Interactions Between Rice Stripe Virus(RSV) P3 Protein And Host Factors

Rice stripe disease has repeatedly broken out and caused extremely heavy losses to rice production in China.The disease is caused by Rice stripe virus(RSV).The viral protein p3,an RNA silencing suppressor encoded by RSV,is thought to play a key role in the viral pathogenesis.In order to investigate the pathogenic mechanism of p3,its two candidate target proteins were screened from rice c DNA library by yeast two-hybrid(YTH)assay.The two candidates were rice WRKY transcription factor 40(Os WRKY40)and rice lipid-related plastid protein.(Plastid lipid-associated protein 2,Os PAP2),and then their full-length encoding regions were cloned,respectively.At the same time,their homologous genes in N.benthamiana,namely Nb PAP and Nb WRKY40,were also cloned.Furthermore,YTH,Pull-down and Bimolecular fluorescence complementation(Bi FC)assays confirmed that Os PAP2,Nb PAP,Os WRKY40 and Nb WRKY40 could interact with p3 in vitro and in vivo.Subcellular localization studies showed that Os PAP2 and Nb PAP proteins were mainly localized in chloroplasts,while Os WRKY40 and Nb WRKY40 proteins were specifically distributed in the nucleus.RSV p3 protein was localized in cytoplasm and nucleus when expressed alone;however,in the Bi FC assays,the interaction of p3 and Os PAP2 or Nb PAP was found to occur mainly in the chloroplast,indicating that the interaction may change the subcellular localization of p3.The expression patterns of Os PAP2 and Os WRKY40 genes were further analyzed using real-time PCR in different tissues(roots,stems,leaves or spikes)of N.benthamiana,and in response to different abiotic stresses,biotic stress(RSV infection)and hormones.The results showed that both Os PAP2 and Os WRKY40 had the highest expression in leaves,and were induced by RSV infection and ethylene(ETH).After Nb PAP and Nb WRKY40 were respectively silenced in N.benthamiana by virus-induced gene silencing(VIGS),the chlorotic and albino phenotype was found in Nb PAP-suppressed plants and dwarfing phenotype in Nb WRKY40-suppressed plants.The photosynthesis-related parameters assay showed that the net photosynthetic rates were decreased significantly in both p3-expressing plants and Nb PAP-suppressed plants.These results indicated that p3 and Nb PAP interaction might inhibit the photosynthesis of plants and that Nb WRKY40 might be involved in the growth and development of plants.In summary,p3 protein could participate in the pathogenesis of RSV by interacting with PAP and WRKY40 of host plants.This study lays a solid foundation for further study on the action mechanism of p3 protein.