Pan-genome-wide analysis ofPantoea ananatisidentified genes linked to pathogenicity in onion
Authors: Agarwal Gaurav , Choudhary Divya , Stice Shaun P. , Myers Brendon K. , Gitaitis Ronald D. , Venter Stephanus N. , Kvitko Brian H. , Dutta Bhabesh 
Published: July 2020

AbstractPantoea ananatisis a member of aPantoeaspp. 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. We conducted a deep pan-genome-wide association study (pan-GWAS) to predict additional genes associated with pathogenicity inP. ananatisusing 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 ofP. 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. This is the first report of using pan-GWAS onP. ananatisfor 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 ofP. 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 inP. ananatiscolonization in onion bulbs. We concluded that HiVir/PASVIL genes are associated with pathogenicP. ananatisstrains and thealtgene cluster alone is not responsible for pathogenicity on onion. The pan-genome also provides clear evidence of constantly evolving accessory genes inP. ananatisthat may contribute to host-range expansion and niche-adaptation.Author summaryPantoea ananatisis a major bacterial pathogen that causes center rot of onion and diseases of a number of other plant species. In order to understand the genome architecture and identify genes responsible for pathogenicity in onion, a pan-genome analysis was performed. We used 81 strains ofP. ananatiscollected over 20 years from different regions of the state of Georgia, USA. The pan-genome study identified a core genome with a conserved set of genes and an accessory genome that displayed variation among strains. We conducted pan-GWAS (pan-genome-wide association study) using presence and absence variants (PAVs) in the genomes and associated onion-pathogenic phenotypes based on a red-onion scale necrosis assay. The study resulted in identification of genes, including a cluster of chromosomal HiVir/PASVIL genes, that are significantly associated with the onion pathogenic phenotype. In addition, we identified 28 novel genes, a majority of which (n= 20) have hypothetical functions. We concluded and further substantiated earlier findings that a cluster of genes is responsible for pathogenicity on onion. 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 bacterial colonization of onion bulbs byP. ananatisstrains. We concluded that HiVir/PASVIL genes are associated with onion-pathogenic strains, and thealtgene cluster alone is not responsible for pathogenicity on onion. This study also provides potential evidence of constantly evolving accessory genes inP. ananatiswhich may help in host range expansion and adaptation to diverse niches.