Pan-Genome of Novel Pantoea stewartii subsp. indologenes Reveals Genes Involved in Onion Pathogenicity and Evidence of Lateral Gene Transfer

Abstract

Pantoea stewartii subsp. indologenes (Psi) is a causative agent of leafspot on foxtail millet and pearl millet; however, novel strains were recently identified that are pathogenic on onions. Our recent host range evaluation study identified two pathovars; P. stewartii subsp. indologenes pv. cepacicola pv. nov. and P. stewartii subsp. indologenes pv. setariae pv. nov. that are pathogenic on onions and millets or on millets only, respectively. In the current study, we developed a pan-genome using the whole genome sequencing of newly identified/classified Psi strains from both pathovars [pv. cepacicola (n = 4) and pv. setariae (n = 13)]. The full spectrum of the pan-genome contained 7030 genes. Among these, 3546 (present in genomes of all 17 strains) were the core genes that were a subset of 3682 soft-core genes (present in ≥16 strains). The accessory genome included 1308 shell genes and 2040 cloud genes (present in ≤2 strains). The pan-genome showed a clear linear progression with >6000 genes, suggesting that the pan-genome of Psi is open. Comparative phylogenetic analysis showed differences in phylogenetic clustering of Pantoea spp. using PAVs/wgMLST approach in comparison with core genome SNPs-based phylogeny. Further, we conducted a horizontal gene transfer (HGT) study using Psi strains from both pathovars along with strains from other Pantoea species, namely, P. stewartii subsp. stewartii LMG 2715^T, P. ananatis LMG 2665^T, P. agglomerans LMG L15, and P. allii LMG 24248^T. A total of 317 HGT events among four Pantoea species were identified with most gene transfer events occurring between Psi pv. cepacicola and Psi pv. setariae. Pan-GWAS analysis predicted a total of 154 genes, including seven gene-clusters, which were associated with the pathogenicity phenotype (necrosis on seedling) on onions. One of the gene-clusters contained 11 genes with known functions and was found to be chromosomally located.

Keywords: accessory genome; core genome; horizontal gene transfer (HGT); pangenome.

Figure 1

Pan-genome analysis of 17 Pantoea stewartii subsp. indologenes genomes. (A) Distribution of gene (cluster) sizes as a function of the number of genomes; (B) Genes contributed by each genome that contributed to the pan-genome.

Figure 2

Theoretical estimation of the core and pan-genome sizes based on the exponential decay model. (A) Estimation of core genome size based on the Willenbrock model fit to OMCL clusters and (B) Estimation of pan-genome size based on the Tettelin model fit to OMCL clusters.

Figure 3

Dendrogram of 17 strains of Pantoea stewartii subsp. indologenes based on the core and accessory genes. (A) Dendrogram based on core genes, i.e., genes present in all 17 strains used in the study; (B) Dendrogram based on soft-core genes, i.e., genes present in at least 95% of the strains; (C) Dendrogram based on shell genes; and (D) Dendrogram based on cloud genes, i.e., the genes specific to each strain or shared by a maximum of two strains.

Figure 4

Phylogeny-based horizontal gene transfer (HGT) among 17 strains of Pantoea stewartii subsp. indologenes strains and four other species of the Pantoea complex, namely, P. ananatis, P. stewartii subsp. stewartii, P. agglomerans, and P. allii. (A) Phylogenetic tree of Pantoea spp. (n = 21) strains based on multiple-sequence alignment. Phylogenetic tree resulted in four clusters (a–d). Size of circles represent the bootstrap values in that order; (B) Predicted gene flow within the four phylogenetic clusters of Pantoea spp. Bands connect donors and recipients, with the width of the band correlating to the number of HGTs and the color corresponding to the donors. Numbers on the circumference of circos plot represent the number of genes that underwent horizontal gene transfers. The four arcs (a–d) of circos represent the four phylogenetic clusters of Pantoea spp. strains; (C–E) Graphical annotations of sequences involved in HGT as per the assigned GO terms: (C) shows the function of genes assigned to biological processes, (D) shows the function of genes assigned to molecular functions, and (E) represents the function of genes assigned to cellular components.

Figure 5

Annotated genes involved in horizontal gene transfer (HGT) with functions assigned under biochemical, metabolic, and physiological pathways operational in Pantoea stewartii subsp. indologenes.

Figure 6

Comparative phylogeny of onion-pathogenic and onion-non-pathogenic strains of Pantoea stewartii subsp. indologenes based on core genome SNPs and presence and absence variations (PAVs). (A) Phylogenetic tree constructed using PAVs using RAxML; (B) Phylogenetic tree constructed using core SNPs using RAxML. Numerical values in decimal represent the branch length. Longer branch length indicates higher genetic divergence.

Figure 7

Phylogenetic tree based on whole genome multi-locus sequence typing (WgMLST). Dendrogram for 21 Pantoea spp. strains was constructed using assembled genome contigs. Size of stars represents the boot strap values and numbers represent the branch length. PSS is Pantoea stewartii subsp. stewartii.