Studies Of The Interactions Of Brassica Yellows Virus P0 With NbSKP1 And NbRAF2 In Nicotiana Benthamiana

RNA silencing is an efficient and versatile mechanism protecting plants from infection of various viruses.To avoid the antiviral silencing,plant viruses have evolved different counter-defense strategies,most notably through the production of viral suppressors of RNA silencing(Viral suppressor of RNA silencing,VSRs).Poleroviruses are worldwide distributed,infect many crops of economic importance,and cause serious plant diseases.The P0 protein encoded by this genus is a VSR,and there are differences in sequence and silencing supreesion functions of P0 proteins.P0 proteins harbor a consensus F-box-like motif reported to be essential for P0-SKP1 interaction,however,Potato leafroll virus P0 that has conserved F-box-like motif can not interact with SKP1.P0-SKP1 interaction in silencing suppression is still controversial.Meanwhile,few studies about the P0-host factors interactions were reported.Therefore,in our study,we mainly focus on the P0BrA encoded by Brassica yellows virus A genotype,identification of crucial amino-acid domain of P0BrA,and the biological significance of P0BrA-SKP1 and P0BrA-NbRAF2 interaction.In our studies,a series of truncated and deleted mutants of the P0BrA were constructed,and essential amino acids were identified.A single amino acid deletion at position 2 significantly attenuated RNA silencing suppression,and completely abolished cell death induction.A single amino acid deletion at position 3 abolished RNA silencing suppression and cell death induction.Deletion of aa 225-249 in the C-terminus of P0BrA was still able to suppress local RNA silencing but abolished cell-death induction.Interestingly,deletion of aa 224-249 at the C-terminus abolished both VSR and cell death induction function of P0BrA.We analyzed the interaction between P0BrA mutants Q2A,LP,and LPK44A with NbSKP1,and the effect of these three mutants on the the degradation of AGO1.We found that Q2A without silencing suppression activity can interact with NbSKP1,but can not degrade AGO1.However,LPK44A with silencing suppression activity can not interact with NbSKP1,but can degrade AGO1.Global mutagenesis and comparative functional analysis showed that P0BrA silencing suppression activity does not depend on P0-SKP1 interaction.The F-box-like motif mutant of P0BrA is destabilized in vivo,and our results indicated the involvement of 26S proteasomes system pathway in destabilization of the mutant protein.We proved that the P0BrA-SKP1 interaction is likely to facilitate protein stability of P0BrA.To further explore the functions of P0 in polerovirus-host interactions,we identified a P0BrA-interacting protein from Nicotiana benthamiana,Rubisco Assembly Factor 2(NbRAF2).The split ubiquitin membrane-bound yeast two-hybrid system and co-immunoprecipitation assays showed that NbRAF2 interacted with P0BrA.Confocol microscopy showed that NbRAF2 localizes in the nucleus,cell periphery,chloroplasts and stromules,and colocalized with P0BrA in the nucleus and cell periphery.In addition,the nuclear pool of NbRAF2 decreased in the presence of P0BrA,and the P0BrA-mediated reduction of nuclear NbRAF2 required dual localization of NbRAF2 in chloroplast and nucleus.The analysis of P0BrA mutants indicated that P0BrA-mediacted the reduction in nucear pool of NbRAF2 did not require the suppression,cell death induction,and SKP1-interaction activities of P0BrA.Silencing NbRAF2 promoted BrYV-A infection in N.benthamiana,and the overexpression of nuclear NbRAF2 inhibited BrYV-A accumulation.Potato leafroll virus P0PL also interacted with NbRAF2 and decrease its nuclear accumulation,indicating that NbRAF2 may be a common target of poleroviruses.Therefore,it was concluded that nuclear NbRAF2 possesses antiviral activity against BrYV-A infection,and that BrYV-A P0BrA interacts with NbRAF2 and inhibits its nuclear accumulation to facilitate BrYV-A infection.Our results showed that P0BrA-SKP1 interaction is likely to facilitate protein stability of P0BrA;P0BrA-RAF2 interaction showed that P0 impairs host resistance through interacting with host chloroplast/nuclear protein to facilitate virus infection.Our results improve our understanding of multifunctional P0.