Identification And Preliminary Fuctional Studyof WBPH Proteins Interacted With SRBSDV S-2827

The white backed planthopper(WBPH),Sogatella furcifera,is an important agricultural pest,and it threats the rice by sucking of plant sap and spreading the rice virus,Southern rice black-streaked dwarf virus(SRBSDV),in a persistent and propagative manner.The outbreak of viral disease has caused huge economic losses to the rice production in China.The screening and identifying of proteins interacting with SRBSDV in WBPH cells,and further identifying of the key genes affecting the acquisition,replication and transmission of SRBSDV,cannot only provide a deep understanding of the interaction mechanism between WBPH and SRBSDV,but also facilitate to identify new target molecules suitable for the management of rice virus transmission through biological control of the vector insects,which has important scientific and practical application value.Through bioinformatics analysis,the negative chain of S1 segment of SRBSDV was predicted to encode a protein with a length of 100 amino acids and a size of approximately 11 kDa.We temporarily named this protein as S-2827 in this study and 189 proteins interacting with S-2827 were screened by tandem affinity purification(TAP)and mass spectrometry.Finally,three WBPH proteins involved in endocytic pathway,CathD,Vps29 and Cct3,were identified as the interacting proteins with the S-2827 protein through the co-immunoprecipitation(Co-IP).And the three genes had high expression levels in the midgut and hindgut tissues of the WBPH,indicating that the three genes may play important roles in the intestinal tissues when SRBSDV infects the WBPH.To investigate the roles of these interacting genes after the SRBSDV infection,knockdown of each gene was performed through RNA interference(RNAi)in WBPH,followd by feeding the treated WBPH on SRBSDV-infected rice plants and then investigating the replication levels of SRBSDV in these WBPH.The results showed that the viruliferous rate and replication level of SRBSDV were both decreased in WBPH after knocked down of CathD and Cct3 genes.At the fourth day of the incubation period,the virus replication level of the WBPH in the dsCathD and dsCct3 groups were decreased to 32.2%and 8.9%compared with the GFP group,indicating that CathD and Cct3 may paly a role in assisting of the virus replication through endocytosis.However,after knocked down of Vps29 gene,there is no significant change of the viruliferous rate in the WBPH,while the replication level of SRBSDV was significantly increased.At the fourth day of the virus incubation period,the virus replication level of WBPH in dsVps29 group was increased to 2.8 times of that in GFP group,indicating that Vps29 may have an antiviral effect.Taken together,we identified the S-2827 protein expressed by SRBSDV through bioinformatics analysis.The binding of S-2827 to Vps29,CathD and Cct3 proteins involved in the claritin-dependent endocytosis pathway was identified by tandem affinity chromatography,mass spectrometry and co-immunoprecipitation.The effects of the three interacting proteins on the replication and transmission of SRBSDV in WBPH were analyzed,providing a crucial basis and direction for further research on the function of S-2827 protein and the interaction between virus protein and insect host.