Abstract:
Pantoea ananatis, a gram negative and facultative anaerobic bacterium is a member
of a Pantoea spp. complex that causes center rot of onion, which significantly affects
onion yield and quality. This pathogen does not have typical virulence factors like type
II or type III secretion systems but appears to require a biosynthetic gene-cluster,
HiVir/PASVIL (located chromosomally comprised of 14 genes), for a phosphonate
secondary metabolite, and the ‘alt’ gene cluster (located in plasmid and comprised
of 11 genes) that aids in bacterial colonization in onion bulbs by imparting tolerance
to thiosulfinates. We conducted a deep pan-genome-wide association study (pan-
GWAS) to predict additional genes associated with pathogenicity in P. ananatis using
a panel of diverse strains (n = 81). We utilized a red-onion scale necrosis assay
as an indicator of pathogenicity. Based on this assay, we differentiated pathogenic
(n = 51)- vs. non-pathogenic (n = 30)-strains phenotypically. Pan-genome analysis
revealed a large core genome of 3,153 genes and a flexible accessory genome. Pan-
GWAS using the presence and absence variants (PAVs) predicted 42 genes, including
14 from the previously identified HiVir/PASVIL cluster associated with pathogenicity,
and 28 novel genes that were not previously associated with pathogenicity in onion.
Of the 28 novel genes identified, eight have annotated functions of site-specific
tyrosine kinase, N-acetylmuramoyl-L-alanine amidase, conjugal transfer, and HTH-type
transcriptional regulator. The remaining 20 genes are currently hypothetical. Further, a
core-genome SNPs-based phylogeny and horizontal gene transfer (HGT) studies were
also conducted to assess the extent of lateral gene transfer among diverse P. ananatis
strains. Phylogenetic analysis based on PAVs and whole genome multi locus sequence typing (wgMLST) rather than core-genome SNPs distinguished red-scale necrosis
inducing (pathogenic) strains from non-scale necrosis inducing (non-pathogenic) strains
of P. ananatis. A total of 1182 HGT events including the HiVir/PASVIL and alt cluster
genes were identified. These events could be regarded as a major contributing factor to
the diversification, niche-adaptation and potential acquisition of pathogenicity/virulence
genes in P. ananatis.
