A Single Amino Acid Mutation in thePlum pox virusHelper Component-Proteinase Gene Abolishes Both Synergistic and RNA Silencing Suppression Activities

Abstract

The effects on symptom expression of single amino acid mutations in the central region of the Plum pox virus (PPV) helper component-proteinase (HC-Pro) gene were analyzed in Nicotiana benthamiana using Potato virus X (PVX) recombinant viruses. PVX recombinant virus expressing the wild-type variant of PPV HC-Pro induced the expected enhancement of PVX pathogenicity, manifested as necrosis and plant death. Recombinant virus expressing a variant of PPV HC-Pro containing a single point mutation ( HCL(134)H) was unable to induce this synergistic phenotype. The RNA silencing suppressor activity of PPV HC-Pro was demonstrated in a transient silencing suppression assay. In contrast, the HCL(134)H mutant showed no such activity. These results indicate that a unique point mutation in PPV HC-Pro impaired its ability to suppress RNA silencing and abolished its capacity to induce synergism, and clearly shows for the first time the link between these two functions in potyvirus HC-Pro. Additionally, we compared the effects on virus accumulation in N. benthamiana plants infected with either the PVX recombinant constructs or with native viruses in double infection experiments. PVX (+) and (-) strand genomic RNA accumulated at similar levels in plants infected with PVX recombinants, leading to an increase in PVX pathology, compared with plants infected with PVX alone. This finding confirms that the enhancement of pathogenicity associated with synergistic interaction is not a consequence of more efficient PVX replication due to RNA silencing suppression by PPV HC-Pro.

The P25 Protein of Potato Virus X (PVX) Is the Main Pathogenicity Determinant Responsible for Systemic Necrosis in PVX-Associated Synergisms

“…Potyvirus-associated synergistic diseases have been suggested to result from suppression of the host defense mechanism based on RNA silencing by the potyviral VSR, helper component-proteinase (HC) protein (18). However, the accumulation of PVX genomic RNA (gRNA) did not increase greatly in N. benthamiana when doubly infected with PVX and either Plum pox virus (PPV) or Tobacco etch virus (TEV), despite the extreme enhancement of symptoms in this host that lead to SN, i.e., synergism in pathology (16,19). PVX-mediated expression of viral genes to determine whether a protein would function as a pathogenicity determinant is a wellestablished technique (17,20).…”

The Conserved FRNK Box in HC-Pro, a Plant Viral Suppressor of Gene Silencing, Is Required for Small RNA Binding and Mediates Symptom Development

“…A mutation in Plum pox potyvirus (PPV) in the HC-Pro central region can affect symptom severity without affecting virus accumulation (K 107 Q or S 231 G) (52). Loss of both Potato virus X/potyviral synergism and suppression of gene silencing were associated with the mutation L 134 H in PPV (21), and a loss of suppression together with loss of synergism (with a comovirus) was also found in Soybean mosaic virus (K 142 I) and TEV (K 144 I) mutants which are relatively conserved (20). Thus, the FRNK box is not the only motif governing HC-Pro RSS function and symptom expression.…”

From Hypo- to Hypersuppression: Effect of Amino Acid Substitutions on the RNA-Silencing Suppressor Activity of the Tobacco etch potyvirus HC-Pro

“…Eleven mutant genotypes, designated as CLA, corresponded to amino acid substitutions at sites that are conserved among all potyviral HC-Pro proteins so far characterized. The mutants T1 and T2 corresponded to amino acid substitutions at the homologous sites of those described by González-Jara et al (2005) as essential for Plum pox virus HCPro suppression activity. The 11 PC mutants corresponded to a single random amino acid substitution and have been described before (Carrasco et al 2007a,b).…”

“…This condition is amply fulfilled by viral silencing suppressors. For example, 1 focusing only on potyviruses, it has been shown that two amino acid replacements in the HC-Pro suppressor of Clover yellow vein virus are sufficient to attenuate the symptoms and reduce virus accumulation (Yambao et al 2008), whereas a single mutation in Plum pox virus HCPro had a similar effect (González-Jara et al 2005). As a final example, mutations in the conserved motifs of Zucchini yellow mosaic virus HC-Pro also produced attenuated viruses on the natural host squash and also abolished the ability to elicit hypersensitive responses in other local lesion hosts (Lin et al 2007).…”