An orphan NtrC-family transcription factor regulates chemotaxis, biofilm formation and virulence in Xanthomonas citri subsp. citri

P. YARYURA; CONFORTE VP; MALAMUD F; ROESCHLIN R; DE PINO V; CASTAGNARO AP; DOW MJ; MARANO MR; VOJNOV AA

La Falda

Workshop; II Workshop Latinoamericano sobre PGPR; 2014

Citrus canker is one of the most important and aggressive bacterial diseases of citrus trees. The causal agent of this disease is Xanthomonas citri subsp. citri (Xcc). This bacterium enters the leaves of the host plant through stomata or tissue lesions. Biofilm formation on citrus leaves plays an important role in epiphytic survival of Xcc. A biofilm-deficient mutant with a transposon insertion in a gene encodes the transcriptional activator of the NtrC family (XAC3733, named ntrC1), whose function is poorly understood in Xanthomonas spp. the second messenger c-di-GMP has also been associated with regulation of biofilm, motility and virulence, we provide data suggesting a possible role of c-di-GMP in NtrC1 regulation of these functions. For in vitro analysis of biofilm formation, we used confocal laser scanning microscopy. Examination of flagella: transmission electron microscope and for flagellum staining subsequent observation by optical microscopy. For pathogenicity assays grapefruits leaves was used as the host plant for Xcc. Gene expression levels was obtained by qRT-PCR. Intracellular alteration in c-di-GMP was achieved by exogenous expression of either the diguanylatecyclase WspR19 from Pseudomonas fluorescens or the c-di-GMP phosphodiesterase PA2567 from P. aeruginosa.The ntrC1 mutant showed impaired structural development of the Xcc biofilm, loss of chemotaxis and reduced virulence in grapefruit plants the absence of a typical biofilm of the wild type with reduced biofilm biomass and thickness. Examination of the flagellar structure of the strains using transmission electron microscopy showed a single polar flagellum in the wild type strain, but no flagellum in the ntrC1 mutant also we found significant decreases in the expression levels of some of the most important genes related to the flagellum synthesis in the ntrC mutant in comparison with the WT also the expression level of a phosphodiesterase XAC0350 was significantly reduced. All defective phenotypes were restored to wild-type levels by the introduction of PA2567. A knock-out of ntrC1 led to a substantial down regulation of fliA, which encodes a sigma 28 transcription factor, as well as fliC and XAC0350 which are potential member of the sigma 28 regulon. XAC0350 encodes an HD-GYP domain cyclic di-GMP phosphodiesterase. These findings suggest that NtrC1 is a key regulator of flagellar-dependent motility and chemotaxis exerting its action through a regulatory pathway that involves FliA and c-di-GMP.