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Discriminating Mutations of HC-Pro of Zucchini yellow mosaic virus with Differential Effects on Small RNA Pathways Involved in Viral Pathogenicity and Symptom Development

¹Laboratory of Plant Molecular Biology, Rockefeller University, 1230 York Ave, New York 10065, U.S.A.; ²Department of Plant Pathology, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 402, Taiwan; ³Institute of Biotechnology, National Taiwan University, 81 Chang-Xing St., Taipei 106, Taiwan

Helper component-proteinase (HC-Pro), the gene-silencing suppressor of Potyvirus spp., interferes with microRNA (miRNA) and short-interfering RNA (siRNA) pathways. Our previous studies showed that three mutations of highly conserved amino acids of HC-Pro, R₁₈₀I (mutation A), F₂₀₅L (B), and E₃₉₆N (C), of Zucchini yellow mosaic virus (ZYMV) affect symptom severity and viral pathogenicity. The mutant ZYMV GAC (ZGAC) with double mutations, R₁₈₀I/E₃₉₆N, induces transient leaf mottling in host plants followed by recovery. This mutant confers complete cross protection against subsequent infection by the parental ZYMV (ZG) strain. Here, we sought to obtain molecular evidence on the roles of the three highly conserved amino acids of HC-Pro in miRNA and siRNA pathways using transgenic Arabidopsis plants expressing comparable levels of wild-type and mutant HC-Pro proteins. We demonstrated that amino acid residues 180, 205, and 396 of HC-Pro are critical for suppression of miRNA, trans-acting siRNA (ta-siRNA), and virus-induced gene silencing (VIGS) pathways but not for sense-post transcriptional gene silencing (s-PTGS). Because the HC-Pro double mutant (R₁₈₀I/E₃₉₆N) does not interfere with miRNA and ta-siRNA pathways, the ZGAC mutant virus elicits only attenuated symptoms. Furthermore, the recovery seen on ZGAC-infected plants likely results from the weak VIGS suppression by the HC-Pro double AC mutant. Thus, through manipulating these three conserved amino acids on HC-Pro, symptom severity of diseases caused by Potyvirus spp. can be modulated to generate useful cross protectants for field application. Although some of our mutated HC-Pro proteins do not interfere with miRNA and ta-siRNA pathways, they still retain the ability to suppress s-PTGS.