Table_1_‘Candidatus Liberibacter asiaticus’ Multimeric LotP Mediates Citrus sinensis Defense Response Activation.DOCX

‘Candidatus Liberibacter asiaticus’ is known as the most pathogenic organism associated with citrus greening disease. Since its publicized emergence in Florida in 2005, ‘Ca. L. asiaticus’ remains unculturable. Currently, a limited number of potential disease effectors have been identified through in silico analysis. Therefore, these potential effectors remain poorly characterized and do not fully explain the complexity of symptoms observed in citrus trees infected with ‘Ca. L. asiaticus.’ LotP has been identified as a potential effector and have been partially characterized. This protein retains structural homology to the substrate binding domain of the Lon protease. LotP interacts with chaperones like GroEL, Hsp40, DnaJ, and ClpX and may exercise its biological role through interactions with different proteins involved in proteostasis networks. Here, we evaluate the interactome of LotP—revealing a new protein–protein interaction target (Lon-serine protease) and its effect on citrus plant tissue integrity. We found that via protein–protein interactions, LotP can enhance Lon protease activity, increasing the degradation rate of its specific targets. Infiltration of purified LotP strained citrus plant tissue causing photoinhibition and chlorosis after several days. Proteomics analysis of LotP tissues recovering after the infiltration revealed a large abundance of plant proteins associated with the stabilization and processing of mRNA transcripts, a subset of important transcription factors; and pathways associated with innate plant defense were highly expressed. Furthermore, interactions and substrate binding module of LotP suggest potential interactions with plant proteins, most likely proteases.

候选属东方韧皮部杆菌（‘Candidatus Liberibacter asiaticus’）是目前已知与柑橘黄龙病关联的致病性最强的病原物。自2005年在佛罗里达州被公开报道出现以来，‘Ca. L. asiaticus’至今仍未实现体外培养。当前，研究人员仅通过计算机模拟分析鉴定出少量潜在病害效应因子，因此这类潜在效应因子的功能表征仍十分有限，也无法完全解释受该菌侵染的柑橘植株所表现出的复杂症状。LotP是已被鉴定出的潜在效应因子之一，且已得到部分功能表征。该蛋白与Lon蛋白酶的底物结合结构域存在结构同源性。LotP可与GroEL、Hsp40、DnaJ、ClpX等分子伴侣发生相互作用，推测其通过与蛋白稳态网络中的多种蛋白结合来发挥生物学功能。本研究针对LotP的蛋白互作组展开分析，鉴定出一个全新的蛋白-蛋白互作靶点——Lon丝氨酸蛋白酶，并探究了其对柑橘植株组织完整性的影响。研究结果显示，LotP可通过蛋白相互作用增强Lon蛋白酶的活性，提升其对特定底物的降解速率。将纯化的LotP注射侵染柑橘植株组织后，数天内即可引发光抑制与褪绿症状。对注射后恢复阶段的LotP处理组织进行蛋白质组学分析，结果发现大量与mRNA转录本稳定及加工相关的植物蛋白、一类重要的转录因子，以及与植物先天免疫相关的通路均呈现高表达水平。此外，LotP的互作与底物结合模块提示其可与植物蛋白（极大概率为蛋白酶）产生潜在相互作用。