Bocsanczy Ana M, Achenbach Ute C M, Mangravita-Novo Arianna, Chow Marjorie, Norman David J
Department of Plant Pathology, University of Florida, IFAS, Mid-Florida Research and Education Center, 2725 Binion Rd,, Apopka, FL 32703, USA.
BMC Genomics. 2014 Apr 12;15:280. doi: 10.1186/1471-2164-15-280.
Ralstonia solanacearum, the causal agent of bacterial wilt, is a genetically diverse bacterial plant pathogen present in tropical and subtropical regions of the world that infects more than 200 plant species, including economically important solanaceous crops. Most strains of R. solanacearum are only pathogenic at temperatures between 25 to 30°C with strains that can cause disease below 20°C considered a threat to agriculture in temperate areas. Identifying key molecular factors that distinguish strains virulent at cold temperatures from ones that are not is needed to develop effective management tools for this pathogen. We compared protein profiles of two strains virulent at low temperature and two strains not virulent at low temperature when incubated in the rhizosphere of tomato seedlings at 30 and 18°C using quantitative 2D DIGE gel methods. Spot intensities were quantified and compared, and differentially expressed proteins were sequenced and identified by mass spectrometry (MS/MS).
Four hundred and eighteen (418) differentially expressed protein spots sequenced produced 101 unique proteins. The identified proteins were classified in the Gene Ontology biological processes categories of metabolism, cell processes, stress response, transport, secretion, motility, and virulence. Identified virulence factors included catalase (KatE), exoglucanase A (ChbA), drug efflux pump, and twitching motility porin (PilQ). Other proteins identified included two components of a putative type VI secretion system. We confirmed differential expression of 13 candidate genes using real time PCR techniques. Global regulators HrpB and HrpG also had temperature dependent expression when quantified by real time PCR.
The putative involvement of the identified proteins in virulence at low temperature is discussed. The discovery of a functional type VI secretion system provides a new potential virulence mechanism to explore. The global regulators HrpG and HrpB, and the protein expression profiles identified suggest that virulence at low temperatures can be partially explained by differences in regulation of virulence factors present in all the strains.
青枯雷尔氏菌是细菌性萎蔫病的病原体，是一种遗传多样性的细菌性植物病原体，存在于世界热带和亚热带地区，可感染200多种植物，包括具有重要经济价值的茄科作物。大多数青枯雷尔氏菌菌株仅在25至30°C的温度下具有致病性，而能够在20°C以下引起疾病的菌株被认为对温带地区的农业构成威胁。为了开发针对这种病原体的有效管理工具，需要确定区分低温下有毒力菌株和无毒力菌株的关键分子因素。我们使用定量二维差异凝胶电泳（2D DIGE）方法，比较了在30°C和18°C下番茄幼苗根际中培养的两株低温有毒力菌株和两株低温无毒力菌株的蛋白质谱。对斑点强度进行了定量和比较，并通过质谱（MS/MS）对差异表达的蛋白质进行了测序和鉴定。
对418个差异表达的蛋白质斑点进行测序，产生了101种独特的蛋白质。鉴定出的蛋白质被归类到基因本体生物学过程的代谢、细胞过程、应激反应、运输、分泌、运动性和毒力类别中。鉴定出的毒力因子包括过氧化氢酶（KatE）、外切葡聚糖酶A（ChbA）、药物外排泵和抽动运动孔蛋白（PilQ）。鉴定出的其他蛋白质包括推定的VI型分泌系统的两个组分。我们使用实时PCR技术确认了13个候选基因的差异表达。通过实时PCR定量时，全局调节因子HrpB和HrpG也具有温度依赖性表达。
讨论了鉴定出的蛋白质在低温毒力中的推定作用。功能性VI型分泌系统的发现提供了一种新的潜在毒力机制以供探索。全局调节因子HrpG和HrpB以及鉴定出的蛋白质表达谱表明，低温下的毒力可以部分由所有菌株中毒力因子调节的差异来解释。
