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

Abstract

由Xanthomonas campestris pv. vesicatoria (Xcv)引起的茄科植物細菌性斑點病，是台灣番椒及番茄栽培區之重要病害，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。台灣之Xcv菌株為多樣性的族群，本研究中所採用的14株菌株依據澱粉分解酶之有無，可將菌株區分為A、B兩菌群，而利用增幅限制片段核酸多型性技術(AFLP) 亦可得到相同的結果。從AFLP的分析圖譜中篩選各菌株間有差異性之片段，並選殖出xopE2基因，xopE2基因在X. campestris pv. vesicatoria菌株間具高度保留性，可作為分類之指標。根據胺基酸序列所作樹狀親源分析，顯示A群菌株Xvt122與X. campestris pv. vesicatoria 85-10的親源關係較之B群菌株Xvt45為近。剔除Xcv Xvt45染色體上的xopE2基因會降低病原菌的感染力。然而，Xcv Xvt122的xopE2基因突變株並不會影響其病原性。此外，無論是Xcv Xvt122或Xcv Xvt45的XopE2蛋白(XopE2A和XopE2B)皆能透過第三型分泌系統抑制由HopPsyA所引發的過敏性反應，大量表現XopE2蛋白則會降低其在感病番茄品系的毒性，而這些生物性功能和XopE2蛋白所具有的保留性三元催化胺基酸(consensus catalytic triad )(159th cysteine)與His硫醇蛋白酶胺基酸(thiol-protease His residue)(47th histidine)無關。 2005年X. campestris pv. vesicatoria 85-10基因組解序後發現有Vir/Tra和Icm/DotT4SS兩套第四型分泌系統存在。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，但對於其所具有的第四型分泌系統所扮演的角色所知甚少。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，目前兩菌群所選殖出來的virB/D4基因組並非很完整，但以目前的結果顯示，A群菌株Xvt122的基因數和排列順序和X. campestris pv. campestris ATCC33913 (Xcc ATCC33913)相似，而B群菌株Xvt45的基因數和排列順序則和X. campestris pv. campestris B100 (Xcc B100)和X. axonopodis pv. citrus 306 (Xac 306)最為相似。兩菌株的VirB/D4基因組G+C含量遠較其兩側基因的G+C含量為低，其基因組下游具有IS4 family和ISxac4 transposase序列，可知，Xcv Xvt122和Xcv Xvt45的VirB/D4基因組是由其他菌屬經水平式基因轉移 (horizontal gene transfer) 而來。VirB/D4第四型分泌系統中，VirB4、VirB11及VirD4是保留性最高的組成蛋白，為此系統組合之能量提供者，Xcv Xvt45的virB4和virB11基因突變株，對寄主植物番茄病徴的發展和病原菌的生長並沒有影響，但Xcv Xvt122的virB4、virB11基因突變株，雖然發病指數沒有顯著差異，但對病原菌的生長卻有抑制作用。

The bacterial spot diseases on tomato (Lycopersicon spp.) and pepper (Capsicum spp.) causing by Xanthomonas campestris pv. vesicatoria (Xcv) are very destructive in Taiwan. The mechanism that Xcv causing this disease depends on type III secretion system (T3SS) and T3SS effectors. The recent study showed that strains of Xcv isolated in Taiwan were heterogeneous and could be divided into two genetically distinct groups (group A and group B). In this study, we proved that using the AFLP technique to discriminate Xcv group A from group B is feasible, and consequently, a XopE2 homologue was identified in all fourteen Xcv strains and it also could be used as an index for classification. Phylogenic analysis of XopE2 amino acid sequences indicated that XopE2 of Xcv Xvt122 (group A) has a closer genetic distance to XopE2 of Xcv 85-10 than to that of Xcv Xvt45 (group B). A single copy deletion of xopE2 within the genome caused a substantial reduction in virulence, but no effect of xopE2 mutation on virulence of Xvt122 were observed. Furthermore, our results revealed that XopE2 of Xcv Xvt122 or Xcv Xvt45 was able to suppress HR in a T3SS-dependent manner and the heterologously expressed XopE2 was sufficient to modulate the virulence on susceptible tomato plants. And those biological functions are not dependent on the consensus catalytic triad (159th cysteine) and thiol-protease His residue (47th histidine) of XopE2. Recent genome sequencing projects have identified Vir/Tra and Icm/Dot T4SSs in X. campestris pv. vesicatoria 85-10, but their precise function awaits further investigation. In this study, we identified the VirB/D4 T4SSs from X. campestris pv. vesicatoria group A (Xvt122) and group B (Xvt45). Based on sequence analysis and gene composition, the virB/D4 cluster of Xcv Xvt122 shows the highest similarity with that of X. campestris pv. campestris ATCC33913；but the virB/D4 cluster of Xcv Xvt45 seems more homologous with that of X. campestris pv. campestris B100 and X. axonopodis pv. citrus 306 (Xac 306). Moreover, the G+C content of the virB/D4 cluster of Xcv Xvt122 or Xcv Xvt45 is lower than that of the neighborhood genes, and the presence of IS4 family and ISxac4 transposase in those loci suggests that the virB/D4 cluster may be a result of horizontal gene transfer from other bacteria. Because VirB4, VirB11 and VirD4, are the most conserved components of the T4SS, and they serve as an energy-consuming process for their assembly or function, we also generated the virB4 and virB11 knocked out mutants to assess the virulence of the genes on the host plant tomato. The inoculation assay revealed that the virB4 or virB11 deletion in Xcv Xvt45 retained its ability to develop disease symptoms and the bacterial growth on tomato Bony Best L305. In contrast, the virB4 or virB11mutation in Xcv Xvt122 reduced bacterial growth on its host tomato and pepper, but there were no effect on disease symptoms development.