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Development of the VIGS system towards enhancing the production level of hatching factors for potato cyst nematode using Nicotiana benthamiana

Go Atsumi: National Institute of Advanced Industrial Science and Technology

<div>Cyst nematodes threaten stable productions of economically important crops such as potato. Host potato plants release chemicals such as triterpenoids including solanoeclepin A from the roots to stimulate hatching of the nematode. However, the biosynthetic pathways are unknown. In this study, we developed a system to evaluate a hatching activity towards potato cyst nematode using root exudates from <em>Nicotiana benthamiana</em> which is a suitable host for a rapid analysis of gene functions using technologies such as virus-induced gene silencing (VIGS). We found that root exudates from <em>N. benthamiana</em> have a hatching activity for potato cyst nematode in <em>in vitro</em> hatching assay. To enhance overall triterpenoid synthesis, we down-regulated the competitive pathways leading to cholesterol and/or phytosterols using tobacco rattle virus (TRV) vector-based VIGS in hydroponically grown <em>N. benthamiana</em>. We isolated the genes encoding enzymes, cycloartenol synthase 1 [<em>CAS1</em>], sterol side chain reductase 2 [<em>SSR2</em>], and S-adenosyl-L-Met-dependent C-24 sterol methyltransferase 1 [<em>SMT1</em>] from<em> N. benthamiana</em>, and constructed TRV vectors to down-regulate respective genes. VIGS using the constructed TRVs successfully reduced <em>CAS1</em>, <em>SSR2</em> and <em>SMT1</em> expressions to approximately 14, 11, and 6% in root tissues 16 days after virus inoculation, respectively. Their expressions were similarly down-regulated in leaf and stem tissues. We believe that the TRV-based VIGS system in <em>N. benthamiana</em> will be a powerful tool to elucidate the biosynthetic pathways of hatching factors, eclepins. This work was supported by a grant from the New Energy and Industrial Technology Development Organization (NEDO) of Japan.</div>