探討吉貝素在水稻與徒長病菌交互作用中扮演的角色

徒長病 (bakanae disease) 由鐮孢菌 Fusarium fujikuroi 感染所引起，受感染的水稻產生植株異常抽高、葉片夾角變大、黃化，以及不稔實等病徵，是臺灣重要的水稻病害之一。植物賀爾蒙吉貝素 (gibberellins, GA) 最初由徒長病菌的培養液中純化鑑定而來，能促進種子發芽、枝條生長及果實發育，並參與植物對抗逆境的反應。前人研究指出，感病水稻品種受到徒長病菌感染後會累積大量GA，然而由於病原菌及植物皆會產生GA，兩者在致病過程中各自扮演何種角色仍有待釐清。本研究透過同源互換，成功剔除徒長病菌Ff266菌株在GA生合成路徑上的重要酵素細胞色素P450-1之基因，再透過液相層析串聯質譜儀分析，確認取得GA合成能力低下之p450-1突變株。將Ff266、G3-1 (綠色螢光菌株) 及p450-1突變株分別接種至水稻感病品種Zerawchanica Karatalski及抗病品種臺農67號的種子，發現p450-1突變株顯著降低罹病度，代表徒長病菌產生之GA會幫助徒長病的病徵發展；qPCR分析及徒手切片顯微觀察則顯示，Ff266、G3-1及18-2 (p450-1突變株) 在水稻根部之定殖程度沒有顯著差異。透過新開發的水耕幼苗接種法，以孢子懸浮液接種5天大的幼苗根部，則發現突變株18-2幾乎無法侵染水稻根部，推測徒長病菌GA有助於其侵染根部組織，但在種子接種系統中，可能因為水稻在種子發芽時期會產生大量GA，因此即使是缺乏GA生合成能力的突變株18-2也能夠順利侵染。茉莉酸 (jasmonic acid, JA) 介導的防禦反應是水稻抵禦徒長病菌的主要機制，為了瞭解病原菌是否透過產生GA抑制水稻防禦反應，本研究以即時定量反轉錄聚合酶連鎖反應測試水稻種子接種Ff266及突變株18-2後3天及7天之基因表現，發現phenylalanine ammonia lyase (OsPAL)、allene oxide synthase 2 (OsAOS2) 及coronatine insensitive 1 (OsCOI1) 基因的表現量幾乎不被調控，分別表示徒長病菌的侵染並未影響水稻水楊酸 (salicylic acid, SA) 生合成、JA生合成及JA訊號接收；接種後3天呈現jasmonate ZIM-domain 9 (JAZ9) 基因顯著下調控及pathogenesis-related 1 α (PR1α) 基因的上調控，顯示徒長病菌的侵染啟動了JA相關防禦反應，然而Ff266和18-2接種組之間並無顯著差異，顯示此時間點下徒長病菌GA並不會抑制水稻JA反應。本研究之成果可提供未來探討水稻與徒長病菌交互作用之參考。

關鍵字

水稻；徒長病；徒長病菌；植物防禦；吉貝素；茉莉酸；根部侵染；水耕幼苗接種

並列摘要

Bakanae disease, caused by Fusarium fujikuroi, is one of the most important rice diseases in Taiwan. Infected rice seedlings show symptoms of abnormal elongation, enlarged leaf angle, yellowing, and infertility. The plant hormone gibberellins (GA) were first purified and identified from the liquid culture of F. fujikuroi. GA is known for the effects of promoting seed germination and shoot/fruit development, and was reported to participate in plant responses to stresses. Previous studies indicated that susceptible rice cultivars showed high accumulation of GA after F. fujikuroi infection. Because both the host plant and pathogen produce GA during pathogenesis, the roles of GA in the rice-Fusarium fujikuroi interaction remain to be clarified. In this study, the gene encoding the key enzyme in the biosynthesis of GA in F. fujikuroi Ff266, cytochrome P450-1, was deleted by homologous recombination. Ultra-performance liquid chromatography tandem mass spectrometer (UPLC-MS/MS) analysis was conducted to confirm the loss of GA production in the p450-1 mutants. The susceptible rice cultivar, Zerawchanica Karatalski, and the resistant rice cultivar, Tainung 67, were inoculated with F. fujikuroi isolates Ff266, G3-1 (expressing green fluorescence protein), or p450-1 mutants by the seed inoculation method. The p450-1 mutants significantly reduced disease severity, which indicated that GA produced by F. fujkuroi promotes the development of bakanae disease. Quantitative real-time PCR analysis, hand sectioning, and microscopic observation revealed similar levels of root colonization between Ff266/G3-1 and the p450-1 mutant 18-2. In a newly developed hydroponic-seedling inoculation system, the roots of 5-day-old seedlings were inoculated with the spore suspension, and the mutant 18-2 barely infected the rice root tissue. We speculated that the GA of F. fujikuroi contributes to root infection, however in the seed inoculation system, possibly due to the large amount of GA produced by rice during the seed germination stage, even the mutant 18-2 lacking the ability of GA biosynthesis can successfully infect rice tissues. Jasmonic acid (JA)-mediated response is critical in rice defense against F. fujikuroi. To understand whether the pathogen utilizes its GA to inhibit host defense mechanisms, quantitative real-time reverse transcription PCR was conducted to analyze the gene expression at 3 and 7 days post inoculation of seeds with Ff266 or the mutant 18-2. The expression levels of phenylalanine ammonia lyase (OsPAL), allene oxide synthase 2 (OsAOS2), and coronatine insensitive 1 (OsCOI1) genes were not regulated, which indicated that the infection of F. fujikuroi did not significantly affect salicylic acid (SA) biosynthesis, JA biosynthesis, and JA perception in rice, respectively. At 3 days post inoculation, jasmonate ZIM-domain 9 (OsJAZ9) gene was significantly down-regulated and pathogenesis-related 1 α (PR1α) gene was up-regulated, indicating that the infection of F. fujikuroi led to the induction of JA-related defense responses. However, no significant difference was observed between inoculations of Ff266 or 18-2, which suggested that the pathogen GA is not involved in suppressing the JA-mediated defense response in rice at that time point. The results of this study can provide a basis for future research on the interactions between rice and F. fujikuroi.

並列關鍵字

Oryza sativa ； bakanae disease ； Fusarium fujikuroi ； plant defense ； gibberellins ； jasmonic acid ； root infection ； inoculation of hydroponic seedlings