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Molecular analysis of an attenuated strain of Cucumber green mottle mosaic virus using in vitro infectious cDNA clone: pathogenicity and suppression of RNA silencing

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Abstract

Cucumber green mottle mosaic virus (CGMMV), a tobamovirus, infects cucurbitaceous plants including watermelon. An attenuated strain of CGMMV (CGMMV-SH33b) has been isolated from CGMMV-SH and used for protecting muskmelon against wild CGMMV. There are five of nine base changes that lead to amino acid substitutions in the genome between SH and SH33b: four in replicase and one in coat protein. Using cDNA clones of both strains, two recombinant clones were constructed: first clone (pCG-KK) has two base changes (one silent at nt 5089 and the other P1397S amino acid substitution in the read-through part of SH 186K replicase) and the second clone (pCG-SP) has two silent base changes and three base changes in SH 129K/186K replicase (A1124V, N1157D and P1397S). The symptom severity on Nicotiana benthamiana induced by each recombinant virus were intermediate between those by SH and by SH33b. The accumulation levels of viruses using ELISA in infected plants were coinciding with symptom severity. Northern blot analysis of Nicotiana benthamiana at 10 dpi indicated that the virus accumulations of these recombinants was in the order of SH>pCG-KK>pCG-SP>SH33b. These results suggest that base changes located in the read-through part of 186K replicase as well as other base changes are responsible for the virus attenuation although we cannot excluded the possibility of silent mutations involved in pathogenicity. Using green fluorescent protein gene (GFP) silenced transgenic tobacco; infection with CGMMV-SH significantly suppressed GFP silencing with visible symptoms, whereas CGMMV-SH33b and their chimera viruses resulted in partial suppression. The suppression was further supported by lower level of GFP mRNA and higher siRNA detected in SH33b infected leaves, than those in CGMMV-SH infections, suggesting that the levels of virus accumulation and symptom severity on N. benthamiana were reasonably parallel with the suppression of RNA silencing of GFP mRNA.

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Abbreviations

CGMMV:

Cucumber green mottle mosaic virus

GFP:

Green fluorescent protein gene

siRNA:

Small interfering RNA

RISC:

RNA-Induced silencing complex

dsRNA:

Double-stranded RNA

SH:

Severe strain

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Acknowledgments

We are grateful to M. Nogami and K. Kohara for the plasmids of cDNA cloning. We also thank D. Murphy for checking the English in this manuscript. This work was supported by the Program for Promotion of Basic and Applied Researches in Bio-oriented Industry (BRAIN), the Ministry of Education, Culture, Sports and Technology of Japan [Grant-in-Aid for Scientific Research for Scientific Research (C), No. 24580065 to MN] and the Japan Science and Technology Agency for the A-step (Adaptable & Seamless Technology Transfer Program through Target-driven R & D) to MN.

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Authors and Affiliations

  1. Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, 790-8566, Japan

    Md Emran Ali, Sumyya Waliullah & Masamichi Nishiguchi

  2. Department of Plant Pathology, The Ohio State University, 026 Selby Hall, 1680 Madison Ave, Wooster, OH, 44691, USA

    Md Emran Ali

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  1. Md Emran Ali
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  2. Sumyya Waliullah
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  3. Masamichi Nishiguchi
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Correspondence to Md Emran Ali.

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Ali, M.E., Waliullah, S. & Nishiguchi, M. Molecular analysis of an attenuated strain of Cucumber green mottle mosaic virus using in vitro infectious cDNA clone: pathogenicity and suppression of RNA silencing. J. Plant Biochem. Biotechnol. 25, 79–86 (2016). https://doi.org/10.1007/s13562-015-0312-z

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  • DOI: https://doi.org/10.1007/s13562-015-0312-z

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