Rapid selection of potyviral cross‐protection effective mutants from the local lesion host after nitrous acid mutagenesis

Zucchini yellow mosaic virus (ZYMV) seriously damages cucurbits worldwide. Control of ZYMV by cross‐protection has been practised for decades, but selecting useful mild viruses is time‐consuming and laborious. Most attenuated potyviruses used for cross‐protection do not induce hypersensitive reaction (HR) in Chenopodium quinoa, a local lesion host Chenopodium quinoa. Here, severe ZYMV TW‐TN3 tagged with green fluorescent protein (GFP), designated ZG, was used for nitrous acid mutagenesis. From three trials, 11 mutants were identified from fluorescent spots without HR in inoculated C. quinoa leaves. Five mutants caused attenuated symptoms in squash plants. The genomic sequences of these five mutants revealed that most of the nonsynonymous changes were located in the HC‐Pro gene. The replacement of individual mutated HC‐Pros in the ZG backbone and an RNA silencing suppression (RSS) assay indicated that each mutated HC‐Pro is defective in RSS function and responsible for reduced virulence. Four mutants provided high degrees of protection (84%–100%) against severe virus TW‐TN3 in zucchini squash plants, with ZG 4‐10 being selected for removal of the GFP tag. After removal of the GFP gene, Z 4‐10 induced symptoms similar to ZG 4‐10 and still provided 100% protection against TW‐TN3 in squash, thus is considered not a genetically engineered mutant. Therefore, using a GFP reporter to select non‐HR mutants of ZYMV from C. quinoa leaves is an efficient way to obtain beneficial mild viruses for cross‐protection. This novel approach is being applied to other potyviruses.