香蕉黃葉病菌熱帶第四型生理小種特異性基因之親緣及功能性分析

Abstract

香蕉黃葉病為土壤傳播性病原真菌 Fusarium oxysporum f. sp. cubense（Foc）所引起之重要病害，廣泛分布於全球香蕉產區。該病害對東亞、東南亞、非洲及拉丁美洲的香蕉產業造成嚴重威脅，已成為全球香蕉種植之主要限制因子。然而目前尚無有效殺菌劑或其他防治手段可有效控制此病害，對香蕉產業構成長期挑戰。Foc依其對不同香蕉品種的致病性分為四個生理小種（race），其中熱帶第四型生理小種（Tropical Race 4; TR4）目前已迅速擴散、並對全球香蕉主要產區的所有商業品種最具毀滅性。近年來，隨著對尖鐮孢菌（Fusarium oxysporum）的基因體學與分子生物學的深入研究，發現其基因體中存在譜系專一性染色體（lineage-specific chromosome; LS chromosomes）。這些染色體富含轉座子，並攜帶具特殊演化特徵且與致病力相關之基因，參與決定病原菌的寄主專一性及與寄主植物之交互作用，且可透過水平基因轉移於不同菌株間傳播。而實驗也證明，其中包含SIX （Secreted in Xylem）效應蛋白基因的LS染色體，在尖鐮孢菌菌株間的轉移，可將非致病菌株轉化為病原菌，有助於感染寄主，因此可知LS染色體對病原菌演化之重要性。實驗室先前研究利用自臺灣分離之TR4與Race 1（R1）菌株，進行基因體定序及比較基因體分析。結果顯示於TR4第3號染色體上具一段LS區域，該區域含有一群僅存在於TR4、而在R1缺失的基因。本研究進一步選取其中三個TR4特有基因（TR4_1128950、TR4_1129000與TR4_1129050），進行功能驗證與演化分析，而TR4_1128950與TR4_1129000預測為細胞質效應子（cytoplasmic effectors）。序列比對與親緣分析顯示目標基因於多種尖鐮孢菌群（Fusarium oxysporum species complex, FOSC）中高度保守，並以多重拷貝形式分佈於不同LS染色體上。為探討這些基因的功能，構建目標基因之TR4過量表現轉殖株，致病接種試驗顯示，過量表現株在感病品種北蕉（Pei-Chiao）與抗病品種台蕉7號（Tai-Chiao No. 7, TC7）的致病能力均略高於野生株；此外，為釐清這些LS基因對寄主專一性之影響，將包含上述TR4特有基因之8.5 kb LS區段導入R1菌株中，接種結果顯示R1轉殖株可感染北蕉，惟致病程度有限，顯示該LS區域為致病所需，但尚不足以引起典型病徵。進一步利用表現eGFP標記之特有基因融合蛋白菌株，在不同培養條件下，透過共軛焦顯微鏡觀察目標蛋白於細胞之定位，結果顯示目標蛋白主要分布於液泡或囊泡結構。綜上所述，本研究揭示Foc TR4基因體LS區域中的特異性基因，在致病力、寄主專一性及演化過程中扮演重要的角色，可做為未來發展香蕉黃葉病防治策略的基礎。

Fusarium wilt of banana is an important banana disease which is caused by the soil-borne fungal pathogen Fusarium oxysporum f. sp. cubense (Foc) and also widely distributed across global banana-producing regions. The disease poses a severe threat to banana industries in East and Southeast Asia, Africa, and Latin America, and is considered one of the most significant constraints to banana cultivation worldwide. To date, no effective fungicides or other control strategies are available to efficiently manage this disease, presenting a long-term challenge for global banana production. Foc is classified into four physiological races. Among them, Tropical Race 4 (TR4) has rapidly spread and is currently the most devastating to all commercial banana varieties in major banana-producing regions worldwide. In recent years, advances in genomics and molecular biology of Fusarium oxysporum have revealed the presence of lineage-specific (LS) chromosomes within their genomes. These LS chromosomes are enriched with transposable elements and harbor genes with unique evolutionary characteristics that are associated with pathogenicity. They play critical roles in determining host specificity and mediating pathogen–host interactions, and can be horizontally transferred between strains. Experimental evidence has demonstrated that LS chromosomes carrying SIX (Secreted in Xylem) effector genes can transform non-pathogenic strains into pathogen, highlighting their importance in the evolution of virulence in F. oxysporum. Previous studies in the lab, Foc TR4 and Race 1 (R1) strains isolated from Taiwan were subjected to genome sequencing and comparative genomic analyses. Results revealed an LS region located on chromosome 3 of TR4 genome, comprising a cluster of genes that are present only in TR4 and absent in R1. In this study, three TR4-specific genes within this region, TR4_1128950, TR4_1129000, and TR4_1129050, were selected for functional characterization and evolutionary analysis. Among them, TR4_1128950 and TR4_1129000 were predicted as cytoplasmic effectors. Sequence alignment and phylogenetic analyses revealed that these genes are highly conserved across Fusarium oxysporum species complex (FOSC), and are distributed as multiple copies on their distinct LS chromosomes. To functionally characterize these genes. TR4 transformants overexpressing these target genes were generated. Pathogenicity assays demonstrated that overexpression transformants exhibited enhanced virulence compared to the wild-type strain on both susceptible ‘Pei-Chiao (PC)’ and resistant ‘Tai-Chiao No.7 (TC7)’ varieties. To further assess their role in host specificity, an 8.5 kb fragment containing three TR4-specific genes was introduced into R1 genome. Pathogenicity assays revealed that R1 transformants were capable of infecting PC, although the extent of infection was limited, suggesting an 8.5 kb LS region was required but is not solely sufficient to cause typical symptom. Subcellular localization study using eGFP-tagged candidate proteins revealed their accumulation in vacuoles/vesicle-localization under different cultural conditions. In summary, this study highlights critical roles of TR4-specific genes on a LS regions in Foc virulence, host specificity, and evolution, and may provide the foundation, for the development of future disease control strategies.