Global gene expression analysis of the phytopathogen Xylella fastidiosa at high iron availability. Xylella fastidiosa

Xylella fastidiosa is the etiologic agent of a wide range of plant diseases including citrus variegated chlorosis (CVC), a major threat to the Brazilian citrus industry. Genome sequences of several strains of this phytopathogen are accessible, enabling large-scale functional studies. Transcript levels in different iron availabilities were assessed with DNA microarrays representing 2608 (91.6%) coding sequences (CDS) of X. fastidiosa CVC strain 9a5c. When treated with the iron chelator 2,2-dipyridyl, 193 CDS were considered as up-regulated and 216 as down-regulated. In the presence of 100uM of ferric pyrophosphate, 218 and 256 CDS were considered as up- and down-regulated, respectively. Differential expression for a subset of 44 CDS was further evaluated by reverse transcription - quantitative PCR that showed a Pearson correlation of 0.77 with array results. The CDS differentially expressed upon the iron concentration shift participate in diverse cellular functions. Many CDS involved with regulatory functions, pathogenicity and cell structure, were modulated in both conditions tested suggesting that major changes in cell architecture and metabolism occur when X. fastidiosa cells are exposed to extreme variations in iron concentration. Interestingly, the modulated CDS include those related to colicin V-like bacteriocin synthesis and secretion and to pili/fimbriae functions. We also investigated the contribution of the ferric uptake regulator Fur to the iron regulon of X. fastidiosa. The promoter regions of strain 9a5c genome were screened for putative Fur boxes and candidates were analyzed by electrophoretic mobility shift assays. Taken together, our data support the hypothesis that Fur is not solely responsible for the modulation of the iron regulon of X. fastidiosa and present novel evidence for iron regulation of pathogenicity determinants. Keywords: stress response; response to iron-replete condition Overall design: Direct comparison between high iron content (100uM ferric pyrophosphate) and control condition. Hybridizations are dye-swaped. There are 2 biological replicates (independent harvest) and 2 technical replicates of each array (L - left and R - right).

苛养木杆菌（Xylella fastidiosa）是多种植物病害的病原物，其中包括柑橘杂色褪绿病（CVC）——该病害对巴西柑橘产业构成重大威胁。
目前已公开获取该植物病原物多个菌株的基因组序列，为大规模功能研究奠定了基础。
本研究采用针对苛养木杆菌CVC菌株9a5c的2608个编码序列（CDS，占该菌株总CDS数的91.6%）设计的DNA微阵列，检测了不同铁有效性条件下的转录水平。
当使用铁螯合剂2,2'-联吡啶处理时，共鉴定出193个上调表达的CDS与216个下调表达的CDS。
在添加100μM焦磷酸铁的培养条件下，分别有218个和256个CDS被鉴定为上调与下调表达。
我们通过反转录定量PCR（reverse transcription quantitative PCR，RT-qPCR）对44个CDS子集的差异表达情况进行了验证，结果显示其与微阵列实验结果的Pearson相关系数达0.77。
铁浓度改变诱导的差异表达CDS参与多种细胞生物学功能。
在两种测试条件下，诸多涉及调控功能、致病性及细胞结构的CDS均发生了表达调控，这提示当苛养木杆菌细胞暴露于铁浓度剧烈波动时，其细胞结构与代谢过程会发生显著改变。
值得注意的是，受调控的CDS包含与大肠杆菌素V（Colicin V）类细菌素的合成与分泌、菌毛/纤毛功能相关的基因。
我们还探究了铁摄取调节因子Fur（ferric uptake regulator）在苛养木杆菌铁调节组中的调控作用。
我们对9a5c菌株基因组的启动子区域进行了推定Fur结合框的筛选，并通过电泳迁移率变动分析（electrophoretic mobility shift assay，EMSA）对候选序列进行了验证。
综合以上实验结果，我们的数据支持"Fur并非唯一调控苛养木杆菌铁调节组的因子"这一假说，并为致病性决定因子的铁调控机制提供了新的实验证据。
关键词：胁迫响应；铁充足条件响应
整体实验设计：高铁含量（100μM焦磷酸铁）组与对照组的直接比较。杂交实验采用染料交换（dye-swapped）设计。每组实验包含2次生物学重复（独立收获的样本），以及每块芯片的2次技术重复（L-左侧，R-右侧）。