Research On The Mechanism Of β Tubulin Of Laodelphax Striatellus Regulating The Accumulation Of Rice Stripe Virus In Insect Vector

Rice stripe virus(RSV),as one of the most harmful plant diseases in rice growing areas in East Asia,is mainly replicated and transmitted in its vector insect,Laodelphax striatellus.Previous studies have found that the interaction between α2-tubulin in Laodelphax striatellus and NS3,a non-structural protein of RSV,affects vector transport and promotes the horizontal transmission of the virus.However,there are many subtypes of tubulin in Laodelphax striatellus.The function of different subtypes of tubulin on the transport,replication and accumulation of RS V in mediators is not clear.In this paper,the mechanism of tubulin β3 subtype regulating RSV accumulation in Laodelphax striatellus was studied.Through GeneBank,the amino acid sequences of six insect tubulin families which including Laodelphax striatellus were obtained and a phylogenetic tree was established after homologous comparison,we found that β-tubulin was highly conservative in insects.Then,the open reading frame sequences of one β-tubulin subtypes of Laodelphax striatellus was amplified by PCR.After comparison,it was found that it was β3-tubulin of Laodelphax striatellus.Through RT-qPCR and Westemblot detection,it was found that the expression ofβ3-tubulin mRNA and protein in poisonous Laodelphax striatellus was 300%higher than that of non-toxic Laodelphax striatellus.Furthermore,the interaction between β3-tubulin and RSV non-structural protein NS3 was found by yeast two-hybrid and Co-IP system,and there may be interaction sites with RSV NS3 in the four functional domains of β3-tubulin detected by piecewise yeast two-hybrid.Immunofluorescence confocal observation showed that β-tubulin and RSV NS3 were co-located in midgut,salivary gland and ovary.In conclusion,RSV infection up-regulated the expression of β3-tubulin in Laodelphax striatellus,and there was a direct interaction between β3-tubulin and RSV NS3,indicating that β3-tubulin may play an important role in virus vector transmission through this interaction.In order to explore the function of the interaction between β3-tubulin and RSV NS3 in the transmission of RSV mediators,RNAi interference technique was used to silence the expression of β3-tubulin in infected Laodelphax striatellus.Inhibition of β3-tubulin expression resulted in a 99%decrease in RSV titer in Laodelphax striatellus,and a consistent decrease in protein content of RSV NP.The inhibition of β3-tubulin expression resulted in a decrease of 61%,41%and 42%of RSV titer in midgut,salivary gland and ovary of Laodelphax striatellus,respectively.Immunofluorescence confocal observation also showed that the silencing of β-tubulin expression significantly decreased the distribution of RSV in different tissues of Laodelphax striatellus.Based on the heterodimer formed by eukaryotic a and β-tubulin,we further studied the synergistic effect of β-tubulin and α-tubulin on the transmission of RSV mediators.The expression of β3-tubulin was inhibited by RNAi interference,and it was found that the expression of α1/α2 tubulin and α8 tubulin decreased respectively 52%and 88%.Based on this,the co-inhibition of α8-tubulin and β3-tubulin resulted in a significant decrease 86%in RSV titer.It can be seen that a8-tubulin and β3 tubulin play a synergistic role in the accumulation of RSV.On the basis of previous studies,this paper clarified that RSV infection led to the upregulation of another tubulin β3 subtype of Laodelphax striatellus,and that β3-tubulin promoted the accumulation of RSV in the mediator by interacting with RSVNS3 multiple sites.Further studies showed that α8 tubulin and β3 tubulin of Laodelphax striatellus cooperated with each other to regulate the accumulation of RSV mediators.The research results provide data for explaining the interaction mechanism between plant viruses and vector insects,and provide a new strategy for the development of rice stripe virus control techniques for Laodelphax striatellus β-tubulin.