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Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31910
標題: 利用異源表現探討十字花科黑腐病第三型蛋白質分泌系統致病因子 AvrXccC、XopD、XopN 與 XopX 之生物功能
Using heterologous expression approaches to study the biological functions of Xanthomonas campestris pv. campestris type III effectors AvrXccC, XopD, XopN, and XopX
作者: 曾貞瑜
Tzeng, Jen-Yu
關鍵字: 十字花科黑腐病菌;Xanthomonas campestris pv. campestris;第三型蛋白質分泌系統致病因子;異源表現;type III secretion system effectors;heterologous expression
出版社: 植物病理學系所
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摘要: 
The genus of gram-negative Xanthomonas comprises 27 species that can infect over 400 hosts, including Brassicaceae, Solanaceae, Apiaceae, etc. To infect plants, the bacteria must overcome plant cellular defenses of PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI) by a subset of effector (T3Es) that are translocated from the bacteria into plant cells via the type III secretion system (T3SS). In nonhost plants, however, T3Es can be recognized by resistance protein (R protein) to induce ETI. Previously, researchers have used genetic approaches, e.g. mutagenesis and ecotopic expression, to characterize bacterial t3es functions in planta. Due to genetic redundancy, only a few t3es exhibit quantitative and qualitative mutant phenotypes upon inoculation onto plants. In this study, we applied a heterologous expression approach to characterize X. campestris pv. campestris (Xcc) t3es using X. campestris pv. raphani (Xcr) as a recipient strain. Xcc causes systemic black rot disease on Brassicaceae, whereas Xcr elicits localized bacterial spots on the leaves of Brassicaceae and Solanaceae. We predict that the two closely related xanthomonads may harbor different T3Es to facilitate their interactions with plant cells. The hypothesis was firstly tested by Southern hybridization to show that Xcc t3es xopD, xopN, xopX, and aveXccC are not present in the genome of Xcr Cxsp3. We continued on analyzing the functions of the 4 Xcc T3Es by ecotopic expression, using Xcr as a recipient, to monitor bacterial population in cabbage and tomato leaves. Due to the instability of pBBR1-MCS5 and its derivatives, the ecotopic expression of Xcc xopX and avrXccC in Xcr did not yield discernible difference between wild type Xcr and transconjugants. Therefore, we modified the approach by inserting the gene-of-interest into the Xcr chromosome, and the resultant recombinants were characterized by their multiplication in planta and their involvement in modulating plant cellular responses. Our results revealed that Cxsp3::xopDXcc101, Cxsp3::xopNXcc101, Cxsp3::xopXXcc101, and Cxsp3::avrXccCXcc101 could maintain bacterial population in the leaves of cabbage and tomato, and delay or prevent the onset of disease symptoms. In parallel, Agrobacterium-mediated transient assay was conducted in tobacco (Nicotiana benthamiana and N. tabacum) and tomato (Solanum lycopersicum Mill) to assay for the putative functions of the 4 Xcc T3Es in planta. At 48-hr post infiltration, transiently expressed XopX induced confluent necrosis on Nb and Nt tobacco and tomato, whilst XopD, XopN, and AvrXccC only elicited chlorosis on tomato leaves but no discernible phenotypes on Nb or Nt tobacco. Our results revealed that XopD Xcc101, XopN Xcc101, XopX Xcc101, and AvrXccCXcc101 were virulence factors. The translocation of XopD Xcc101, XopN Xcc101, XopX Xcc101, or AvrXccCXcc101 by Xcr into plant cells delayed the onset of necrotic symptom, which prolongs bacterial survival in comparison with the sudden decline on the wild-type Xcr population upon symptom development. However, overexpression of XopX in N. benthamiana and N. tabacum by Agrobacterium-mediated transient assay induced cell death, suggesting that XopX might be involved in perturbing plant defenses in the later stage of infection to promote symptom development. In summary, the heterologous expression approach using Xcr as a recipient strain to assay for the phenotypes of Xcc T3Es is feasible and may uncover the disguised functions of Xcc T3Es.

Xanthomonas 為革蘭氏陰性菌，依據 DNA gyrase subunit B (gyrB) 核酸序列可分為 27 種，寄主範圍廣泛，包括十字花科、茄科、繖形花科等重要的蔬菜作物。植物病原細菌入侵寄主時須突破兩道植物防禦系統 (plant immune system) ─ PAMP-triggered immunity (PTI) 及effector-triggered immunity (ETI)。革蘭氏陰性病原細菌演化出第三型蛋白質分泌系統 (type III secretion system, T3SS)，利用 Hrp pilus 直接將致病因子 (effector, t3e) 送入寄主細胞中干擾植物防禦，但這些致病因子也可能被非寄主植物的 R (resistance) protein 辨識後快速啟動 ETI，進而限制病原菌增殖。目前研究 t3es 功能可透過細菌基因突變、接種試驗、利用基因轉殖在植物內表現 t3es 後進行性狀分析，或以生物資訊法分析胺基酸特定酵素活性區域及可辨識之 motif 以預測 T3Es 功能。本研究則採用異源表現方法，利用 2 株X. campestris病理小種 (pathovars campestris and raphani) 分析 Xanthomonas T3SEs 的功能。X. campestris pv. campestris (Xcc) 為十字花科黑腐病菌，而 X. campestris pv. raphani (Xcr) 則為十字花科細菌性斑點病菌，兩者雖有相近的分類地位，但在病徵表現與可感染的寄主種類相差甚大，推測兩者所帶的 T3SEs 可能為決定病徵及寄主範圍差異之因子。為確認此假設是否成立，首先以聚合酶連鎖反應與南方雜合法確認 Xcr Cxsp3 不帶有與 Xcc 具同源性之 xopD、xopN、xopX 與 avrXccC；再以 pBBR1-MCS5 選殖目標基因後將重組質體送入 Xcr 中表現，但 Xcr Cxsp3 帶有 pBBR1-MCS5 的轉形菌株在沒有抗生素篩選的培養環境中穩定性不佳，為使 Xcr Cxsp 轉型菌株接種寄主植物後可穩定表現目標基因，故將目標基因以同源互換方式插入 Xcr Cxsp3 染色體中，以 Xcr Cxsp3 重組菌株表現目標基因並接種於甘藍與番茄後發現，XopD、XopN 及 XopX 可抑制 Xcr Cxsp3 於寄主植物上之病徵發展，而AvrXccC 則延緩病徵之發生，且於接種後 12 天內皆可維持細菌族群數達 104 - 106 CFU / cm2。此外，利用Agrobacterium-mediated transient assay 於菸草及番茄葉肉細胞中短暫表現 Xcc XopD、XopN、XopX 及 AvrXccC 時發現XopX 可產生壞疽現象，而 XopD、XopN 及 AvrXccC 則不會在接種區域造成壞疽。根據此次研究結果得知，上述 4個目標基因皆為 Xcc 毒力因子，在Xcr中表現時可影響重組菌株於寄主植物番茄與甘藍上之細菌族群數及病徵之發展。以 Xcr 異源表現 Xcc 之T3Es，可避免傳統基因突變無法觀察之表現型，是目前研究 Xcc 第三型蛋白質分泌系統 t3es 功能另一可行的方法。
URI: http://hdl.handle.net/11455/31910
其他識別: U0005-2408201223502200
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