A novel BLUF photoreceptor modulates the Xanthomonas citri subsp. citri–host plant interaction

Plant–pathogen interaction is influenced by multiple environmental factors, including temperature and light. Recent works have shown that light modulates not only the defense response of plants but also the pathogens virulence. Xanthomonas citri subsp. citri (Xcc) is the bacterium responsible for citrus canker, an important plant disease worldwide. The Xcc genome presents four genes encoding putative photoreceptors: one bacteriophytochrome and three blue light photoreceptors, one LOV and two BLUFs (bluf1: XAC2120 and bluf2: XAC3278). The presence of two BLUFs proteins is an outstanding feature of Xcc. In this work we show that the bluf2 gene is functional. The mutant strain, XccΔbluf2, was constructed demonstrating that BLUF2 regulates swimming-type motility, adhesion to leaves, exopolysaccharide production and biofilm formation, features involved in the Xcc virulence processes. An important aspect during the plant–pathogen interaction is the oxidative response of the host and the consequent reaction of the pathogen. We observed that ROS detoxification is regulated by Xcc bluf2 gene. The phenotypes of disease in orange plants produced by WT and XccΔbluf2 strains were evaluated, observing different phenotypes. Altogether, these results show that BLUF2 negatively regulates virulence during citrus canker. This work constitutes the first report on BLUF-like receptors in plant pathogenic bacteria.


Introduction
Light perception allows living organisms to adapt their growth and development to different environmental conditions. Light sensing is accomplished by photoreceptor This publication is dedicated to Prof. Silvia E. Braslavsky, a pioneer in photobiology and photobiophysics, on the occasion of her 80th birthday.
Analía Carrau and Josefina Tano have contributed equally to this work.
Extended author information available on the last page of the article 1 3 proteins, which can perceive light with different wavelength, intensity, and polarization degree. Photoreceptors can be classified in six families according to the chemical structure of their chromophore: rhodopsins, phytochromes, xanthopsins, cryptochromes, LOV (Light, oxygen and voltage) domain proteins and BLUF (blue light sensing using flavins) domain proteins [1][2][3][4][5][6].
Xanthomonas citri subsp. citri (Xcc) is a Gram-negative bacterium responsible for citrus canker, a severe disease that affects most commercial citrus cultivars, causing significant crop losses worldwide. During the interaction with compatible host plants, Xcc behaves as a hemibiotrophic pathogen entering the plant tissues through stomata and wounds, and it colonizes the apoplast causing tissues hyperplasia [7]. Xcc genome contains four genes encoding photoreceptors: one phytochrome (XAC4293), one LOV (XAC2555) and two BLUF proteins (XAC2120 named BLUF1 and XAC3278 named BLUF2) [8,10]. Our previous studies demonstrated that Xcc-LOV is a functional blue light receptor. Xcc-LOV protein is composed of a LOV-domain, a histidine kinase (HK) and a response regulator, and it was fully characterized by spectroscopic analysis showing similarity to other described bacterial LOV proteins. The light state of the Xcc-LOV protein is the signaling state, probably initiating a signal transduction cascade involved in a physiological response [11]. Through its kinase activity, Xcc-LOV modulates bacterial features related to the pathogenic process, such as motility, adhesion, biofilm formation and oxidative stress resistance. This protein is also involved in the development of disease symptoms during the interaction with host plants [11]. Moreover, an expression profiling of Citrus sinensis leaves showed that genes directly involved in plant defense are upregulated upon infection with an XccΔlov knock-out mutant strain [12]. Xcc-LOV would be involved in controlling the host tissue damage during the infection process. Control of the virulence by light is a mechanism that would allow Xcc to survive and thrive in the host tissue [11]. An important and outstanding finding is that to date Xanthomonas is the only genus of bacterial plant pathogen that has been demonstrated to possess BLUF photoreceptors, X. citri being the only one having two bluf genes. Beside the wide distribution of BLUF proteins in bacteria and algae, their physiological roles are poorly understood. AppA from Rhodobacter sphaeroides was the first identified BLUF protein, and it is involved in the switch between photosynthesis and aerobic respiration via a complex regulation mechanism [9]. At intermediate oxygen concentration, blue light reduces the affinity of AppA for the repressor PpsR that can now bind to its target promoters [4]. FixD, a BLUF protein from Synechocystis sp. PCC6803, participates in the phototaxis control [13]. Tschowri et al. have demonstrated that, in Escherichia coli, the BLUF protein YcgF binds to promoters of genes involved in the regulation of biofilm formation [14]. This protein can also sense temperature, integrating both environmental stimuli. Furthermore, Gomelsky et al. proposed that YcgF can be regulated by the cell redox state through its GGDEF-and EAL-coupled domains [5,15]. Another example is a BLUF protein of Acinetobacter baumannii involved in controlling biofilm, motility and virulence functions [16][17][18][19][20][21]. The relationship between light and virulence has recently been studied in many phytopathogens such as Agrobacterium tumefaciens [22], Agrobacterium fabrum [23], Xanthomonas citri subsp. citri [11], Xanthomonas campestris pv. campestris [24], Pseudomonas syringae pv. tomato DC3000 [25][26][27][28][29][30], Pseudomonas syringae pv. syringae B728a [31,32] and Pseudomonas cichorii JBC1 [33]. Nevertheless, there are no functions reported for BLUF proteins in the physiology or virulence of plant pathogens. The aim of the present work was to study the role of the photoreceptor BLUF2, inserted in a horizontal gene transfer region of the Xcc genome, in the physiology and virulence of this phytopathogen. Herein, we observed that this photoreceptor is expressed equally under light and dark conditions. On the other hand, we demonstrated that BLUF2 protein is implicated in different pathogenicity mechanisms, such as bacterial motility, adhesion to biotic surfaces, biofilm formation, exopolysaccharide (EPS) production, reactive oxygen species (ROS) detoxification and in the regulation of catalase activity. The most relevant result in this work is that the BLUF2 and light modulate the interaction between Xcc and orange plants. Likewise, this work describes a functional BLUF photoreceptor in a bacterial phytopathogen for the first time.

