請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/95769
標題: 芒果炭疽病菌之組胺酸激酶基因CgHK5p與CgHK6功能性分析
Functional analysis of histidine kinase genes CgHK5p and CgHK6 in mango pathogen Colletotrichum gloeosporioides
作者: 陳原諄
Yuan-Jhun Chen
關鍵字: Colletotrichum gloeosporioides
組胺酸激酶
HAMP repeat
芒果炭疽病
環境逆境
致病性
Colletotrichum gloeosporioides
Histidine kinases
HAMP repeat
mango anthracnose
environmental stress
pathogenicity
引用: 安寶貞, 2003. 檬果炭疽病. 植物保護圖鑑系列10, 76-81. 吳雅芳等, 2012. 芒果綜合管理技術. 臺灣芒果產業發展研討會專刊: 63-72. 林?哲, 2016. 芒果炭疽病菌組氨酸激酶基因群之研究. 國立中興大學植物病理所學位論文. 林聰明等, 2008. 芒果病蟲害管理. 節能減碳與作物病害管理研討會專刊: 205-206. 張家綺, 2014. 以正向遺傳學方法鑑別芒果炭疽病菌之致病因子. 國立中興大學植物病理所學位論文. 陳立儀, 2012. 台灣芒果產業現況及輔導措施. 臺灣芒果產業發展研討會專刊: 1-8. Bahn YS, Jung KW, 2013. Stress signaling pathways for the pathogenicity of Cryptococcus. Eukaryot Cell 12, 1564-1577. Bahn YS, Kojima, K., Cox, G.M., and Heitman, J., 2006. A unique fungal two-component system regulates stress responses, drug sensitivity, sexual development, and virulence of Cryptococcus neoformans. Molecular Biology of the Cell 17, 3122–3135. Bahn YS, Xue C, Idnurm A, Rutherford JC, Heitman J, Cardenas ME, 2007. Sensing the environment: Lessons from fungi. Nature Reviews Microbiology 5, 57-69. Bayram O, Braus GH, Fischer R, Rodriguez-Romero J, 2010. Spotlight on Aspergillus nidulans photosensory systems. Fungal Genetics and Biology 47, 900-918. Buschart A, Gremmer K, El-Mowafy M, Van Den Heuvel J, Mueller PP, Bilitewski U, 2012. A novel functional assay for fungal histidine kinases group III reveals the role of HAMP domains for fungicide sensitivity. Biotechnol 157, 268-277. Catlett NL, Yoder OC, Turgeon BG, 2003. Whole-genome analysis of two-component signal transduction genes in fungal pathogens. Eukaryotic Cell 2, 1151-1161. Defosse TA, Sharma A, Mondal AK, et al., 2015. Hybrid histidine kinases in pathogenic fungi. Molecular Microbiology 95, 914-924. Duan Y, Ge C, Liu S, Wang J, Zhou M, 2013. A two-component histidine kinase Shk1 controls stress response, sclerotial formation and fungicide resistance in Sclerotinia sclerotiorum. Molecular Plant Pathology 14, 708-718. Duran R, Cary JW, Calvo AM, 2010. Role of the osmotic stress regulatory pathway in morphogenesis and secondary metabolism in filamentous fungi. Toxins (Basel) 2, 367-381. Eisenman HC, Casadevall A, 2012. Synthesis and assembly of fungal melanin. Applied Microbiology and Biotechnology 93, 931-40. Fillinger S, Ajouz S, Nicot PC, Leroux P, Bardin M, 2012. Functional and structural comparison of pyrrolnitrin- and iprodione-induced modifications in the class III histidine-kinase Bos1 of Botrytis cinerea. PLoS One 7. Foureau E, Clastre M, Montoya EJ, et al., 2014. Subcellular localization of the histidine kinase receptors Sln1p, Nik1p and Chk1p in the yeast CTG clade species Candida guilliermondii. Fungal Genetics and Biology 65, 25-36. Hoch JA, 2000. Two-component and phosphorelay signal transduction. Microbiol. 3:165–170. Hu Y, He J, Wang Y, et al., 2014. Disruption of a phytochrome-like histidine kinase gene by homologous recombination leads to a significant reduction in vegetative growth, sclerotia production, and the pathogenicity of Botrytis cinerea. Physiological and Molecular Plant Pathology 85, 25-33. Jacob S, Foster AJ, Yemelin A, Thines E, 2014. Histidine kinases mediate differentiation, stress response, and pathogenicity in Magnaporthe oryzae. Microbiologyopen 3, 668-687. Klein M, Swinnen S, Thevelein JM, Nevoigt E, 2017. Glycerol metabolism and transport in yeast and fungi: Established knowledge and ambiguities. Environmental Microbiology 19, 878-893. Lee JK, Jung HM, Kim SY, 2003. 