Molecular Mechanisms Underlying Suppression Of RNA Silencing By Brassica Yellows Virus P0 Protein

The poleroviruses are worldwide distributed infecting many crops of economic importance and causing severe plant diseases and yield losses.Brassica yellows virus(BrYV),a newly identified polerovirus in Chinese crucifer,harbors six open reading frames(ORFs)and encodes PO protein by its ORFO.As previously reported,PO proteins of several poleroviruses were viral suppressors of RNA silencing(VSR).However,the VSR activities of different PO proteins and the mechanism of how PO suppresses RNA silencing remain unascertained.Here,this dissertation examined the VSR functions of PO encoded by BrYV genotype A(P0BrA)and analyzed the related molecular mechanisms.Agrobacterium mediated transient co-expression assay revealed that P0BrA suppressed both local and systemic RNA silencing induced by single-stranded RNAs(ssRNAs)as well as local RNA silencing induced by double-stranded RNAs(dsRNAs).Through northern blot analysis,P0BrA was proved to inhibit the accumulation of both primary and secondary siRNAs.We further used several point mutations of P0BrA to identify key amino acids required for VSR activities.The results showed that suppression of systemic RNA silencing and inhibition of the accumulation of 24 nt siRNAs were abolished by the Ala substitution of the residue 184 Leu in POBrA.In contrast,the Ala substitutions of the F-box like motif signature residues(63 Leu/64 Pro)in POBrA destroyed the suppression activities on either the accumulation of 21/22 nt siRNAs or local RNA silencing.These results also imply that 21/22 nt and 24 nt siRNAs induce local silencing and systemic silencing respectively.Collectively,the data demonstrate that P0BrA is a strong VSR that inhibits the accumulations of both primary and secondary siRNAs of length 21 nt,22 nt and 24 nt.According to the above genetic results,we carried out some preliminary studies to investigate the molecular mechanism underling the strong VSR activities of P0BrA.Consistent with previous studies of other PO proteins,P0BrA could also mediated AGO1 degradation.However,point mutant assay showed that some unknown mechanism,other than AGO1 degradation,was required for the suppression of systemic RNA silencing by P0BrA.Here,we found that,a down regulation of DCL3 expression in N.benthamiana leaves transiently overexpressed P0BrA or its mutant protein L63A/P64A.Meanwhile,the transiently expressed P0BrA or its L63A/P64 mediated an increased dsRNA cleavage activity of N.benthamiana DCLs in vitro but decreased the accumulations of cleavage products.The electrophoretic mobility shift assay(EMSA)showed that P0BrA didn't exhibit binding activities to long dsRNAs.Collectively,these findings revealed that P0BrA might inhibit the transcription level and dicing process in a long dsRNA independent manner.To clarify the subcellular localization of P0BrA,N.benthamiana leaves expressing P0BrA were separated into nuclear and cytoplasmic fractions for western blot analysis,showing that P0BrA was present mainly in cytoplasmic fraction rather than nuclear fraction.The subcellular localization of transiently expressed GFP fusion P0BrA was examined in N.benthamiana leaves.The results showed a localization of P0BrA in granules in the cytoplasm and co-localized with RNA-dependent RNA polymerase 6(RDR6)or suppressor of gene silencing 3(SGS3).In a transient over-expression system,the SGS3 from Arabidopsis thaliana could be co-immunoprecipitated with P0BrA-3FLAG in N.benthamiana leaves.Besides,the biogenesis of trans-acting siRNA(tasiRNA)in a reconstruction system using N.benthamiana was abolished by P0BrA.These results indicated that P0BrA might interfere with the siRNA amplification process.Besides the RNA silencing suppressor activity,transiently expressed P0BrA in N.benthamiana leaves also induced hypersensitive response(HR),which was accompanied with the accumulation of Reactive oxygen species(ROS)and induction of pathogenesis-related gene 1(PR1)expression.Meanwhile,this necrosis phenotype induced by P0BrA in N.benthamiana leaves could be delayed by salicylic acid(SA)pretreatment.We further use the GAL4 yeast two-hybrid system to screen host factor interacting with POBrA from an Arabidopsis thaliana cDNA library,and obtained five candidates.Immunoprecipitation assay combined with mass spectrometric analysis(IP/MS)were also performed in N.benthamiana,and six candidate proteins were found to be interact/associate with P0BrA.In a separate project,a new carlavirus,tentatively named as Potato virus H(PVH),was found on potato plants in China,Hohhot.Complete genomic sequences of two isolates of PVH were determined using reverse transcription-PCR(RT-PCR)and the 5' rapid amplification of cDNA ends(5' RACE)method.Sequence analysis revealed that PVH had the typical genomic organization of members of the genus Carlavirus,However,PVH shared coat protein(CP)and replicase amino acid sequence identities lower than 70%with those reported carlaviruses.Phylogenetic analyses based on the amino acid sequences of replicase and CP showed that PVH formed a distinct branch,which was related only distantly to other carlaviruses.After prokaryotic expression and purification,CP protein of PVH was prepared to raise the polyclonal antiserum against PVH in a New Zealand rabbit.Western blotting assays showed that PVH was not related serologically to other potato carlaviruses.Besides,PVH has a distinct host range in contrast with other potato carlaviruses,and was capable of co-infecting potato plants with several potato viruses.The PVH particles were filamentous and slightly curved,with a modal length of 570 nm.All these results support the classification of PVH as a novel species in the genus Carlavirus.Preliminary results also indicated that a cysteine-rich protein encoded by the smallest ORF located in the 3' proximal region of the genome suppressed local RNA silencing and enhanced the pathogenicity of the recombinant PVX.