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標題: 利用轉基因植物探討十字花科黑腐病菌Xanthomonas campestris pv. campestris第三型致病性蛋白分子之致病機制
Functional analysis of Xanthomonas campestris pv. campestris type III effectors using transgenic plant approach
作者: 何宜屏
He, Yi-Ping
關鍵字: Xanthomonas campestris pv. campestris;十字花科黑腐病菌
出版社: 生物化學研究所
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A bacterial inhibitor of host programmed cell death defenses is an E3 ubiquitin ligase. Science 311, 222-226. Kim, J.G., Taylor, K.W., Hotson, A., Keegan, M., Schmelz, E.A., and Mudgett, M.B. (2008). XopD SUMO protease affects host transcription, promotes pathogen growth, and delays symptom development in xanthomonas-infected tomato leaves. Plant Cell 20, 1915-1929. Kim, J.G., Li, X., Roden, J.A., Taylor, K.W., Aakre, C.D., Su, B., Lalonde, S., Kirik, A., Chen, Y., Baranage, G., McLane, H., Martin, G.B., and Mudgett, M.B. (2009). Xanthomonas T3S Effector XopN Suppresses PAMP-Triggered Immunity and Interacts with a Tomato Atypical Receptor-Like Kinase and TFT1. Plant Cell 21, 1305-1323. Kinch, L.N., Yarbrough, M.L., Orth, K., and Grishin, N.V. (2009). Fido, a Novel AMPylation Domain Common to Fic, Doc, and AvrB. PLoS One 4. Kvitko, B.H., Park, D.H., Velasquez, A.C., Wei, C.F., Russell, A.B., Martin, G.B., Schneider, D.J., and Collmer, A. (2009). Deletions in the Repertoire of Pseudomonas syringae pv. tomato DC3000 Type III Secretion Effector Genes Reveal Functional Overlap among Effectors. Plos Pathog 5. Levine, B., and Yuan, J. (2005). Autophagy in cell death: an innocent convict? J Clin Invest 115, 2679-2688. Lin, C.S., Liu, N.T., Jane, W.N., Tsay, H.S., Wu, H., and Chang, W.C. (2007). Chloroplast genome aberration in micropropagation-derived albino Bambusa edulis mutants, ab1 and ab2. Plant Cell Tiss Org 88, 147-156. Metz, M., Dahlbeck, D., Morales, C.Q., Al Sady, B., Clark, E.T., and Staskawicz, B.J. (2005). The conserved Xanthomonas campestris pv. vesicatoria effector protein XopX is a virulence factor and suppresses host defense in Nicotiana benthamiana. Plant J 41, 801-814. Nomura, K., Debroy, S., Lee, Y.H., Pumplin, N., Jones, J., and He, S.Y. (2006). A bacterial virulence protein suppresses host innate immunity to cause plant disease. Science 313, 220-223. Ntoukakis, V., Mucyn, T.S., Gimenez-Ibanez, S., Chapman, H.C., Gutierrez, J.R., Balmuth, A.L., Jones, A.M., and Rathjen, J.P. (2009). Host inhibition of a bacterial virulence effector triggers immunity to infection. Science 324, 784-787. Records, A.R., and Gross, D.C. (2010). Sensor kinases RetS and LadS regulate Pseudomonas syringae type VI secretion and virulence factors. J Bacteriol 192, 3584-3596. Shan, L., He, P., Li, J., Heese, A., Peck, S.C., Nurnberger, T., Martin, G.B., and Sheen, J. (2008). Bacterial effectors target the common signaling partner BAK1 to disrupt multiple MAMP receptor-signaling complexes and impede plant immunity. Cell Host Microbe 4, 17-27. Stebbins, C.E., and Galan, J.E. (2001). Structural mimicry in bacterial virulence. Nature 412, 701-705. Wang, L., Tang, X., and He, C. (2007). The bifunctional effector AvrXccC of Xanthomonas campestris pv. campestris requires plasma membrane-anchoring for host recognition. Mol Plant Pathol 8, 491-501. Williams PH. (1980). Black rot: a continuing threat to world crucifers. Plant Disease 64.8, 736-742. Xiang, T., Zong, N., Zou, Y., Wu, Y., Zhang, J., Xing, W., Li, Y., Tang, X., Zhu, L., Chai, J., and Zhou, J.M. (2008). Pseudomonas syringae effector AvrPto blocks innate immunity by targeting receptor kinases. Curr Biol 18, 74-80.
