Modifications of the helper component-protease of Zucchini yellow mosaic virus for generation of attenuated mutants for cross protection against severe infection

A nonpathogenic mild strain is essential for control of plant viruses by cross protection. Three amino acid changes, Arg(180)-> Ile(180) (GA mutation), Phe(205)-> Leu(205) (GB mutation), and Glu(396)-> Asn(396) (GC mutation), of the conserved motifs of the helper component-protease (HC-Pro) of a severe strain TW-TN3 of Zucchini yellow mosaic virus (ZYMV), a member of the genus Potyvirus, were generated from an infectious cDNA clone that carried a green fluorescent protein reporter. The infectivity of individual mutants containing single, double, or triple mutations was assayed on local and systemic hosts. On Chenopodium quinoa plants, the GB mutant induced necrotic lesions; the GA, GC, and GBC mutants induced chlorotic spots; and the GAB and GAC mutants induced local infection only visualized by fluorescence microscopy. On squash plants, the GA, GB, GC, and GBC mutants caused milder mosaic; the GAC mutant induced slight leaf mottling followed by recovering; and the GAB mutant did not induce conspicuous symptoms. Also, the GAC mutant, but not the GAB mutant, conferred complete cross protection against the parental virus carrying a mite allergen as a reporter. When tested on transgene-silenced transgenic squash, the ability of posttranscriptional gene silencing suppression of the mutated HC-Pro of GAC was not significantly affected. We concluded that the mutations of the HC-Pro of ZYMV reduce the degrees of pathogenicity on squash and also abolish the ability for eliciting the hypersensitive reaction on C. quinoa, and that the mutant GAC is a useful mild strain for cross protection.