Gene expression in Citrus sinensis (L.) Osbeck following infection with the bacterial pathogen Candidatus Liberibacter asiaticus causing Huanglongbing in Florida. Citrus sinensis

Huanglongbing (HLB) (=citrus greening) is a destructive disease of citrus which is caused by a fastidious, phloem-inhabiting bacterium of the genus Candidatus Liberibacter. Large-scale analysis of gene expression changes in ‘Valencia’ orange leaves were studied during the course of 19 weeks after inoculation with Ca. L. asiaticus using the Affymetrix GeneChip® citrus genome array to provide new insights into the molecular basis of citrus response to this pathogen. Of the more than 33,000 probe sets on the microarray 21,067 were expressed in the leaves, of which 279 and 515 were differentially expressed (FDR ≤ 0.05) five to nine and 13-17 weeks after inoculation, respectively. Results from semi-quantitative RT-PCR analysis performed on 14 selected genes were highly correlated with those observed with the microarray. Gene expression changes involved a variety of different processes including cell defense, transport, cellular organization, photosynthesis, and carbohydrate metabolism. Notable was the pathogen-induced accumulation of transcripts for a phloem-specific lectin PP2-like protein. Transcriptional changes and their relation to disease symptom development are discussed. This is the first study of transcriptional profiling in citrus in response to liberibacter infection using microarray technology. Huanglongbing (HLB) is a destructive disease of citrus which is suspected to be caused by a phloem-inhabiting bacterium of the genus Candidatus Liberibacter. Large-scale analysis of gene expression changes in ‘Valencia’ orange (C. sinensis) leaves were studied during the course of 19 weeks after inoculation with Ca. L. asiaticus (Las), the pathogen associated with HLB in Florida, using the Affymetrix GeneChip® citrus genome array to provide new insights into the molecular basis of citrus response to this pathogen. Overall design: Three year-old 'Valencia' orange (C. sinensis) scions on Cleopatra mandarin (C. reticulata Blanco) rootstocks were graft-inoculated with non-infected (control) or Las-infected tissue from greenhouse- (control) or field-grown 'Lisbon' lemon (C. limon) trees. Fully expanded leaves were collected at 5, 9, 13, and 17 weeks after inoculation (wai). Total RNA was extracted from two control plants and from three Las-inoculated plants per time point. Equal amounts of RNA were combined from samples collected 5 wai and 9 wai and from samples collected 13 wai and 17 wai, resulting in two non-infected and three infected biological replicates for both early (5-9 wai) and late (13-17 wai) time points and used for hybridization on Affymetrix citrus microarrays.

柑橘黄龙病（Huanglongbing, HLB，又称柑橘绿斑病）是一种毁灭性柑橘病害，其病原为候选韧杆菌属（Candidatus Liberibacter）下的一类韧皮部定植、难培养的细菌。本研究采用Affymetrix柑橘基因组基因芯片®（Affymetrix GeneChip® citrus genome array），对接种候选韧杆菌亚洲亚种（Ca. L. asiaticus）后19周内的瓦伦西亚橙叶片基因表达变化开展大规模分析，以期为解析柑橘应对该病原菌的分子机制提供新视角。该微阵列芯片共包含33000余个探针组（probe sets），其中21067个可在叶片中检测到表达；接种后5~9周及13~17周时，分别有279个和515个基因呈现差异表达（错误发现率False Discovery Rate, FDR ≤ 0.05）。对14个筛选出的基因进行半定量逆转录聚合酶链式反应（semi-quantitative RT-PCR）分析，所得结果与微阵列检测结果高度吻合。基因表达变化涉及细胞防御、物质转运、细胞结构组织、光合作用以及碳水化合物代谢等多个生物学过程。值得注意的是，病原菌诱导了韧皮部特异性凝集素PP2样蛋白编码转录本的积累。本文还讨论了转录组变化与病害症状发育的关联。本研究是首次采用微阵列技术开展柑橘应对候选韧杆菌侵染的转录组分析（transcriptional profiling）研究。柑橘黄龙病（HLB）是一种毁灭性柑橘病害，其病原被认为是候选韧杆菌属（Candidatus Liberibacter）下的一类韧皮部定植细菌。本研究采用Affymetrix柑橘基因组基因芯片®（Affymetrix GeneChip® citrus genome array），对接种与佛罗里达州柑橘黄龙病相关的候选韧杆菌亚洲亚种（Ca. L. asiaticus, Las）后19周内的瓦伦西亚橙（C. sinensis）叶片基因表达变化开展大规模分析，以期为解析柑橘应对该病原菌的分子机制提供新视角。整体实验设计：将种植3年的瓦伦西亚橙（C. sinensis）接穗嫁接于克里曼丁红橘（C. reticulata Blanco）砧木上，分别采用未侵染的（对照组）或携带有Las的组织进行嫁接接种，其中对照组织来源于温室或田间种植的里斯本柠檬（C. limon）植株。分别于接种后5、9、13和17周（wai，weeks after inoculation的缩写）采集完全展开的叶片。每个时间点分别从2株对照植株和3株接种Las的植株中提取总RNA，将5wai与9wai的样品、13wai与17wai的样品分别混合等量RNA，由此获得早期（5~9wai）和晚期（13~17wai）两个时期的未侵染组生物学重复各2份、侵染组生物学重复各3份，用于Affymetrix柑橘微阵列的杂交实验。