Modification of the N-terminal FWKG-alpha H1 element of potyviral HC-Pro affects its multiple functions and generates effective attenuated mutants for cross-protection

Abstract

Control of plant viruses by cross-protection is limited by the availability of effective protective strains. Incorporation of an NIa-protease processing site in the extreme N-terminal region of the helper component protease (HC-Pro) of turnip mosaic virus (TuMV) resulted in a mutant virus TuHN(D)I that induced highly attenuated symptoms. Recombination analysis verified that two variations, F7I mutation and amino acid 7-upstream-deletion, in HC-Pro co-determined TuHN(D)I attenuation. TuHN(D)I provided complete protection to Nicotiana benthamiana and Brassica campestris subsp. chinensis plants against infection by the severe parental strain. Aphid transmission tests revealed that TuHN(D)I was not aphid-transmissible. An RNA silencing suppression (RSS) assay by agroinfiltration suggested the RSS-defective nature of the mutant HC-Pro. In the context (amino acids 3-17) encompassing the two variations of HC-Pro, we uncovered an FWKG-alpha-helix 1 (alpha H1) element that influenced the functions of aphid transmission and RSS, whose motifs were located far downstream. We further demonstrated that HC-Pro F7 was a critical residue on alpha H1 for HC-Pro functions and that reinstating alpha H1 in the RSS-defective HC-Pro of TuHN(D)I restored the protein's RSS function. Yeast two-hybrid and bimolecular fluorescence complementation assays indicated the FWKG-alpha H1 element as an integral part of the HC-Pro self-interaction domain. The possibility of regulation of the mechanistically independent functions of RSS and aphid transmission by the FWKG-alpha H1 element is discussed. Extension of TuMV HC-Pro FWKG-alpha H1 variations to another potyvirus, zucchini yellow mosaic virus, also generated nonaphid-transmissible cross-protective mutant viruses. Hence, the modification of the FWKG-alpha H1 element can generate effective attenuated viruses for the control of potyviruses by cross-protection.