus inoculation; H. T. 28
performed rice transforming experiments; J. H. and J. W. provided assistance in data analysis; Y. B. 29
and W. C. conceived the project and wrote the article; Y. B. and H. Z. supervised and 30
complemented the writing. 31
Plant Physiology Preview. Published on January 7, 2019, as DOI:10.1104/pp.18.01013
Copyright 2019 by the American Society of Plant Biologists
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Short title: OsSYP121 mediates host resistance to rice blast 33
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One sentence summary: OsSYP121 accumulates at fungal penetration sites and plays an 35
important role in rice blast resistance 36
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Abstract 38
Magnaporthe oryzae is a fungal pathogen that causes rice (Oryza sativa) blast. SNAREs 39
(soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are key components in 40
vesicle trafficking in eukaryotic cells and are known to contribute to fungal pathogen resistance. 41
Syntaxin of Plants 121 (SYP121), a Qa-SNARE, has been reported to function in non-host 42
resistance in Arabidopsis thaliana. However, the functions of SYP121 in host resistance to rice 43
blast are largely unknown. Here we report that the rice SYP121 protein, OsSYP121, accumulates 44
at fungal penetration sites and mediates host resistance to rice blast. OsSYP121 is plasma 45
membrane-localized and its expression was obviously induced by the rice blast in both the 46
blast-resistant rice landrace Hei (Heikezijing) and the blast-susceptible landrace Su (Suyunuo). 47
Overexpression of OsSYP121 in Su resulted in enhanced resistance to blast. Knockdown of 48
OsSYP121 expression in Su resulted in a more susceptible phenotype. However, knockdown of 49
OsSYP121 expression in the resistant cultivar Hei resulted in susceptibility to the blast fungus. The 50
POsSYP121::GFP-OsSYP121 accumulated at rice blast penetration sites in transgenic rice, as 51
observed by confocal microscopy. Yeast two-hybrid results showed that OsSYP121 can interact 52
with OsSNAP32 (Synaptosome-associated protein of 32 kDa) and OsVAMP714/724 53
(Vesicle-associated membrane protein714/724). The interaction between OsSYP121 and 54
OsSNAP32 may contribute to host resistance to rice blast. Our study reveals that OsSYP121 plays 55
an important role in rice blast resistance as it is a key component in vesicle trafficking. 56
57
Introduction 58
Vesicle trafficking plays crucial roles in plant development and immune responses 59
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(Somerville et al., 2004; Samaj et al., 2006; Lipka et al., 2007; Kwon et al., 2008a; Van Damme 60
and Geelen, 2008; Meyer et al., 2009). SNAREs (soluble N-ethylmaleimide-sensitive factor 61
attachment protein receptors) are key components in vesicle trafficking in eukaryotic cells (Heese 62
et al., 2001; Wick et al., 2003) and play a universal role in diverse biological processes including 63
cytokinesis, defense response, pollen tube and root hair tip growth, root formation and hormone 64
response in plants (Dacks and Doolittle, 2002; Lipka et al., 2007; Enami et al., 2009). Four 65
different types of SNAREs form a SNARE complex through their R-, Qa-, Qb- and Qc-SNARE 66
domains to determine the specificity of intracellular fusion (Antonin et al., 2000; Fukuda et al., 67
2000). Syntaxins (Qa-SNAREs) and interacting SNARE proteins (R-, Qb- and Qc-SNAREs) 68
contribute to the fusion of intracellular transport vesicles with acceptor membranes in diverse 69
trafficking pathways (Pajonk et al., 2008; Reichardt et al., 2011).The SYP1 (syntaxin of plant 1) 70
subfamily is a plant-specific syntaxin family that belongs to the Qa-SNARE family. Nine SYP1 71
genes, SYP111, SYP112, SYP121, SYP122, SYP123, SYP124, SYP125, SYP131 and SYP132, are 72
found in Arabidopsis (divided into three groups) and all localized on the plasma membrane 73
(Uemura et al., 2004). The expression of SYP1s is tissue-specific, only SYP132 ubiquitously 74
expressed in various tissues throughout plant development (Enami et al., 2009). SYP111/ 75
KNOLLE is well known as a cytokinesis-specific syntaxin that is specifically expressed during 76
mitosis and localizes to the forming cell plate (Lukowitz et al., 1996; Heese et al., 2001). SYP112 77
can functionally replace the cell-cycle-regulated KNOLLE protein (Sanderfoot et al., 2000; Muller 78
et al., 2003). As a calcium-dependent phosphorylation protein in Arabidopsis, SYP122 has 79
redundant functions with its closest homologue SYP121 in the secretion of cell wall deposits 80
(Nuhse et al., 2003; Assaad et al., 2004; Zhang et al., 2007). SYP123, which is predominately 81
expressed in root hairs and localizes to the tip region of root hairs, can function with SYP132 to 82
mediate tip-focused membrane trafficking for root hair tip growth (Ichikawa et al., 2014). SYP124, 83
SYP125 and SYP131 are pollen-specific syntaxins involved in pollen tube growth (Kato et al., 84
2010; Silva et al., 2010; Ul-Rehman et al., 2011). NbSYP132 in Nicotiana benthamiana acts as the 85
cognate target-SNARE for the exocytosis of vesicles containing PR proteins in plant basal and 86
salicylate-associated defense (Kalde et al., 2007). 87
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SYP121 is the most intensively studied and well-characterized syntaxin (Collins et al., 2007; 88
Kwon et al., 2008b). SYP121/SYR1 was originally identified in tobacco, and it can prevent the 89
potassium and chloride ion channel response to ABA in stomatal guard cells (Leyman et al., 1999; 90
Leyman et al., 2000). SYP121/PEN1 in Arabidopsis was also shown to directly interact with the 91
potassium and chloride ion channel through an FxRF motif to facilitate solute uptake for cell 92
expansion and plant growth (Sutter et al., 2006; Honsbein et al., 2009; Grefen et al., 2010; 93
Honsbein et al., 2011). SYP121/PEN1 has been verified to contribute to penetration resistance in 94
Arabidopsis (Collins et al., 2003; Kwon et al., 2008a; Kwon et al., 2008b; Kwon et al., 2008c). 95
SYP121/ROR2 in barley was localized at the plasma membrane in non-pathogen challenged 96
epidermal cells but accumulate focally near the papilla structure below the penetration sites 97
infected by powdery mildew (Assaad et al., 2004; Bhat et al., 2005; Collins et al., 2007). SYP121 98
is believed to act in mediating vesicle fusion events in an extracellular defense pathway by 99
specifically forming a ternary SNARE complex with SNAP33 (Synaptosome-associated protein of 100
33 kDa) and the VAMP721/722 (Vesicle-associated membrane protein721/722) to deliver defense 101
components to the space between the plasma membrane and the plant cell wall where fungus is 102
attacking (Collins et al., 2003; Kwon et al., 2008b). 103
As a major food crop, rice has a genome encoding 57 SNARE proteins (Sanderfoot, 2007), 104
