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

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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.

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Acknowledgments

We are grateful to T. Okuno (Kyoto University) for providing the pBIC-P19. We also thank K. Goto (Hokkaido University) for helpful discussions. This study was supported in part by Grants-in-Aid for Scientific Research (S) and Young Scientists (A) from the Ministry of Science, Education, Culture, and Technology of Japan.

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Authors and Affiliations

  1. Pathogen-Plant Interactions Group, Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589, Japan

    Go Atsumi, Kenji S. Nakahara, Tomoko Sugikawa Wada, Sun Hee Choi & Ichiro Uyeda

  2. Laboratory of Cell Biology and Manipulation, Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589, Japan

    Chikara Masuta

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  1. Go Atsumi
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  2. Kenji S. Nakahara
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  6. Ichiro Uyeda
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Correspondence to Ichiro Uyeda.

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705_2012_1281_MOESM1_ESM.pdf

Supplementary material 1 (PDF 3724 kb). Supplementary Fig. 1 Schematic representation of the ClYVV infectious cDNA used in this study. Full-length cDNA of ClYVV No. 30 isolate was fused to the cauliflower mosaic virus 35S promoter [11, 25, 30]. Cl-D193Y encodes a point mutation, aspartic acid to tyrosine, at aa position 193 in HC-Pro [33]. Either the TBSV p19 or the CMV 2b coding sequence was introduced into the Cl-D193Y genome [16, 23, 26]. p19m has point mutations at the aa positions 75 (arginine to glycine) and 78 (arginine to glycine) [16]; 2bm has an arginine-to-cysteine mutation at aa position 46 [5]. Supplementary Fig. 2 ClYVV accumulation in upper non-inoculated leaves. Virus CP accumulation at 8 dpi was compared using DAS-ELISA. The signal value of healthy plants was zero. Supplementary Fig. 3 Comparison of RNA silencing suppression activity of FLAG-tagged TBSV p19 and CMV 2b (wild type or mutant). (a) GFP, an inverted repeat sequence of GFP (IR-GFP), and suppressor of RNA silencing (p19, p19m, 2b, or 2bm tagged with FLAG) were expressed using an Agrobacterium-mediated transient assay in N. benthamiana leaves. GFP fluorescence was detected at 4 days after infiltration. (b) Total RNA was extracted from the infiltrated area indicated in (a), and mRNA levels of GFP were compared by northern hybridization. (c) Total proteins were extracted from the infiltrated area indicated in (a), and levels of suppressor proteins were compared by western blotting using an antibody against FLAG. (d) Comparison of RNA-silencing suppression activity between wild-type and mutant p19 proteins. The amount of Agrobacterium carrying p19 was diluted to 1/4 or 1/8 of the amount of Agrobacterium carrying p19m. GFP fluorescence was detected at 4 days after infiltration. (e) Total RNA was extracted from the infiltrated area indicated in (d), and mRNA levels of GFP were compared by northern hybridization. (f) Total proteins were extracted from the infiltrated area indicated in (d), and levels of suppressor proteins were compared by western blotting using an antibody against FLAG. Supplementary Fig. 4 Protein stability of P19 and 2b mutants. (a) Mutant p19 protein was expressed with wild-type p19 or GUS (negative control) using an Agrobacterium-mediated transient assay in N. benthamiana leaves. Total proteins were extracted from the infiltrated area, and the level of p19 mutant protein was compared with that of wild-type p19 by western blotting using an antibody against FLAG. (b) Mutant 2b protein was expressed with p19, ClYVV P1/HC-Pro, or GUS (negative control), and the amount of mutant 2b protein was compared with that of wild-type 2b as in (a). (c) p19 wild-type or mutant protein was expressed via Cl-WT, and the level of protein was compared by western blotting using an antibody against FLAG. The level of ClYVV CP protein accumulation was used as a control. ClYVV CP was detected by western blotting using an antibody against ClYVV CP. (d) The levels of 2b wild-type or mutant protein were compared as in (c). Supplementary Fig. 5 Effect of TBSV p19 expression from ClYVV, which has an R-to-I mutation in the HC-Pro FRNK motif, on symptom expression in PI 118501. Symptoms were monitored, and photographs were taken at 11 dpi. Supplementary Fig. 6 Expression of either TBSV p19 or CMV 2b using the white clover mosaic virus vector. Photographs of upper non-inoculated leaves were taken at 14 dpi

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Atsumi, G., Nakahara, K.S., Wada, T.S. et al. Heterologous expression of viral suppressors of RNA silencing complements virulence of the HC-Pro mutant of clover yellow vein virus in pea. Arch Virol 157, 1019–1028 (2012). https://doi.org/10.1007/s00705-012-1281-3

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