Comparative Secretome Analyses of Toxigenic and Atoxigenic Rathayibacter Species

Phytopathogenic Rathayibacter species are unique bacterial plant pathogens because they are obligately vectored by plant parasitic anguinid nematodes to the developing seedheads of forage grasses and cereals. This understudied group of plant-associated Actinomycetes includes the neurotoxigenic plant pathogen R. toxicus, which causes annual ryegrass toxicity in grazing livestock. R. toxicus is currently endemic to Australia and is listed as a plant pathogen select agent by the U.S. Department of Agriculture-Animal and Plant Health Inspection Service. The complex Rathayibacter disease cycle requires intimate interactions with the nematode vector and plant hosts, which warrants an increased understanding of the secretory and surface-associated proteins that mediate these diverse eukaryotic interactions. Here we present the first comparative secretome analysis for this complex, nematode-vectored Rathayibacter genus that compares the three agronomically damaging toxigenic and atoxigenic Rathayibacter species, R. toxicus, R. iranicus, and R. tritici. The exoproteomic comparison identified 1,423 unique proteins between the three species via liquid chromatography-tandem mass spectrometry, leading to the identification of putative pathogenicity-related proteins and proteins that may mediate nematode attachment. Of the uniquely identified proteins, 94 homologous proteins were conserved between the three Rathayibacter exoproteomes and comprised between 43.4 and 58.6% of total protein abundance. Comparative analyses revealed both conserved and uniquely expressed extracellular proteins, which, interestingly, had more similarities to extracellular proteins commonly associated with bacterial animal pathogens than classic plant pathogens. This comparative exoproteome analysis will facilitate the characterization of proteins essential for vector attachment and host colonization and assist in the development of serological diagnostic assays.