The Role Of SNARE Protein Gene OsSYP121 In Resistance To The Blast In Rice (Oryza Sativa L.)

SNARE (soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) is one of the proteins involves in membrane fusion, which plays important roles in regulation of growth and development, and in response to biotic and abiotic stresses in plant. The genomic sequences of both the blast (Magnaporthe oryzae) and rice (Oryza sativa L.) are available, providing a unique opportunity to study a host-pathogen interaction from both sides using function genomics approaches.The previous study of our lab has demonstrated that SNARE protein gene OsSYP121 (syntaxin of plant 121) is induciable by M. Oryzae through the gene expression profiles analysis of M. Oryzae interaction with rice using Microarray. In this paper, six SYP1 genes were identified in rice genome. We preliminarily studied the bioinformatics characteristics, expression profile and subcellular localization. Moreover, the roles of OsSYP121 in resistance to rice blast are mainly analyzed. The results are as follows:Six OsSYPl genes OsSYP111, OsSYP121, OsSYP124, OsSYP125, OsSYP131 and OsSYP132 were isolated using RT-PCR from rice seedling. Domain assay showed that three signature motifs of the SYP1 proteins are a SyN domain consists of Habc a-helices, a Qa-SNARE motif and a transmembrane domain, respresently. Based on the splicing pattern, the SYP1 subfamily from rice and Arabidopsis were divided into three groups: SYP11, SYP12 and SYP13 by The phylogenetic analysis. Exon-intron analysis indicated that SYP11s and SYP12s have either no or one intron, but SYP13s have multiple introns from eight to twelve. These results suggest that it is an ancient and conserved subfamily and occurs prior to the dicot-monocot split, but the differential splicing pattern reflect functional divergence.Semi-quantitative RT-PCR analysis shows that four genes have different tissue expression patterns. OsSYP121 and OsSYP132 were constitutively expressed, OsSYP111 and OsSYP124 were shown to be tissue-specific, OsSYP111 only expressed in tissues-containing dividing cells, e. g. roots and young panicles, OsSYP124 expressed highly in young panicles, and also expressed in leaf blades and sheaths. It is suggesting that each of four genes plays unique functions in growth and development of rice. Expression profile analysis of four genes in rice varieties resistant (Heikezijing) and susceptible (Suyunuo) to rice blast shows that OsSYPls were induced with various degrees, and especially the expression of OsSYP121 was significantly increased. The expression of OsSYP121 was increased and reached peak at 8 hpi, then decreased gradually in Suyunuo, in contrast, that was gradually and significantly increased within 24 hpi in Heikezijing. It’s demonstrated that OsSYP121 plays a role in resstance to rice blast in rice. Subcellular localization analysis showed that in the rice protoplast, OsSYP121 and OsSYP132 proteins were only localized on the plasma membrane, but OsSYP111 and OsSYP124 were localized on the plasma membrane and Golgi apparatus.Overexpression vector pCAMBIA-1304-OsSYP121 and suppression vector pTCK303-OsSYP121 were constructed and transformed using Agrobacterium-mediated transformation into Suyunuo and Heikezijing, respectively. Three overexpression (OE-5, OE-8, OE-11) and two suppression (R1, R57) transgentic lines were obtained. The phenotypic observation shows that the plant height of OsSYP121-OX lines significantly ’decreased in comparison of wild type (Suyunuo) due to the length of each internode shorted in OsSYP121-OX lines, meanwhile, seed setting rate and 100-seed weight decreased. Whereas, there was no difference of plant height between OsSYP121-RNAl lines and wild type (Heikezijing).The effect of OsSYP121 trangentic rice plants in response to M. Oryzae demonstrated that OsSYP121 plays an important role in resistance to M. Oryzae in rice. After 7 day post-inoculation, the symptom on leaves of Suyunuo were serious with gray spots and even coalescing with yellowing outside the reddish borders of lesion. By contrast, disease severity and the numbers of lesion decreased in OsSYP121-OX plants. Statistic analysis indicated that compared with Suyunuo, numbers of lesion reduced, but lesion length had no change in OsSYP121-OX plants. The germination and infection process of the spores were observed in OsSYP121-OX and Suyunuo plants after Uvitex staining, the result showed that the most of spores stasis at appressoriun phase and unable to penetration the cells of OsSYP121-OX plants until 24 hpi, the ratio of this phase was up to 62.65-70.01% and significantly higher than Suyunuo (25.61%). It is suggesting that over expression of OsSYP121 inhibit infection by M. Oryzae and decrease infection rate to increase disease resistanc of susceptible variety Suyunuo. Moreover, the expression of OsSYPl21 inhibited in different degree resulted in decreasing resistance to M. Oryzae in resistant variety Heikezijing with the emergence of gray spots, typically elliptical with gray to whitish centers.The coding region and promoter sequence of OsSYP121 from 32 rice varieties were analyzed for sequence difference. The sequencing results showed that there was no difference among coding region of OsSYP1 21, but had differences in its promoter region, including 38 SNP,2 base insertions and 2 base deletion. Multialignment analysis showed there are five types of the promotor of OsSYP121, so 32 rice varieties were classified into five groups based on the promotor sequence. Combined analysis of the cis-acting element in the promotor of OsSYP121, it was found that five base mutation resulted in the change of cis-acting elements including three light responsive elements and two abiotic stress responsive elements (heat stress responsiveness, HSE; MYB binding site involved in drought-inducibility, MBS).Using OsSYP121 as bait to screen cDNA library of Heikezijing via DUALmembrane system, three different OsSYP121-interacting proteins (121P1-3) were identified. Through BLAST and protein domain ananlysis, three OsSYPl21-interacting proteins were characterized as follow:121P1(Heat shock protein 82),121P2 (Photosystem II 10 kDa polypeptide,chloroplast precursor); 121P3 (Pyruvate dehydrogenase kinase isoforml.By yeast two-hybrid system and DUALmembrane system, it’s demonstrated that OsSYP121 is interacting with OsSNAP32 and OsVAMP714/724. So the further analyses in theory and experiment will be continued.These results suggest that the subfamily of OsSYPl is an ancient and highly conserved family, and each member has special functions in response to biotic and abiotic stresses in rice. Moreover, OsSYP121 involves in resistance to the blast in rice through vesicle trafficking.