The Functional Study Of AC2 And AC4 Encoded By Tomato Leaf Curl Hsinchu Virus

Begomovirus is the largest genus of the family Geminiviridae and contains more than 400 formally approved virus species.In recent decades,begomoviruses have exploded throughout the world,posing a serious threat to agricultural production.Studying the gene function of begomovirus helps to understand the mechanism of begomovirus infection,and thus provides theoretical support for the prevention and control of disease related to begomovirus.Tomato leaf curl Hsinchu virus(ToLCHsV)is a begomovirus widely distributed in Taiwan,Hainan,Guangdong,Fujian,Zhejiang and Jiangsu in China.In this study,the functional analysis of AC2 and AC4 of ToLCHsV was investigated.The main experiments and results are as follows:(1)The role of AC2 and AC4 in ToLCHsV infection: Three ToLCHsV mutants were constructed by site-directed mutagenesis: ToLCHsV-?AC2(cannot express AC2),ToLCHsV-?AC4(cannot express AC4),and ToLCHsV-?AC2-AC4(cannot expresst AC2 and AC4).The infectivity of these three mutants was investigated by inoculating each of them to N.benthamiana.The three ToLCHsV mutants were found to be able to infect N.benthamiana and cause severe symptoms.However,the time of symptom appearance was delayed for 1-2 days for ToLCHsV-? AC2 and 2-3 days for ToLCHsV-? AC4 and ToLCHsV-? AC2-AC4 compared to wildtype ToLCHsV.In the dynamics of viral DNA accumulation,the three ToLCHsV mutants showed significant differences: on the 9th day,their accumulation levels in N.benthamiana from high to low were ToLCHsV-?AC4,ToLCHsV-?AC2-AC4 and ToLCHsV-?AC2,on the 18 th day,was ToLCHsV-?AC2-AC4,ToLCHsV-?AC4,and ToLCHsV-?AC2;on the 27 th day,the accumulation level of ToLCHsV-?AC2-AC4 was the lowest,while ToLCHsV-?AC2 and ToLCHsV-?AC4 did not differ significantly.However,at all three time points,the accumulation levels of the three ToLCHsV mutants were lower than the wild-type ToLCHsV.(2)Post-Transcriptional Gene Silencing(PTGS)and Transcriptional Level Gene Silencing(TGS)suppression activity of AC2 and AC4: To determine whether AC2 and AC4 of ToLCHsV have PTGS suppression activity,the ORF of the two proteins were each cloned into the transient expression vector pEarlyGate100,creating pEarlyGate100-AC2 and pEarlyGate100-AC4.GFP fluorescence was observed after injecting pEarlyGate100-AC2 and pEarlyGate100-AC4 into leaves of 16 c N.benthamianatogether with a binary vector(35S-GFP)expressing a GFP mRNA,and the level of GFP mRNA accumulation was detected.The results show that AC4 can significantly inhibit the occurrence of PTGS,while AC2 does not have this function.(3)Subcellular localization of AC2 and AC4 and their ability to enhance heterologous virus infection: To clarify the subcellular localization of ToLCHsV AC2 and AC4,the ORFs of AC2/AC4 were constructed into pEarlyGate101,creating pEarlyGate101-AC2/AC4.pEarlyGate101-AC2 and pEarlyGate101-AC4 were separately introduced into N.benthamiana epidermal cells by agroinfiltration,and the distribution of YFP fluorescence in the cells was observed by confocal microscopy on two days after agroinfiltration.The results showed that ToLCHsV AC2-YFP was distributed in both cytoplasm and nucleus,while AC4-YFP was mainly localized to chloroplasts.To investigate whether ToLCHsV AC2 and AC4 can enhance the infection of a heterologous virus,this study used VX-AC2 and PVX-AC4 to inoculate N.benthamiana.The results showed that ToLCHsV AC2 and AC4 significantly enhanced the pathogenicity of PVX and significantly increased the accumulation of PVX in N.benthamiana.AC2 is more active than AC4 in enhancing PVX pathogenicity and accumulation levels.In summary,this study deepened our understanding of the function of ToLCHsV AC2 and AC4 genes,and laid the foundation for further analysis of the infection mechanism of ToLCHsV.In addition,this study found that AC2 is not necessary for ToLCHsV infection,and that AC4 of ToLCHsV is localized to chloroplasts.These two findings are different from other previously reported begomoviruses.Therefore,this study also provides a new perspective for a comprehensive understanding of the gene function of begomovirus AC2 and AC4.