利用寄主誘導基因靜默策略抑制Fusarium fujikuroi鈣調磷酸酶基因表現以增進水稻抗徒長病

Abstract

水稻徒長病(bakanae disease)是由Fusarium fujikuroi所引起的真菌性病害，普遍發生於全球各水稻產區。目前對於水稻徒長病的防治多以化學藥劑為主，然而此方法可能會對環境造成不良影響或導致病原菌抗藥性的產生。寄主誘導基因靜默(host-induced gene silencing, HIGS)是近年來開發之生物技術為背景的防治方法，即利用寄主產生的小片段RNA (siRNA)作為分子標靶，針對病原菌或有害生物的特定基因進行RNA干擾(RNAi)，導致該基因靜默。鈣調磷酸酶(calcineurin)是一種由催化(catalytic, Cna1)及調控(regulatory, Cnb1)次單元所組成的蛋白質二聚體複合體。研究指出，鈣調磷酸酶會調控真菌的鈣離子訊息傳遞，並在生長、逆境耐受性及毒力上扮演重要角色。因此本研究首先利用實驗室已建立之番茄萎凋病菌Cna1及Cnb1兩個次單元的單基因突變菌株(single deletion mutant)進行基因功能分析，探討鈣調磷酸酶是否也在重要植物病原真菌之一的Fusarium屬中扮演重要角色。經由菌絲型態構造分析、酵素活性試驗(calcineurin phosphatase activity test)、菌株產孢及接種試驗，初步證明鈣調磷酸酶活性對於番茄萎凋病菌的生長、厚膜孢子形成及致病力是必需的。經實驗發現，鈣調磷酸酶抑制劑FK506及Cyclosporin A能夠抑制徒長病菌的生長。接著本研究進一步利用HIGS策略抑制F. fujikuroi CNA1及CNB1基因表現，促進水稻對徒長病的抗性。利用農桿菌基因轉殖的方式將徒長病菌鈣調磷酸酶的專一性RNA干擾 (RNAi)構築導入水稻中，建立轉殖株並以南方墨漬法(Southern blot) 篩選出單套轉基因之水稻。根據接種試驗及病原菌質量測定結果顯示，帶有FfCNA1-Ri 及FfCNB1-Ri表現載體的水稻轉殖株皆能提高對徒長病之抗性。本研究證明HIGS策略能有效靜默病原真菌鈣調磷酸酶表現，進而抑制徒長病菌的生長，同時提供未來水稻或其他重要作物病害防治上的新方向。

Bakanae or foolish seedling disease of rice is caused by the ascomycetous fungus Fusarium fujikuroi, of which is widespread in many rice growing areas. Current protection strategies rely on the fungicides, but this results in chemical-resistant F. fujikuroi and other undesirable environmental effects. An alternative approach involving RNAi, termed host-induced gene silencing (HIGS), of which generating siRNA molecules in plants that target to the specific mRNAs of pathogens and thereby resulted in their degradation. Calcineurin belongs to a Ca2+/calmodulin-dependent phosphatase, which is comprised of heterodimer with a catalytic (Cna1) and a regulatory (Cnb1) subunits. There have been known that calcineurin mediates Ca2+ signaling that regulates the growth, stress responses and virulence in fungal kingdom. Based on pharmacological inhibition of calcineurin in F. fujikuroi, we found that calcineurin inhibitor FK506 or cyclosporinA can strongly inhibit the growth of F. fujikuroi. We therefore aim to determine whether repressing the expression of CNA1 or CNB1 gene in F. fujikuroi by the HIGS strategy in rice can enhance resistance against bakanae disease. By using Agrobacterium-mediated gene transformation strategy, the FfCNA1-Ri and FfCNB1-Ri constructs have been introduced into rice plants, respectively, and the copy number of transgenes were analyzed by Southern blot hybridization in the transgenic lines. Based on the pathogen inoculation assay and fungal biomass quantification, results showed that the transgenic rice plants that carry the FfCNA1-Ri or FfCNB1-Ri constructs increased resistance against bakanae disease, promising its potential use in the future.