Detection And Infectious Clone Construction Of ToCV And Analysis Of Its Interaction With Key Proteins Of Host Plant

Tomato chlorosis virus(ToCV),belonging to genus crinivirus of Closteroviridae,was severely epidemic threaten of important crops of Solanaceae in Shangdong province,China.Recently,ToCV was rapidly spread to most of Solanaceae crops planting region,and threated the planting safety of Solanaceae crops especially tomato.So the rapid detection method,molecular mechanisms of virus-host interaction was urgently manipulated to understand the distribution of ToCV,and its potential threaten to Solanaceae crops.The results would scientifically contribute to control ToCV in field.The polyantibodies of ToCV CP and HSP70 protein were prepared using prokaryotic expressed and purified ToCV CP and HSP70 protein as antigen.Western blotting and ELISA detection resulted showed diluted 10,000 × of polyantibodies of ToCV CP and HSP70 protein still recognize the ToCV.The ToCV full-genomic cDNA infectious clone was constructed using the seamless cloning.The ToCV infectious clone could not infect BY2 cell line of Nicotiana benthamiana,and tomato using Agrobacteria infiltration,N.benthamiana and tomato by particle bombardment.The ToCV infectious clone infected of 15% N.benthamiana plant using Agrobacteria infiltration.The infection ratio of the ToCV infectious clone was the highest upto 60% to BY2 cell line of N.benthamiana using PEG mediated method.The ToCV distribution in tomato plant was quantified by qRT-PCR.ToCV was inoculated to tomato plant by whitefly fed with ToCV infected tomato,and qRT-PCR results showed ToCV locate only in inoculated leafs in 10 dpi,and move to upper leafs in 15 dpi,move to lower leafs in 20 dpi.The highest concentration of ToCV was located in new shoots.The autophagy pathway possesses critical functions in plant viruses infecting host.The effect of ToCV influencing to tomato autophagy was tested by MDS staining and LC3 protein biosynthesis.The results showed ToCV could activate tomato autophagy,and further experiments testified inhibiting autophagosome vesicles mature by 3-MA,not inhibiting autophagosome biogenesis by BFA enhances ToCV replication.Yeast two-hybrid test verified ToCV CP and CPm could interact with autophagy ATG1 protein.Together,it could be deduced ToCV inhibits autophagosome vesicles mature,and uses double-membrane of unmatured autophagosome vesicles as replication cites to enhance ToCV replication.The unequivocal molecular mechanisms of ToCV interacting with host autophagy pathway w ould be uncovered by testifying the veracity of ToCV CP/CPm interacting with ATG1,which are key proteins involving in host autophagy pathway,and medulating ToCV replicaiton by ex pressing of ATG1.