but none of them has been well-characterized. In our previous work, we cloned five SNAREs 105
genes, including OsSNAP32 (Bao et al., 2008; Luo et al., 2016), OsSYP71 (Bao et al., 2012) and 106
OsNPSN11-13 (Bao et al., 2008). The expression of the SNAP25-type gene OsSNAP32 was 107
induced by H2O2, PEG6000, low temperature and rice blast fungus inoculation treatments in rice 108
seedlings (Bao et al., 2008). The overexpression of OsSNAP32 and OsSYP71 in rice showed 109
enhanced tolerance to oxidative stress and rice blast (Bao et al., 2012; Luo et al., 2016). 110
In this paper, we isolated and analyzed the expression of OsSYP111, OsSYP121 and 111
OsSYP132 distributed in three SYP1 subgroups from rice, and only the expression of OsSYP121 112
was induced by the blast fungus. To elucidate the function of OsSYP121 in rice resistance to blast, 113
we overexpressed and knocked down expressed of OsSYP121 in transgenic rice and observed the 114
location of PSYP121: GFP- SYP121 in transgenic rice inoculated by blast fungus by microscopy. 115
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116 117
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Results 118
119
Expression of OsSYP121 is induced by the blast fungus 120
The expression profiles of OsSYP111, OsSYP121 and OsSYP132 in rice landrace Hei were 121
detected in various tissues. OsSYP121 and OsSYP132 was predominantly detected in leaf blades 122
and leaf sheaths (Fig. 1A). In order to determine three genes’ expression in Hei and Su after blast 123
fungus inoculation, the expression of SYP121 at 48 h in Hei and 8 h in Su with same expression 124
level were normalized as “1” and relative expression of these genes were detected. It was found 125
that OsSYP121 in Hei was continually increased after the blast fungus inoculation until 48 h, while 126
the expression of OsSYP121 was increased to the peak at 8 h in Su and dropped back to a lower 127
level at 24 h (Fig. 1B). The expressions of OsSYP132 were induced at 8 h with lower expression 128
level both in Hei and Su, while the expressions of OsSYP111 were rarely detected. A phylogenetic 129
analysis of SYP1s proteins from Arabidopsis and rice revealed that all of these proteins were 130
clustered into three subgroups: SYP11s, SYP12s and SYP13s (Supplemental Fig. S1A, Uemura et 131
al., 2004). Three genes OsSYP111, OsSYP121 and OsSYP132 distributed in three subgroups were 132
cloned from rice (Supplemental Table S1), and protoplast subcellular localization results showed 133
that GFP-OsSYP111 were mainly localized in the plasma membrane and cytoplasm, while 134
GFP-OsSYP121 and GFP-OsSYP132 were localized at the plasma membrane comparing with 135
GFP control that was globally localized in the cytoplasm and the nucleus (Supplemental Fig. S1B- 136
S1I). The syntaxin domain of SYP121 proteins in different organisms contains three α-helix 137
domains: Ha, Hb and Hc at the N-terminus (Supplemental Fig. S2). 138
OsSYP121 is associated with penetration resistance to rice blast fungus 139
Three OsSYP121 overexpression transgenic lines (OE5-Su, OE8-Su, and OE11-Su) and two 140
knockdown lines (RI3-Su and RI7-Su) in Su, and two OsSYP121 knockdown transgenic lines 141
(RI1-Hei and RI57-Hei) in Hei were obtained using an Agrobacterium-mediated method (drived 142
by 35S promotor) (Supplemental Fig. S3, S4 and S5). The OE5-Su, OE8-Su, and OE11-Su 143
transgenic lines showed significantly dwarf phenotype compared with wild-type Su, while other 144
agronomic traits showed no difference (Supplemental Fig. S6). All the knockdown transgenic 145
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lines (RI3-Su, RI7-Su, RI1-Hei and RI57-Hei) showed the same agronomic traits with their 146
wild-type controls (Supplemental Fig. S6). After inoculated with rice blast fungus(strain Hoku1) 147
at the 3-4 leaf stage, OE5-Su, OE8-Su, and OE11-Su showed more resistance than wild-type Su, 148
with less lesions, whereas RI3-Su, RI7-Su, RI1-Hei and RI57-Hei were more susceptible to blast 149
than their wild-type controls (Fig. 2, A and B). The lesion length of all transgenic plants showed 150
no differences (Fig. 2B). 151
To gain a mechanistic insight into the enhanced blast resistance in the OsSYP121-OE lines 152
and the susceptible phenotype in the OsSYP121-RI lines, we observed the penetration process of 153
blast fungus to classify rice defense responses through a quantitative microscopic assessment of 154
the interaction of rice and M. oryzae (Nakao et al., 2011). In wild-type Su, 48.09% of the 155
penetrated cells were in Type IV stage, 58.08-64.1% of penetrated cells in RI3-Su and RI7-Su 156
were in the Type IV stage, and 54.20-76.74% of penetrated cells in the OE5-Su, OE8-Su and 157
OE11-Su were unable to develop into differentiated appressoria (Type II) (Fig. 2, C and D). In 158
wild-type Hei, more than 90% of the cells were in the Type I stage, and 19.3-25.9% of the 159
penetrated cells in RI1-Hei and RI57-Hei were in the Type III and Type IV stages. Thus, 160
overexpression of OsSYP121 in transgenic plants more frequently prevented the penetration of 161
rice blast fungus and the establishment of infection hyphae. 162
OsSYP121 accumulates at pathogen penetration sites 163
In non-inoculated leaf sheaths, either GFP-OsSYP121 or GFP-OsSYP132 was exclusively 164
distributed in the plasma membrane (Fig. 3, A and E), in agreement with the results in rice 165
protoplasts (Supplemental Fig. S1, C and E). After inoculated with the compatible strain Hoku 1 166
for 30 h, the accumulation of GFP-OsSYP121 as cup-shape structures was observed beneath the 167
appressoria of M. oryzae (Fig. 3, B-D), while no difference in GFP-OsSYP132 distribution was 168
observed between non-inoculated and inoculated leaf sheath (Fig. 3F). The observed cup-shape 169
structures were specifically caused by the accumulation of GFP-OsSYP121 but not 170
auto-fluorescence because the fluorescence was not observed in non-transgenic Su plants (Fig. 3, 171
F-H). 172
OsSYP121 can interact with OsSNAP32 and mediates the host resistance to rice blast fungus 173
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To explore the ternary SNARE complexes composed of OsSYP121, seven genes 174
OsVAMP711, OsVAMP714, OsVAMP721, OsVAMP722, OsVAMP724, OsVAMP727 and 175
OsSNAP32 were cloned from rice as candidates to identify any interactions (Fig. 4A). The yeast 176
two-hybrid results showed that OsSYP121 could interact with OsSNAP32, OsVAMP714 and 177
OsVAMP724 (Fig. 4A). The interaction of OsSYP121 and OsSNAP32 was also confirmed using a 178
Bimolecular Fluorescence Complementation (BiFC) assay in the N. benthamiana transient 179
expression system (Fig. 4B). 180
In this study, OsSNAP32 RNAi transgenic lines OsSNAP32RI in Su showed more 181
susceptible phenotype (Fig. 5), which is consistent with our previous results that OsSNAP32 182
