Structural and functional analyses of D199A mutation provide new insights into XccBphP bacteriophytochrome signalling in Xanthomonas campestris pv. campestris (Póster)

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

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias 2017; 2017

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

Manyorganisms possess photoreceptors that are capable of detecting lightwavelengths and transduce this information within cells. Phytochromesconstitute a major superfamily of light-sensing proteins that are reversiblyphotoconverted between a red-absorbing (Pr) and a far-red-absorbing (Pfr)state. Xanthomonas campestris pv. campestris (Xcc), the causal agent of blackrot disease, codes for a functional bacteriophytochrome (XccBphP). Previouswork of our group have demonstrated that XccBphP is a bathyphytochrome thatacts as a negative regulator of virulence. Here, we go deeply into the study ofthe XccBphP structure and function. In this work, we analyze the role of theconserved residue Asp199 located in the photosensory domain, which is crucialfor photochemical Pr-to-Pfr conversion. For this aim, we purified therecombinant XccBphP-D199A full-length mutant, crystalized it and solved thecrystal structure, revealing a Pr state identical to the wild-type Prstructure. We also evaluated its UV-Vis absorption spectroscopic propertiesshowing that this mutation locks XccBphP in Pr state. Once the effect of the mutationwas established, we tested the effect of D199A during infection. We firstcomplemented an XccbphP null mutant strain with XccBphP-D199A (D199A). Weinoculated 10-day-old Arabidopsis plant seedlings with bacterial strainscultured under red, far-red or dark conditions. After 2 days p.i., bacterialCFU per plant mg were determined. We found that D199A was less virulent thanthe wildtype and the XccbphP strains in all cases regardless of the light treatment.D199A ability to infect plants remained comparable to null mutant straincarrying the plasmid with an intact XccbphP copy. Exopolysaccharide production,a known virulence factor, correlated with the infection results. Takentogether, these results indicate that D199A mutation locks XccBphP in anactivated Pr-like state, irresponsive to light, inhibiting virulence factorsand plant infectivity.