1,8-Dihydroxynaphthalene (DHN)-melanin biosynthesis inhibitors increase erythritol production in torula corallina, and dhn-melanin inhibits erythrose reductase. Applied and Environmental Microbiology 69, 427-434. Li D, Agrellos OA, Calderone R, 2010. Histidine kinases keep fungi safe and vigorous. Current Opinion in Microbiology 13, 424-430. Lin CH, Chung KR, 2010. Specialized and shared functions of the histidine kinase- and HOG1 MAP kinase-mediated signaling pathways in Alternaria alternata, a filamentous fungal pathogen of citrus. Fungal Genetics and Biology 47, 818-827. Liu Z, Friesen TL, 2012. Polyethylene glycol (PEG)-mediated transformation in filamentous fungal pathogens. Methods in Molecular Biology 835, 365-375. Ludwig N, Lohrer M, Hempel M, et al., 2014. Melanin is not required for turgor generation but enhances cell-wall rigidity in appressoria of the corn pathogen Colletotrichum graminicola. Molecular Plant-Microbe Interactions Journal 27, 315-327. Masayuki Matsushita KDJ, 2002. Histidine kinases as targets for new antimicrobial agents. Bioorganic & Medicinal Chemistry 10, 855–867 Mascher T, Helmann JD, Unden G, 2006. Stimulus perception in bacterial signal-transducing histidine kinases. Microbiology and Molecular Biology Reviews 70, 910-938. Meena N, Kaur H, Mondal AK, 2010. Interactions among HAMP domain repeats act as an osmosensing molecular switch in group III hybrid histidine kinases from fungi. Biological Chemistry 285, 12, 121-132. Nongpiur R, Soni P, Karan R, Singla-Pareek SL, Pareek A, 2012. Histidine kinases in plants: Cross talk between hormone and stress responses. Plant Signaling & Behavior 7, 1230-1237. Ota IM, And Varshavsky, A, 1993. A yeast protein similar to bacterial two-component regulators. Science 262: 566–569. Perraud A-L, Weiss V, Gross R, 1999. Signalling pathways in two-component phosphorelay systems. Trends in Microbiology 7, 115-120. Posas F, Wurgler-Murphy SM, Maeda T, 1996. Yeast HOG1 MAP kinase cascade is regulated by a multistep phosphorelay mechanism in the SLN1–YPD1–SSK1 Two-Component Osmosensor. Cell 86, 865–875. Qiu L, Wang JJ, Chu ZJ, Ying SH, Feng MG, 2014. Phytochrome controls conidiation in response to red/far-red light and daylight length and regulates multistress tolerance in Beauveria bassiana. Environmental Microbiology 16, 2316-2328. Radice S, Ferraris M, Marabini L, Grande S, Chiesara E, 2001. Effect of iprodione, a dicarboximide fungicide, on primary cultured rainbow trout (Oncorhynchus mykiss) hepatocytes. Aquatic Toxicology 54, 51-68. Singleton CK, Xiong Y, 2013. Loss of the histidine kinase DhkD results in mobile mounds during development of Dictyostelium discoideum. PLoS One 8, 756-818. Spadinger A, Ebel F, 2017. Molecular characterization of Aspergillus fumigatus TcsC, a characteristic type III hybrid histidine kinase of filamentous fungi harboring six HAMP domains. International Journal of Medical Microbiology 307, 200-208. W H, 1980. Calcofluor white and Congo red inhibit chitin microfibril assembly of Poterioochromonas evidence for a gap between polymerization and microfibril formation. Journal of Cell biology 87. Wang F, Tao J, Qian Z, et al., 2009. A histidine kinase PmHHK1 regulates polar growth, sporulation and cell wall composition in the dimorphic fungus Penicillium marneffei. Mycological Research 113, 915-923. Wiedemann A, Spadinger A, Lowe A, Seeger A, Ebel F, 2016. Agents that activate the high osmolarity glycerol pathway as a means to combat pathogenic molds. International Journal of Medical Microbiology 306, 642-651. Yamada-Okabe T, Mio, T., Ono, N., Kashima, Y., Matsui, M., Arisawa, M., and Yamada-Okabe, H, 1999. Roles of three histidine kinase genes in hyphal development and virulence of the pathogenic fungus Candida albicans. Bacteriol 181: 7243–7247. Yu PL, Chen LH, Chung KR, 2016. How the Pathogenic Fungus Alternaria alternata copes with stress via the response regulators ssk1 and sho1. PLoS One 11, 149-153. Zhang H, Liu K, Zhang X, et al., 2010. A two-component histidine kinase, MoSLN1, is required for cell wall integrity and pathogenicity of the rice blast fungus, Magnaporthe oryzae. Current Genetics 56, 517-528. Zhou G, Wang J, Qiu L, Feng MG, 2012. A Group III histidine kinase (mhk1) upstream of high-osmolarity glycerol pathway regulates sporulation, multi-stress tolerance and virulence of Metarhizium robertsii, a fungal entomopathogen. Environmental Microbiology 14, 817-829.