RNAi transgenic lines in Heikezijing decreased resistance to blast (Luo et al., 2016). In order to 183
study the genetic interaction between OsSYP121 and OsSNAP32, OsSYP121RI transgenic plants 184
in Su were used to cross with OsSNAP32RI transgenic plants in Su to generate 185
OsSYP121RIOsSNAP32RI double knock-down transgenic plants. The rice blast disease assay 186
showed that the susceptibilities of OsSYP121RI, OsSNAP32RI, and OsSYP121RIOsSNAP32RI 187
were similar, with more lesion and higher percentage of Type Ⅳ infected cells than wild type Su 188
(Fig. 5). These results indicate OsSYP121 may genetically interact with OsSNAP32 and mediates 189
host resistance in rice. 190
OsSYP121 promotes rice defense response to blast fungus 191
To identify the genes probably affected by OsSYP121, we compared the transcriptomes of 192
R1-Hei, R57-Hei and wild-type Hei through microarray analysis. Compared with the Hei 193
background, 51 genes were down-regulated by <0.66 folds changes both in RI1-Hei and RI57-Hei, 194
and 89 genes were up-regulated by > 1.5 folds both in RI1-Hei and RI57-Hei (Fig.6A, 195
Supplemental table S3-S4). To identify genes related to metabolic reconfiguration in the different 196
combinations, the AGRIGO and MapMan tools were used to conduct the Go enrichment and 197
display the significantly regulated pathways. By AGRIGO Go enrichment analysis, only the GO 198
term “cellular component” were identified with default significance levels (FDR＜0.05), and 20% 199
of down-regulated and up-regulated DEGs were associated with “cytoplasmic membrane-bounded 200
vesicle”, “membrane-bounded vesicle”, “cytoplasmic vesicles” and “vesicles”(Fig.6, B and C). By 201
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MapMan analysis, we found that 2 down-regulated genes were associated with vesicle trafficking 202
(OsSNAP32) and auxin trafficking (OsPILS7a) in the transport overview (Supplemental Fig. S7). 203
One down-regulated gene and three up-regulated genes were associated with biotic stress, and one 204
up-regulated gene was associated with development and two genes were associated with abiotic 205
stress in the cellular response pathway (Supplemental Fig. S7B). Twelve down-regulated genes 206
and twenty six up-regulated genes were related to pathogen/pest attack pathways (Supplemental 207
Fig. S7C). We further investigated the expression of six down regulated genes OsSNAP32 208
(Os02g0437200), OsPILS7a (Os09g38130), OsMYB20 (Os02g49986), OsWRKY21 209
(Os01g60640), OsRbohF (Os08g35210) and OsHSP90 (Os09g0482610) and OsSGT1 210
(Os01g0624500) in OsSYP121 overexpression and knock down expression transgenic plants. 211
These results suggest that OsSYP121 can affect the expressions of OsSNAP32, OsPILS7a, 212
OsMYB20, OsWRKY21, OsRbohF and OsHSP90 to trigger plant immunity responses (Fig. 7). 213
214 215
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DISCUSSION 216
Compared with yeast and mammals, which only have two and four syntaxins, there are 18 217
syntaxins in Arabidopsis and 14 syntaxins in rice (Uemura et al., 2004; Lipka et al., 2007; 218
Sanderfoot et al., 2007; Reichardt et al., 2011). In SYP1 subgroup of syntaxin, there are nine 219
AtSYP1s in Arabidopsis and six OsSYP1s in rice. In contrast to Arabidopsis, less OsSYP1s were 220
detected in rice and the roles of OsSYP1s proteins in rice host resistance were largely unknown. 221
Subcellular localization analysis of OsSYP111, OsSYP121 and OsSYP132 distributed in three 222
OsSYP1s subgroups showed that OsSYP121 and OsSYP132 were localized to plasma membrane, 223
while OsSYP111 was localized to plasma membrane and cytoplasm. The subcellular localization 224
of OsSYP111, OsSYP121 and OsSYP132 are similar as their homologs in Arabidopsis (Uemura et 225
al., 2004). The expression of these three genes in response to M. oryzae showed that only 226
OsSYP121 was significantly induced by M. oryzae. In resistant landrace Hei, the expression of 227
OsSYP121 was obviously and stably induced until 48 h upon blast fungus inoculation. In 228
susceptible landrace Su, the expression of OsSYP121 was induced at 8 h and then declined. 229
Overexpression of OsSYP121 in Su leads to enhanced resistant and knock down expression of 230
OsSYP121 in Hei and Su showed more susceptible. These data suggest that the expression level of 231
OsSYP121 is correlated with the susceptible and resistant phenotype and OsSYP121 might play an 232
important role in the rice defense response to M. oryzae attack. 233
Overexpression of OsSYP121 in Su significantly decreased the number of lesions but not 234
lesion length in transgenic rice, indicating that pathogen penetration was prevented in the early 235
stages. Furthermore, microscopic observation of the blast fungus infection process in the 236
transgenic plants revealed that penetration-stage defense was induced in OsSYP121-OE rice, 237
which indicates that OsSYP121 may function during M. oryzae penetration into rice epidermal 238
cells. In Arabidopsis, knockout PEN1 leads to enhanced penetration of non-host powdery mildew 239
pathogen, but results in enhanced resistance to adapted powdery mildew (Kwon et al., 2008b; 240
Zhang et al., 2007). Silencing of MdSYP121 increased resistance to Botryosphaeria dothidea (He 241
et al., 2018). In our study, it is interesting that knocking down of OsSYP121 in the resistant 242
landrace Hei and susceptible landrace Su leads to susceptibility. This indicates that the SYP121 243
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may play different roles among phytopathosystem of biotroph, necrotroph and seminecotroph. 244
While SYP121 plays positive role in penetration resistance, it also play a negative role in 245
SA-signaling that is required for resistance against biotrophic pathogens. However, SA signaling is 246
generally antagonistic to JA- and ET-signalings that are required for resistance against 247
necrotrophic pathogens. Powdery mildew fungi are biotroph, Botryosphaeria dothidea is a 248
necrotroph, whereas, Magnaporthe oryzae is a semi-necrotroph. All these fungi have to penetrate 249
the host cell wall, but post-penetration resistance in the host requires different hormone signaling. 250
Both JA- and ET-signalings play positive roles in blast-disease resistance. Therefore, SYP121 251
shows conserved penetration resistance but different in post-penetration resistance. 252
Overexpression of OsSYP121 showed enhanced resistance and dwarfism phenotype. It is not clear 253
that there is the relationship between resistance and dwarfism phenotypes and whether OsSYP121 254
can induce a constitutive defense response. Loss of PEN genes in Arabidopsis affects not only 255
penetration resistance against non-adapted powdery mildew but also HR induced after recognition 256
of pathogenic effectors (Johansson et al., 2014). In the further research, we would identify the SA 257
