探究徒長病對水稻苗期轉錄體之影響

Abstract

水稻徒長病 (bakanae disease) 為Fusarium fujikuroi所引起的種子及土壤傳播性真菌病害，其侵染水稻後會造成植株徒長及/或葉片夾角加大等不正常生理現象，嚴重時可導致植株死亡或穀粒不充實。據報導，水稻徒長病曾在許多大規模水稻種植國家如日本、印度、泰國等地造成嚴重損失；臺灣近年則於東部有較嚴重發病之情形。由於水稻抵禦徒長病菌的相關機制目前研究甚少，本研究利用Illumina次世代定序技術，分析水稻抗病品系Tainung 67 (TNG67)及感病品系Zerawchanica karatals (ZK) 之幼苗基部受徒長病菌感染7天後之轉錄體，透過比對水稻與阿拉伯芥資料庫進行基因功能預測，探討與賀爾蒙及防禦相關之基因表現變化。結果顯示，F. fujikuroi接種後有顯著表現差異之基因 (differential expression genes, DEGs)，在抗病品系TNG67有118個 [包括WRKY、plant pattern recognition receptor (PRR)及peroxidase]，在感病品系ZK有169個 (包括cytochrome P450、WRKY、PRR、glycoside hydrolase、thaumatin-like protein及peroxidase)。檢視賀爾蒙相關之基因網路，發現吉貝素生合成、茉莉酸生合成、茉莉酸訊號傳遞路徑於接種前後並無整體上升或下降趨勢；而在抗性品系TNG67中，參與吉貝素訊號傳遞之OsGID1 (吉貝素受體；促進DELLA蛋白之降解) 表現量下降、OsSLR1 (DELLA蛋白；吉貝素訊息傳遞抑制因子) 則顯著上升，顯示抗病品種對吉貝素有負調控的現象。針對16個被註解為抗性相關之DEGs，利用real-time qRT-PCR測定額外三次獨立實驗中，TNG67與ZK兩品種接種7、14、21天後之基因表現，其中八個基因在接種後7天與轉錄體分析結果具有一致性，且於不同次獨立實驗之趨勢一致。本研究選擇以CRISPR/Cas9技術進行基因功能喪失驗證，首先以實驗室前人研究所得之三個抗稻熱病候選基因為對象，建立水稻CRISPR/Cas9系統，再應用於四個抗徒長病候選基因之突變。上述候選基因之single guide RNA (sgRNA) 質體，經T7核酸內切酶I進行水稻原生質體短暫表現之突變體檢測後，篩選出sgRNA突變率超過10%者，繼續進行水稻轉殖株之建立。目前已獲得其中一個基因之五個T0轉殖品系，經基因型測定後，確認有四個品系於目標序列被成功突變。期望透過本研究瞭解水稻對徒長病之抗性網路及有潛力的抗性基因，為水稻育種及病害防治找到新的發展方向。

Bakanae disease is a seedborne and soilborne fungal disease caused by Fusarium fujikuroi. The plants infected with the pathogen show symptoms such as abnormal stem elongation and/or wider angle of the leaf with the stem. Severe infection can cause seedling blight and sterile grains. In rice-growing countries such as Japan, India, and Thailand, there are reports on bakanae disease causing yield losses and great impact on rice production. In recent years, outbreaks of bakanae disease were observed in Taiwan, particularly in the eastern areas. Since little is known about the mechanisms involved in rice against F. fujikuroi, the research focuses on applying Illumina next-generation sequencing technology to analyze the transcriptome profiles of a resistant cultivar Tainung 67 (TNG67) and a susceptible cultivar Zerawchanica karatals (ZK), using the basal stems of rice seedlings collected after 7 days post inoculation (dpi) of F. fujikuroi. Genes were annotated according to the reference databases of Oryzae sativa (MSU7) and Arabidopsis thaliana (TAIR10). Expression profiles of genes associated with phytohormones and defense responses were examined. There were 118 differential expression genes (DEGs) in the resistant TNG67 [including WRKYs, plant pattern recognition receptor (PRR), and peroxidases] and 169 DEGs in the susceptible ZK (including cytochrome P450, WRKY, PRR, glycoside hydrolase, thaumatin-like protein, and peroxidase). For phytohormone biosynthesis and signaling pathways, overall up- or down-regulations were not observed in gibberellin (GA) biosynthesis, jasmonic acid (JA) biosynthesis, and JA signaling pathways. In TNG67, the GA signaling pathway seemed to be suppressed according to the down-regulation of OsGID1 (gibberellin receptor; induced degradation of DELLA proteins) and up-regulation of OsSLR1 (DELLA protein; GA-insensitive gene homolog) after F. fujikuroi inoculation. Real-time quantitative RT-PCR was conducted to evaluate the expression levels of 16 defense-related DEGs at 7, 14, and 21 dpi in additional three independent inoculation trials. A set of 8 candidate genes, with 7-dpi expression patterns similar to the transcriptome data and consistency among different replicated trials, were selected for loss-of-function verification using CRISPR/Cas9 system. Three blast-resistance candidate genes identified in our another study were used as targets to set up CRISPR/Cas9 system, which was subsequently applied to four candidate genes associated with bakanae resistance. Mutation efficiencies of single guide RNA (sgRNA) vectors were tested in rice protoplasts using T7 endonuclease I (T7E1) assay, and the constructs showed mutation efficiencies of >10% were sent for generating transgenic rice plants. Currently five T0 mutant lines for one candidate gene were obtained. These lines have been genotyped and four lines were successfully mutated at the target site. Uncovering potential resistance-related genes and mechanisms will provide new insights into resistance breeding and disease control of bakanae disease.