由X. campestris pv. campestris所引起的黑腐病是十字花科作物的主要病害之一,然而其致病力則依賴於第三型分泌系統,因此透過第三型致病性蛋白分子,探討病原菌與植物的交互作用是防治X .campestris pv. campestris 的關鍵。但由於致病性蛋白分子具有的功能性重疊以及結構模仿的特性,因此無法單純藉由突變菌株以及序列比對來分析或預測致病性蛋白分子的功能。本實驗利用基因轉殖植物對X. campestris pv. campestris第三型致病性蛋白進行全面探討,結果發現經由誘導劑β-estradiol處理後XVE::AvrXccC8004、XVE::XopX8004轉殖株發芽後就停止生長,其葉片出現萎縮和死亡;XVE::AvrBs18004轉殖株在發芽後仍然可以生長,但植株成長緩慢,其葉片較為細長、彎曲且有黃化現象;XVE::XopD8004轉殖株發芽後則出現胚軸延長、子葉向下彎曲、葉綠素缺失的白化苗外表型。根據文獻指出AvrXccC8004和P. syringae 第三型致病性蛋白分子AvrB有60%以上相似度,而AvrB在結構上具有類似於AMPylation活性的Fido domain。為了釐清AvrXccC8004誘發細胞死亡的機制,本實驗建構AvrXccC8004不同缺失片段的轉基因植株,結果只有完整的AvrB-like domain之AvrXccC8004(110-440a.a.)才能誘發細胞死亡,同時也發現和AvrXccC8004相差一個胺基酸的AvrXcc1701也只需要AvrB-like domain就可誘發細胞死亡,而且AvrXcc1701 (110-440a.a.)-YFP會分佈於細胞質中並聚集成小點,因此AvrXccC1701誘發細胞死亡並不需要嵌入細胞膜上。透過細胞切片和電子顯微鏡的觀察得知,AvrXccC8004誘導後植物細胞的葉綠體趨向於扁平細長並有大量油滴出現,且出現類似自體吞噬的囊泡。由於植物自體吞噬的作用和免疫反應之間的關係尚未清楚,未來將朝此方向釐清AvrXccC8004造成細胞死亡的機制。

The black rot disease, caused by X. campestris pv. campestris, is one of the most important disease of the cruciferous crops. Its virulence is dependent on the type III secretion system to inject effector proteins. In order to control the disease caused by X.campestris pv.campestris, it is important to study the interaction between plants and pathogens through type III effector protein. However, due to functional redundancy and structural mimics of type III effector protein, it is difficult to analyze and pre-dict the functions of effector proteins simply by mutation studies or sequence alignment analysis. Here, ten type III effector proteins of X. campestris pv. campestris 8004 were analyzed by transgenic Arabidopsis approach. After β-estradiol treatment, XVE::AvrXccC8004 and XVE::XopX8004 transgenic plants stop growth after germination and show cell death phenotypes;XVE::AvrBs18004 transgenic plants show slowly growth phenotypes after germination and with slender and yellow color leaves;XVE::XopD8004 transgenic plants show etiolated seedling with long hypocotyl and chlorophyll-less symptoms. AvrXccC8004 has been show to have more than 60% similarity with P. syringae type III effector-AvrB. Structurely, AvrB was predicated to have a Fido domain with AMPylation-like activity. In order to understand the mechanism of cell death induced by AvrXccC-8004, transgenic plants with different truncated fragments of AvrXccC8004 were created. Howere, only AvrXccC8004 (110-440 a.a.), a intact AvrB-like domain, can induce cell death.The same result was observed in AvrXccC1701, a single amino acid different with AvrXccC8004. Morover, the influoresent signal of AvrXccC1701 (110-440 a.a.)-YFP was observed as foci in the cytoplasm. Therefore AvrXccC1701 induced cell death was not embedded in the cell membrane. The autophagosome-like vesicles and flatted chloroplasts with oil droplets were observed in XVE::AvrXccC8004 transgenic plants using cellular sections and electron microscopy. However, it is still not clear about the interaction between auto-phagy and immune response in plants. We will continuous to study the mechanism of cell death trigged by AvrXccC.
其他識別: U0005-2908201111081700
Appears in Collections:生物化學研究所

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