concentration and HR phenotype to learn more about the functions of OsSYP121 in the defense 258
response. 259
Microscopic observation of GFP-OsSYP121 transgenic plants clearly showed the 260
accumulation of OsSYP121 in penetration sites at 24-48 h after inoculation, while OsSYP132 261
remained localized in the plasma membrane after inoculation. It provides evidence that OsSYP121 262
contributes to penetration resistance in rice–M. oryzae interaction. As the first line of plant defense 263
against fungi, penetration resistance is achieved by localized cell wall appositions or papillae at 264
fungal penetration sites and functions as physical and chemical barriers to cell penetration (Aist, 265
1976; Schmelzer, 2002; Aist, 2003; Hardham et al., 2007; Yang et al., 2014). Penetration 266
resistance of Arabidopsis against powdery mildew fungi relies on PEN1 as well as PEN2/PEN3, 267
which can contribute to the synthesis and secretion of antimicrobial proteins and metabolites 268
(Collins et al., 2003; Lipka et al., 2005; Stein et al., 2006; Bednarek et al., 2009). The syntaxin 269
PEN1 in Arabidopsis has been identified as important molecular components in nonhost resistance 270
to Bgh (Collins et al., 2003; Thordal-Christensen, 2003; Zhang et al., 2007). We found that 271
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OsSYP121 plays a critical role in rice penetration resistance against M. oryzae and that the 272
OsSYP121 accumulated at rice blast fungi penetration sites and mediates host resistance in rice. 273
Some clues showed that the rice–M. oryzae system is a good system for the study of fungus 274
penetration and pre-invasion resistance (Robatzek, 2007; Ribot et al., 2008; Faivre-Rampant et al., 275
2008). Although the relocalization and concentration of SYP121 proteins at penetrations sites to 276
powdery mildew in Arabidopsis and barley are well studied, and the SYP121 proteins appeared to 277
be actively recruited to papillae at the penetrations sites of powdery mildew fungus (Assaad et al. 278
2004; Bhat et al. 2005). However, the function of OsSYP121 in the rice-blast fungi interaction 279
system is still not well known. It is well known that there are no papillae in the blast fungi 280
penetration sites. It is worth to study the function and location of OsSYP121 in rice, a staple food 281
crop. 282
In this study, we cloned the candidate Qb-SNAREs and OsVAMPs and used yeast two-hybrid 283
systems to check the interactions between OsSYP121 and the protein candidates. It was found the 284
OsSNAP32 and OsVAMP714/724 can interact with OsSYP121, whereas the AtSYP121 in 285
Arabidopsis can interact with AtSNAP33 and AtVAMP721/722 (Kwon et al., 2008b). This 286
suggests that there may be different elements in the OsSYP121 SNARE complex in rice and 287
Arabidopsis. Sugano et al (2016) reported that the OsVAMP714-mediated trafficking pathway 288
plays an important role in rice blast resistance. Overexpression of OsVAMP714 in rice leads to 289
enhanced resistance, while knock down expression of OsVAMP714 in rice showed serious 290
susceptibility. In our previous study, OsSNAP32 have been proved to function in rice blast 291
resistance (Luo et al., 2016). In this study, the working model for OsSYP121 could be speculated 292
as: OsSYP121 can interact with OsSNAP32, VAMP714/724 to form the SNARE complex; in the 293
blast fungi invasion phase, OsSYP121 can accumulate at fungi penetration sites; the vesicle 294
trafficking and defense associated genes OsMYB20, OsWRKY21, OsRbohF and OsHSP90 could 295
be affected by knock known expression of OsSYP121 (Fig.8). 296
In summary, our study demonstrates that OsSYP121 functions in fungi penetration, and 297
OsSYP121 can interact with OsSNAP32 and mediate host resistance to rice blast. This indicates 298
OsSYP121 might play an important role in the rice defense response to M. oryzae attack. 299
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300
MATERIALS AND METHODS 301
Plant materials and growth 302
Two rice landraces (Oryza sativa subsp. japonica), Heikezijing (Hei) and Suyunuo (Su)with 303
resistance and susceptibility to the blast fungus strain Hoku1, respectively (Wang et al., 2002), and 304
seven OsSYP121 overexpression and knock down expression transgenic lines (T2) generated 305
including OE5-Su, OE8-Su, OE11-Su, RI3-Su, RI7-Su, RI1-Hei and RI57-Hei were used in this 306
study. 307
Rice seeds of two landraces and transgenic lines were sown in plastic pots (diameter=10 cm 308
and height=10 cm) containing garden soil (75 % ordinary garden soil and 25 % nutrient soil) and 309
grown in a greenhouse (16 h light/8 h dark period at 25±3 °C) three weeks for the blast fungus 310
inoculation and induction expression analysis of target genes. Some landraces seedlings were 311
transplanted in the fields in Nanjing. At the flowering stage of Hei, root, stem, leaf blade, leaf 312
sheath, immature panicle (5-6cm) and flowering panicle samples were collected for tissue-specific 313
expression analysis of target genes. N. benthamiana plants were grown in the greenhouse at 24°C 314
for 4-5 weeks for BiFC transient expression assay (Waadt et al., 2008). 315
Pathogen inoculation and disease evaluation 316
The blast strain Hoku1 (provided by Prof. Zhiyi Chen, JAAS, China) was used for blast 317
fungus inoculation in this study. The three weeks rice seedlings were inoculated by spraying with 318
spore suspension (1×105 spores per mL in 0.025% (w/v) Tween-20) as previously reported (Wang 319
et al., 2002). The inoculated seedlings were kept in dark incubation room with 100% relative 320
humidity and 26 °C for 24 h, then moved to the greenhouse for the disease inducing. Seven days 321
after inoculation, OsSYP121 transgenic plants and OsSYP121RI OsSNAP32RI crossed plants were 322
assessed for lesion number each inoculated leaf and lesion length according to the methods of Shi 323
et al. (2010) and Mackill et al. (1992). 324
qPCR and RT-qPCR analysis 325
Total RNA was extracted from various rice tissues using the Trizol reagent (Invitrogen, 326
USA), according to the manufacturer’s instructions. First-strand cDNA was synthesized with 2 μg 327
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of purified total RNA using the RT-PCR system (Promega, USA). Leaves of Hei and Su were 328
sampled at 0 h, 8 h, 24 h, 48 h and 72 h after inoculation, frozen in liquid nitrogen immediately, 329
and then stored at -80 °C. The leaves of transgenic lines were collected and stored at -80 °C for 330
RNA extraction and qPCR analysis and RT-qPCR analysis. All the primers are shown in 331
Supplemental table S2. 332
The qPCR was performed using FastStart Universal SYBR Green Mastermix (ROX) (Roche, 333
USA) and an Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems, USA). 334
Reactions were set up with the following program: 1 min at 95 °C, followed by 40 cycles of 95 °C 335
