Department of Plant Pathology, Michigan State University, East Lansing, MI 48824, USA.
Mol Plant Pathol. 2012 Feb;13(2):160-73. doi: 10.1111/j.1364-3703.2011.00738.x. Epub 2011 Aug 10.
The bacterial pathogen Erwinia amylovora is the causal agent of fire blight, an economically significant disease of apple and pear. Disease initiation by E. amylovora requires the translocation of effector proteins into host cells via the hypersensitive response and pathogenicity (hrp) type III secretion system (T3SS). The alternative sigma factor HrpL positively regulates the transcription of structural and translocated components of the T3SS via hrp promoter elements. To characterize genome-wide HrpL-dependent gene expression in E. amylovora Ea1189, wild-type and Ea1189ΔhrpL strains were cultured in hrp-inducing minimal medium, and total RNA was compared using a custom microarray designed to represent the annotated genes of E. amylovora ATCC 49946. The results revealed 24 genes differentially regulated in Ea1189ΔhrpL relative to Ea1189 with fold-change expression ratios greater than 1.5; of these, 19 genes exhibited decreased transcript abundance and five genes showed increased transcript abundance relative to Ea1189. To expand our understanding of the HrpL regulon and to elucidate direct versus indirect HrpL-mediated effects on gene expression, the genome of E. amylovora ATCC 49946 was examined in silico using a hidden Markov model assembled from known Erwinia spp. hrp promoters. This technique identified 15 putative type III novel hrp promoters, seven of which were validated with quantitative polymerase chain reaction based on expression analyses. It was found that HrpL-regulated genes encode all known components of the hrp T3SS, as well as five putative type III effectors. Eight genes displayed apparent indirect HrpL regulation, suggesting that the HrpL regulon is connected to downstream signalling networks. The construction of deletion mutants of three novel HrpL-regulated genes resulted in the identification of additional virulence factors as well as mutants displaying abnormal motility and biofilm phenotypes.
细菌病原体韧皮部坏死杆菌是火疫病的病原体，火疫病是苹果和梨的一种具有重要经济意义的疾病。韧皮部坏死杆菌的疾病起始需要通过过敏反应和致病性（hrp）III 型分泌系统（T3SS）将效应蛋白易位到宿主细胞中。替代σ因子 HrpL 通过 hrp 启动子元件正向调节 T3SS 的结构和易位成分的转录。为了表征韧皮部坏死杆菌 Ea1189 中 HrpL 依赖性全基因组基因表达，野生型和 Ea1189ΔhrpL 菌株在 hrp 诱导的最小培养基中培养，并用定制的微阵列比较总 RNA，该微阵列旨在代表韧皮部坏死杆菌 ATCC 49946 的注释基因。结果显示，与 Ea1189 相比，Ea1189ΔhrpL 中有 24 个基因差异调节，其 fold-change 表达比率大于 1.5；其中 19 个基因的转录丰度降低，5 个基因的转录丰度增加。为了扩大对 HrpL 调控子的理解，并阐明 HrpL 对基因表达的直接和间接影响，使用从已知的欧文氏菌属 hrp 启动子组装的隐马尔可夫模型，对韧皮部坏死杆菌 ATCC 49946 的基因组进行了计算机分析。该技术鉴定了 15 个推定的 III 型新型 hrp 启动子，其中 7 个通过基于表达分析的定量聚合酶链反应进行了验证。结果发现，HrpL 调节的基因编码 hrp T3SS 的所有已知成分，以及 5 个推定的 III 型效应子。有 8 个基因显示出明显的间接 HrpL 调节，表明 HrpL 调控子与下游信号网络相连。三个新型 HrpL 调节基因缺失突变体的构建导致鉴定出更多的毒力因子以及表现出异常运动和生物膜表型的突变体。
