Soft-rot Enterobacteriaceae (SRE), typified by Pectobacterium and Dickeya genera, are phytopathogenic bacteria inflicting soft-rot disease in crops worldwide. By combining genomic information from 100 SRE with whole-transcriptome data sets, we identified novel genomic and transcriptional associations among key pathogenicity themes in this group. Comparative genomics revealed solid linkage between the type I secretion system (T1SS) and the carotovoricin bacteriophage (Ctv) conserved in 96.7% of Pectobacterium genomes. Moreover, their coactivation during infection indicates a novel functional association involving T1SS and Ctv. Another bacteriophage-borne genomic region, mostly confined to less than 10% of Pectobacterium strains, was found, presumably comprising a novel lineage-specific prophage in the genus. We also detected the transcriptional coregulation of a previously predicted toxin/immunity pair (WHH and SMI1_KNR4 families), along with the type VI secretion system (T6SS), which includes hcp and/or vgrG genes, suggesting a role in disease development as T6SS-dependent effectors. Further, we showed that another predicted T6SS-dependent endonuclease (AHH family) exhibited toxicity in ectopic expression assays, indicating antibacterial activity. Additionally, we report the striking conservation of the group 4 capsule (GFC) cluster in 100 SRE strains which consistently features adjacently conserved serotype-specific gene arrays comprising a previously unknown organization in GFC clusters. Also, extensive sequence variations found in gfcA orthologs suggest a serotype-specific role in the GfcABCD machinery. IMPORTANCE Despite the considerable loss inflicted on important crops yearly by Pectobacterium and Dickeya diseases, investigations on key virulence and interbacterial competition assets relying on extensive comparative genomics are still surprisingly lacking for these genera. Such approaches become more powerful over time, underpinned by the growing amount of genomic information in public databases. In particular, our findings point to new functional associations among well-known genomic themes enabling alternative means of neutralizing SRE diseases through disruption of pivotal virulence programs. By elucidating novel transcriptional and genomic associations, this study adds valuable information on virulence candidates that could be decisive in molecular applications in the near future. The utilization of 100 genomes of Pectobacterium and Dickeya strains in this study is unprecedented for comparative analyses in these taxa, and it provides novel insights on the biology of economically important plant pathogens.

21Soft-rot Enterobacteriaceae (SRE) typified by Pectobacterium and Dickeya genera are 22 phytopathogenic bacteria inflicting soft-rot disease in crops worldwide. By combining 23 genomic information from 100 SRE with whole-transcriptome datasets, we identified novel 24 genomic and transcriptional associations amongst key pathogenicity themes in this group. 25Comparative genomics revealed solid linkage between the type I secretion system (T1SS) 26 and the carotovoricin bacteriophage (Ctv) conserved in 96.7% of Pectobacterium genomes. 27Moreover, their co-activation during infection might indicate a novel functional association 28 involving T1SS/Ctv. Another bacteriophage-borne genomic region mostly confined in less 29 than 10% of Pectobacterium organisms was found, presumably comprising a novel lineage-30 specific prophage in the genus. We also detected the transcriptional co-regulation of a 31 previously predicted toxin/immunity pair (WHH and SMI1_KNR4 families) along with type VI 32 secretion system (T6SS) and hcp/vgrG genes suggesting a role in disease development as 33 T6SS-dependent effectors. Further, we showed that another predicted T6SS-dependent 34 endonuclease (AHH-family) exhibited toxicity in ectopic expression assays indicating 35 antibacterial activity. Additionally, we report the striking conservation of group-4-capsule 36 (GFC) cluster in 100 SRE strains which consistently features adjacently conserved serotype-37 specific gene-arrays comprising a previously unknown organization in GFC clusters. Also, 38 extensive sequence variations found in gfcA orthologs suggest a serotype-specific role in the 39 GfcABCD machinery. 40