Novel Role of Blufs Proteins from Xanthomonas citri subsp. citri in Plant Infectivity

MOYANO, L.; MACHINANDIARENA, F.; BARBALHO FERREIRA, C.; KRAISELBURD, I.; TIRABOCHI FERRO, M.I.; FRANCO DE OLIVEIRA, J.C.; DAURELIO, L.D.; ORELLANO, E.G.

Córdoba

Congreso; 16th International Congress on Photobiology; 2014

International Union of Photobiology

Xanthomonas citri subsp. citri (Xcc) is a Gram-negative bacterium responsible for citrus canker, a severe disease that affects most commercial citrus cultivars, causing significant crop losses worldwide. The Xcc genome contains four genes encoding putative photoreceptors: one phytochrome, one LOV (Light, Oxygen and Voltage)- and two BLUF (Blue Light sensing Using Flavins)-proteins. BLUF proteins use FAD (Flavin adenine dinucleotide) as chromophore. In these proteins, a transient and reversible redshifted state (BLUF RED ) is generated upon blue-light irradiation, dictated by a hydrogen bonding switch reaction involving N (5), O (4) and two conserved tyrosine and glutamine residues. BLUF RED formation seems to involve a light driven electron and photon transfer from a conserved tyrosine residue to FAD, followed by hydrogen bonding rearrangement and radical pair recombination [1]. In the present work, we studied bluf1 (Xcc2120) and bluf2 (Xcc3278) genes from Xcc. First, we performed an in silico analysis comparing these BLUF domains with those present in homologous proteins by amino acid sequence alignments. Considering Xanthomonas whose genomes have been sequenced (around 130 species), only ten have putative BLUF-proteins and a phylogenetic analysis revealed that all of them integrate the same monophyletic group. In order to perform a spectroscopic characterization of these proteins, we constructed BLUF1 and BLUF2 over-expressing strains in Escherichia coli. Both proteins were successfully over-expressed. Although most of the expressed protein was found in the insoluble phase, a small amount protein remained soluble. We also constructed a bluf2 gene knockout mutant strain of Xcc. Upon the interaction with host plants, this strain showed a remarkable reduction in infectivity compared with the wild type strain. This result suggests that bluf2 gene is functional and probably implicated in the bacterial pathogenesis process. [1] Mandalari C., Losi A., Gärtner W. Photochem Photobiol Sci 2013 12: 1144-1157.