A Small Protein Directly Represses Phage Lytic Cycle Genes in " Liberibacter asiaticus" within Psyllids.

Huanglongbing (HLB) is a severe disease of citrus caused by an uncultured alphaproteobacterium " Liberibacter asiaticus" and transmitted by Asian citrus psyllids (). Two prophage genomes, SC1 and SC2, integrated in ". Liberibacter asiaticus" strain UF506 were described previously, and very similar prophages are found resident in the majority of ". Liberibacter asiaticus" strains described worldwide. The SC1 lytic cycle is marked by upregulation of prophage late genes, including a functional holin (SC1_gp110); these late genes are activated when ". Liberibacter asiaticus" is , but not when infecting the psyllid host. We previously reported that the holin promoter is strongly and constitutively active in (a cultured proxy for uncultured ". Liberibacter asiaticus") but is suppressed in a dose-dependent manner by crude aqueous extracts from applied exogenously. Here we report that the suppressor activity of the crude psyllid extract was heat labile and abolished by proteinase K treatment, indicating a proteinaceous repressor and of a size smaller than 30 kDa. The repressor was affinity captured from aqueous extracts using biotinylated holin promoter DNA immobilized on magnetic beads and subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein database interrogation was used to identify a small DNA-binding protein encoded by a gene carried by strain wDi, a resident endosymbiont of as the repressor. The -translated repressor protein was able to penetrate . cells, bind to ". Liberibacter asiaticus" promoter DNA, and partially suppress holin promoter-driven β-glucuronidase (GUS) activity, indicating potential involvement of an additional interacting partner(s) or posttranslational modification(s) for complete suppression. Expression of the repressor protein appeared to be constitutive irrespective of ". Liberibacter asiaticus" infection status of the insect host. Host acquisition of a new microbial species can readily perturb the dynamics of preexisting microbial associations. Molecular cross talk between microbial associates may be necessary for efficient resource allocation and enhanced survival. Classic examples involve quorum sensing (QS), which detects population densities and is both used and coopted to control expression of bacterial genes, including host adaptation factors. We report that a 56-amino-acid repressor protein made by the resident psyllid endosymbiont can enter cells of , a cultured proxy for the uncultured psyllid endosymbiont ". Liberibacter asiaticus" and repress ". Liberibacter asiaticus" phage lytic cycle genes. Such repression in ". Liberibacter asiaticus" may be critical to survival of both endosymbionts, since phage-mediated lysis would likely breach the immunogenic threshold of the psyllid, invoking a systemic and nonspecific innate immune reaction.