探討番茄 12g690 調控之病害反應與抗青枯病番茄 Hawaii 7998 之特性

Abstract

青枯病是由病原細菌 Ralstonia solanacearum (Rs) 引起之土壤傳播性病害，對眾多重要經濟作物造成嚴重危害，因此其抗病育種工作急迫且重要。在抗青枯病之番茄品系Hawaii 7996 (H7996) 中主導抗第一型 Rs 菌系之量化性狀基因座 (quantitative trait locus，簡稱QTL) 為Bwr12；我們先前研究顯示，位於此區之 12g690 可能會透過調控水楊酸 (salicylic acid, SA) 與茉莉酸 (jasmoic acid, JA) 訊息傳遞而在番茄抵抗青枯病中扮演負向調控者角色。此外， Hawaii 7998 (H7998) 為另一青枯病抗病番茄品系，但其抗病特性與防禦機制尚未闡明。本研究之第一部份為針對 12g690 在多種重要病害之角色與其基因特性進行進一步探討。以轉殖菸草驗證 12g690 在茄科植物抵抗青枯病反應中扮演負向調控者，但12g690 之轉錄表現在番茄莖基部與根部可能未受 Rs 感染調控，且在抗病 H7996 與感病 WVa700 番茄品系中之表現亦無明顯差異。此外，利用短暫基因過量表現、短暫基因靜默及基因轉殖植物等策略所得之分析結果指出 12g690 在番茄與菸草防禦 Pseudomonas syringae pv. tomato (Pst)、 Pectobacterium carotovorum subsp. carotovorum (Pcc) 及 Botrytis cinerea (Bc) 的反應中可能並非主要調控者。分析 H7996 與 WVa700 之 12g690之 genomic DNA 與 cDNA序列發現，H7996 序列中有數個非同義差異 (non-synonymous SNPs) 與一個24-bp insertion，在此兩種品系番茄之莖基部與根部中 12g690 mRNA 主要是以保留兩個內含子 (intron) 形式存在，且剪切情況並未因感染 Rs 而有明顯改變；在植體中表現重組蛋白質所得結果也一致指出，因 12g690 RNA 之第一個 intron 被保留而導致轉譯時造成 early termination 以致於產生僅有36個胺基酸之蛋白質產物 (12g6901-36)。蛋白質定位結果顯示 12g6901-36-GFP 座落於細胞膜，而全長之12g690 (12g690FL)-GFP 則無法被表現出來。本研究之第二部份分析 H7998抗病範圍所得結果顯示，H7998 不僅對 Pcc、 Pst 及 Bc 都有良好抗性，且對 Pcc與 Pst之抗性較 H7996 更佳，且其 H2O2 累積與 callose deposition等初級免疫反應也比 H7996 稍強。以上研究結果提供了番茄防禦相關之重要訊息。

Bacterial wilt (BW) caused by Ralstonia solanacearum (Rs) is a devastating soil-borne disease of many crops, and breeding for durable resistance is urgent and important for disease control. The major QTL associated with tomato cultivar Hawaii 7996 (H7996) resistance against Rs phylotype I strains is named Bwr12. Our previous studies suggested that 12g690 may have a negative role in tomato resistance to BW via regulating SA and JA pathways. In addition, the disease responses and defense nature of another BW-resistant tomato cultivar Hawaii 7998 (H7998) are not characterized. The first part of this study aimed to further determine the roles of 12g690 in several important diseases and its molecular characteristics. Characterization of transgenic tobacco plants confirmed the negative role of 12g690 in BW-defense. However, 12g690 transcription in tomato root and stem-base might not be regulated by Rs infection nor differentially regulated in H7996 and susceptible cultivar WVa700. In addition, results of transient and transgenic functional genetic analyses suggested that 12g690 may not have a major role in tomato and tobacco against Pseudomonas syringae pv. tomato (Pst), Pectobacterium carotovorum subsp. carotovorum (Pcc) and Botrytis cinerea (Bc). Furthermore, sequence analysis of 12g690 genomic DNA in H7996 and WVa700 revealed non-synonymous SNPs and a 24-bp insertion are present in H7996. Analyses of 12g690 transcripts showed that most transcripts retain both introns in both cultivars and this intron-retention phenomenon is not altered by Rs infection. Intron retention in 12g690 transcripts would lead to early termination with a predicted protein product of 36 amino acids (12g6901-36) and in planta expression of 12g690 recombinant protein showed consistent results. Protein subcellular localization assay further showed that 12g6901-36-GFP localizes on plasma membrane, while 12g690FL-GFP was not detectable. The second part of this study aimed to determine the disease responses and defense nature. The results showed that H7998 confers similar or higher degrees of resistance to Pcc, Pst and Bc compared to H7996. Moreover, H7998 confers slightly higher levels in H2O2 accumulation and callose deposition, two key characteristics of innate immunity. Together, these results provide important information regarding the nature of tomato defense.