Independent Evolution with the Gene Flux Originating from Multiple Xanthomonas Species Explains Genomic Heterogeneity in Xanthomonas perforans

Abstract

Xanthomonas perforans is the predominant pathogen responsible for bacterial leaf spot of tomato and X. euvesicatoria for that of pepper in the southeast United States. Previous studies have indicated significant changes in the X. perforans population collected from Florida tomato fields over the span of 2 decades, including a shift in race and diversification into three phylogenetic groups driven by genome-wide homologous-recombination events derived from X. euvesicatoria In our sampling of Xanthomonas strains associated with bacterial spot disease in Alabama, we were readily able to isolate X. perforans from symptomatic pepper plants grown in several Alabama counties, indicating a recent shift in the host range of the pathogen. To investigate the diversity of these pepper-pathogenic strains and their relation to populations associated with tomatoes grown in the southeast United States, we sequenced the genomes of eight X. perforans strains isolated from tomatoes and peppers grown in Alabama and compared them with previously published genome data available from GenBank. Surprisingly, reconstruction of the X. perforans core genome revealed the presence of two novel genetic groups in Alabama that each harbored a different transcription activation-like effector (TALE). While one TALE, AvrHah1, was associated with an emergent lineage pathogenic to both tomato and pepper, the other was identified as a new class within the AvrBs3 family, here designated PthXp1, and was associated with enhanced symptom development on tomato. Examination of patterns of homologous recombination across the larger X. euvesicatoria species complex revealed a dynamic pattern of gene flow, with multiple donors of Xanthomonas spp. associated with diverse hosts of isolation.IMPORTANCE Bacterial leaf spot of tomato and pepper is an endemic plant disease with a global distribution. In this study, we investigated the evolutionary processes leading to the emergence of novel X. perforans lineages identified in Alabama. While one lineage was isolated from symptomatic tomato and pepper plants, confirming the host range expansion of X. perforans, the other lineage was isolated from tomato and acquired a novel transcription activation-like effector, here designated PthXp1. Functional analysis of PthXp1 indicated that it does not induce Bs4-mediated resistance in tomato and contributes to virulence, providing an adaptive advantage to strains on tomato. Our findings also show that different phylogenetic groups of the pathogen have experienced independent recombination events originating from multiple Xanthomonas species. This suggests a continuous gene flux between related xanthomonads associated with diverse plant hosts that results in the emergence of novel pathogen lineages and associated phenotypes, including host range.

Keywords: TAL effector; Xanthomonas; emerging; host specificity; novel lineages; recombination.

FIG 1

Midpoint-rooted maximum-likelihood phylogeny of 41 X. perforans strains isolated from tomato and pepper plants grown in Florida and Alabama based on a concatenated alignment of 16,501 core genome SNPs. The tips are color coded according to the SCs identified in the first level of the HierBAPS hierarchy (24). The phylogroup designations of strains described previously (18, 22) are shown in parentheses, and strains sequenced in this study are highlighted in red. The scale bar indicates the number of substitutions per site.

FIG 2

X. perforans strains collected in Alabama carry a plasmid nearly identical to one found in X. cynarae pv. gardneri, which harbors the transcription activation-like effector AvrHah1, and are pathogenic to pepper. (A) Alignment of contigs assembled with plasmidSPAdes software from X. perforans strain AL65 and the X. cynarae pv. gardneri (Xcg) plasmid JS749-3.2. The inner ring shows the GC content of the plasmid, and the annotations of coding sequences (CDS) (shown in black) are provided where available. (B) Bacterial spot symptoms produced by two strains from SC6, with and without AvrHah1, shown 4 days postinoculation (DPI). Leaves of the susceptible pepper cultivar ECW were infiltrated with a needleless syringe using a bacterial suspension adjusted to 10⁴ CFU ml⁻¹. The individual strains used for inoculation are shown in parentheses. (C) Compatible and incompatible interactions on pepper cultivar ECW30R with Bs3 resistance, shown 2 days postinoculation with a bacterial suspension raised to 10⁸ CFU ml⁻¹. Hypersensitive resistance mediated by the recognition of AvrHah1 in strain AL65 is shown on the top left portion of the leaf. Pepper strains ALS7B and ALS7E, from SC6 and SC3, are shown on the bottom left and bottom right portions of the leaf, respectively.

