Quantitative Proteomics Analysis Of Rice Leaves Infected By Rice Stripe Virus And RNAi-mediated Transgenic Rice Resistance To Rsv And Rice Black Streaked Dwarf Virus

Rice is one of the most important cereal crops in the world, especially in Asian counties. Many plant viruses result in the yield loss of rice, including Rice stripe virus(RSV) and Rice black-streaked dwarf virus(RBSDV), which are two members of economically important viruses. RSV-induced typical symptoms are chlorotic stripes and necrosis of newly emerged leaves. However, the mechanism of RSV-induced symptoms was not clear. As it is impossible to cure the infected plants, choosing resistant cultivars is both economic and effective in controlling the plant viral diseases. As the conventional resistant resource of rice cultivars is currently limited, improving viral resistance of rice is one of the current issues of breeding program. Therefore, studying the mechanism of RSV-induced symptoms will improve our knowledge in the breeding process.In this study, using an i TRAQ(isobaric tags for relative and absolute quantitation) approach, 681 differentially expressed proteins were identified in the leaves of RSV-infected and noninfected rice. A bioinformatics analysis indicated that over 70% of downregulated differentially expressed proteins were located in the chloroplast and took part in the chlorophyll metabolism. Magnesium chelatase subunits CHLI(magnesium chelatase subunit I) and CHLD(magnesium chelatase subunit D), which were involved in chlorophyll biosynthesis, expressed reduction on the m RNA and protein level, suggesting that chlorophyll biosynthesis of rice leaves was inhibited during RSV infection. In addition, Aspartic protease(ASP) was one of the differentially expressed proteins with elevated expression after RSV invasion. During the normal development of rice, Aspartic protease triggered the programmed cell death. The accumulation of Aspartic protease during RSV infection may lead to chlorosis and wither on the leaves. After RSV infection, plant defense was triggered by the expression of Bet v1 allergen family proteins, HSP70(Hot shock protein 70 k Da), and superoxide dismutase. Our yeast two-hybrid assay showed an interaction between protein RAP and RSV SP, which suggested that SP accumulation induced the upregualtion of RAP after RSV infection. Together, these findings may yield new insights into mechanisms underlying rice stripe disease symptom development.Using transgenic technology, the transgenic rice lines containing RSV-pc3-hp RNA derived from RSV nucleocapsid was generated, and manifested resistance to RSV to increase genetic resource. Small RNA(s RNA) blot analysis showed that the 21 nt-24 nt small interfering RNAs(si RNAs) complementary to the RSV nucleocapsid sequences conferred resistant activity, suggesting that the si RNAs derived from the RSV-pc3-hp RNA and RNAi mediated the resistance to RSV. Compared to that of the nontransgenic rice line, the expression of Argonaute(AGO) 1B, AGO1 C, AGO1 D, RNA-dependent RNA polymerase(RDR) 4, SHOOTLESS2(SHL2) and Dicer-like(DCL) 1A upregulated in the transgenic rice, while the expression of AGO1 A, AGO18 and SHOOT ORGANIZATION1(SHO1) downregulated. After RSV infection, AGO2 expression increased in the transgenic rice, compared to nontransgenic rice, while AGO1 B, AGO1 C, AGO1 D, AGO11, AGO16, RDR2, RDR4, SHL2, DCL1 A, DCL1 C and DCL3 B expressed reduction. It indicated that RSV-pc3-hp RNA was associated with the expression pattern of AGOs, RDRs and DCLs.Further, the RSV-HCP-RBSDV-HS10 chimeric fragment, which was comprised of part segments of the RSV pc3 and RBSDV S10, was inserted into p MCG161 for construction of a hairpin RNA(RSV-HCP-RBSDV-HS10-hp RNA) structure. Then the hp RNA was inserted into p CAMBIA1301(without hygromycin gene deleted previously) to obtain the p CAMBIA1301-DB. Transgenic rice was obtained using Agrobacteria-mediated transgenic technology. Basing on these results, RBSDV and RSV resistant to transgenic rice lines was selected after evaluation of resistance to the viruses in the field. In addition, the transgenic rice lines containing not hygromycin gene but RSV-HCP-RBSDV-HS10 gene was acquired by PCR screening, so the transgenic rice markerless gene conferred to resistant to both RSV and RBSDV. Southern blot analyses showed that the transgenic rice contained the multiple copies of the target gene, indicating that RSV-HCP-RBSDV-HS10 genes already were incorporated into the genome of rice. s RNA blot analyses showed that the 21 nt-24 nt si RNAs complementary sequence of the target gene existed in the transgenic rice, suggesting that the si RNAs derived from the hp RNA. So RNAi mediated the resistance to both RSV and RBSDV.