Structural and Functional Analysis of Pr-induced Variants of XccBphP Bacteriophytochrome From Xanthomonas campestris

CONFORTE, VALERIA P.; SIRIGU, SERENA; KLINKE, SEBASTIÁN; GOLDBAUM, FERNANDO ALBERTO; OTERO, LISANDRO HORACIO; ANTELO, GIULIANO TOMÁS; CHAVAS, LEONARD MICHEL GABRIEL; MALAMUD, FLORENCIA; TOUM, LAILA; RINALDI, JIMENA; VOJNOV, ADRIAN A.; BONOMI, HERNÁN RUY

Buenos Aires

Congreso; SAIB-SAMIGE Joint Meeting 2020 ? Online; 2020

SAIB - SAMIGE

Photoreceptors are able to detect light and transduce that signal generating a cellular response. Among them are the red/farred light sensing bacteriophytochromes (BphP). These bilin-binding proteins have the ability to photoswitch between twostates, a red-absorbing (Pr) and a far-red-absorbing (Pfr), by the isomerization of the bilin chromophore and generatingstructural changes that result in the transduction of the light signal into biochemical signaling. The genome of Xanthomonascampestris pv. campestris (Xcc), the causative agent of black rot in crucifers, codes for a functional BphP (XccBphP) whichwas extensively described in previous studies. It has been defined as a negative regulator of several light-mediated mechanismsinvolved in its virulence. Here, we deepened the analysis of the XccBphP structure and function. In this work, we designedand constructed three different variants with single amino acid changes that affect XccBphP photocycle favoring its Pr state:L193Q, L193N and D199A. We purified the recombinants full-length mutants, crystalized them and solved their crystalstructures, showing a Pr conformation almost identical to the wild-type previously obtained. We also examined their UV-Visabsorption spectroscopic properties, proving that Pr is their preferred state. After establishing the effect of each mutation onthe structure of XccBphP, we tested the effects of altering the XccBphP photocycle using exopolysaccharide (EPS) productionand stomatal aperture assays as indicators of its bacterial signaling pathway. Null mutant complementation assays showed thatL193Q or L193N Pr-stabilized versions decreased bacterial EPS production in darkness or under far-red light and increased itunder red light in an amplified manner compared to the complementation with the wild-type version. Furthermore, strainsexpressing Pr-favored XccBphP versions could not promote stomatal reopening at Xcc null mutant levels when tested inArabidopsis epidermis. Taken together, our results highlight the relevance of the XccBphP Pr-Pfr balance in physiologicalprocesses in Xcc.