Discriminating mutations of HC-Pro of zucchini yellow mosaic virus with differential effects on small RNA pathways involved in viral pathogenicity and symptom development.

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, R(180)I (mutation A), F(205)L (B), and E(396)N (C), of Zucchini yellow mosaic virus (ZYMV) affect symptom severity and viral pathogenicity. The mutant ZYMV GAC (ZGAC) with double mutations, R(180)I/E(396)N, 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 (R(180)I/E(396)N) 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.