The ?-tubulin Of Laodelphax Striatellus Interacts With Non-Structural Protein NS3 Of RSV To Assist Virus Transmission

Rice stripe virus(RSV),is responsible for significant yield losses in rice production in temperate and subtropical East Asia.RSV is transmitted by the small brown planthopper(SBPH),Laodelphax striatellus,in a persistent-propagative and transovarial manner.RSV has filamentous ribonucleoprotein particles(RNPs)that contain four single-stranded RNAs encoding seven proteins.The nonstructural protein 3(NS3)is the most abundant transcript in viruliferous SBPH.It is suspected that NS3 can participate in suppressing the host immune response in both plants and the insect vector.In order to explore the role of RSV NS3 in virus transmission,we have successfully constructed a cDNA library of SBPH in the early stage,and screened out the interacting proteins by the pGBKT7-NS3 bait vector.Among these proteins,the mRNA expression levels of ?-tubulin was significantly higher in the salivary glands or whole body of viruliferous L.striatellus,compared with the naive vectors in the previous studies.Here,we used yeast two hybrid system,RNAi silencing,immunofluorescence confocal,Western blot to investigate the effect of interaction between a-tubulin and RSV NS3 on virus transmission.The results were as follows.(1)It was confirmed that NS3 interacted with LsTUB by Y2H and Pull-down assays.NS3 and LsTUB were co-located in salivary gland,midgut and ovary.Subsequent Q-PCR and Western Blot experiments showed that suppression of LsTUB expression resulted in decreased mRNA level and proteomic level of RSV.These findings indicated that LsTUB interacts with RSV NS 3 and may have an important role in RSV propagation in the vector.(2)The interaction site of RSV NS3 and LsTUB was verified by yeast two-hybrid.It was found that the binding site of NS3 and LsTUB located in amino acid residues at positions 74-76 and 80-82,which is a region necessary for NS3 interaction and silencing suppressor activity.Further,the BiFc experiment was used to detect the RSV NS3 silencing inhibitory activity in tobacco,and the result indicated that the presence of LsTUB didn't affect the silencing suppressor activity of RSV NS3.In Sf9 cells,LsTUB didn't affect the efficient replication of RSV within 24 hours after RSV infection.However,suppression of LsTUB strongly inhibited the spread of RSV from the initially infected cells of the midgut to the salivary glands in SBPHs.In summary,tubulin in SBPH affected the accumulation and transmission efficiency of RSV and it was directly caused by the slower RSV transmition in the SBPH processed by dsLsTUB.These studies provide a basis for elucidating the interaction between plant viruses and insect vectors,and may lead to new disease control strategies.