Characterization of EfhX, a calcium sensor protein in Xanthomonas required for elicitation of the hypersensitive response in Solanum species

Abstract

We have identified a gene in several strains of Xanthomonas that, when expressed, can elicit a hypersensitive response (hr) in tomato plants. Hr elicitation is normally limited to specific bacterial‐host pairings, but introduction and expression of this gene by bacterial species that do not normally infect tomato serves to elicit hr, indicating that the encoded 170 amino acid protein is crucial to the infectious process. Bioinformatic analysis of the protein, which we have named EfhX ( EF‐ hand containing protein from Xanthomonas) reveals that the protein is predicted to contain a domain of unknown function spanning the amino terminal region, a single transmembrane alpha‐helix spanning amino acids 60 to 81 and two putative EF‐hand calcium binding domains in the carboxy‐terminal domain of the protein. In order to better understand the function of this novel protein, we have cloned the EfhX gene from Xanthomonas aurantifolia and expressed it in Escherichia coli. We have successfully purified the protein to homogeneity as determined by SDS‐PAGE, anti‐hexahistidine Western Blot and MALDI‐TOF mass spectrometry. Using the purified protein, we have used isothermal titration calorimetry to confirm that the protein has two calcium binding sites, with one site having an affinity (Kd) for calcium of 6.75 mM and the second site having an affinity of 1 uM. We are currently performing circular dichroism spectropolarimetry and gel filtration studies to determine if the quaternary state of the protein changes upon calcium binding. Simultaneously, we have initiated crystallization trials of the purified protein to identify solution conditions suitable for growth of crystals for x‐ray diffraction experiments so that the structure of the protein can be determined.Support or Funding InformationThis work is supported by grants from the National Center for Research Resources (5 P20 RR16461) and the National Institute of General Medical Sciences (8 P20 GM103499) from the National Institutes of Health.