Identification of putative bluf genes in Xcc
From the nucleotides FASTA sequence of bluf1 (XAC2120), previously identified in the Xcc genome, an internal search of putative paralogous copies was established to verify the presence of other genes with similar domains presence, using for this BLASTn (default parameters) in the Kegg repository [34]. Once the putative paralogous copy was identified, confirmation of the putative functional domains was established using Pfam [35] and Interpro [36].

Secondary and three-dimensional structure prediction of BLUF1 and BLUF2
From the FASTAS amino acid sequences of the bluf1 gene and their paralogous copy named bluf2 (XAC3278), the 3D structure prediction analyses were performed by Phyre2 tool, using default parameters [37]. Measures of confidentiality, identity, alignment of residues and positioning of atoms in the Cartesian plane (xyz coordinates), provided as output results, were tabulated and compared. The superposition of 3D structure was done using DALI [38], using the .pdb files generated by the Phyre2 tool for both proteins (Bluf1 and Bluf2). The percentage value emitted was used as a superposition quality attribute.

Prediction of bluf genes in HGT regions and gene cluster analysis
To verify whether the XAC3278 gene was inserted into a putative genomic island, the Xcc genome was loaded into the IslandViewer tool [39]. The results presented by the tool allow identifying which of the three algorithms the tool was able to identify the putative genomic island (Orange-SIGI-HMM, Blue-IslandPath-DIMOB, Aquamarine-Islander).
Once the starting and ending position of the putative island where XAC3278 was inserted was identified, an analysis of the synteny of the genes in this region was performed in an attempt to identify the island as a possible product of HGT (Horizontal Gene Transfer) events, using the comparison tool provided by the KEGG SPS:refid::bib40 [40] and IMG (Integrated Microbial Genomes) [41].

Phylogenetic analysis
For each target bluf copy of Xcc, a BLASTp search using the PSI-Blast online algorithm [42,43] was performed with the maximum allowed number of hits of 500, to be conservative. Subsequent multiple alignment of sequences from both genes altogether was done in Mafft v7.25 [44] using the thorough G-INS-i algorithm for better accuracy. TrimAl v1.4 [45] was used with the option '-gappyout' to automatically remove sites with an excess of gaps. IQTree v1.4.4 [46] was employed for ML searches, with branch support assessed by 1000 pseudoreplicates of ultrafast bootstrap replicates [47].
Best-fit protein model for each phylogenetic analysis was chosen by a previous ML round in IQTree with the option '-m TESTNEW', which also allows for the possibility of 'FreeRate' models [48] in which R rate classes and their number are inferred from the data without relying on any parametric distribution; the best model was chosen by the Bayesian information criterion (BIC). Figtree v1.4 (available at http:// tree. bio. ed. ac. uk/ softw are/ figtr ee/) was used for tree visualization, midpoint re-rooting (due to presence of distant bacteria clades that may appear in different parts of the gene trees), and coloring of branches.

