Inter-species expression of an EF-HAND CALCIUM BINDING PROTEIN in Xanthomonas perforans leads to reduced virulence and decreased immune evasion in tomato plants.

Many phytopathogenic bacteria require a type three secretion system (TTSS) to activate effector triggered immunity (ETI). We identified a calcium binding protein, EfhX_Xfa, in the citrus pathogen, X. citri subsp. aurantifolii, that does not require a TTSS to activate reactive oxygen species (ROS) and elicit a hypersensitive reaction (HR) in tomato leaves following infection. Purified, recombinant EfhX_Xfa was shown to bind two moles of calcium per mole of protein, whereas mutation of the first of two EF-hands did not bind calcium . EfhX_Xfa expression was determined to be inducible in hrp-inducing medium. Additionally, growth of X. perforans transconjugants with and without the efhX_Xfa gene in hrp-inducing medium differed in intracellular calcium concentration; the transconjugant without efhX_Xfa yielded higher cell pellet masses and higher increased intracellular calcium concentrations relative to cells expressing EfhX_Xfa. An EfhX_Xfa homolog, EfhX_Xe, present in the pepper pathogen, X. euvesicatoria, when expressed in the tomato pathogen, X. perforans, triggered ROS production and an HR in tomato leaves and is a host-limiting factor. Interestingly, all tested X. perforans and X. euvesicatoria strains pathogenic on tomato contain a stop codon immediately upstream of the first EF-hand domain in the efhX_Xe gene, whereas most X. euvesicatoria strains pathogenic on pepper do not.