Diversity and plasticity of the intracellular plant pathogen and insect symbiont "Candidatus Liberibacter asiaticus" as revealed by hypervariable prophage genes with intragenic tandem repeats

Abstract

"Candidatus Liberibacter asiaticus" is a psyllid-transmitted, phloem-limited alphaproteobacterium and the most prevalent species of "Ca. Liberibacter" associated with a devastating worldwide citrus disease known as huanglongbing (HLB). Two related and hypervariable genes (hyv(I) and hyv(II)) were identified in the prophage regions of the Psy62 "Ca. Liberibacter asiaticus" genome. Sequence analyses of the hyv(I) and hyv(II) genes in 35 "Ca. Liberibacter asiaticus" DNA isolates collected globally revealed that the hyv(I) gene contains up to 12 nearly identical tandem repeats (NITRs, 132 bp) and 4 partial repeats, while hyv(II) contains up to 2 NITRs and 4 partial repeats and shares homology with hyv(I). Frequent deletions or insertions of these repeats within the hyv(I) and hyv(II) genes were observed, none of which disrupted the open reading frames. Sequence conservation within the individual repeats but an extensive variation in repeat numbers, rearrangement, and the sequences flanking the repeat region indicate the diversity and plasticity of "Ca. Liberibacter asiaticus" bacterial populations in the world. These differences were found not only in samples of distinct geographical origins but also in samples from a single origin and even from a single "Ca. Liberibacter asiaticus"-infected sample. This is the first evidence of different "Ca. Liberibacter asiaticus" populations coexisting in a single HLB-affected sample. The Florida "Ca. Liberibacter asiaticus" isolates contain both hyv(I) and hyv(II), while all other global "Ca. Liberibacter asiaticus" isolates contain either one or the other. Interclade assignments of the putative Hyv(I) and Hyv(II) proteins from Florida isolates with other global isolates in phylogenetic trees imply multiple "Ca. Liberibacter asiaticus" populations in the world and a multisource introduction of the "Ca. Liberibacter asiaticus" bacterium into Florida.

Fig. 1.

General features of hyv_I and hyv_II genes (scaled) in the genome of “Candidatus Liberibacter asiaticus” from various geographic isolates. The black arrow boxes in A1 to A4 and B1 to B3 represent the open reading frames of hyv_I and hyv_II genes with different numbers of full or partial repeat units in the middle. Each light grey box in the middle of the gene represents a 132-bp full tandem repeat unit, and each deep grey box stands for partial repeat unit. The numbers above the hyv_I and hyv_II genes indicate the gene size and the start and end positions of repeat regions in the hyv_I and hyv_II genes. A1 and B1 are hyv_I and hyv_II genes with two flanking genes on both 5′ and 3′ ends in the FL-Psy62 genome. Dark horizotal- and vertical-arrow boxes in A1 and B1 represent two hypothetical genes neighboring the 3′ end of the hyv_I (A1) and hyv_II (B1) genes; zigzag and large-confetti arrow boxes with dashed outline boxes represent two other hypothetical genes neighboring the 5′ ends of the hyv_I and hyv_II genes (not to scale). The spotted box or arrow in B1 to B3 represents the sequence variations (similarity of less than 80%) compared to the sequence in same region within the hyv_I gene or the flanking genes.

Fig. 2.

PCR amplification of the hyv_I and hyv_II gene target regions in isolates of “Candidatus Liberibacter asiaticus” from Florida and other global origins, using primer sets LJ788/LJ729 for hyv_I (A) and LJ812/LJ1089 for hyv_II (B). Lane M: 1-kb DNA ladder (Promega); lanes 1 to 16, DNA from “Ca. Liberibacter asiaticus”-infected plants or psyllids of global origins outside the United States; lanes 17 to 38, DNA from “Ca. Liberibacter asiaticus”-infected different hosts in Florida; lanes 17 to 19, DNA from “Ca. Liberibacter asiaticus”-positive psyllid from field; lane 20, DNA from “Ca. Liberibacter asiaticus”-free psyllid control from greenhouse; lanes 20 to 23, DNA from “Ca. Liberibacter asiaticus”-positive citrus from field; lanes 24 to 26, DNA from “Ca. Liberibacter asiaticus”-positive citrus from greenhouse, CG8-S/CG8-R leaf samples collected from scion/rootstock of grafted citrus CG8; lane 27, “Ca. Liberibacter asiaticus”-free citrus control from greenhouse; lanes 28 to 32, “Ca. Liberibacter asiaticus”-positive periwinkle from greening, B1 and B2 from branch 1 and branch 2 in dodder-transmitted periwinkle plants P1 (lanes 28 and 29) and PP11 (lanes 30 and 31), PG from grafted-transmitted periwinkle plant; lane 33, “Ca. Liberibacter asiaticus”-free periwinkle control; lanes 34 to 37, “Ca. Liberibacter asiaticus”-positive dodder collected from citrus, periwinkle, tobacco, and potato; lane 38, “Ca. Liberibacter asiaticus”-free dodder control.

Fig. 3.

Majority rule consensus tree based on maximum parsimony analyses of 29 amino acid sequences representing hyv_I gene from global isolates of “Candidatus Liberibacter asiaticus.” The leucine-rich repeat protein sequence from Colwellia psychrerythraea is used as the outgroup. Branch support values represent maximum-parsimony bootstrap (≥70%) before the slash and RAxML (≥70%) bootstrap values after the slash. Each branch label reflects the identification listed in Table 2 and indicates the geographic origin of the sample.

Fig. 4.

Majority rule consensus tree based on maximum parsimony analyses of 17-amino-acid sequences representing the hyv_II gene from global isolates of “Candidatus Liberibacter asiaticus.” The leucine-rich repeat protein sequence from Colwellia psychrerythraea is used as the outgroup. Branch support values represent maximum-parsimony bootstrap (≥70%) before the slash and RAxML (≥70%) bootstrap values after the slash. Each branch label reflects the identification listed in Table 2 and indicates the geographic origin of the sample.