Plasmids, bacterial strains, and growth conditions
The bacterial strains and plasmids used in this study are listed in Table 1. Xcc cells were grown aerobically at 28 °C with shaking at 200 rpm in Silva Buddenhagen (SB) medium [49], Nutrient Broth (NB) medium [50] or in the XVM2 medium [51], supplemented with the corresponding antibiotics. All Xcc strains were derivatives of the strain Xcc 99-1330 (Hasse) Dowson type A [52], which was kindly provided by Blanca I. Canteros. E. coli cells were grown aerobically at 37 °C with shaking at 200 rpm in Luria Bertani (LB) medium [53]. Antibiotics were used at the following final concentrations: ampicillin (Amp), 25 μg/mL for Xcc and 100 μg/mL for E. coli, streptomycin (Sm) 50 μg/mL and gentamycin (Gm) 40 μg/mL. The assays were performed under different light conditions. For light conditions, bacteria were grown under continuous white light at an intensity of 20 μE/m 2 s 1 from a fluorescent lamp (cool daylight) or continuous blue light provided by LEDs (Macroled ® Model No. SMD 5050) with an intensity of 21 μE/m 2 s. Incubation in darkness was

Construction of the bluf2 mutant strain (XccΔbluf2) and the complemented strain (cXccΔbluf2)
The XccΔbluf2 mutant strain was obtained by replacing the bluf2 gene for an antibiotic resistance cassette (ARC). The upstream (U) and downstream (D) regions of bluf2 were amplified from Xcc WT genomic DNA using the primer pairs Bluf3278UPfw/Bluf3278UPrv and Bluf-3278DOWNfw/ Bluf3278DOWNrv (see Supplementary Information Table S1). The sequences obtained were cloned in the high-copy-number vector pBluescript KS+, in E. coli JM109. Between these regions, the ARC was ligated. The full construction U-ARC-D was sub-cloned into a mobilizable plasmid pKmobGII. This plasmid was then transferred to E. coli S17 conjugant strain. By conjugation, the plasmid was transferred to Xcc WT where a double homology recombination event replaced the bluf2 gene with the ARC. The bluf2 mutant was selected using 40 μg/mL Km. Bluf2 knock-out was confirmed by PCR with bluf2-specific primers, BLUF3278fw and BLUF3278rv (see Supplementary  Information Table S1), using genomic DNA from Bluf2 knock-out strain as template. Details of the construction are explained in the Supplementary Information S1. For mutant complementation, the bluf2 gene was amplified from Xcc WT genomic DNA using BLUF3278fw and BLUF3278rv primers (see Supplementary Information  Table S1), and the PCR product was confirmed by automated DNA sequencing. The amplified sequence was cloned in the wide spectrum host vector, pBBR1MCS-3 [54]. The amplified region included the bluf2 putative-promoter sequence, predicted with SoftBerry (http:// www. softb erry. com). Then, the recombinant plasmid pBBR/bluf2 was transferred by conjugation to XccΔbluf2 strain, rendering cXccΔbluf2 strain.

RNA preparation and gene expression analysis
Xcc WT, XccΔbluf2 and cXccΔbluf2 cultures were grown under white light, blue light, and darkness for 6 h in XVM2 medium, and bacterial total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's recommendations. After extraction, the RNA was treated with RNase-free DNase (Promega) and its integrity was checked by agarose gel electrophoresis 1.5% (w/v). cDNA first strand was synthesized from 1 mg of total RNA as template using 200 U M-MLV Reverse Transcriptase (Promega, USA), 0.5 mM dNTP mixture, 0.5 mg gene-specific primers (BLUF3278rt or rpoBrv, see Table S1), and incubating for 60 min at 42 °C.
The PCR reactions were conducted in a total volume of 25 μL containing 5 μL of 1:10 diluted cDNA, 0.5 U Taq polymerase (Invitrogen), 0.2 mM dNTP, 0.1 mM for a specific sense and anti-sense primers (BLUF3278fw/BLUF3278rt and rpoBfw/rpoBrv, see Table S1), PCR buffer (Invitrogen) 1X and 0.5 mM MgCl 2 . The thermal cycle conditions used were: 94 °C for 3 min, 94 °C for 30 s, 58 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min. The numbers of cycles were specific for each pair of primers. The endogenous normalization was performed using the rpoB gene. RT-PCR products were resolved on 1.5% agarose gels, and densitometrically quantified using Gel-Pro Analyzer Software 3.1 (Media Cybernetics). The assay was conducted in three independent experiments.

