Single amino acid substitutions in the coat protein and RNA-dependent RNA polymerase alleviated the virulence of Cucumber green mottle mosaic virus and conferred cross protection against severe infection

The objective of this paper was to screen attenuated mutants of CGMMV and evaluate their potential in cross protection. An infectious cDNA clone of CGMMV, pCGMMV, was obtained by cloning intron-containing CGMMV genome to modified pCambia0390 vector with theCauliflower mosaic virus 35S promoter. Five pCGMMV-derived mutants were obtained via site-directed mutagenesis and inoculated toNicotiana benthamiana plants for symptom observation. The attenuated CGMMV mutants were evaluated for their efficiency in cross protection. The intron-containing clone pCGMMV induced similar disease symptoms and accumulated similar titres of virus inN. benthamiana plants as wild-type CGMMV. Mutations of aspartic acid at position 89 in the coat protein to alanine (D89A) or glutamic acid at position 1069 in the ORF1/2 read-through protein, in the RNA-dependent RNA polymerase domain to alanine (E1069A) alleviated the symptoms of pCGMMV inN. benthamiana plants significantly. In cross protection assay, the two mutants pCGMMV-CP-D89A and pCGMMV-RdRp-E1069A could prevent the superinfection of CGMMV, with protection efficiency of 91.7% and 100%, respectively. The intron-containing clone pCGMMV was stable and highly infectious. The D89 in the coat protein and E1069 in the RNA-dependent RNA polymerase played an important role in regulating the virulence of CGMMV. Mutants pCGMMV-CP-D89A and pCGMMV-RdRp-E1069A were of great potential in the control of CGMMV via cross protection.

Source: Virus Genes - December 31, 2019 Category: Genetics & Stem Cells Source Type: research