for 10 s, 60-62 °C for 15 s, and 72 °C for 40 s. The relative expression levels of each gene were 336
calculated using the 2–△△CT method (Livak and Schmittgen, 2001). Three biological replicates were 337
performed for each qPCR reaction. The expression level of 18S-rRNA was used as an internal 338
control (Jain et al., 2006). RT-qPCR reactions were set up with the following program: 1 min at 339
95 °C, followed by 27-36 cycles of 95 °C for 30 s; 58-62 °C for 30 s; and 72 °C for 45 s. The 340
expression level of Actin gene in rice was used as an internal control (Martin, 1999). 341
Bioinformatics analysis of OsSYP1s 342
The phylogenetic analysis of SYP1s in rice and Arabidopsis was performed using MEGA6 343
software (Tamura et al., 2013). Full-length amino acid sequences of 15 SYP1 proteins, AtSYP111, 344
AtSYP112, AtSYP121, AtSYP122, AtSYP123, AtSYP124, AtSYP125, AtSYP131, AtSYP132, 345
OsSYP111, OsSYP121, OsSYP124, OsSYP125, OsSYP131 and OsSYP132, were used to 346
generate a bootstrap neighbor-joining phylogenetic tree. Bootstrap probabilities were obtained 347
from 1000 replicates. Multiple sequence alignment of SYP1s proteins was carried out by Clustal X 348
1.8 (Thompson et al., 1997), and the results were edited by GENEDOC 349
(http://www.psc.edu/biomed/genedoc). Pfam (http://pfam.xfam.org/) and TMHMM 350
(http://www.cbs.dtu.dk/ services/TMHMM/) were used to annotate the protein domain of SYP1 351
proteins. 352
Subcellular localization of OsSYP1s in protoplast 353
Full-length cDNA fragments of OsSYP111, OsSYP121 and OsSYP132 were amplified from 354
Hei cDNA and cloned into the pGEM-T vector (Takara). To construct the transient expression 355
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15
plasmids, the full-length cDNA fragments were inserted into the pUC18 vector, N- terminal of the 356
fragments inframed with GFP. 357
Protoplast extraction of young rice seedlings (Landrace Hei) and plasmid transient 358
transformation were performed as described (Chen et al., 2006). A total of 10 μg plasmid DNA for 359
each construction were mixed with 200 μL of suspended protoplasts (1×106 cells/mL) and then 360
incubated in the dark at 28 °C. The transformed cells were observed by Zeiss 710 Laser Confocal 361
microscopy after 12 and 16 h. 362
Generation and identification of transgenic plants 363
Full-length OsSYP121 was inserted into the pCAMBIA1300S vector to generate the 364
overexpression transgenic vector pCAMBIA1300S-OsSYP121. A 246-bp OsSYP121-specific 365
fragment was used to generate the knockdown expression transgenic vector pTCK303-OsSYP121, 366
as described by Wang et al. (2004). The fragments with native promoter and coding regions of 367
OsSYP121 or OsSYP132 were inserted to pCAMBIA1304 and in framed with GFP to generate the 368
final vectors POsSYP121::GFP-OsSYP121 or POsSYP132::GFP-OsSYP132 (Supplemental Fig.S8). 369
These vectors were transformed into rice plants using Agrobacterium-mediated methods (Toki et 370
al., 2006) 371
Southern blotting was conducted to identify the transgenic plants using DIG High Prime 372
DNA Labeling and Detection Starter Kit I (Version 10.0) (Roche) according to the manufacturer’s 373
instructions. Twenty micrograms of EcoRI-digested genomic DNA were hybridized to the hpt 374
(hygromycin phosphotransferase)-specific fragment probe. 375
Microscopy observation of inoculated leaves 376
As previously described (Chen et al., 2010), the inoculated leaves were sampled at 24 h after 377
inoculation (hpi) and submerged in lactophenol-ethanol (1:2 v/v) solution for 1-2 days. The 378
samples were treated with Uvitex-2B staining. According to Nakao et al (2011) methods, the 379
fungal growth was observed under fluorescence microscope (Nikon Eclipse 80i). Four types of 380
fungal growth stage: Type I, M. oryzae conidium (CO) without germ tubes; Type II, differentiated 381
appressorium (APP) formation; Type III, establishment of infection hypha (primary hypha, PHY); 382
Type IV, branch formation on infection hypha (secondary hypha, SHY) were identified, and the 383
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16
percentage of each type in the total observed cells was calculated. At least nine leaves from three 384
plants each transgenic line or landrace were sampled. 385
Localization of OsSYP121-GFP and OsSYP132-GFP in cell 386
To identify the localization of OsSYP121-GFP and OsSYP132-GFP in transgenic plants, the 387
sixth leaf sheaths were placed in blast fungus conidial suspension (1 × 105 conidia mL-1) and 388
incubated for 30 h at 25 °C in the dark, then the epidermal cells of leaf sheaths were sampled for 389
microscopy as described by Tanabe et al. (2009). 390
Yeast two-hybrid assay 391
Based on Arabidopsis’ report that the AtSYP121 interacting proteins are AtSNAP33 and 392
AtVAMP721/722, seven SNARE members homologous proteins in rice were selected for Yeast 393
two-hybrid assay. Full-length cDNA of OsSYP121 was inserted into the pBT3-N vector (Bait), 394
and full-length cDNA of OsSNAP32, and OsVAMP711, OsVAMP714, OsVAMP721, OsVAMP722, 395
OsVAMP724 and OsVAMP727 were inserted into pPR3-N (Prey)(Dualsystems Biotech AG). The 396
constructs were transformed into yeast strain NMY51 according to the protocol for the DUAL 397
membrane Kit 1. The positive clones on SD (-Leu, -Trp) medium, were transferred to SD (-Trp, 398
-Leu, -His, -Ade) medium containing X-α-Gal (20 μg mL-1) and 3-Amino-1,2,4-Triazole (3AT, 5 399
mM) to identify protein–protein interactions. The interaction between Cub-OsSYP121 and NubI 400
served as a positive control, whereas co-expression of Cub-OsSYP121 and NubG served as a 401
negative control. Yeast NMY51 cells harbored the C-terminal half of ubiquitin (Cub) and an 402
artificial transcription factor (LexA-VP16) fusion construct and the mutated N-terminal half of 403
ubiquitin (NubG) fusion constructs. The yeast cells were spotted on SD medium without Leu and 404
Trp (-LW; selection for positive transformants), and 10-fold dilutions of the yeast cells were 405
spotted on SD medium without Leu, Trp, His, and Ade (-LWHA and 5 mM 3-amino-1,2,4-triazole; 406
selection for interaction) and incubated for 5 d at 30 °C. 407
BiFC assay 408
A previously described protocol (Waadt et al., 2008) was followed to observe BiFC signals 409
with some modification. The full-length cDNA of OsSYP121 was cloned into the pSPYNE173 410
vector to generate OsSYP121:YFPN, and OsSNAP32 was inserted into the pSPYCE vector to 411
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17
generate OsSNAP32:YFPC. The constructs were transformed into the Agrobacterium strain 412
EHA105, respectively. Overnight cell cultures were collected and re-suspended in 1 mL of AS 413
medium (1 mL of 1 M MES-KOH pH 5.6, 333 μL of 3 M MgCl2, 100 μL of 150 mM 414
acetosyringone) to OD600 at 0.7–0.8. The working suspensions were prepared by mixing at a 1:1:1 415
