Functional characterization of LotP from Liberibacter asiaticus

Abstract

Summary Liberibacter asiaticus is an unculturable parasitic bacterium of the alphaproteobacteria group hosted by both citrus plants and a psyllid insect vector (Diaphorina citri). In the citrus tree, the bacteria thrive only inside the phloem, causing a systemically incurable and deadly plant disease named citrus greening or Huanglongbing. Currently, all commercial citrus cultivars in production are susceptible to L. asiaticus, representing a serious threat to the citrus industry worldwide. The technical inability to isolate and culture L. asiaticus has hindered progress in understanding the biology of this bacterium directly. Consequently, a deep understanding of the biological pathways involved in the regulation of host–pathogen interactions becomes critical to rationally design future and necessary strategies of control. In this work, we used surrogate strains to evaluate the biochemical characteristics and biological significance of CLIBASIA_03135. This gene, highly induced during early stages of plant infection, encodes a 23 kDa protein and was renamed in this work as LotP. This protein belongs to an uncharacterized family of proteins with an overall structure resembling the LON protease N‐terminus. Co‐immunoprecipitation assays allowed us to identify the Liberibacter chaperonin GroEL as the main LotP‐interacting protein. The specific interaction between LotP and GroEL was reconstructed and confirmed using a two‐hybrid system in Escherichia coli. Furthermore, it was demonstrated that LotP has a native molecular weight of 44 kDa, corresponding to a dimer in solution with ATPase activity in vitro. In Liberibacter crescens, LotP is strongly induced in response to conditions with high osmolarity but repressed at high temperatures. Electrophoretic mobility shift assay (EMSA) results suggest that LotP is a member of the LdtR regulon and could play an important role in tolerance to osmotic stress.

Understanding the Physiology of <b><i>Liberibacter asiaticus</i></b>: An Overview of the Demonstrated Molecular Mechanisms

“…This protein was previously misannotated as an LA1 aminopeptidase, but structural analysis suggested that LotP actually belongs to a different, uncharacterized family of proteins. Co-immunoprecipitation assays identified GroEL as the main interacting protein, and a physical interaction between GroEL and LotP was confirmed via two-hybrid assay [Loto et al, 2017]. LotP is predicted to be critical to the pathogenesis in planta through this GroEL interaction and may have a role in masking Gro-EL from the plant immune defense system.…”

“…Further, J Mol Microbiol Biotechnol 2018;28:116-127 DOI: 10.1159/000492386 to determine its biological significance. In silico analysis of LotP revealed that the sequence is highly conserved among the Rhizobiaceae family (56-86% identity), and it is vastly distributed in α-proteobacteria (35% plant-associated bacteria, 1/2 of these are plant pathogens) [Loto et al, 2017]. This protein was previously misannotated as an LA1 aminopeptidase, but structural analysis suggested that LotP actually belongs to a different, uncharacterized family of proteins.…”

“…LotP is predicted to be critical to the pathogenesis in planta through this GroEL interaction and may have a role in masking Gro-EL from the plant immune defense system. GroEL is very important in many insect-vectored pathosystems, and it is hypothesized that LotP participates in GroEL moonlighting activity to enhance the pathogenicity of L. asiaticus; however, further investigation is necessary to unveil the precise role of LotP in the HLB-citrus pathosystem [Loto et al, 2017].…”

“…Analysis of the transcriptome data generated by Yan et al [2013] revealed that a large number of genes that were significantly induced in planta encoded hypothetical proteins, including the 3 genes displaying the largest induction (> 7-fold) [Loto et al, 2017;Yan et al, 2013]. One of these genes, encoding CLIBASIA_03135 (renamed LotP), was recently biochemically characterized…”

A Review of the Citrus Greening Research and Development Efforts Supported by the Citrus Research and Development Foundation

“…The genome-enabled ability to analyze CLas gene expression in the plant and vector led to the identification of gene activation patterns and regulatory elements in the bacterium . A LuxR receptor of an unknown citrus signal increased CLas symptoms in transgenic citrus (Gabriel, 2017), and the LdtR transcriptional regulator was shown to be a master regulator of global gene expression, including that of the stress response gene LotP (Pagliai et al, , 2017Loto et al, 2017). The genome also revealed that CLas likely has a functional Sec (Type II) secretion system for transporting proteins from the bacterial cell to the extracellular environment.…”