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

Cross protection is a promising alternate to control Cucumber green mottle mosaic virus (CGMMV) which is of increasing economic importance to cucurbit production worldwide. One major factor confronting the application of cross protection to control CGMMV is the scarcity of available mild mutants. 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 the Cauliflower mosaic virus 35S promoter. Five pCGMMV-derived mutants were obtained via site-directed mutagenesis and inoculated to Nicotiana 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 in N. 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 in N. 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.