Molecular basis of Citrus Greening and related diseases gleaned from genome analyses of hosts and pathogens

We completed computational comparative analysis of the three genome categories: Liberibacter, Citrus and Psyllid. The results are available at the website . For each protein in each genome we predicted its various properties from its sequence, from local features such as order/disorder, secondary structure, transmembrane segments, coiled coils and signal peptides, to 3D structure and functional annotation that followed from all the predictions. This resource can be used by researchers who pursue studies of individual proteins and we welcome any questions and requests for additional information and analysis. Most interesting results were obtained by comparative analysis of pathogenic Liberibacter species with a non-pathogentic L. crescens, and through comparison of Liberibacter and citrus proteins. For instance, we analyzed of Liberibacter metabolic enzymes to detect those from pathogenic species that are missing in non-pathogen. We found one such enzyme not in a prophage region that functions in terpenoid biosynthesis: CLIBASIA_05065 encodes a geranyltranstransferase. While all of the Liberibacters encode enzymes to produce isopentenyl pyrophosphate (IPP), only the pathogenic strains possess a geranyltranstransferase that elongates the IPP chain. The products of the geranyltransferase enzyme (geranyl-PP and farnesyl-PP) provide the building blocks for monoterpenoid biosynthesis, which is specific to plants. E.g., the plant terpine limonene responsible for the strong smell of oranges is formed by cyclization of geranyl-PP. The steroid biosynthetic pathway from farnesyl-PP in plants generates the hormone brassinosteroid as well as other phytosteroids. Modification of this plant metabolic pathway by Las might contribute to pathogenesis. Overall, we found 70 genes unique to the pathogenic strains Candidatus Liberibacter americanus str. Sao Paulo (Lam) and Candidatus Liberibacter asiaticus str. psy62 (Las) that are missing from non L. crescens BT-1. 40% of the genes unique to the pathogens are found in the prophage regions. Two of such proteins should have signal peptides: a putative guanylate kinase (CLIBASIA_00055) and a hypothetic protein of unknown function found only in Liberibacter and the SC1/SC2 prophages (CLIBASIA_05560). These proteins are likely secreted and may be host virulence factors. However, the N-terminal sequence region of CLIBASIA_00055 does not likely serve as a secretion signal, as it forms the first hydrophobic strand of the guanylate kinase domain. The Las genome contains an additional core guanylate kinase (Gmk) (CLIBASIA_04045) that is orthologous to the Gmk of Liberibacter crescens and likely functions in purine metabolism. The presence of a second unique GMK encoded by the prophage remains unclear. Additionally, prophage contains a Xre-Bro protein pair (CLIBASIA_05625 and CLIBASIA_0002) similar to phage repressor-antirepressors that determine lytic state. The Las genome contains an additional gene (CLIBASIA_04440) that has potentially migrated from the prophage with sequence similar to the C-terminus of the CLIBASIA_00020-encoded Bro protein. CLIBASIA_04440 is probably not unique to Las, because searching the Lam nucleotide records identified a potential open reading frame that encodes the entire Bro domain-containing protein sequence (from an alternate start codon: CUG instead of AUG). Further inspection of the Las genome upstream of CLIBASIA_04440 yielded another missed open reading frame encoding the N-terminal Bro domain. Together, these genes may regulate expression of genes that induce the lytic cycle. The Xfas53 prophage contains a CI repressor upstream from the conserved gene neighborhood. The Xfas53 CI repressor includes a xenobiotic response element (XRE)-type HTH domain, followed by a S24 LexA-type peptidase. Similar CI repressors are involved in the regulation of the choice of phage lysogenic or lytic life cycle. An XRE-type HTH containing gene (CLIBASIA_05625) is found upstream from the Liberibacter asiaticus conserved BroN neighborhood that might also function as a repressor that controls the phage lytic life cycle.