摘要: 芒果炭疽病菌Colletotrichum gloeosporioides造成芒果炭疽病害使芒果產量下降,貯藏期縮短,造成經濟上極大的損失。真菌是如何感知環境訊號以及對入侵寄主植物的過程進行反應是我們所想了解的。混合組胺酸激酶 (Hybrid histidine kinase;HHK) 具有感知真菌細胞內與細胞外環境訊號的功能並且調控真菌的生理反應。於前人研究中,由芒果炭疽病菌強毒力菌株TYC-2之基因組中發現12個HHK,其中三個基因經過基因功能分析後只發現參與部分環境逆境的感知,不參與在致病過程。本研究的目的為對TYC-2所帶有的HHK 基因CgHK6與CgHK5p進行功能性分析。利用農感菌轉殖法 (Agrobacterium tumefaciens-mediated transformation;ATMT) 結合split marker的技術對CgHK6與CgHK5p進行基因剔除,經過PCR與南方墨點法確認兩CgHK6基因剔除株ΔCgHK6A1與ΔCgHK6A2以及三CgHK5p基因剔除株ΔCgHK5p1、ΔCgHK5p2與ΔCgHK5p3進行後續試驗。在芒果未成熟與成熟之切離葉上的致病性測試結果顯示ΔCgHK6與TYC-2無顯著差異。ΔCgHK6A1與ΔCgHK6A2於PDA上有較TYC-2更為深黑色的菌落型態,ΔCgHK6A1與ΔCgHK6A2生長於MS培養基上有較TYC-2快的現象。處理1M 甘油之滲透壓逆境時ΔCgHK6A1與ΔCgHK6A2生長較TYC-2佳,然而處理1.5 M山梨糖醇之滲透壓逆境時ΔCgHK6A1與ΔCgHK6A2的耐受性較TYC-2低。處理氧化逆境時ΔCgHK6A1與ΔCgHK6A2相較於TYC-2對於0.1 mM H2O2有更好的耐受性。處理殺菌劑依普同時ΔCgHK6A1與ΔCgHK6A2之菌落直徑約為TYC-2的兩倍。ΔCgHK5p1、ΔCgHK5p2與ΔCgHK5p3在致病能力、一般生長、產孢、滲透壓逆境、金屬離子逆境、細胞壁穩定度以及處理殺菌劑時未發現與TYC-2有性狀上的顯著差異。結果顯示,CgHK5p可能不參與在測試之環境訊號的感知或是導因於基因功能互補 (Gene redundancy)。本研究亦證實CgHK6參與TYC-2對多種環境逆境的抗性與黑色素的生合成。
Colletotrichum gloeosporioides is a fungal pathogen causing mango anthracnose disease and limiting the production of mongo fruit. How this pathogen senses the environmental and host cues during pathogenesis is what we attempt to investigate. Hybrid histidine kinase (HHK) can perform a function of receiving intra- and extracellular environmental signals to regulate physiological processes in fungi. In a previous study, 12 genes encoding HHK have been identified in the genome of TYC-2 and three of them have been functional analysis but no function was identified among the three genes in TYC-2 pathogenicity. The objective of this study is to analyze the function of two additional HKKs in TYC-2, CgHK6 and CgHK5p. Agrobacterium tumefaciens-mediated transformation combined with split marker strategy was used to knockout the target gene. After confirming by PCR and Southern blotting, two CgHK6 knockout mutants, ΔCgHK6A1 and ΔCgHK6A2, and three CgHK5p knockout mutants, ΔCgHK5p1, ΔCgHK5p2 and ΔCgHK5p3, were generated and selected for further phenotyping. Pathogenicity tests on young mango leaf and mature fruit showed that no significant differences were observed between TYC-2 and CgHK6-null mutants. ΔCgHK6A1 and ΔCgHK6A2 developed darker colonies than TYC-2 did on PDA medium. Mycelial growth rate of the mutants increased when compared with TYC-2 on MS medium. Under osmotic stresses, ΔCgHK6 strains had better growth on medium containing 1M glycerol and worse growth on medium containing 1.5 M sorbitol than TYC-2. When exposed to oxidative stress, ΔCgHK6A1 and ΔCgHK6A2 showed higher tolerance to 0.1 mM H2O2 than TYC-2. After treated with fungicide Iprodione, colony diameters ofΔCgHK6 strains were almost two times larger as that of TYC-2. For the phenotyping of ΔCgHK5p strains, no significant differences were found between ΔCgHK5p strains and TYC-2 on pathogenicity, mycelial growth and sporulation under regular conditions, and mycelial growth under cell wall integrity, metal ions, osmotic and fungicides stresses. These results suggest that CgHK5p might not be involved in the response to those tested environmental signals or it might be due to gene redundancy. Our results conclude that CgHK6 plays important roles in TYC-2 responding to multiple stresses and melanin synthesis.
URI: http://hdl.handle.net/11455/95769
文章公開時間: 2020-08-24
顯示於類別:植物病理學系

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