番茄青枯病菌毒力相關因子之研究

Abstract

由青枯病菌(Ralstonia solanacearum)所造成的細菌性萎凋病（又稱為青枯病）是全球許多作物重要且極為複雜的病害。青枯病菌具有相當廣泛的寄主範圍且具不同程度的毒力表現，高毒力的菌株甚至可造成番茄抗病品系Hawaii 7996 (H7996)的完全萎凋，因此研究青枯病菌毒力表現關鍵因子對於研擬此病害之有效防治管理策略是非常重要的。本研究之目標為探討各種致病相關因子的表現與毒力之相關性，並進一步研究高或低毒力菌株特有的基因在毒力所扮演的角色。首先，針對不同毒力的青枯病菌野生型菌株進行特性分析，結果顯示生物膜形成能力(biofilm formation)可能與毒力表現具有正相關性；然而，各菌株毒力的表現與生長速度、泳動力(swimming motility)、及對氧化逆境(oxidative stress)的敏感性並無明顯的相關性，因此推測毒力的表現是由許多基因構成之複雜網絡所調控，是許多生理生化特性的微小差異累加而成之結果。其次，先前利用生物微陣列晶片(microarray)已找到一群青枯病菌高毒力或低毒力菌株特有的基因，但這些基因是否確實與毒力表現相關及其具體功能則尚未知。本研究將高毒力菌株特有的RSc2322-RSc2325基因群(gene cluster)在高毒力菌株Pss1308及Pss749中依其操縱子(operon)之排列組成分成三個轉錄單位，以質體插入破壞個別基因，並分析其各項特性，結果顯示突變株的泳動能力下降，對於氧化逆境也較為敏感，且在Pss1308背景中的RSc2325突變株及在Pss749背景中的RSc2324突變株在抗病品系番茄H7996的毒力表現下降。同時，將RSc2322-RSc2325全長基因群轉移到低毒力菌株Pss216後，基因轉移表現株相較於載體轉型株有較高的根部附著能力及較低的泳動力，且在感病品系L390番茄也有較高的毒力表現。綜合突變株及基因轉移表現株的特性及毒力表現的結果可推測RSc2322-RSc2325基因群確實與青枯病菌高毒力的表現有關，且具有加成效果。此外，本研究亦針對低毒力特有之第三型分泌蛋白 (type III effectors, T3Es)基因RSp0213和RSc3174進行相關研究；首先利用啟動子活性分析驗證此兩基因確實受第三型分泌系統的轉錄調控因子HrpG調控；將RSp0213和RSc3174送入高毒力菌Pss190和Pss1308表現後，所得基因轉移表現株在本研究檢測生理特性的表現皆未受影響，但高毒力菌株Pss190在抗病品系H7996的毒力表現則明顯降低，因此這兩個低毒力特有之T3Es的存在確實與青枯病菌低毒力表現有關聯。透過這些分子層面的研究使得我們對青枯病菌毒力相關機制有初步的了解，且預期對研擬青枯病之有效防治管理策略將有所啟發。

Ralstonia solanacearum is a soil-borne pathogen which causes the devastating bacterial wilt disease (BW) worldwide. R. solanacearum race 1 strains have an unusual wide host range. Moreover, they are highly diverse both in their genotypes and virulence on tomato. Tomato cv. Hawaii 7996 (H7996), known to be the most stable resistance source, can be totally broken down by a group of R. solanacearum strains with high virulence. Therefore, gaining insight of virulence determinants of R. solanacearum and elucidation of their functions are expected to provide important information leading to new strategies for effective disease management of BW. The aims of this study were to characterize R. solanacearum strains with various levels of virulence and to study the function of R. solanacearum candidate virulence determinants. Characterization of wild-type strains of various virulence levels suggests that the differential capability of biofim formation may be related to the virulence levels. However, the variable growth rate, capability of swimming motility, and sensitivity of oxidative stress implies that virulence is complex and may require the coordination of multiple facets. Furthermore, previous microarray analysis had identified groups of genes unique to either high- or low-virulent strains. Single-gene knock-out mutants in the background of high-virulence strains Pss1308 or Pss749 of RSc2322-RSc2325 gene cluster, which is unique to high-virulent strains, displayed decreased motility, increased sensitivity to oxidative stress, and decreased virulence in H7996. Consistently, a low virulent-strain expressing the RSc2322-RSc2325 gene cluster displayed decreased motility, increased capability of root attachment, and increased virulence in a susceptible tomato cultivar L390. These findings together indicate the RSc2322-RSc2325 gene cluster additively contributes to high virulence. Moreover, promoter activity assays also confirmed the type III effectors RSp0213 and RSc3174, which are unique to low virulent strains, are regulated by HrpG. Most importantly, a high-virulent strain carrying RSp0213 or RSc3174 conferred decreased virulence in H7996, revealing these effectors do play roles in the low virulence level of R. solanacearum strains. These studies together are expected to pave the way not only for elucidating mechanisms and determinants involved in R. solanacearum virulence but also potentially establishing useful disease control means.