Pan-Genomic Analysis Permits Differentiation of Virulent and Non-virulent Strains of Xanthomonas arboricola That Cohabit Prunus spp. and Elucidate Bacterial Virulence Factors

Abstract

Xanthomonas arboricola is a plant-associated bacterial species that causes diseases on several plant hosts. One of the most virulent pathovars within this species is X. arboricola pv. pruni (Xap), the causal agent of bacterial spot disease of stone fruit trees and almond. Recently, a non-virulent Xap-look-a-like strain isolated from Prunus was characterized and its genome compared to pathogenic strains of Xap, revealing differences in the profile of virulence factors, such as the genes related to the type III secretion system (T3SS) and type III effectors (T3Es). The existence of this atypical strain arouses several questions associated with the abundance, the pathogenicity, and the evolutionary context of X. arboricola on Prunus hosts. After an initial characterization of a collection of Xanthomonas strains isolated from Prunus bacterial spot outbreaks in Spain during the past decade, six Xap-look-a-like strains, that did not clustered with the pathogenic strains of Xap according to a multi locus sequence analysis, were identified. Pathogenicity of these strains was analyzed and the genome sequences of two Xap-look-a-like strains, CITA 14 and CITA 124, non-virulent to Prunus spp., were obtained and compared to those available genomes of X. arboricola associated with this host plant. Differences were found among the genomes of the virulent and the Prunus non-virulent strains in several characters related to the pathogenesis process. Additionally, a pan-genomic analysis that included the available genomes of X. arboricola, revealed that the atypical strains associated with Prunus were related to a group of non-virulent or low virulent strains isolated from a wide host range. The repertoire of the genes related to T3SS and T3Es varied among the strains of this cluster and those strains related to the most virulent pathovars of the species, corylina, juglandis, and pruni. This variability provides information about the potential evolutionary process associated to the acquisition of pathogenicity and host specificity in X. arboricola. Finally, based in the genomic differences observed between the virulent and the non-virulent strains isolated from Prunus, a sensitive and specific real-time PCR protocol was designed to detect and identify Xap strains. This method avoids miss-identifications due to atypical strains of X. arboricola that can cohabit Prunus.

Keywords: almond; bacterial spot disease; comparative genomics; stone fruit trees.

Figure 1

Maximum likelihood tree of concatenated sequences of the genes dnaK, fyuA, gyrB and rpoD of non-virulent Xanthomonas arboricola strains isolated from Prunus spp. For comparative purposes pathogenic strains of X. arboricola pv. pruni isolated from Prunus spp. and X. arboricola strains isolated from other hosts were included. X. citri subsp. citri was used as an outgroup. Bootstrap values of 1,000 replicates are represented over or below the branches. Selected strains for subsequent whole genome sequencing are in bold.

Figure 2

Graphical circular representation of the draft genome of the Prunus-non-virulent strains of Xanthomonas arboricola CITA 14 and CITA 124. The contigs were arranged by Mauve, using the genome sequence of X. arboricola pv. juglandis strain Xaj417 as reference. COG categories were assigned to predicted genes using the NCBI's conserved domain database. Circular map was constructed using CGview. From outside to center: Genes on forward strand; genes on reverse strand; GC content; GC skew.

Figure 3

Potential groups of orthologous genes present in Xanthomonas arboricola. Core, shell and cloud groups of orthologous genes shared by 17 genome sequences of X. arboricola (A). Venn diagram showing the groups of orthologous genes shared by five genome sequences of pathogenic (CITA 33 and IVIA 2626.1) and non-virulent (CITA 14, CITA 44, and CITA 124) strains of X. arboricola isolated from Prunus spp. (B).

Figure 4

Pan-genome tree for 17 strains of Xanthomonas arboricola with a variable virulence. Tree construction was based in the distance between genomes according to the Manhattan distance. Bootstrap values over 50% are showed at the branch points.

Figure 5

Calibration curves for detection of xopE3 in Xanthomonas arboricola pv. pruni. Calibration curves have been obtained from dilution series of purified DNA (A) and bacterial cells (B) of X. arboricola pv. pruni strain CITA 33. Real-time PCR amplification was performed in three independent assays using the primers XopE3F/R and the TaqMan probe XopE3p.