Amylase-associated genetic pattern in Xanthomonas euvesicatoria on pepper

Abstract

Bacterial leaf spot of pepper (BSP), primarily caused by Xanthomonas euvesicatoria (Xe), poses a significant challenge to pepper production worldwide. Despite its impact, the genetic diversity of this pathogen remains underexplored, which limits our understanding of its population structure. To bridge this knowledge gap, we conducted a comprehensive analysis using 103 Xe strains isolated from pepper in southwest Florida to characterize genomic and type III effector (T3E) variation in this population. Phylogenetic analysis of core genomes revealed a major distinct genetic lineage associated with amylolytic activity. This amylolytic lineage was represented in Xe strains globally. Molecular clock analysis dated the emergence of amylolytic strains in Xe to around 1972. Notably, non-amylolytic strains possessed a single base pair frameshift deletion in the ⍺-amylase gene yet retained a conserved C-terminus. GUS assay revealed the expression of two open reading frames in non-amylolytic strains, one at the N-terminus and another that starts 136 base pairs upstream of the ⍺-amylase gene. Analysis of T3Es in the Florida Xe population identified variation in 12 effectors, including two classes of mutations in avrBs2 that prevent AvrBs2 from triggering a hypersensitive response in Bs2-resistant pepper plants. Knowledge of T3E variation could be used for effector-targeted disease management. This study revealed previously undescribed population structure in this economically important pathogen.IMPORTANCEBacterial leaf spot (BSP), a significant threat to pepper production globally, is primarily caused by Xanthomonas euvesicatoria (Xe). Limited genomic data has hindered detailed studies on its population diversity. This study analyzed the whole-genome sequences of 103 Xe strains from peppers in southwest Florida, along with additional global strains, to explore the pathogen's diversity. The study revealed two major distinct genetic groups based on their amylolytic activity, the ability to break down starch, with non-amylolytic strains having a mutation in the ⍺-amylase gene. Additionally, two classes of mutations in the avrBs2 gene were found, leading to susceptibility in pepper plants with the Bs2 resistance gene, a commercially available resistance gene for BSP. These findings highlight the need to forecast the emergence of such strains, identify genetic factors for innovative disease management, and understand how this pathogen evolves and spreads.

Keywords: Pepper; Xanthomonas euvesicatoria; amylase; frameshift; population genomics; type III effectors.

Fig 1

Maximum likelihood phylogenetic tree of X. euvesicatoria based on 4,005 core genes of 155 strains. Clades are highlighted based on association with amylolytic activity. The circle outside the tree represents amylase phenotype, host, and location of isolation, moving from inner to outer circles.

Fig 2

Dated phylogenetic tree of X. euvesicatoria strains. The most recent common ancestor of amylolytic strains was estimated to emerge in 1972 (95% confidence interval, 1958–1982). The redness and thickness of the branches are proportional to the posterior probability for a significant change in phenotype on the given branch. The timescale is indicated at bottom.

Fig 3

Cloning and deletion mutants of ⍺-amylase gene. (A) Confirmation of an amylolytic phenotype in non-amylolytic strains 20_20_B2 and 85–10. 20_20_B2 and 85–10 are wild-type strains; 20_20_B2_Amy and 85–10_Amy are transconjugants with ⍺-amylase clone from an amylolytic strain 19_200_E2. (B) Deletion of ⍺-amylase gene and its C-terminus results in loss of amylolytic activity. ΔAmy and ΔAmyC represent the deletion of the whole gene and C-terminus only, respectively, of ⍺-amylase gene, in the amylolytic wild-type strain 19_200_E2. ΔAmy:Amy represents complemented with whole gene.

Fig 4

Frameshift mutation in ⍺-amylase gene. (A) Intact ⍺-amylase gene sequences for amylolytic strains predicted to produce a single open reading frame (ORF) of 475 aa. (B) Deletion of one bp (T) at position 453 causes a frameshift in non-amylolytic strains. The frameshift leads an earlier stop codon and two ORFs (ORF1-222 aa and ORF2-242 aa) in which the secretion signal is in ORF1 and amylase domain is out of frame in ORF2. (C) An alternative ORF in non-amylolytic strains starts 136 bp upstream of wild-type ⍺-amylase gene and frameshift mutation puts the C-terminus back in frame leading to a bigger product ORF3 of 520 aa that lacks a secretion signal. Locations of GUS fusions for ORFs 1–3 are also shown.

Fig 5

Gus activity (β-glucuronidase) of three predicted ORFs (Fig. 4) from the nonfunctional ⍺-amylase gene containing a frameshift of the non-amylolytic strain 20_20_B2 when expressed in non-amylolytic strain 20_20_B2 and grown in nutrient agar or nutrient agar amended with starch. pL3uidA represents positive control, pL6uidA represents negative control, and ORF1, ORF2, and ORF3 represent three predicted ORFs. ORF1 and ORF2 represent the predicted ORFs due to frameshift leading to an earlier stop codon (see Fig. 4B). ORF3 represents additional larger predicted ORF starting 136 base pairs upstream of the intact gene (see Fig. 4C). The error bars indicate standard errors.

Fig 6

Wild-type (WT) avrBs2 sequence as compared to mutations that are associated with susceptible reaction in pepper genotypes with the Bs2 resistance gene. (A) Point mutation at 1100 bp position of avrBs2 causing change in amino acid from Glu to Lys. (B) Insertion of transposon (AF077016; IS1646) in the promotor region of avrBs2.