結論 - 利用AFLP分析茄科細菌性斑點病菌之變異性並選殖與致病性相關之基因產物

由 Xanthomonas campestris pv. vesicatoria (Xcv)引起的茄科細菌性斑點病，需透過 第三型分泌系統的調控機制和分泌的有效蛋白(effectors)引發其病原性。病原菌感染植物後所分泌的effector 蛋白與植物交互作用的結果，可呈現感病性或抗病性等不同

反應，因此尋找 effector 蛋白及探討其功能，是目前植物病理學的主流之一。

Xanthomonas 不同病原型的基因體內個別所包含的 effector 基因至少為 15 種以上 (Thieme et al., 2007)，這些基因除了少數集中分布於病原島嶼區域外，其餘則分散於染色體其他區域及質體上 (Joardar et al., 2005)。本研究中利用 AFLP 分析選殖出 XopE2 effector 蛋白，xopE2 在 Xanthomonas spp.中為高度保留的基因。有效蛋白的退 化代表其在致病過程中不被需要，或是有其他功能相近的蛋白可以相互取代、因此不同病原型菌株所保留的完整有效蛋白，應對其病原性或寄主範圍有效度不等的影響。

實驗結果顯示(1) xopE2 突變會影響 Xcv B 群菌株對番茄的毒性，(2)大量表現 A 群或 B 群的 XopE2 蛋白皆能降低細菌的繁殖與病徴的嚴重性，(3) A 群或 B 群的 XopE2 蛋白皆能抑制 P. syirngae pv, syringae 在煙草上引發的過敏性反應。此外，本研究中，

所有B 群菌株的 xopE2 基因不只存在於染色體上，亦存在於質體上，而 xopE1 基因

似乎只存在於A 群菌株而不存在 B 群菌株。有效蛋白的消長可能反應病原菌於寄主

上的適應性，是兩者共同演化的結果，兩菌群之間 xopE1 基因存在與否以及質體上是 否能獲得 xopE2 基因等現象，說明彼此之間的演化可能是由不同的路徑來進行。

茄科細菌性斑點病菌在分類上一直是有爭議的，目前已將 Xcv 之菌株重新分類

為四群：A 群 X. euvesicatoria (=X. axonopodis pv. vesicatoria)、B 群 X. vesicatoria

、

C 群 X. perforans 和 D 群 X. gardneri (Jones et al., 2000; 2004)。本研究中採用的 A 群供

試菌株屬於不同的生理小種，從AFLP 分析圖譜結果顯示，彼此之間差異性極大，利

用生物資訊分析，應有助於探討這些差異是否和生理小種不同所致，以及台灣是否也有C 群菌株的分佈問題。此外，本研究所使用 Xcv 兩群的 XopE2 蛋白的胺基酸序列

和兩側區域的基因組成具差異性，所以根據 xopE2 基因兩側的序列設計適合的引子

對，利用PCR 的技術來區分兩群菌株是值得嘗試且可行之法。

病原細菌在自然界中為了生存，會發展出多種分泌系統，使其能夠順利的侵入寄主，並成功的在寄主體內繁殖寄生，目前認為第三型分泌系統是由鞭毛 (flagellum) 系統演變而來 (Galan and Collmer, 1999)，而第四型分泌系統則由纖毛 (conjugative pilus)系統演變而來 (Covacci et al., 1999)，雖然在組成分子的序列相似度上並不明 顯，兩者間仍具有許多共通處。如 Helicobacter pylori 的 CagY 蛋白是和 VirB10 同源 的蛋白，根據共軛焦免疫電子顯微鏡觀察結果顯示，CagY 所構成的表面突出物和第三型分泌系統的針狀結構類似 (Rohde et al., 2003)。剔除 Mesorhizobium lotir R7A 的第四型分泌系統會造成其對不同的寄主植物根瘤的產生有不同的反應，有趣的是，很 多 M. loti 的品系具有第四型分泌系統，但缺乏第三型分泌系統，可能對某些細菌而 言，這兩種系統的功能具重疊性，因此不需兩種系統同時存在即可達到傳送受質的功能 (Llosa and O’Callaghan, 2004)。

X. campestris pv. vesicatoria 的第三型分泌系統為致病性上不可或缺的分泌胞 器，對其致病機制也有深入之瞭解 (Gűrlebeck et al., 2006; Kay and Bonas, 2009)，但所具有的第四型分泌系統的功能與寄主的相互關係大部份仍不清楚，有待深入探討方知其在不同菌株所扮演的角色 (Marques et al., 2001; Brunings and Gabriel, 2003)。本研 究從建構好的 X. campestris pv. vesicatoria A 群菌株 Xvt122 和 B 群菌株 Xvt45 基因庫 中選殖出與VirB/D4 系統相關的基因，在 VirB/D4 基因組的模式中，VirB11 進行 ATP 水解過程後，會改變本身的構形，將受質傳遞至VirB6/VirB8 的核心構造 (Cascales and Christie, 2004a)，除此之外，VirB11 對 T-pilus 的組成也是必要的(Sagulenko et al., 2001)。而 VirB4 雖然具有 NTP-binding motif，但目前沒有相當的證據支持其具有 ATP 水解功能，但其對受質傳遞是必要的因子(Yuan et al., 2005)。將 Mesorhizobium loti R7A 共生島嶼 (symbiosis island)上的 VirB/D4 基因組突變後，發現其在不同寄主有不同的 反應，對 Lotus corniculatus 植物會延遲根瘤作用，但對 Liucaena leucocephala 則能有 效的產生根瘤作用 (Hubber et al., 2004)。本研究中不同菌株 virB4 和 virB11 基因缺失

株對寄主有不同反應，甚至同一菌株對不同寄主反應也略有差異，是因必須有其他的第四型分泌系統共同作用，或是其傳送的受質不同所致，需進一步求證。

Alegria 等人(2005)利用酵母菌雙雜合系統 (yeast two-hybrid) 分析顯示 X.

axonopodis pv. citri 的 VirD4 和十二個未知功能的蛋白間有相互關係，這十二個未知 蛋白在C 端皆具有約 120 個胺基酸的相似性區域，包括保留性序列 GLxRIDHV 和 GxxDPAHxRAHV (x 為任意胺基酸) 和高含量的麩胺酸 (glutamine-rich) 特性，推測有可能是第四型分泌系統的受質，將這些蛋白命名為XVIPs (Xanthomonas VirD4 interacting proteins)。除了酵母菌雙雜合系統外，亦可嘗試利用第二章所採用的 AFLP 技術或二維蛋白質電泳分析技術，篩選出接種 virB/D4 基因缺失菌株所誘導表現的寄 主植物基因，進而探討這些基因的生物功能與 virB/D4 基因的相互關係，以瞭解第四 型分泌系統與寄主間的交互作用。

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在文檔中利用AFLP分析茄科細菌性斑點病菌之變異性並選殖與致病性相關之基因產物 (頁 105-120)