Huanglongbing (HLB) is considered the most devastating bacterial disease in citriculture. It is caused by the phloem-limited α-proteobacterium ‘Candidatus Liberibacter asiaticus’ (CLas). All citrus varieties are susceptible to HLB and many studies have been conducted in an attempt to identify potential targets involved in bacterial virulence and pathogenicity mechanisms. One of the most important virulence factors of bacterial pathogens is the effector molecule that can modify host cell structures, facilitate infection, and/or trigger host defense responses. In this study, 14 CLas candidates for secreted hypothetical proteins were predicted by a bioinformatics pipeline, one of which (NSEC01) was predicted to carry a nuclear localization sequence (NLS). Putative effectors HLBSP04 and HLBSP21 were conserved in all genomes evaluated, whereas HLBSP01, HLBSP19, and HLBSP30 were exclusively found in CLas. Expression analysis of candidate effector genes was performed in citrus leaves exhibiting five distinct HLB symptoms (asymptomatic, blotchy mottle, zinc deficiency, yellowed veins, and vein corky) and used as a guide for the selection of effectors for further functional studies. Genes coding for candidate effectors HLBSP13, HLBSP20, HLBSP28, and NSEC01 had high expression levels in blotchy mottle leaves, suggesting they could be used as marker genes that may facilitate detection of the pathogen in trees with this HLB symptom. As NSEC01 was exclusively found in this work, it was selected for transient expression in Nicotiana benthamiana leaves, but further investigations need to be carried out. Also, other candidate effectors predicted in this work still need to be functionally characterized. This study allowed discussing and proposing hypotheses about CLas classification according to virulence and lifestyle, which can help understand the citrus-CLas interaction.

中文翻译：

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'Candidatus Liberibacter asiaticus' 推定效应物：表现出明显黄龙病症状的柑橘叶中的计算机分析和基因表达

黄龙病（HLB）被认为是柑橘养殖中最具破坏性的细菌病害。它是由韧皮部限制的α-变形菌“Candidatus Liberibacter asiaticus”（CLas）引起的。所有柑橘品种都对 HLB 敏感，并且已经进行了许多研究以试图确定涉及细菌毒力和致病机制的潜在目标。细菌病原体最重要的毒力因子之一是效应分子，它可以改变宿主细胞结构、促进感染和/或触发宿主防御反应。在这项研究中，通过生物信息学管道预测了 14 个分泌假设蛋白质的 CLas 候选者，其中一个（NSEC01）被预测为携带核定位序列（NLS）。假定的效应子 HLBSP04 和 HLBSP21 在所有评估的基因组中都是保守的，而 HLBSP01、HLBSP19 和 HLBSP30 仅在 CLas 中发现。候选效应基因的表达分析在表现出五种不同 HLB 症状（无症状、斑点斑驳、缺锌、黄脉和脉软木塞）的柑橘叶中进行，并用作选择效应子以进行进一步功能研究的指南。编码候选效应子 HLBSP13、HLBSP20、HLBSP28 和 NSEC01 的基因在斑点斑驳叶中具有高表达水平，表明它们可以用作标记基因，可能有助于在具有这种 HLB 症状的树木中检测病原体。由于 NSEC01 仅在这项工作中被发现，因此它被选为在本氏烟草叶中进行瞬时表达，但需要进一步研究。此外，这项工作中预测的其他候选效应器仍需要进行功能表征。