Structural and function analyses of D199A mutation provide new insight into XccBphP bacteriophytochrome signaling in Xanthomonas campestris pv. campestris

BONOMI, HERNAN R.; KLINKE, SEBASTIÁN; MALAMUD, FLORENCIA; CONFORTE, VALERIA; RINALDI, JIMENA; VOJNOV, ADRIAN; OTERO, LISANDRO HORACIO; GOLDBAUM, FERNANDO A.

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de BioCiencias; 2017

SAIB, SAIC, SAI, SAA, SAB, SAB, SAFE, SAFIS, SAH, SAP

Many organisms possess photoreceptors that are capable of detecting light wavelengths and transduce this information within cells. Phytochromes constitute a major superfamily of light-sensing proteins that are reversibly photoconverted between a red-absorbing (Pr) and a far-red-absorbing (Pfr) state. Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot disease, codes for a functional bacteriophytochrome (XccBphP). Previous work of our group have demonstrated that XccBphP is a bathyphytochrome that acts as a negative regulator of virulence. Here, we go deeply into the study of the XccBphP structure and function. In this work, we analyze the role of the conserved residue Asp 199 located in the pho tosensory domain, which is crucial for photochemical Pr-to-Pfr conversion. For this aim, we purified the recombinant XccBphP-D199A full-length mutant, crystalized it and solved the crystal structure, revealing a Pr state identical to the wild-type Pr structure. We also evaluated its UV-Vis absorption spectroscopic properties showing that this mutation locks XccBphP in Pr state. Once the effect of the mutation was established, we tested the effect of D199A during infection. We first complemented an XccbphP null mutant strain with XccBphP-D199A (D199A). We inoculated 10-day-old Arabidopsis plant seedlings with bacterial strains cultured under red, far-red or dark conditions. After 2 days p.i., bacterial CFU per plant mg were determined. We found that D199A was less virulent than the wild-type and the XccbphP strains in all cases regardless of the light treatment. D199A ability to infect plants remained comparable to null mutant strain carrying the plasmid with an intact XccbphP copy. Exopolysaccharide production, a known virulence factor, correlated with the infection results. Taken together, these results indicate that D199A mutation locks XccBphP in an activated Pr-like state, irresponsive to light, inhibiting virulence factors and plant infectivity.