Molecular Pathogenesis Of Cucumber Green Mottle Mosaic Virus

Cucumber green mottle mosaic virus(CGMMV)is a member of the genus Tobamovirus(family Virgaviridae).CGMMV is seed transmitted and causes serious economic losses in cucurbit crops,especially for grafted watermelon.CGMMV has been listed as a quarantine pest in China,and it was very difficult to control the disease caused by CGMMV.In order to investigate pathogenic molecular determinants in CGMMV genome,infectious clone was constructed,and further mutational analysis was employed.The study will help us better understand molecular pathogenesis of CGMMV and provide us new ideas and methods for the control of CGMMV.Major results as follows:(1)The construction of the infectious clones for CGMMV.Because the construction of viral infectious clone as a key technology was remained as a bottleneck in the research of virology,in this study,three infectious clones for CGMMV,Zucchini yellow mosaic virus(ZYMV)and Watermelon mosaic virus(WMV)were simultaneously constructed to ensure that the technology was better practiced.PXT1-CGMMV,the infectious clone for CGMMV,was constructed using conversional restriction endonuclease-T4 DNA ligase method.For pXT1-ZYMV and pXT1-WMV,the infectious clones for ZYMV and WMV,homologous recombination method was employed.By inoculation on hosts and subsequent detections including DAS-ELISA,RT-PCR and Northern Blot,three infectious clones were competent to systemically infecting plants,suggesting infectious clones for three viruses had been successfully constructed.(2)Defining the effect of coding region of CGMMV viral protein on viral pathogenicity and symptom induction.The pXT1-CGMMV was assayed using site-directed mutagenesis.Based on mutational locations,37 mutants for replication protein,21 mutants for Move Protein(MP)and 22 mutants for Coat Protein(CP)were obtained,respectively.The results of inoculations on Nicotiana benthamiana showed the mutations of replication protein and MP are associated with viral pathogenicity.The mutations of CP not only associated with viral pathogenicity,but also could change the symptom induction.The potential role of Internal Region(IR)and RNA Helicase(HEL)of viral replication protein,MP and CP on viral infection were detected by using Potato virus X(PVX)expression system.The results showed that the expression of IR and HEL attenuated the pathogenicity of PVX,implying their relationship with CGMMV pathogenicity;MP and CP could cause changes of symptom induced by PVX,suggesting MP and CP maybe anticipate in symptom emerge of diseases caused by CGMMV.(3)The discovery of the phenomenon of reverse mutation resulting from Arg at residue 284 of the replication protein and clarifying the influence from the reverse of Arg284 and the production regularity of reverse mutation.Arg 284 of the replication protein of CGMMV was found to have the characteristic of reverse mutation in inoculation experiments and the reversion of Arg284 associated with the recovery of viral pathogenicity.Mutational analysis of aa284 was carried out,and the results showed that viral pathogenicity,the number of base mutation and the type of base mutation associated with the reversionof Arg284.Further,the Arg to Cys mutation frome other Arg residues of CGMMV replication protein were carried out,and the revival phenomenon did not happen.Transient expression system and co-infection inoculation were employed to analyze the reason of the reverse-mutation emerge,the results indicated that the activity from silencing suppression was not the determining factor in Arg284 reverse mutation,it remains to be further study.