Tn5-induced mutations in Agrobacterium vitis F2/5 resulted in both altered grape necrosis and tobacco leaf panel collapse phenotypes, suggesting that the underlying mechanisms of the reactions are related. The reaction on tobacco resembles the classical hypersensitive response (HR) caused by several plant pathogenic bacteria in that it is observable within 14 h, is inhibited by treatment of plants with metabolic inhibitors, and results in the inability to recover the pathogen from the necrotic zone. Strains of A. vitis differ with regard to their efficiency of causing the reaction on tobacco. An EcoRI fragment from one mutant, M6, which is necrosis-altered and HR-minus, was cloned and sequenced. Sequence analysis revealed that the Tn5 insertion occurred in a region that shares significant homology with genes involved in long chain fatty acid production by the marine bacteria Shewanella spp. and Moritella marina. Complementation of M6 with a cosmid clone from an F2/5 DNA library restored the tobacco HR and grape necrosis phenotypes.

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Beneficial plant root-associated microorganisms carry out a range of functions that are essential for plant performance. To study bacterial adaptation in the rhizosphere, we employed experimental evolution to track the physiological and genetic dynamics of root-dwelling Pseudomonas protegens in the Arabidopsis thaliana rhizosphere under axenic conditions. We identified 35 mutations in 28 genes, including those important for cell surface decoration. Four mutations in three OBC3 (O-antigenic polysaccharide biosynthesis cluster 3) genes, i.e., oafA, galE and RS09880, were identified that can be associated with the decoration of the O-antigen, a major constituent of lipopolysaccharide (LPS). Enhanced root colonization of oafA and RS09880 mutants is linked to enhanced root attachment in a plant. OafA encodes a two-domain O-antigen acetyltransferase, and the mutations introduce a premature stop codon halfway directly between both domains. Intriguingly, we observed similar mutations in OBC3 of publicly available environmental P. protegens strain genomes, including several non-plant associated ones. Two OBC3 mutants, i.e., in galE and RS09880, showed altered LPS structure and reduced sensitivity towards the environmental phage ΦGP100. Altogether, our results highlight a role for O-antigen decoration in root colonization, likely by impacting bacterial root attachment, and reveal OBC3 mutations in nature, which occasionally can contribute to resistance to environmental phages.