Divergent Symptoms Caused By Geminivirus-encoded C4 Proteins Is Determined By Their Ability To NbSK?

Geminiviruses are small single-stranded plant DNA(ssDNA)viruses that occurs worldwide and can cause severe damages to crop productions.The C4 proteins encoded by geminiviruses can induce viral symptoms.Three geminiviruses were used in this study,including Tomato leaf curl Yunnan virus(TLCYnV),Tomato yellow leaf curl China Virus(TYLCCNV)and Tobacco curly shoot virus(TbCSV),molecular mechanisms of geminivirus-encoded C4 proteins inducing different symptoms were explored.We expressed C4 proteins through the heterologous expression system of Potato X virus(PVX),and demonstrated that the C4 proteins encoded by different geminiviruses can induce different symptoms.Using Yeast two hybrid technology and co-expression laser confocal technology,it is proved that the symptom determinant capability of C4 proteins are tightly coupled to their binding ability with NbSK?.TLCYnV C4 has the strongest binding ability with NbSK? and induces the most serious symptom.It was confirmed that the mini domain of TLCYnV C4(amino acids at positions 28-37)is critical for the interaction with NbSK?.After replacing the mini domain of TLCYnV C4 with TYLCCNV C4 and TbCSV C4,these two kinds of geminivirus-encoded C4 s induced severe symptoms.Subcellular structure separation experiment showed that the severity of symptoms induced by recombinant TYLCCNV C4 or TbCSV C4 correlate with the amount of NbSK? tethered to the membrane by the C4 protein.In addition,we constructed a full-length infectious clone of the recombinant virus to infect N.benthamiana,and the recombinant TYLCCNV harboring the mini domain of TLCYnV C4 induces more severe symptoms compared with wild-type TYLCCNV.Thus,this study provides new insights into the mechanism by which different geminivirus-encoded C4 proteins possess divergent symptom determinant capabilities.