AUTHOR=Myers Brendon K. , Shin Gi Yoon , Agarwal Gaurav , Stice Shaun P. , Gitaitis Ronald D. , Kvitko Brian H. , Dutta Bhabesh 

TITLE=Genome-wide association and dissociation studies in Pantoea ananatis reveal potential virulence factors affecting Allium porrum and Allium fistulosum  ×  Allium cepa hybrid

JOURNAL=Frontiers in Microbiology

VOLUME=13

YEAR=2023

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.1094155

DOI=10.3389/fmicb.2022.1094155

ISSN=1664-302X

ABSTRACT=<p><italic>Pantoea ananatis</italic> is a member of a <italic>Pantoea</italic> species complex that causes center rot of bulb onions (<italic>A. cepa</italic>) and also infects other <italic>Allium</italic> crops like leeks (<italic>Allium porrum</italic>), chives (<italic>Allium schoenoprasum</italic>), bunching onion or Welsh onion (<italic>Allium fistulosum</italic>), and garlic (<italic>Allium sativum</italic>). This pathogen relies on a chromosomal phosphonate biosynthetic gene cluster (HiVir) and a plasmid-borne thiosulfinate tolerance cluster (<italic>alt</italic>) for onion pathogenicity and virulence, respectively. However, pathogenicity and virulence factors associated with other <italic>Allium</italic> species remain unknown. We used phenotype-dependent genome-wide association (GWAS) and phenotype-independent gene-pair coincidence (GPC) analyses on a panel of diverse 92 <italic>P. ananatis</italic> strains, which were inoculated on <italic>A. porrum</italic> and <italic>A. fistulosum</italic> × <italic>A. cepa</italic> under greenhouse conditions. Phenotypic assays showed that, in general, these strains were more aggressive on <italic>A. fistulosum</italic> × <italic>A. cepa</italic> as opposed to <italic>A. porrum</italic>. Of the 92 strains, only six showed highly aggressive foliar lesions on <italic>A. porrum</italic> compared to <italic>A. fistulosum</italic> × <italic>A. cepa</italic>. Conversely, nine strains showed highly aggressive foliar lesions on <italic>A. fistulosum</italic> × <italic>A. cepa</italic> compared to <italic>A. porrum</italic>. These results indicate that there are underlying genetic components in <italic>P. ananatis</italic> that may drive pathogenicity in these two <italic>Allium</italic> spp. Based on GWAS for foliar pathogenicity, 835 genes were associated with <italic>P. ananatis</italic>’ pathogenicity on <italic>A. fistulosum</italic> × <italic>A. cepa</italic> whereas 243 genes were associated with bacterial pathogenicity on <italic>A. porrum</italic>. The Hivir as well as the <italic>alt</italic> gene clusters were identified among these genes. Besides the ‘HiVir’ and the <italic>alt</italic> gene clusters that are known to contribute to pathogenicity and virulence from previous studies, genes annotated with functions related to stress responses, a potential toxin-antitoxin system, flagellar-motility, quorum sensing, and a previously described phosphonoglycan biosynthesis (pgb) cluster were identified. The GPC analysis resulted in the identification of 165 individual genes sorted into 39 significant gene-pair association components and 255 genes sorted into 50 significant gene-pair dissociation components. Within the coincident gene clusters, several genes that occurred on the GWAS outputs were associated with each other but dissociated with genes that did not appear in their respective GWAS output. To focus on candidate genes that could explain the difference in virulence between hosts, a comparative genomics analysis was performed on five <italic>P. ananatis</italic> strains that were differentially pathogenic on <italic>A. porrum</italic> or <italic>A. fistulosum</italic> × <italic>A. cepa</italic>. Here, we found a putative type III secretion system, and several other genes that occurred on both GWAS outputs of both <italic>Allium</italic> hosts. Further, we also demonstrated utilizing mutational analysis that the <italic>pepM</italic> gene in the HiVir cluster is important than the <italic>pepM</italic> gene in the pgb cluster for <italic>P. ananatis</italic> pathogenicity in <italic>A. fistulosum</italic> × <italic>A. cepa</italic> and <italic>A. porrum</italic>. Overall, our results support that <italic>P. ananatis</italic> may utilize a common set of genes or gene clusters to induce symptoms on <italic>A. fistulosum × A. cepa</italic> foliar tissue as well as <italic>A. cepa</italic> but implicates additional genes for infection on <italic>A. porrum</italic>.</p>