Pan-Genome-Wide Analysis of Pantoea Ananatis Identified Genes Linked to Pathogenicity in Onion

Abstract

Background: Pantoea ananatis 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), for a phosphonate secondary metabolite, and the onion-virulence regions, OVR (localized on a megaplasmid), for onion pathogenicity and virulence, respectively. Results: 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-GWAS revealed a large core genome of 3,153 genes and a flexible accessory genome of ≤5,065 genes. Phylogenomic analysis using pan-GWAS and presence and absence variants (PAVs) distinguished red-scale necrosis inducing (pathogenic) strains from non-scale necrosis inducing (non-pathogenic) strains of P. ananatis. The pan-GWAS also 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, TraR/DksA family transcriptional regulator, and HTH-type transcriptional regulator. The remaining 20 genes are currently hypothetical. Conclusions: This is the first report of using pan-GWAS on P. ananatis for the prediction of novel genes contributing to pathogenicity in onion, which will be utilized for further functional analyses. Pan-genomic differences (using PAVs) differentiated onion pathogenic from non-pathogenic strains of P. ananatis, which has been difficult to achieve using single or multiple gene-based phylogenetic analyses. The pan-genome analysis also allowed us to evaluate the presence and absence of HiVir/PASVIL genes and 11 megaplasmid-borne OVR-A genes regarded as the ‘alt’ cluster that aid in P. ananatis colonization in onion bulbs. We concluded that HiVir/PASVIL genes are associated with pathogenic P. ananatis strains and the alt gene cluster alone is not responsible for pathogenicity on onion. The pan-genome also provides clear evidence of constantly evolving accessory genes in P. ananatis that may contribute to host-range expansion and niche-adaptation.

Pan-genome of Novel <em>Pantoea stewartii</em> subsp. <em>indologenes</em> Reveal Genes Involved in Onion Pathogenicity and Evidence of Lateral Gene Transfer

“…cepacicola PNA 03-3 is more closely related to P. ananatis (LMG 2665 T ), which is reflected in the core SNP-based phylogeny. The WgMLST, an extended concept of MLST is complementary to PAV based phylogenetic analysis [8]. As expected, we observed similar pattern of phylogenetic classification of strains both with PAVs-and wgMLST-based phylogeny.…”

“…In the current study we identified 3,546 core genes, 5 to 382 cloud genes and 394 to 703 shell genes. A similar study using 81 P. ananatis strains identified 3,153 core genes and cloud genes ranging from 1000 to 6,808 [8]. We observed a stark difference in the number of cloud genes when compared to our earlier study on P. ananatis.…”

“…The open feature of Psi is consistent with P. ananatis and Geobacillus spp. dataset [8,17,31] as opposed to other species, such as Bifidobacterium breve and Staphylococcus lugdunensis, which depicted a closed trend [32,33].…”

“…As a result, several studies have been conducted to 2 comprehensively explore features specific to each genome. Most widely explored genomic variants used in genome-wide studies are SNPs both in prokaryotes [8] and eukaryotes [9][10][11][12][13][14]. Another most widely explored variations in prokaryotic genomes are presence and absence variants (PAVs).…”

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

“…As a result, several studies have been conducted to comprehensively explore features specific to each genome. The most widely explored genomic variants used in genome-wide studies are single nucleotide polymorphisms (SNPs) that are utilized in both prokaryotes [ 8 ] and eukaryotes [ 9 , 10 , 11 , 12 , 13 , 14 ]. Another widely used technique to explore variations in prokaryotic genomes is the use of presence and absence variants (PAVs).…”