A Preliminary Study On The Pathogenicity And Molecular Mechanism Of Tomato Chlorotic Virus P7 Protein

Tomato chlorosis virus(ToCV),a member of the genus Crinivirus,is transmitted by the insect vector whitefly.ToCV is epidemic threat to tomato due to no resistance of tomato varieties at present.Toelucidate the pathogencity and its molecular mechanims would scientifically contriute to resistant breeding of tomato varieties agaisnt ToCV.The P7 protein encoded by Tomato chlorotic virus is a small protein with unknown function and contains a transmembrane domain.In this study,P7 protein was selected as a potential pathogenicity deteminant to uncover the function of its pathogenicity and mechenism in the process of ToCV infection.The research findings were as following:The ToCV P7 gene was cloned by specific RT-PCR and recombinanted into the Potato virus X(PVX)vector to generate the recombinant virus PVX:P7.Using technology,ToCV P7 protein was expressed heterologously in Nicotiana benthamiana plants by Agrobacterium-mediated infiltration.The symptoms of severe mottled mosaics and shrinkage appeared in the leaves of inoculated N.benthamiana at 12 dpi.The quatification of PVX using western blotting and RT-q PCR showed that ToCV P7 protein could significantly increase the accumulation of protein and mRNA of PVX CP in N.benthemiana plants.This result suggested P7 protein has function of pathoenicity determinant for promoting PVX to infect N.benthamiana.In addition,the phynotype of transgenic tomato plants with overexpressing of ToCV P7 gene appeared showed short and shrunken leaves,and low seed setting rate.This result reconfirmed that the P7 protein funtions as viral pathogenicity determinant.The P7 was divided into two domains of N^1-33-terminal,which containing a transmembrane domain and C^34-65-terminal evenly.The two domains of P7 protein were heterologous expressed in N.benthamiana by PVX vector.The results of host phynotypes and PVX quantification suggested the C^34-65-terminus of P7 protien was essential for its pathogenicity deteminant.The results of yeast two-hybrid,bimolecular fluorescence complementation(BiFC)and luciferase complementation imaging(LCI)revealed that the P7 protein can interact with itself.BiFC alsos showed that the subcellular location of P7 protein self-interaction was existing in the cytoplasm,which was similar as the subcellular location of P7 protein alone.The P7 protein was segmented,and the results of the yeast two-hybrid demonstrated the N^1-33-terminus was dispensable for the P7protein self-interaction.For exploring the molecular mechanism of the pathogenicity of P7 protein,the recombinant P7tagged with GFP was transient overexpressed in N.benthamina,and the host protein interacted with P7 protein was pulled down by affinity chromatography,the host proteins were identified by liquid chromatography tandem mass spectrometry(LC-MS/MS).Two enzymes of Adenosine kinase(ADK)and S-adenosylmethionine synthase(SAMS)involving in the methyl cycle of plants have been screened to interact with P7 protien,which hinted P7 protein probably mediate the DNA methylation of plants to function as pathogenicity deteminant.The recombinant PVX virus vector PVX:P7 was innoculated into the N.benthamiana transgene line 16c-TGS(the 35S promoter was DNA methylated,and resulted in silencing of GFP gene),the P7 protien could restore the expressing of GFP gene through reversing the inhibition of GFP gene expressing by reducing the methylation level of the 35S promoter region in 16c-TGS.