Functional Analysis Of SNARE Protein OsSNAP32 In Resistance To The Blast In Rice(Oryza Sativa L.)

The membrane fusion in vesicle trafficking in plant cells is mediated by SNARE(soluble N-ethylmaleimide-sensitive-factor attachment protein receptors)proteins,which could be involved in cytokinesis,regulation of plant growth and development,interaction with ion channel proteins,or response to abiotic stress and disease resistance.Rice SNARE protein gene OsSNAP32 was in silico cloned by our lab in japonica rice landrace Heikezijing resistance to rice blast The OsSNAP32 encodes a protein containing both Qb-SNARE and Qc-SNARE domains.The function of OsSNAP32 in resistance to the blast in rice(Oryza sativa L.)was analyzed in this research,and the main results were as follows:The full-length gene sequence of OsSNAP32 was relatively conserved between resistant-blast Heikezijing and susceptible-blast japonica rice landrace Suyunuo.Tissue expression analysis showed similar expression pattern of OsSNAP32 between Heikezijing and Suyunuo.OsSNAP32 constitutively expressed in various rice tissues,with higher expression in the leaves and flowering panicle,lower expression in root and stem,and with slightly higher expression in Heikezijing than in Suyunuo.The expression of OsSNAP32 showed a rapid increase in both Heikezijing and Suyunuo at 2 hpi with rice blast Hoku 1,then decreased,with greater induction of OsSNAP32 expression in Heikezijing than that in Suyunuo.By rice protoplast expression system it was found that the subcellular localization of OsSNAP32::GFP fusion protein was located in the plasma membrane and cytoplasm.The full-length gene sequence of OsSNAP32 was cloned in resistant-blast rice landrace Heikezijing,using bioinformatics and Agrobacterium-medidted transgenic methods it was obtained six OsSNAP32 overexpressing transgenic lines in Suyunuo(OE6,j OE9,OE13,OE15,OE17 and OE26),four OsSNAP32 RNAi transgenic lines in Suyunuo(RS1,RS16,RS18 and RS20),and five OsSNAP32 RNAi transgenic lines in Heikezijing(RH2,RH4,RH7,RH10 and RH13).These OsSNAP32 transgenic lines(T3)were inoculated by one Japanese blast race Hoku 1 and six Chinese blast races(191ZB13,97-2ZC15,35-1ZD1,55-1ZE3,42-2ZF1 and 113ZG1)at the three-leaf stage,respectively.The results showed,a week after inoculated with Hoku 1,compared with non-transgenic control Suyunuo,the lesions number on the leaves of OsSNAP32 overexpressing transgenic lines was significantly reduced,but no significant difference in the lesion size,the lesions number on the leaves of OsSNAP32 RNAi transgenic lines was increased,but no significant difference in the lesion size;no lesions were observed on the inoculated leaves of the resistant control Heikezijing as reported previously,however,some typical lesions were observed on those leaves of OsSNAP32 RNAi transgenic lines.Results similar to those with Hoku 1 were obtained after inoculated with mixed six Chinese races.The OsSNAP32 transgenic lines(T2)were inoculated with mixed six Chinese rice blast races(191ZB13,97-2ZC15,35-1ZD1,55-1ZE3,42-2ZF1 and 113ZG1)at booting stage in order to identify the panicle blast resistance.The results showed,compared with non-transgenic control Suyunuo,the lesion area on the flag leaf sheathes of OsSNAP32 overexpressing transgenic lines was significantly reduced,up to 9.39?23.50%,the lesion area on the flag leaf sheathes of OsSNAP32 RNAi transgenic lines was significantly increased,up to 16.44-18.25%.Compared with non-transgenic control Heikezijing,the incidence of panicle blast in OsSNAP32 RNAi transgenic lines was increased in different degrees.Using GAL4 yeast two-hybrid system OsSNAP32 was used as "bait" protein,three interacting proteins were screened in rice young panicle cDNA library,named S32IP3,S32IP5 and S32IP6.S32IP3 contains one KIP1 conserved domain and may be involved in rice disease resistance by KIP1 domain interacting with some kind of rice receptor-like kinase.S32IP5 contains Adaptin N and B2-adapt-app_C conserved domains,may be involved in intracellular vesicle transport.S32IP6 contains a N-terminal C2 domain,may be involved in regulating endocytic vesicles circulation,as well as rice growth.Therefore,we hypothesized that OsSNAP32 may be involved in rice desease resistance by interacting with S32IP3,intracellular vesicle transport by interacting with S32IP5,and regulating endocytic vesicles circulation,as well as rice growth by interacting with S32IP6.In summary,it was supposed that rice SNARE protein OsSNAP32 possibly played a role in the process of rice blast resistance to penetration through interacting with other proteins,which provided a foundation for molecular mechnism research on rice-Magnaporthe oryzae interaction in future.