Functional Analysis Of Brassica Yellows Virus P3a Protein In Virus Systemic Infection

Viral long-distance movement is a key process during the viral systemic infection.During the past few decades,lots of efforts were devoted to determining the viral factors involved in systemic infection of virus and to elucidating the molecular mechanisms underlying this process.Recently,a newly identified small open reading frame(ORF)in poleroviruses,termed ORF3a,was reported to encode a P3a protein which is participating in the systemic infection of poleroviruses.However,the function of P3a is not completely understood,and little is known about the mechanism for the P3a-dependent systemic infection.In this dissertation,molecular biology and biological chemistry analysis were conducted to further understand the role of P3a in systemic infection.Sequencing of a brassica yellows virus(BrYV)mutant defective in systemic infection revealed two-nucleotide variation at positions 3406 and 3467 in the genome.Subsequent nucleotide substitution analysis proved that only the nonsynonymous substitution(C?U)at position 3406,resulting in P3aP18L abolished the systemic infection of BrYV.Preliminary investigation showed that the wild type BrYV was able to load into the petiole of the agroinfiltrated Nicotiana henthamiana leaves,whereas the mutant were unable.Further experiments revealed that the P3a and its mutant P3ap18L localized to the Golgi apparatus and near the plasmodesmata,as well as the endoplasmic reticulum.Both P3a and P3aP18L were able to self-interact in vivo,however,the mutant P3aP18L seemed to form more stable dimer than wild type.More interestingly,it is confirmed firstly that the ectopic expression of P3a of other poleroviruses and luteoviruses,as well as co-infection with Pea enation mosaic virus2(PEMV 2),restored the ability of systemic movement of BrYV P3a defective mutant,indicating that the P3a is functionally conserved in poleroviruses and luteoviruses and is redundant when BrYV co-infects with PEMV 2.These observations provide a novel insight into the conserved function of P3a and its underlying mechanism in virus systemic infection.The BrYVs were divided into three genotypes(BrYV-A,-B,and-C)which have greater divergence in ORF0 than other ORFs.The sequence identity between the three POs is 86.7-89.2%.Here,agrobacterium mediated transient co-expression assay revealed that P0BrB and P0BrC also have local and systemic RNA silencing suppressor activities.Transient expression of P0BrB or P0BrC caused weaker necrotic symptom than P0BrA.In this study,several key amino acid sites in P0BrA that affect the pathogenicity of the protein were confirmed.Transcriptomic analysis showed the differentially expressed genes were unique to BrYV+PEMV 2 infection in the early stage.The accumulation of BrYV was not obviously increased in PEORF4 transgenic N.benthamiana.The antiserum against the PEMV 2 MP were prepared and can be well used for detection of PEMV 2.