INVESTIGADORES
KLINKE Sebastian
congresos y reuniones científicas
Título:
Structure and function of the bacteriophytochrome from the phytopathogen Xanthomonas campestris (Comunicación Oral)
Autor/es:
FLORENCIA MALAMUD; LISANDRO OTERO; SEBASTIÁN KLINKE; JIMENA RINALDI; LAILA TAUM; FRANCISCO VELÁZQUEZ-ESCOBAR; MARÍA FERNÁNDEZ LÓPEZ; GABRIELA SYCZ; RODRIGO SIEIRA; ANDRÉS TOSCANI; GUSTAVO GUDESBLAT; FEDERICO COLUCCIO LESKOW; MARÍA ANDREA MROGINSKI; PETER HILDEBRANDT; ADRIAN A. VOJNOV; FERNANDO A. GOLDBAUM; HERNÁN R. BONOMI
Lugar:
Tucumán
Reunión:
Congreso; III Reunión de Fotobiólogos Moleculares Argentinos "GRAFOB del Bicentenario"; 2016
Institución organizadora:
Grupo Argentino de Fotobiología GRAFOB
Resumen:
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. Bacteriophytochromes (BphPs) are found among bacteria, including pathogens. In this work we aimed to study the structural changes propagated from the photosensory module to the output module during the signal transduction event and the physiological role of the BphP from the plant pathogen Xanthomonas campestris (XccBphP).Using X-ray protein crystallography, we have obtained the structure of the full-length XccBphP bearing its photosensor and its complete output module, a PAS9 domain [1]. In the crystals, the protein was found to be in the Pr state, and using resonance Raman spectroscopy and QM/MM calculations, the chromophore was determined as a mixture of Pr and Meta-R states. The quaternary assembly in the crystal reveals a parallel dimer where the output module contributes to the helical dimer interface. Static light scattering measurements show that in solution the output module or the photosensor behave as monomeric species, while the full-length XccBphP is a dimer. UV-Vis spectroscopy determinations show that XccBphP is a bathy-type BphP and that the presence of the PAS9 domain slows 10-fold the Pr-Pfr dark conversion. Based on a structural comparison between our structure in Pr and RpBphP1 in Pfr, we propose that the Pr-Pfr photoconversion may involve changes in the relative positioning of the output module. We then tested the hypothesis that XccBphP plays a role in bacterial pathogenesis. Illumination of Xcc cultures prior to plant infections generates lower CFU counts per plant mg at day 2 p.i.; this effect is abolished in a XccbphP null-mutant, which always displays enhanced virulence. Xanthan and biofilm formation, both virulence factors, are down-regulated by illuminating cultures and are dependent on XccBphP presence. Extracellular xanthan content in culture supernatants was measured after EtOH precipitations; biofilm structures observed in a confocal laser scanning microscopy setup and analyzed by COMSTAT software. Stomatal aperture regulation (measured using an optical microscope with the aid of an eyepiece micrometer) and callose deposition (assessed by differential dyeing with aniline blue and fluorescence microscopy), both well-established plant defense mechanisms against bacterial pathogens, are overridden by the XccbphP strain. Additionally, RNA-Seq from far-red irradiated Xcc or XccBphP overexpressing strain produce genome-wide transcriptional changes, including the inhibition of several Xcc virulence systems. Our findings show that Xcc senses light through XccBphP, eliciting bacterial virulence attenuation via down-regulation of bacterial virulence factors. Taken together, our work provides insight into (i) the XccBphP light sensory mechanisms at the atomic scale and (ii) its physiological role in the plant-pathogen interaction.