請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/95777
標題: 台灣柑橘潰瘍病菌果膠分解酵素基因之表現與功能分析暨基因體分析
Expression and functional analysis of pectolytic enzyme genes and genome analysis of Xanthomonas citri subsp. citri Taiwan strains
作者: 張世杰
Shih-Chieh Chang
關鍵字: 柑橘潰瘍病菌
果膠分解酵素
啟動子活性
基因缺失
非典型病徵
基因體解序
致病因子
Xanthomonas citri subsp. citri
pectolytic enzyme
promoter acitivity
gene deletion
pectate lyase activity
atypical symptom
whole genome sequencing
virulence factor
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Characterization of phenotypically distinct strains of Xanthomonas axonopodis pv. citri from Southwest Asia. Eur. J. Plant Pathol. 104, 477-487.
摘要: 柑橘潰瘍病 (citrus canker) 為全球柑橘產業之重要病害,其病原菌 Xanthomonas citri subsp. citri (簡稱 Xcc) 為革蘭氏陰性、好氣之桿狀細菌,可感染芸香科各柑橘品種;果膠分解酵素可分解植物細胞壁,造成植物組織軟化、細胞質滲漏,並導致細胞死亡,為植物病原細菌之重要致病因子。本研究利用 β-glucuronidase (GUS) 做為報導蛋白,研究 Xcc strain XW19果膠分解酵素基因 pel1、pel2、pel3、pglA 及 peh-1之啟動子表現能力與其調控因子,並以基因缺失法針對 pel1、pel3、pglA 及 peh-1 等基因進行突變,探討其功能及對 Xcc strain XW19 致病過程的影響,發現 pel1、pel3、pglA 及 peh-1 基因啟動子之表現受 sodium polypectate、XVM2 培養基及柑橘葉片誘導且表現能力與細菌族群生長呈正相關,顯示 pel1、pel3、pglA 及 peh-1 可能與 Xcc strain XW19 之致病能力有關,然而只有 pel1 基因之表現受葡萄糖引起之分解代謝物抑制作用所抑制;pel3 基因啟動子於所有測試條件下皆可表現出最高之 GUS 活性,而 pel2 基因啟動子不論於培養條件下或植物體內皆測不到其表現量。接種突變菌株 XW19 ΔpglA 及 XW19 Δpeh-1 於柑橘葉片中,其病斑形態與病徵發展情形皆不受影響,推測 pglA 與 peh-1 基因可能與潰瘍病徵之發生無關,其功能尚待驗證;pel1 基因缺失會降低果膠裂解酵素活性,但其突變菌株 XW19 Δpel1並未造成柑橘葉片上病斑形態與病徵發展情形之變化,顯示 pel1 基因雖具分解果膠質的能力,但非影響病斑水浸狀邊緣之唯一因子;接種突變菌株 XW19 Δpel3 於柑橘葉片中所產生之病斑較小且發病時間較晚,顯示Pel3 為 Xcc strain XW19 致病過程中之毒力因子,影響潰瘍病病徵之誘發及細菌在寄主植物體中族群生長情形。此外,為探討限制引起非典型病徵之 Xcc strain XW121 於寄主植物內生長的因素,利用 PacBio RSII sequencing platform 進行基因體解序,獲得 4 個重疊群,序列資料以 Magnifying Genome (MaGe) Annotation Platform 分析發現 Xcc strain XW121 具有 1 個大小為 5,194,029 bp 之染色體及 2 個大小分別為 88,415 bp 與 16,610 bp 之質體,基因體全長為 5,299,054 bp,G+C content 為 64.7%,基因體含 5,168 個 putative CDS,具 2 套 rRNA operons、56 個 tRNA 及 6 個 misc_RNA,其中 3,539 個 CDS 可被歸類於 COG functional classes,佔基因體之 68.48%,尚有 1,629 個 CDS 無法被定義。利用 Roary pipeline 及 SiLiX software 分析顯示 Xcc strain XW121 在演化上屬於 A 病原型且較接近分離自中國大陸之柑橘潰瘍病菌株,與 A、Aw 及 A* 病原型進行比對分析後發現,Xcc strain XW121 具有 368 個 CDS 為其菌株所獨有,其中 334 個為未知功能基因、3 個為 transposase 及 31 個具預測功能之基因,其重要性尚待了解;此外,Xcc strain XW121 相較於 Xcc strain 306 缺少 XynB2、PthA1、PthA2、XopC2、XopAK 及 Hpa1 等致病因子,這些基因與非典型病徵之產生及細菌族群生長受限之關聯性尚待進一步實驗證實。
Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is one of the most destructive diseases of citrus. The pectolytic enzymes produced by phytobacteria are important virulence factors involved in tissue maceration, electrolyte loss and cell death of host plants. In this study, the promoter activity of the pectolytic enzyme genes pel1, pel2, pel3, pglA, and peh-1 were investigated in Xcc strain XW19 using the β-glucuronidase (GUS) gene as a reporter, and the function of pel1, pel3, pglA, and peh-1 were investigated using the gene deletion mutagenesis. GUS activity expressed under the control of the pel1, pel3, pglA, and peh-1 gene promoters positively correlated with bacterial growth, and displayed high GUS activity in the presence of sodium polypectate and induced in XVM2 medium and host plant. However, only pel1 was subjected to catabolite repression by glucose. GUS activity was significantly enhanced in the XW19-derived reporter strains after they were inoculated into the leaves of Mexican lime and grapefruit, suggesting the involvement of the pel1, pel3, pglA, and peh-1 genes in XW19 pathogenesis. The pel3 promoter produced the highest GUS activity under all test conditions, whereas no GUS activity was detected using the pel2 promoter in vitro and in planta. In comparison with wild type XW19, the canker lesions elicited by the pel1, pglA or peh-1 mutant was the same on the leaves of Mexican lime and grapefruit. Although, the pel3 mutant displayed reduced growth and induced smaller canker lesions on the leaves of Mexican lime or grapefruit compared with the wild type XW19, we demonstrate that Pel3 of Xcc strain XW19 is a virulence factor, and affects the canker elicitation and bacterial growth in host plant. In addition, the PacBio RSII sequencing platform was used to obtain a complete sequence of Xcc strain XW121, an Ar pathogenic variant in Taiwan. Based on Magnifying Genome (MaGe) Annotation Platform analysis, Xcc strain XW121 has a single, circular chromosome and 2 plasmids. The genome size is 5,299,054 bp and the G+C content averages 64.7%. The Xcc strain XW121 genome contains 5,168 putative CDSs, 2 rRNA operons, 56 tRNAs, and 6 misc_RNAs. There were 3,539 CDSs (68.48%) could be assigned to one or more COG functional classes, whereas there was not enough evidence for 1,629 CDSs to be assigned to any COG category. Phylogenetic analysis revealed that Xcc strain XW121 was grouped into the same cluster with A pathotype and more closely related with strains isolated from China. We also compared Xcc strain XW121 to the genome of Xcc strains 306, NCPPB 3608, and JJ238-24. Comparative genomic analysis showed 368 CDSs are unique to XW121, of which 334 are hypothetical genes, 3 are transposase, and 31 are singletons with predicted functions. In addition, XynB2, PthA1, PthA2, XopC2, XopAK, and Hpa1 were absent from Xcc strain XW121. These virulence factors might be responsible for the atypical symptom-producing and bacterial growth in host plant.
URI: http://hdl.handle.net/11455/95777
文章公開時間: 2020-02-06
顯示於類別:植物病理學系

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