Type three effector gene distribution and sequence analysis provide new insights into the pathogenicity of plant-pathogenic Xanthomonas arboricola

Abstract

Xanthomonas arboricola is a complex bacterial species which mainly attacks fruit trees and is responsible for emerging diseases in Europe. It comprises seven pathovars (X. arboricola pv. pruni, X. arboricola pv. corylina, X. arboricola pv. juglandis, X. arboricola pv. populi, X. arboricola pv. poinsettiicola, X. arboricola pv. celebensis, and X. arboricola pv. fragariae), each exhibiting characteristic disease symptoms and distinct host specificities. To better understand the factors underlying this ecological trait, we first assessed the phylogenetic relationships among a worldwide collection of X. arboricola strains by sequencing the housekeeping gene rpoD. This analysis revealed that strains of X. arboricola pathovar populi are divergent from the main X. arboricola cluster formed by all other strains. Then, we investigated the distribution of 53 type III effector (T3E) genes in a collection of 57 X. arboricola strains that are representative of the main X. arboricola cluster. Our results showed that T3E repertoires vary greatly between X. arboricola pathovars in terms of size. Indeed, X. arboricola pathovars pruni, corylina, and juglandis, which are responsible for economically important stone fruit and nut diseases in Europe, harbored the largest T3E repertoires, whereas pathovars poinsettiicola, celebensis, and fragariae harbored the smallest. We also identified several differences in T3E gene content between X. arboricola pathovars pruni, corylina, and juglandis which may account for their differing host specificities. Further, we examined the allelic diversity of eight T3E genes from X. arboricola pathovars. This analysis revealed very limited allelic variations at the different loci. Altogether, the data presented here provide new insights into the evolution of pathogenicity and host range of X. arboricola and are discussed in terms of emergence of new diseases within this bacterial species.

Fig 1

Phylogeny of the species of X. arboricola based on rpoD gene sequences. WB and VOC refer to strains of X. arboricola pv. juglandis causing walnut blight and vertical oozing canker, respectively. The phylogenetic tree was constructed using the neighbor-joining method. The confidence of nodes was tested with 1,000 bootstrap replicates. Bootstrap values under 50 are not shown. The scale represents the number of substitutions per site. The tree is rooted with the rpoD gene sequence of strain CFBP5241 of X. campestris pv. campestris. X. populi strains were also included in the phylogenetic analysis.

Fig 2

Distribution of 53 T3E genes among 57 strains belonging to six pathovars of X. arboricola. The nomenclature and classification scheme used for T3E genes in xanthomonads is available at the Xanthomonas resource website (http://www.xanthomonas.org/t3e.html). The distribution of 11 genes (3 are hrp, and 8 are hrp conserved [hrc]) coding for the structural and regulatory components of the T3SS of the Hrp2 family is also presented. The presence or absence of an orthologue of each selected gene was determined by PCR. Black squares represent the presence of the corresponding gene; white squares represent the absence of the gene. X. arboricola pv. juglandis strains that belong to the VOC cluster on the basis of f-AFLP analysis (24) are shown with an asterisk.

Fig 3

Phylogenetic analyses of eight T3E genes from X. arboricola pathovars. Trees were inferred using the neighbor-joining method from aligned nucleotide sequences of T3E genes from X. arboricola pathovars. Bootstrapping was performed with 1,000 replicates. The scale represents the number of substitutions per site. The trees are rooted with T3E gene sequences from Xanthomonas sequenced genomes. Abbreviations for strains used as outgroups are as follows: Xcc CFBP5241, Xanthomonas campestris pv. campestris; Xoo MAFF311018, Xanthomonas oryzae pv. oryzae; Xac 306, Xanthomonas axonopodis pv. citri; Xav CFBP5618, Xanthomonas axonopodis pv. vesicatoria; Xau ICPB10535, Xanthomonas fuscans subsp. aurantifolii; Xg ATCC 19865, Xanthomonas gardneri.