Genomic survey of pathogenicity determinants and VNTR markers in the cassava bacterial pathogen Xanthomonas axonopodis pv. Manihotis strain CIO151

Abstract

Xanthomonas axonopodis pv. manihotis (Xam) is the causal agent of bacterial blight of cassava, which is among the main components of human diet in Africa and South America. Current information about the molecular pathogenicity factors involved in the infection process of this organism is limited. Previous studies in other bacteria in this genus suggest that advanced draft genome sequences are valuable resources for molecular studies on their interaction with plants and could provide valuable tools for diagnostics and detection. Here we have generated the first manually annotated high-quality draft genome sequence of Xam strain CIO151. Its genomic structure is similar to that of other xanthomonads, especially Xanthomonas euvesicatoria and Xanthomonas citri pv. citri species. Several putative pathogenicity factors were identified, including type III effectors, cell wall-degrading enzymes and clusters encoding protein secretion systems. Specific characteristics in this genome include changes in the xanthomonadin cluster that could explain the lack of typical yellow color in all strains of this pathovar and the presence of 50 regions in the genome with atypical nucleotide composition. The genome sequence was used to predict and evaluate 22 variable number of tandem repeat (VNTR) loci that were subsequently demonstrated as polymorphic in representative Xam strains. Our results demonstrate that Xanthomonas axonopodis pv. manihotis strain CIO151 possesses ten clusters of pathogenicity factors conserved within the genus Xanthomonas. We report 126 genes that are potentially unique to Xam, as well as potential horizontal transfer events in the history of the genome. The relation of these regions with virulence and pathogenicity could explain several aspects of the biology of this pathogen, including its ability to colonize both vascular and non-vascular tissues of cassava plants. A set of 16 robust, polymorphic VNTR loci will be useful to develop a multi-locus VNTR analysis scheme for epidemiological surveillance of this disease.

Figure 1. Comparison of the genomic structure of Xam CIO151 with that of closely related members from the genus Xanthomonas.

Scaffolds of Xam CIO151 were ordered based on the alignment with the complete genome sequence of X. euvesicatoria, Xeu, and then genome comparisons were performed using MUMmer (A). Alignment of ordered scaffolds of Xam CIO151 with the complete genome sequences of X. axonopodis pv. citri str. 306, Xac (B); X. campestris pv. campestris str. 8004, Xcc (C); X. albilineans, Xal (D); and Xanthomonas oryzae pv. oryzae PXO99^A, Xoo (E) chromosomes. Scaffolds classified as parts of the chromosome of Xam CIO151 are shown in the y-axis. Red dots represent conserved segments while blue dots represent inverted regions.

Figure 2. Circular representation of the genome sequence of Xam CIO151.

From outside to inside: first circle in blue indicates CDS predicted in the positive strands for the scaffolds classified as probable chromosomal regions. Second circle in red indicates the CDS predicted in the negative strand. Red spots in the black third circle indicate the region identified with atypical nucleotide composition. The fourth circle indicates the deviation pattern from the average G+C content. Inner circle shows GC skew values, positive values are shown in purple and negative values are shown in orange. Numbers correspond to scaffold IDs.

Figure 3. Organization of pathogenicity-related gene clusters in the Xam CIO151 genome.

Open arrows with labels indicate genes with assigned functions, black arrows indicate genes with early stop codons, open arrows without labels indicate conserved hypothetical proteins and grey arrows indicate non-conserved hypothetical proteins. Graphs above clusters show the G+C content and deviations from the average value. A. Xanthomonadin gene cluster; * indicates genes related to pigB genomic region and ** indicates genes reported as important for cluster functionality. Abbreviations used are: H = halogenase (xanmn_chr15_0075), BP = xanthomonadin biosynthesis protein (xanmn_chr15_0079), E = xanthomonadin exporter (xanmn_chr15_0373), PSP = putative secreted protein (xanmn_chr15_0080), BACPD = xanthomonadin biosynthesis acyl carrier protein dehydratase (xanmn_chr15_0082), BA = putative xanthomonadin biosynthesis acyltransferase (xanmn_chr15_0081 and xanmn_chr15_0083), BMP = putative xanthomonadin biosynthesis membrane protein (xanmn_chr15_0084), ACP = acyl carrier protein (xanmn_chr15_0085), XanB1 = putative reductase/halogenase (xanmn_chr15_0086), XanB2 = putative pteridine-dependent deoxygenase like protein (xanmn_chr15_0087), AMP-l = AMP-ligase (xanmn_chr15_0088), DP = dipeptidyl peptidase (xanmn_chr15_0090). B. Cluster implicated in xanthan production (gum). C. Regulation of pathogenicity factors (rpf) cluster. D. Type III secretion system (T3SS) cluster.

Figure 4. Phylogeny of conserved effectors in the genus Xanthomonas.

Phylogenetic tree of concatenated conserved effector protein sequences of AvrBs2, XopK, XopL, XopN, XopQ XopR families and the Hpa1 protein, obtained with a Bayesian approach. Numbers on branches indicate Bayesian support values. Length of branches indicates the number of amino acid substitutions per site.

Figure 5. Molecular analysis of selected VNTR loci of Xam.

PCR amplicons of VNTR loci of Xam were separated by agarose gel electrophoresis. A, XaG1_02 (362 bp); B, XaG1_29 (251 bp); C, XaG1_58 (192 bp); D, XaG2_50 (119 bp); E, XaG1_12 (111 bp). For comparison, expected sizes for Xam strain CIO151 are given in brackets.