Pr-favoured variants of the bacteriophytochrome from theplant pathogenXanthomonas campestrishint on lightregulation of virulence-associated mechanisms

VALERIA CONFORTE; LISANDRO OTERO; LAILA TOUM; SERENA SIRIGU; GIULIANO ANTELO; JIMENA RINALDI; SABRINA FOSCALDI; SEBASTIAN KLINKE; LEONARD MICHEL GABRIEL CHAVAS; ADRIAN VOJNOV; FERNANDO A GOLDBAUM; FLORENCIA MALAMUD; HERNÁN BONOMI

FEBS JOURNAL

WILEY-BLACKWELL PUBLISHING, INC

Año: 2021

1742-464X

Red/far-red light-sensing bacteriophytochrome photoreceptor (BphP) path-ways play key roles in bacterial physiology and ecology. These bilin-bind-ing proteins photoswitch between two states, Pr (red absorbing) and Pfr(far-red absorbing). The isomerization of the chromophore and the down-stream structural changes result in the light signal transduction. The agri-cultural pathogenXanthomonas campestrispv.campestris(Xcc) code for asingle bathy-like type BphP (XccBphP), previously shown to negatively reg-ulate several light-mediated biological processes involved in virulence.Here, we generated three different full-length variants with single aminoacid changes within its GAF domain that affect theXccBphP photocyclefavouring its Pr state: L193Q, L193N and D199A. While D199A recombi-nant protein locksXccBphP in a Pr-like state, L193Q and L193N exhibit asignificant enrichment of the Pr form in thermal equilibrium. The X-raycrystal structures of the three variants were solved, resembling the wild-type protein in the Pr state. Finally, we studied the effects of altering theXccBphP photocycle on the exopolysaccharide xanthan production andstomatal aperture assays as readouts of its bacterial signalling pathway.Null-mutant complementation assays show that the photoactive Pr-favouredXccBphP variants L193Q and L193N tend to negatively regulatexanthan productionin vivo. In addition, our results indicate that strainsexpressing these variants also promote stomatal apertures in challengedplant epidermal peels, compared to wild-typeXcc. The findings presentedin this work provide new evidence on the Pr state ofXccBphP as a nega-tive regulator of the virulence-associated mechanisms by light inXcc.AbbreviationsBphP, bacterial phytochrome; BV, biliverdin Ixα; EPS, exopolysaccharide; MW, molecular weight; OM, output module; Pfr, far-red-absorbingstate; Pr, red-absorbing state; PSM, photosensory module; SEC, size-exclusion chromatography; SLS, static light scattering;Xcc,Xanthomonas campestrispv.campestris;XccBphP, bacterial phytochrome fromXanthomonas campestrispv.campestris.1The FEBS Journal (2021)ª2021 Federation of European Biochemical Societies