ratio with three Agrobacterium strains carrying the YFPN fusion construct, the YFPC fusion 416
construct, and the gene silencing inhibitor p19 strain (Voinnet et al., 2003), respectively. The 417
mixture was standing for 2–4 hours. The Agrobacterium suspensions were then co-infiltrated onto 418
the abaxial surface of 4-5weeks N. benthamiana plant leaves. Fluorescence of the epidermal cell 419
layer of the lower leaf surface was examined at 2-4 days after infiltration. Images were captured 420
with a Zeiss 710 Laser Scanning Confocal Microscope, with excitation wavelengths of 488 nm 421
and 496 nm and an emission wavelength between 520-535 nm for YFP signals. 422
Microarray and pathway analyses 423
Three-week seedlings of OsSYP121-RI lines R1 and R57 and Hei were sampled and three 424
biological replicates were used for the microarray assay. RNA isolation, purification and 425
hybridization of Affymetrix microarrays were conducted by the Biotechnology Group 426
(Biotechnology Corporation, Shanghai). We used the ordinary Student’s t-test (P < 0.05) to 427
identify significantly differentially expressed genes. Probe sets showing more than 1.5-fold 428
changes for up-regulation and less than 0.66-fold changes for down-regulation in expression were 429
considered to be DEGs (differentially expressed genes). Functional enrichment analysis of DEGs 430
using the GO domains “molecular function”, “biological process” and “cellular component” was 431
performed by AGRIGO (http://bioinfo.cau.edu.cn/agriGO/ndex.php) with default significance 432
levels (FDR< 0.05). The MapMan tool (Thimm et al., 2004) was employed to analyze the 433
metabolic and signaling changes in the microarray data based on the expression value of each 434
DEG. A metabolic pathway overview was produced by loading the DEGs with their expression 435
values into the locally-installed MapMan program and shown using color intensity. 436
Accession Numbers 437
Sequence data from this article can be found in the GenBank data libraries under accession 438
numbers: OsSY121 (BAS86738.1); OsSYP132 (BAT00191.1); OsSYP111 (BAS86268.1); 439
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18
OsSYP124 (BAD32916.1); OsSYP125 (BAD25019.1); OsSYP131 (BAS96357.1); AtSYP111 440
(AEE28306.1); AtSYP112 (AEC06747.1); AtSYP121 (AAF23198.1); AtSYP122 (AEE78943.1); 441
AtSYP123 (AEE82307.1); AtSYP124 (AEE33817.1); AtSYP125 (AEE28704.1); AtSYP131 442
(AEE73995.2); AtSYP132 (AED91242.1); NtSYP121 (AAD11808.1); HvSYP121 (AAP75621.1); 443
ZmSYP121(ACG40338.1); OsVAMP727 (BAD13129.1); OsVAMP724(BAD30660.1); 444
OsVAMP722(BAD30158.1); OsVAMP721(BAS86911.1); OsVAMP714 (BAT09923.1); 445
OsVAMP711 (BAA95814.1). 446
Supplemental Data 447
Supplemental Figure S1. Phylogenetic analysis of SYP1s proteins and subcellular localization of 448
OsSYP111, OsSYP121 and OsSYP132. 449
Supplemental Figure S2. Multiple sequence alignment of SYP121 proteins in different 450
organisms. 451
Supplemental Figure S3. Identification of OsSYP121 overexpression transgenic plants in Su. 452
Supplemental Figure S4. Identification of OsSYP121 knock down expression transgenic plants 453
in Su. 454
Supplemental Figure S5. Identification of OsSYP121 knock down expression transgenic plants 455
in Hei. 456
Supplemental Figure S6. Agronomic traits of OsSYP121-OE and OsSYP121-RI transgenic 457
plants. 458
Supplemental Figure S7. Microarray analysis showed that OsSYP121 can trigger the plant 459
immunity response. 460
Supplemental Figure S8. The transgenic vector constructions of POsSYP121::GFP-OsSYP121 and 461
POsSYP132::GFP-OsSYP132. 462
Supplemental Table S1. Sequence characteristics of OsSYP111, OsSYP121 and OsSYP132. 463
Supplemental Table S2. Primers used in this study. 464
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19
Supplemental Table S3. Up-regulated genes in RI57-Hei and RI1-Hei compared to wild-type Hei 465
by microarray analysis. 466
Supplemental Table S4. Down-regulated genes in RI57-Hei and RI1-Hei compared to wild-type 467
Hei by microarray analysis. 468
469
FIGURE LEGENDS 470
Figure 1 Expression of OsSYP121 is induced by blast fungus inoculation. A, Tissue specific 471
expression assays of SYP1s in rice landrace Hei. The expressions of OsSYP111, OsSYP121 and 472
OsSYP132 were detected by RT-qPCR. Rice Actin was used as an internal control. B, The 473
expression patterns of four OsSYP1s genes in landraces Hei and Su inoculated with the M. oryzae 474
strain Hoku1 were investigated by qPCR. The seedlings of Hei and Su were collected after 475
inoculation for 0h, 8h, 24h, 48h and 72h. The expressions of OsSYP121 at 48hpi in Hei and at 476
8hpi in Su were the same and defined as 1. The amplification of the rice 18s-rRNA was used as an 477
internal control. Error bars represent standard deviations (SD) of three technical replicates. 478
479
Figure 2 OsSYP121 was associated with penetration resistance to rice blast fungus. A, The rice 480
blast resistant phenotypes of OsSYP121-OE lines (OE5, OE8, and OE11), OsSYP121-RI lines 481
(RI3 and RI7) and their wild-type plants Su, OsSYP121-RI lines (RI1 and RI57) and their 482
wild-type plants Hei inoculated by M. oryzae strain Hoku1. The leaves with lesions were shown 483
here. Bar, 1 cm. B, The lesion number per leaf and the lesion length of transgenic lines and 484
wild-type plants. Lesion number and lesion length were measured at 7 dpi. Each bar indicates the 485
average and standard deviation of at least 30 seedlings. Significantly different values compared 486
with wild type plants are denoted by asterisks (*P < 0.05, **P < 0.01 by Dunnett’s test). C, Four 487
types of individual conidia were classified by microscopy using Uvitex-2B staining: Type I, M. 488
oryzae conidium (CO) without germ tubes; Type II, differentiated appressorium (APP) formation; 489
Type III, establishment of infection hypha (primary hypha, PHY); Type IV, branch formation on 490
infection hypha (secondary hypha, SHY). Bars, 20 μm. D, The percentages of rice–M. oryzae 491
interactions in each of the four types were detected in transgenic plants. At least 150 penetration 492
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20
sites were observed in each sample. Error bars represent standard deviations (SD) of three 493
technical replicates. 494
495
Figure 3 OsSYP121 accumulated at rice blast fungus penetration sites. Microscopy analysis of 496
GFP -OsSYP121 and GFP-OsSYP132 localization in transgenic plants inoculated with compatible 497
M. oryzae strain Hoku1. A, The GFP-OsSYP121 was localized at the plasma membrane before 498
inoculation. B-D, GFP-OsSYP121 accumulated at rice blast fungus penetration sites in PSYP121: 499
GFP- SYP121- transgenic plants. E-F, GFP- OsSYP132 was localized at the plasma membrane in 500
PSYP132: GFP-SYP132 transgenic plants before (E) or after inoculation (F). G-H, No 501
