A Tobacco Rattle virus-induced Gene Silencing System for a soil-borne Vascular Pathogen Ralstonia solanacearum

由Ralstonia solanacearum引起的茄科作物青枯病是一具全球重要性的土壤傳播性維管束病害，然而茄科作物對青枯病菌防禦機制的研究及資料卻尚闕如。在此研究中，為了應用病毒誘導基因靜默系統(VIGS)於青枯病及其他土壤傳播性維管束病害相關研究，我們首先評估農桿菌菌株、植物株齡、接種方法及茄科植物品種對phytoene desaturase (PDS)基因靜默效率的影響，將煙草脆裂病毒的VIGS系統最佳化，並發展出子葉注射法。以此方法靜默PDS，發現在所有供試番茄品種上均有良好靜默效果，而部分番椒及所有茄子品種卻無明顯靜默效果。於9天大的番茄幼苗靜默植物抗病相關基因TGA2.2的測試發現，靜默效應在接種5天後已經在莖基部開始發生，接著在根部而後在幼葉呈現；同時將不同標的基因之片段建構於同一載體中所達到之多基因靜默效果，比混合數個各自帶有不同標的基因片段農桿菌菌株的方式更佳。利用本文建立之靜默系統進行茉莉酸防禦訊息傳遞途徑關鍵基因COI1之靜默，結果發現青枯病菌在番茄抗病品種'Hawaii 7996'莖基部及中段內的菌量顯著增加，顯示茉莉酸防禦訊息傳遞途徑在番茄對青枯病的防禦機制中扮演正面角色，此發現與阿拉伯芥對青枯病的防禦機制研究所知的相反；文中並討論其他防禦訊息傳遞途徑可能如何共同參與番茄青枯病防禦網絡。

關鍵字

青枯病；茉莉酸防禦訊息傳遞途徑；青枯病菌；病毒誘導基因靜默

並列摘要

Bacterial wilt (BW), caused by Ralstonia solanacearum, is a devastating soil-borne vascular disease of solanaceous crops worldwide. However, information on the defense mechanisms of Solanaceae against this bacterium is limited. In this study, we optimized a virus-induced gene silencing (VIGS) system to broaden the application of VIGS for the study of plant response to soil-borne vascular pathogens such as R. solanacearum. Due to the soil-borne and rapid xylem-routed infection features of BW, factors involved in Tobacco rattle virus-based VIGS system, such as Agrobacterium tumefaciens strains, plant ages, inoculation methods and plant genotypes, were evaluated. An optimized cotyledon agroinfiltration method was developed using phytoene desaturase gene as the marker. All tomato cultivars, but a few pepper and no eggplant cultivars tested showed good competence in gene silencing using the optimized VIGS protocol. Because tomato responses at spatial and temporal levels are critical for the outcome of R. solanacearum infection, spatial and temporal assessments of VIGS efficiency were carried out. The results showed that silencing of TGA2.2 in 9-day-old tomato plants initiated 5 days post inoculation in stembases and later in roots and then in young leaves. Moreover, a chimeric approach was shown to be effective for multi-gene silencing than mixing multiple Agrobacterium strains carrying individual target genes. With the established protocol, we were able to show that silencing COI1, an essential key player in the jasmonic acid (JA) signaling pathway, led to increase of R. solanacearum proliferation in stembases and mid-stems of 'Hawaii 7996', a tomato cultivar with durable resistance to BW. Our study provides the first demonstration for a positive role of the JA signaling pathway in tomato resistance to BW, and notably, is inconsistent with the reports in Arabidopsis. The involvement and possible interplays of other known defense signaling pathways in the tomato BW-defense network to R. solanacearum are discussed.

並列關鍵字

Bacterial wilt ； Jasmonic acid defense signaling pathway ； Ralstonia solanacearum ； Virus-induced gene silencing

國際替代計量

A Tobacco Rattle virus-induced Gene Silencing System for a soil-borne Vascular Pathogen Ralstonia solanacearum

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