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|標題:||桃褐腐病菌 MfAP1 基因選殖及功能分析
Cloning and functional analysis of an oxidative stress-regulated gene MfAP1 from Monilinia fructicola
|關鍵字:||桃褐腐病菌;Monilinia fructicola;激活蛋白;氧化還原;activator protein 1;Redox||出版社:||植物病理學系所||引用:||參考文獻 Annis SL, Goodwin PH, 1997. Recent advances in the molecular genetics of plant cell wall-degrading enzymes produced by plant pathogenic fungi. European Journal of Plant Pathology 103, 1-14. Aguirre J, Rios-Momberg M, Hewitt D, Hansberg W, 2005. Reactive oxygen species and development in microbial eukaryotes. Trends in Microbiology 13, 111-8. Alarco AM, Raymond M, 1999. The bZip transcription factor Cap1p is involved in multidrug resistance and oxidative stress response in Candida albicans. Journal of Bacteriology 181, 700-8. Apel K, Hirt H, 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annual Review of Plant Biology 55, 373-99. Beckman KB, Ames BN, 1998. The free radical theory of aging matures. Physiological Reviews 78, 548-71. Biggs AR, Northover J, 1988. Early and late-season susceptibility of peach fruits to Monilinia fructicola. 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Physiological and Molecular Plant Pathology 74, 100-10.||摘要:||
植物病原真菌感染過程中需要面對寄主為抵抗病原菌侵入所產生的氧化壓力 (reactive oxygen species, ROS )，其中參與氧化還原壓力調控一重要因子為轉錄因子activator protein 1又稱做AP-1，此因子已被研究指出於哺乳類動物及真菌中作為抗ROS的調控因子。本實驗對桃核腐病菌MfAP1基因進行分離解序，證實其於感染玫瑰花瓣過程中有表現的情形。除了MfAP1基因亦針對桃核腐病菌其他與氧化還原調控相關的基因MfGR1、MfGPx1及 MfG6PD1以及毒力因子MfCUT1 進行表現量分析，結果顯示這些基因隨著氧化還原狀態的改變基因表現亦有變化。欲了解MfAP1在桃核腐病菌感染桃果樹過程中所扮演的角色，利用原生質體轉殖系統及農桿菌轉殖系統分別產生MfAP1 silenced transformants 及MfAP1 overexpression transfomants。分析MfAP1 silenced transformants 特性，將轉殖株接種於玫瑰花瓣上顯示MfAP1 基因表現量下降使得桃核腐病菌毒力下降，且對氧化壓力較野生型菌株為敏感。另一方面，針對MfAP1 overexpression transformants進行特性分析，將轉殖株接種於桃果實上結果顯示MfAP1 基因表現量上升，可促進桃褐腐病病斑的擴展且較野生型菌株更能抵抗氧化壓力。綜合以上實驗結果證實MfAP1對桃褐腐病菌抵抗氧化壓力為重要的因子，且在感染過程中扮演毒力因子的角色。
Redox sensing is a common mechanism regulating celluar activity. Fungal pathogens have to face oxidative stress produced by the host plant via oxidative burst during plant-microbe interactions. The transcription factor activator protein 1 (AP-1), which has been characterized as a regulator in reactive oxygen species resistance in mammalian and fungi, has a crucial role in fungal virulence. An MfAP1 gene in Monilinia fructicola has been cloned and sequenced, which encoded a polypeptide that resembles many yeast AP1-like transcription factors of yeasts and fungi. Expression of MfAP1 gene is varied in different infection stages. Quantitative rt-PCR analysis of redox related genes of Monilinia fructicola after treated with caffeic acid and hydrogen peroxide shows that gene expression of these genes is in a redox-dependent manner. To investigate the role of MfAP1 in the infection process , two strategies were used in this experiment. One is PEG/CaCl2 mediated protoplast transformation to develop the MfAP1 silencing transformants; the other is Agrobacterium mediated T-DNA transformation to generate the MfAP1 overexpression transformants. When pathogenicity assay of MfAP1 silenced transformants was analyzedon rose petals, a significant reduction on brown rot lesion development was observed. On the other hand, the ability to develop lesion of MfAP1 overexpression transformants was promoted. When MfAP1 gene expression was silenced, these transformants were less effective in detoxifying H2O2. On the contrary, MfAP1 overexpression transformants were more resist to the oxidative stress. Our results demonstrate that MfAP1 is important in response to oxidative stress in M. fructicola and is a virulence factor of M. fructicola during peach fruit infection.
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