FIG 3

Identification of a new class of transcription activation-like effector, PthXp1, in X. perforans. (A) Dendrogram (left) constructed from an alignment of repeat variable diresidue sequences (right) found among the previously described transcription activation-like effectors carried by bacterial spot pathogens and PthXp1 from X. perforans strains AL37 and AL57 (shown in red). The GenBank accession numbers of the sequences are shown in parentheses, and the scale bar indicates the number of substitutions per site. (B) Differential reactions in tomato cultivar MoneyMaker with Bs4 resistance and pepper cultivar ECW30R with Bs3 resistance. Leaves were infiltrated with a bacterial suspension adjusted to 10⁸ CFU ml⁻¹ using X. perforans strain ALS7B, pathogenic to tomato and pepper, and the nearly isogenic strain ALS7B^pthXp1 carrying the pthXp1 gene on a broad-host-range cosmid. Hypersensitive resistance is indicated by the appearance of collapsed/necrotic leaf tissue. X. euvesicatoria strain 87-7 with avrBs4 and X. perforans strain AL65 with avrHah1 were included as positive controls for Bs4 and Bs3 recognition, respectively. (C) Symptom development associated with PthXp1 on tomato cultivar MoneyMaker leaves infiltrated with a bacterial suspension raised to 10⁸ CFU ml⁻¹, shown 3 days postinoculation (DPI).

FIG 4

Landscape of homologous recombination within the X. euvesicatoria species complex. (A) Phylogeny of 67 X. perforans, X. euvesicatoria, and closely related X. euvesicatoria pathovars based on a core genome alignment of 3.98 Mb. The branches corresponding to the sequence clusters inferred within X. perforans (Fig. 1) are labeled at the nodes, and lineages predicted by the FastGear software are color coded at the right of the tree. (B) Patterns of recent recombination across the core genome of the X. euvesicatoria species complex. The colors indicate the donor lineages of the recent recombination events, and the line graph above shows the frequency of recombination within X. perforans at a particular site in the alignment. Recombination events originating from outside the sampled Xanthomonas population are shown in red. (C) Gene contents of core genome loci with elevated recombination frequencies (numbered i to vii) within X. perforans. Genes are color coded according to their annotation, with hypothetical proteins shown in purple. Gene names/functional annotations are shown where available. TBDR, putative TonB-dependent receptor.

FIG 5

Summary of the proportions and origins of recent recombinations among lineages within the X. euvesicatoria (Xeu) species complex and X. perforans SCs as predicted by FastGear. “Outside of X. euvesicatoria complex” indicates the proportion of recombinant sequences donated from outside the sampled Xanthomonas population. The error bars indicate standard deviations of the mean.

FIG 6

Model illustrating the putative origins of recent recombination events (solid arrows) and horizontal gene transfers (dotted arrows) leading to the emergence of X. perforans SC3 to SC6, along with the corresponding host range. The thickness of the arrows showing recent recombination is proportional to the overall amount of sequence donated to each X. perforans lineage. The specific genes or functional pathways associated with recombination/horizontal-transfer events are labeled where available; Xps TIISS, Xps type II secretion system; TonB, TonB transduction system; xopE3^Tnp, xopE3 disrupted by a transposon insertion; avrBsT-like, putative type 3 secreted effector gene with 71% amino acid identity to AvrBsT. The chromosomes (ovals) and plasmids (smaller circles) of donor lineages are color coded accordingly. We currently do not know if the pthXp1 gene was acquired via plasmid or Tn3-like transposase-mediated horizontal transfer or the origin of this novel transcription activation-like effector. A question mark was placed after the host range of SC4, as pathogenicity to pepper has been documented experimentally for only one strain from the group (22).