‘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.
AbstractIn this study, newly identified small molecules were examined for efficacy against ‘Candidatus Liberibacter asiaticus’ in commercial groves of sweet orange (Citrus sinensis) and white grapefruit (Citrus paradisi) trees. We used benzbromarone and/or tolfenamic acid delivered by trunk injection. We evaluated safety and efficacy parameters by performing RNAseq of the citrus host responses, 16S rRNA gene sequencing to characterize citrus-associated microbial communities during treatment, and qRT-PCR as an indirect determination of ‘Ca. L. asiaticus’ viability. Analyses of the C. sinensis transcriptome indicated that each treatment consistently induced genes associated with normal metabolism and growth, without compromising tree viability or negatively affecting the indigenous citrus-associated microbiota. It was found that treatment-associated reduction in ‘Ca. L. asiaticus’ was positively correlated with the proliferation of several core taxa related with citrus health. No symptoms of phytotoxicity were observed in any of the treated trees. Trials were also performed in commercial groves to examine the effect of each treatment on fruit productivity, juice quality and efficacy against ‘Ca. L. asiaticus’. Increased fruit production (15%) was observed in C. paradisi following twelve months of treatment with benzbromarone and tolfenamic acid. These results were positively correlated with decreased ‘Ca. L. asiaticus’ transcriptional activity in root samples.