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

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.

黄龙病（HLB，又称柑橘黄龙病）是一种毁灭性的柑橘病害，其病原体为一种对营养要求严格的、寄生于韧皮部的 Candidatus Liberibacter 属细菌。本研究对‘Valencia’橙树叶在接种 Ca. L. asiaticus（亚洲类伯克霍尔德氏菌）后19周内的基因表达变化进行了大规模分析，利用 Affymetrix GeneChip® 柑橘基因组芯片进行，以期为柑橘对这种病原体的分子响应机制提供新的见解。在超过33,000个探针集中，有21,067个在叶片中表达，其中在接种后第五至九周和第十三至十七周，分别有279个和515个探针集表现出差异表达（FDR ≤ 0.05）。对14个选定的基因进行的半定量RT-PCR分析结果与微阵列观察到的结果高度相关。基因表达变化涉及多种不同的过程，包括细胞防御、运输、细胞组织、光合作用和碳水化合物代谢。值得注意的是，病原体诱导韧皮部特异性凝集素 PP2-like 蛋白的转录本积累。转录变化及其与疾病症状发展的关系得到了讨论。这是首次使用微阵列技术对柑橘响应 liberibacter 感染的转录组学进行的研究。黄龙病（HLB）是一种毁灭性的柑橘病害，据推测其病原体为一种寄生于韧皮部的 Candidatus Liberibacter 属细菌。本研究对‘Valencia’橙树（C. sinensis）在接种与佛罗里达州黄龙病相关的病原体 Ca. L. asiaticus（Las）后19周内的基因表达变化进行了大规模分析，利用 Affymetrix GeneChip® 柑橘基因组芯片进行，以期为柑橘对这种病原体的分子响应机制提供新的见解。总体设计：将3岁的‘Valencia’橙树（C. sinensis）嫁接在 Cleopatra 柑橘（C. reticulata Blanco）砧木上，以温室（对照）或田间种植的‘Lisbon’柠檬（C. limon）树的未感染（对照）或 Las 感染的组织进行嫁接接种。在接种后第5、9、13和17周收集充分展开的叶片。从每个时间点各选取两株未感染植物和三株 Las 接种植物提取总RNA。将第5周和第9周以及第13周和第17周收集的样本RNA等量混合，从而得到两个未感染和三个感染生物重复样本，用于在 Affymetrix 柑橘微阵列上进行杂交。