auto-fluorescence was detected in wild type plants Su (S) before (G) or after inoculation (H). 502
Arrows marked the appressorium of M.oryzae. Bars, 10 μm. GFP: Green Fluorescent Protein 503
(green). BF: Bright Field. 504
505
Figure 4 Characterization of OsSYP121 interaction with OsSNAP32 protein. A, Yeast two-hybrid 506
assays indicate interactions of OsSYP121 with OsSNAP32 and OsVAMP714/724. B, BiFC assay 507
for OsSYP121 and OsSNAP32 interaction in N. benthamiana leaves. The chlorophyll 508
autofluorescence (red), YFP fluorescence (yellow), bright field, and combined images were taken 509
with a confocal microscope 2-4 d after transfection. Bars, 20 μm. PM: Plasma membrane; YFPC: 510
Yellow Fluorescent Protein C-terminal; YFPN: Yellow Fluorescent Protein N-terminal. 511
512
Figure 5 OsSYP121 interact with OsSNAP32 to mediate penetration resistance to rice blast 513
fungus. A, The phenotype of OsSYP121RI, OsSNAP32RI and OsSYP121RI OsSNAP32RI lines 514
and wild-type plants Su infected by M. oryzae. The leaves with lesions were shown here. Bar, 1 515
cm. B, The lesion number per leaf of transgenic lines and wild-type plants. Lesion number were 516
measured at 7 dpi. Each bar indicates the average and standard deviation of at least 30 seedlings. 517
Significantly different values compared with wild type plants are denoted by asterisks (*P < 0.05, 518
**P < 0.01 by Dunnett’s test). C, Histograms show the percentages of rice–M. oryzae interactions 519
in each of the four types represented in transgenic plants. At least 150 penetration sites were 520
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21
observed and categorized into the four types. D, The lesion length of transgenic lines and 521
wild-type plants were measured. Lesion length were measured at 7 dpi. Each bar indicates the 522
average and standard deviation of at least 30 seedlings. 523
524
Figure 6 GO enrichment analysis of microarray showed that OsSYP121 can trigger vesicle 525
trafficking response. A, Venn diagram of the genes from different comparisons. Three biological 526
replicates and two transgenic lines were used for microarray analysis. The genes with 1.5 folds 527
changes compared with control were considered as the different expression genes. B, GO 528
enrichment analysis were carried by AGRIGO GO terms, such as “biological process”, “molecular 529
function” and “cellular component”, were identified using AGRIGO 530
(http://bioinfo.cau.edu.cn/agriGO/ndex.php) with default significance levels (FDR< 0.05). 531
532
Figure 7 Expression patterns of differential expressed genes in microarray and reported plant 533
immunity pathway associated genes in transgenic lines OE8-Su, OE11-Su, RI1-Hei, RI57-Hei and 534
wild-types Su and Hei. The expressions of all genes in the microarray (transgenic line RI57-Hei 535
and Hei) are also shown. Three biological replicates were performed both in microarray and 536
RT-PCR experiments. Significantly different expressions compared with those of the wild-type 537
controls are denoted by asterisks (*P < 0.05, **P < 0.01 by Dunnett’s test). 538
539
Figure 8 Working model for the roles of OsSYP121 in rice-blast fungus interaction. In rice cell, 540
OsSYP121 can interact with OsSNAP32, VAMP714/724 to form the SNARE complex. In the 541
blast fungi invasion phase, OsSYP121 can accumulate at blast fungi penetration sites. The vesicle 542
trafficking and defense associated genes could be affected by knock known expression of 543
OsSYP121. 544
545 546
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0.00
0.20
0.40
0.60
0.80
1.00
1.20
OsSYP111 OsSYP121 OsSYP132
Rela
tive e
xp
ressio
n
0h
8h
24h
48h
72h
0.00
0.20
0.40
0.60
0.80
1.00
1.20
OsSYP111 OsSYP121 OsSYP132
Rela
tive e
xp
ressio
n
0h
8h
24h
48h
72h
Figure 1
Figure 1. Expression of OsSYP121 is induced by blast inoculation.
A, Tissue specific expression assays of SYP1s in rice variety Heikezijing (Hei). The expressions of OsSYP111,
OsSYP121 and OsSYP132 were detected by RT-qPCR. Actin was used as an internal control.
B, The expression patterns of four OsSYP1s genes in Heikezijing (Hei) and Suyunuo (Su) inoculated with the M.
oryzae strain Hoku1 were investigated by qPCR. The seedlings of Hei and Su were collected after inoculation for 0h,
8h, 24h, 48h and 72h. The expressions of OsSYP121 at 48hpi in Hei and at 8hpi in Su were the same and defined as
1. The amplification of the rice 18s-rRNA gene was used as an internal control. Error bars represent standard
deviations (SD) of three technical replicates.
B A
OsSYP111
OsSYP121
OsSYP132
Actin
0.2
0.4
0.6
0.8
Rela
tive e
xp
ressio
n
0.0
1.0
1.2
0.2
0.4
0.6
0.8 R
ela
tive e
xp
ressio
n
0.0
1.0
1.2 OsSYP111 OsSYP121 OsSYP132
OsSYP111 OsSYP121 OsSYP132
C
Hei
Su
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0
0.5
1
1.5
2
Figure 2
Figure 2. OsSYP121 was associated with penetration resistance to rice blast.
A, The blast resistant phenotypes of OsSYP121-OE lines (OE5-Su, OE8-Su, and OE11-Su) , OsSYP121-RI lines
(RI3-Su and RI7-Su) and their wild-type plants Su (Suyunuo), OsSYP121-RI lines (RI1-Hei and RI57-Hei) and their
wild-type plants Hei (Heikezijing) inoculated by M. oryzae strain Hoku1. The leaves with lesions were shown here. Bar,
1cm. 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 dpi. Each bar indicates the average and standard deviation of at least 30 seedlings.
Significantly different values compared with wild type plants are denoted by asterisks (*P < 0.05, **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 standard
deviations (SD) of three technical replicates.
0
0.5
1.0
1.5
2.0
0
10
20
30
40
Len
gth
of
lesio
n (
mm
)
Perc
en
tag
e o
f ty
pes (
%)
A B
C D
Nu
mb
er
of
lesio
n p
er
leaf
0.00
20.00
40.00
60.00
80.00
100.00
120.00
S OE5 OE8 OE11 RI3 RI7 H RI1 RI57
Type I
Type II
Type III
Type IV
0
0.2
0.4
0.6
0.8
1.0
1.2
OE5
-Su OE8
-Su
OE11
-Su
RI3
-Su
RI7
-Su
Su RI1
-Hei
RI57
-Hei Hei
OE5
-Su OE8
-Su
OE11
-Su
RI3
-Su
RI7
-Su
Su RI1
-Hei RI57
-Hei
Hei
OE5
-Su OE8
-Su
OE11
-Su
RI3
-Su
RI7
-Su
Su RI1
-Hei
RI57
-Hei Hei
OE5
-Su OE8
-Su
OE11
-Su
RI3
-Su
RI7
-Su Su
RI1
-Hei
RI57
-Hei Hei
10
20
0
Rela
tive e
xp
ressio
n
OsSYP121
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Figure 3
Figure 3. OsSYP121 accumulated at rice blast penetration sites.
Microscopic analysis of GFP-OsSYP121 and GFP-OsSYP132 localization in transgenic plants inoculated with M. oryzae
strain Hoku1. A, The GFP-OsSYP121 was localized at the plasma membrane before inoculation. B-D, GFP-OsSYP121
accumulated at rice blast penetration sites in PSYP121:GFP-SYP121 transgenic plants. E-F, GFP-OsSYP132 was
localized at the plasma membrane in PSYP132:GFP-SYP132 transgenic plants before(E) and after inoculation(F). G-H, No
auto-fluorescence was detected in wild type plants Suyunuo(S) before (G) or after inoculation (H). Arrows marked the
appressorium of M.oryzae. Bars, 10 μm. GFP: Green Fluorescent Protein (green); BF: Bright Field.
