Functional Study On AV2 Of Tomato Leaf Curl Hsinchu Virus

Begomoviruses are widely distributed in the world,posing a serious threat to the production of staple or economic crop plants including tomato,cassava,cotton and tobacco.A better understanding of the function of the proteins encoded by begomoviruses will provide new insights into the pathogenesis of begomoviruses and lay the basis for sustainable control of this group of viruses.Tomato leaf curl Hsinchu virus?ToLCHsV?is a begomovirus widely distributed in southern China.The function of ToLCHsV AV2 was dissected in this study.The main experiments and results are as follows:?1?Preparation of AV2 polyclonal antibody and detection of AV2 in host plants:The open reading frame?ORF?of ToLCHsV AV2 was cloned into PEASY?-Blunt E1 vector,and then the recombinant plasmid was introduced into E.coli BL21.By optimizing various experimental conditions,the AV2 protein successfully expressed in the bacteria.The bacterially expressed protein was used to immunize mice to obtain a polyclonal antibody to AV2.The accumulation of AV2 in ramie and tobacco plants infected by ToLCHsV was then confirmed by Western Blot experiments with the polyclonal antibody obtained here.?2?The importance of AV2 in the infection of ToLCHsV:A ToLCHsV mutant named ToLCHsV-?AV2,which is unable to express AV2,was created by site-directed mutagenesis.ToLCHsV-?AV2 was inoculated to Nicotiana benthamiana and N.tabacum using agroinfiltration.The results showed that ToLCHsV-?AV2 can infect both plants systemically and cause symptoms as severe as those caused by wild type ToLCHsV.However,the time of symptom appearance was delayed by 2 or 9 days in N.benthamiana and N.tabacum,respectively.the accumulation of ToLCHsV-?AV2 in infected plants was also significantly lower than ToLCHsV.?3?The ability of ToLCHsV AV2 to suppress transcriptional gene silencing?TGS?:The ORF of AV2 was cloned into the vector pGR107and the resulting recombinant Potato virus X,PVX-AV2,was inoculated to 16c-TGS N.benthamiana.The results showed that AV2 could greatly enhance the pathogenicity of PVX and decrease the cytosine methylation level of the 35S promoter driving the expression of a GFP mRNA in 16C-TGS N.benthamiana.This indicated that AV2 of ToLCHsV can suppress TGS.We confirmed this by showing that the cytosine methylation level of ToLCHsV-?AV2 is much higher than ToLCHsV in infected N.benthamiana.?4?The ability of ToLCHsV AV2 to suppress post transcriptional gene silencing?PTGS?:The ORF of AV2 was cloned into the transient expression vector pEarlyGate100,obtaining pEarlyGate100-AV2.Agrobacterium GV3101 harboring pEarlyGate100-AV2 was mixed with those harboring a binary vector expressing a green fluorescent protein?GFP?gene.The mixture was infiltrated into leaves of N.benthamiana 16C,which expresses GFP from a transgene.In leaf patches co-expressing GFP and AV2,GFP fluorescence was strong at 5 dpi and the accumulation level of GFP was substantially higher than in leaf patches infiltrated with the empty vector?Plus GFP?.This indicated that ToLCHsV AV2 could suppress PTGS.?5?Identification of a key residue for the TGS and PTGS suppressor activity of AV2:The cysteine at amino acid position 89 in AV2 was changed to serine,creating AV2^C89S.The ability of AV2^C89S89S to suppress TGS and PTGS was investigated as described above.The results showed that AV2^C89S89S lost the ability to suppress TGS and PTGS.The cellular localization and self-interaction of AV2^C89S89S were also investigated.The results showed that the C89S mutation did not affect the cellular localization and self-interaction of AV2.?6?The importance of the TGS and PTGS suppression activity for the action of AV2:The AV2 of ToLCHsV was changed for AV2^C89S89S by a single nucleotide mutation which did not affect the amino acid sequence of AV1,resulting in ToLCHsV-AV2^C89S.ToLCHsV-AV2^C89S89S was inoculated to N.benthamiana and N.tabacum.The results showed that ToLCHsV-AV2^C89S89S behaves like ToLCHsV-?AV2:it can infect N.benthamiana and N.tabacum but the symptom appearance was delayed and viral DNA accumulation was reduced compared to wild type ToLCHsV.In summary,this study shows that AV2 is not essential for ToLCHsV.However,AV2 can significantly promotes the infection of ToLCHsV.The function of AV2 is closely related to its TGS and PTGS suppressor activity.The data here deepened our understanding of plant-ToLCHsV interaction.They are also valuable for a thorough understanding of the function of the AV2 in begomoviruses.