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p2 of Rice grassy stunt virus (RGSV) and p6 and p9 of Rice ragged stunt virus (RRSV) isolates from Vietnam exert suppressor activity on the RNA silencing pathway

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Abstract

In Vietnam, the two main viruses that cause disease in rice are the Rice grassy stunt virus (RGSV) and the Rice ragged stunt virus (RRSV). Outbreaks of these two viruses have dramatically decreased rice production in Vietnam. Because natural resistance genes are unknown, an RNAi strategy may be an alternative method to develop resistance to RGSV and RRSV. However, this strategy will be efficient only if putative silencing suppressors encoded by the two viruses are neutralized. To identify these suppressors, we used the classical green fluorescent protein (GFP) agroinfiltration method in Nicotiana benthamiana. Then, we investigated the effects of viral candidate proteins on GFP expression and GFP siRNA accumulation and their interference with the short- or long-range signal of silencing. RGSV genes s2gp1, s5gp2, and s6gp1 and RRSV genes s5gp1, s6gp1, s9gp1, and s10gp1 were selected for viral silencing suppressor investigation according to their small molecular weight, the presence of cysteines, or the presence of a GW motif in related protein products. We confirmed that protein p6 of RRSV displays mild silencing suppressor activity and affects long-range silencing by delaying the systemic silencing signal. In addition, we identified two new silencing suppressors that displayed mild activity: p2 of RGSV and p9 of RRSV.

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Acknowledgments

We greatly thank Prof. David Baulcombe, the Gatsby Charitable Foundation and PBL (Norwich), for providing the N. benthamiana 16c line. We also thank Dr. Thomas Kroj, INRA for very useful suggestion regarding GFP scanning technique. We are very grateful to Dr. D Sérémé for statistical analysis. TDN was supported by an IRD ARTS PhD fellowship.

Author contributions

SL and CB designed the research; TN, SL, and MB performed viral genes cloning, siRNA expression and transient expression of 35S::candidate gene, and the silencing inducer 35S::GFP in N. benthamiana. TN performed experimentation described in Fig. 4 and Tables 1 and 2. HT, DV, and PG contributed for analysis data and discussion. TN, SL, CB, and PG have written the manuscript.

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Correspondence to Christophe Brugidou.

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Edited by Thomas Hohn.

Thanh Duc Nguyen and Séverine Lacombe contributed equally to this work.

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11262_2015_1229_MOESM1_ESM.docx

Supplemental data Table S1: RGSV and RRSV viral proteins. The ones selected for analysis are noted in bold.Supplemental data Table S2: List of primers used for virus gene amplification. Supplementary material 1 (DOCX 21 kb)

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Nguyen, T.D., Lacombe, S., Bangratz, M. et al. p2 of Rice grassy stunt virus (RGSV) and p6 and p9 of Rice ragged stunt virus (RRSV) isolates from Vietnam exert suppressor activity on the RNA silencing pathway. Virus Genes 51, 267–275 (2015). https://doi.org/10.1007/s11262-015-1229-2

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