PS
YP
121::
GF
P-S
YP
121
PS
YP
132::
GF
P-S
YP
132
S
u
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Figure 4
Figure 4. Characterization of OsSYP121 interaction with OsSNAP32 protein.
A, Yeast two-hybrid assays indicate interactions of OsSYP121 with OsSNAP32 and OsVAMP714/724.
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 2-4 d after transfection. Bars, 20 μm. PM: Plasma membrane; YFPC: Yellow
Fluorescent Protein C-terminal; YFPN: Yellow Fluorescent Protein N-terminal.
Cub-OsSYP121+NubI
Cub+NubG
Cub-OsSYP121+NubG
Cub-OsSYP121+NubG-OsVAMP727
Cub+NubG-OsVAMP727
Cub-OsSYP121+NubG-OsVAMP724
Cub+NubG-OsVAMP724
Cub-OsSYP121+NubG-OsVAMP714
Cub+NubG-OsVAMP714
Cub-OsSYP121+NubG-OsVAMP711
Cub+NubG-OsVAMP711
SD(-LW) SD(-LWAH)+5mM 3-AT
Cub-OsSYP121+NubI
Cub+NubG
Cub-OsSYP121+NubG
Cub-OsSYP121+NubG-OsSNAP32
Cub+NubG-OsSNAP32
Cub-OsSYP121+NubG-OsVAMP721
Cub+NubG-OsVAMP721
Cub-OsSYP121+NubG-OsVAMP722
Cub+NubG-OsVAMP722
A
B
121:YFPC
32:YFPN Merged with Bright Field Chlorophy Ⅱ PM
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0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
Su OsSYP121RI OsSNAP32RIOsSYP121RIOsNAP32RI
Type I
Type II
Type III
Type IV
Figure 5
Figure 5. OsSYP121 interact with OsSNAP32 to mediate penetration resistance to rice blast.
A, The phenotype of OsSYP121RI, OsSNAP32RI and OsSYP121RI OsSNAP32RI lines and wild-type plants Su
infected by M. oryzae. The leaves with lesions were shown here. Bar, 1 cm. B, The lesion number per leaf of
transgenic lines and wild-type plants. Lesion number were measured at 7 dpi. Each bar indicates the average and
standard deviation of at least 30 seedlings. Significantly different values compared with wild type plants are denoted
by asterisks (*P < 0.05, **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 length of transgenic lines and wild-type plants were measured.
Lesion length were measured at 7 dpi. Each bar indicates the average and standard deviation of at least 30 seedlings.
Perc
en
tag
e o
f ty
pes (
%)
C
Nu
mb
er
of
lesio
n p
er
leaf
A B
Su OsSYP121RI OsSNAP32RI OsSYP121RI OsSNAP32RI
Su OsSYP121RI OsSNAP32RI
OsSNAP32RI
OsSYP121RI
Su OsSYP121RI OsSNAP32RI
OsSNAP32RI
OsSYP121RI
Len
gth
of
lesio
n (
mm
)
Su OsSYP121RI OsSNAP32RI
OsSNAP32RI
OsSYP121RI
D
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A Down regulated Up regulated
R57 VS Hei R1 VS Hei R57 VS Hei R1 VS Hei
0
10
20
30
40
50
60B
0
10
20
30
40
50
60
70
GO enrichment
C
Perc
en
t o
f g
en
es(%
) P
erc
en
t o
f g
en
es(%
)
Down regulated
Up regulated
GO enrichment
Figure 6
Figure 6. GO enrichment analysis of microarray showed that OsSYP121 can trigger vesicle trafficking response. A, Venn
diagram of the genes from different comparisons. Three biological replicates and two transgenic lines were used for microarray
analysis. The genes with 1.5 folds changes compared with control were considered as the different expression genes. B, GO
enrichment analysis were 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 (FDR< 0.05)
411 51 178 490 89 191
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0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
Su OE-8 OE-11 Hei R-1 R-57
OsWRKY21
0.0
1.0
2.0
3.0
4.0
5.0
6.0
Su OE-8 OE-11 Hei R-1 R-57
OsMYB20
0.0
0.5
1.0
1.5
2.0
2.5
Su OE-8 OE-11 Hei R-1 R-57
Re
lati
ve
Ex
pre
ss
ion
OsPILS7a (Auxin efflux carrier component)
Figure 7
0
0.5
1
1.5
2
2.5
3
3.5
4
Su OE-8 OE-11 Hei R-1 R-57
Re
lati
ve
Ex
pre
ss
ion
OsHSP90
0
0.5
1
1.5
2
2.5
Su OE-8 OE-11 Hei R-1 R-57
Rela
tive E
xp
ressio
n
OsRbohF
A B
D
E F
0.49 1.00
RI57 Hei
0.47 1.00
RI57 Hei
0.50 1.00
RI57 Hei
Os.7051.1.S1_at
Os02g49986 Os.30568.1.S1_at Os01g60640
Os.15679.1.S1_S_at
Os08g35210
0.62 1.00
RI57 Hei
Os.57460.1.S1_at
Os09g0482610
** **
** **
** **
** **
** **
* **
** **
Re
lati
ve E
xp
ressio
n
Su OE8-Su OE11-Su Hei RI1-Hei RI57-Hei Su OE8-Su OE11-Su Hei RI1-Hei RI57-Hei
Re
lati
ve E
xp
ressio
n
Re
lati
ve E
xp
ressio
n
Su OE8-Su OE11-Su Hei RI1-Hei RI57-Hei
Re
lati
ve E
xp
ressio
n
Su OE8-Su OE11-Su Hei RI1-Hei RI57-Hei
**
Figure. 7 Expression patterns of differential expressed genes in microarray and reported plant immunity pathway
associated genes in transgenic lines OE8-Su, OE11-Su, RI1-Hei, 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 qPCR experiments. Significantly different expressions compared with
those of the wild-type controls are denoted by asterisks (*P < 0.05, **P < 0.01 by Dunnett’s test).
0.0
2.0
4.0
6.0
8.0
Su OE-8 OE-11 Hei R-1 R-57
OsSNAP32
Su OE8-Su OE11-Su Hei RI1-Hei RI57-Hei
** **
** **
Re
lati
ve E
xp
ressio
n
OS.21394.1.S1_at
Os02g0437200
0.34 1.00
RI57 Hei ** **
** **
OS.47814.1.a1_s_at
Os09g38130
0.54 1.00
RI57 Hei
C
**
Su OE8-Su OE11-Su Hei RI1-Hei RI57-Hei
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OsSYP121-OsSNAP32-
VAMP714/724
Vesicle Trafficking
Rice cell
Blast fungus
Plasma membrane
Defense
Figure 8. Working model for the roles of OsSYP121 in rice-blast interaction.
In rice cell, OsSYP121 can interact with OsSNAP32, 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 knock known expression of OsSYP121.
Figure 8
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