Heterologous expression of viral suppressors of RNA silencing complements virulence of the HC-Pro mutant of clover yellow vein virus in pea

Abstract

Many plant viruses encode suppressors of RNA silencing, including the helper component-proteinase (HC-Pro) of potyviruses. Our previous studies showed that a D-to-Y mutation at amino acid position 193 in HC-Pro (HC-Pro-D193Y) drastically attenuated the virulence of clover yellow vein virus (ClYVV) in legume plants. Furthermore, RNA-silencing suppression (RSS) activity of HC-Pro-D193Y was significantly reduced in Nicotiana benthamiana. Here, we examine the effect of expression of heterologous suppressors of RNA silencing, i.e., tomato bushy stunt virus p19, cucumber mosaic virus 2b, and their mutants, on the virulence of the ClYVV point mutant with D193Y (Cl-D193Y) in pea. P19 and 2b fully and partially complemented Cl-D193Y multiplication and virulence, including lethal systemic HR in pea, respectively, but the P19 and 2b mutants with defects in their RSS activity did not. Our findings strongly suggest that the D193Y mutation exclusively affects RSS activity of HC-Pro and that RSS activity is necessary for ClYVV multiplication and virulence in pea.

Quantitative and Qualitative Involvement of P3N-PIPO in Overcoming Recessive Resistance against Clover Yellow Vein Virus in Pea Carrying the cyv1 Gene

“…The GST-fused PIPO was produced and subjected to SDS-PAGE as described above. The band, which migrated at around 33 kDa, was specifically detected by immunoblotting with the antibody as described previously (48), indicating that this antibody is able to detect PIPO (see Fig. 6B).…”

“…Uninoculated upper leaves with symptoms were harvested at 9 dpi, ground in liquid nitrogen, and denatured in 12-fold (wt/vol) urea denaturing buffer containing 4.5 M urea, 1% (vol/vol) Triton X-100, 0.5% dithiothreitol (DTT), 0.00625 M Tris-HCl (pH 6.8), 2% (wt/vol) SDS, 5% mercaptoethanol, 5% sucrose, and 0.002% bromophenol blue. The plant lysates were analyzed by SDS-PAGE, as described by Atsumi et al (48).…”

P3N-PIPO, a Frameshift Product from the P3 Gene, Pleiotropically Determines the Virulence of Clover Yellow Vein Virus in both Resistant and Susceptible Peas

“…1G). In previous studies, we revealed that the HC-Pro gene is indirectly involved in the induction of lethal systemic cell death in PI 118501 (20,22,32). We found that a Cl-No.30 mutant with a D-to-Y substitution at amino acid position 193 of HC-Pro (Cl-D193Y) loses the ability to induce cell death in PI 118501 (20).…”

“…Western blots were conducted as described previously (30,32). Proteins were resolved in 12% NuPAGE Bis-Tris gels (Thermo Fisher Scientific) using MES-SDS buffer (FLAG-tagged protein detection) or in 10% SDS-PAGE using Tris-glycine buffer (coat protein [CP] detection [33]), followed by electrotransfer onto a polyvinylidene difluoride (PVDF) membrane.…”

“…A double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was conducted according to methods described in our previous report (32). A single GFP focus derived from the virus was excised from inoculated leaves and used for antigens.…”