Bacterial motility assays
To analyze swimming motility, saturated cultures of Xcc WT, XccΔbluf2 and cXccΔbluf2 were sub-cultured into fresh SB medium at 2% inoculum and grown to late-exponential phase (15 h) in dark condition. Later, the cultures were adjusted to an OD600 = 1. Aliquots of 2 µL were inoculated on the center of 0.3% w/v SB-agar plates and incubated at 28 °C in a moist chamber under white light, blue light or in dark conditions. After two days of growth the migration zones were photographed under standardized conditions. Migration diameters were quantified using ImageJ software (v1.41; National Institutes of Health, Bethesda, MD, USA). The data were obtained averaging the diameters measured in the north-south and east-west directions in each plate. Three plates were prepared for each strain and one of these plates was incubated in one of the illumination conditions tested (white light, blue light and darkness). Three independent experiments were carried out with three replicates per experiment [11].

Examination of flagella
Flagella of Xcc WT and XccΔbluf2 grown under white light, blue light and dark conditions were evaluated. Three-day-old swarming bacteria from the border of migration zones were picked with a sterile toothpick and fixed in 2% (v/v) formaldehyde. The samples were prepared by placing a bacterial suspension drop on a copper grid (400 meshes) covered by collodion for 5 min. Then, the excess was removed by supporting the grid on a filter paper. Those bacteria attached to the grid were contrasted with 2% (v/v) phospho-tungstic acid for 40 s. Samples were examined with a transmission electron microscope JEM 1200 EX II (JEOL Ltd., Tokyo, Japan). Digital images were captured with a camera Erlangshen ES1000W, Model 785 (Gatan Inc., Pleasanton, California, USA). Each image is a representative result of three independent experiments.

In planta adhesion assay
Overnight cultures of Xcc WT, XccΔbluf2 and cXccΔbluf2 were sub-cultured at 2% inoculum in XVM2 media and grown in constant agitation. After 16 h cultures were harvested by centrifugation and cell pellets were washed and adjusted to OD 600 = 1 in fresh XVM2 media, 20 μL of each bacterial suspension were placed on the abaxial face of the leaves and incubated for 6 h at 28 °C in a humidified chamber in white light, blue light or dark conditions. Bacterial adhesion was analyzed by Crystal Violet staining of the leaves for 15 min at room temperature, and the unbound dye was removed by washing with distilled water [11]. For all plant bioassays three independent experiments were performed, using three leaves from different plants in each experiment.

Xanthan production
To quantify the exopolysaccharide production, overnight cultures of Xcc strains were adjusted to the same CFU/mL and sub-cultured in fresh SB medium. The cultures were grown at 28 °C with shaking at 200 rpm for 3 days under white light, blue light and dark conditions. Bacteria were harvested by centrifugation, and EPS was precipitated by the addition of two volumes of ethanol and 1 M NaCl. The precipitate was vacuum filtrated and weighed. The assay was conducted in three independent experiments with three replicates per experiment.

Biofilm formation assay
We analyze the biofilm formation on glass tubes. After an ON growth, Xcc WT, XccΔbluf2 and cXccΔbluf2 were adjusted to 10 7 CFU/mL and sub-cultured in 2 mL of SB broth. The tubes were incubated statically at 28 °C under white light, blue light and in darkness. After 3 days, the growth medium was removed, and the adhered cells were stained with Crystal Violet 0.1% (w/v) for 15 min. The unbound dye was removed and washed with distilled water. To quantify the biofilm formation, we added 3 mL of ethanol 95% (v/v) to solubilize the dye and the absorbance at 540 nm was measured [11]. The biofilm assays were conducted in three independent experiments with three replicates per experiment.

Hydrogen peroxide tolerance assay
Xcc strains were cultured in SB medium until saturation at 28 °C in constant agitation under dark condition. Inoculums of 200 μL of the cells were spread with a swab on PYMagar plates (1.8% w/v) containing paper discs imbibed in 100 mM H 2 O 2 . Paper discs imbibed in distilled sterile water were used as negative control. The plates were incubated in a moist chamber under white light, blue light and dark conditions for 48 h at 28 °C. Growth inhibition was observed as clear halos formed around de paper discs [55]. The hydrogen Peroxide tolerance assay was conducted in three independent experiments with three replicates per experiment.

Catalase activity assay
Protein extracts were prepared from 10 mL cultures of Xcc WT, XccΔbluf2 and cXccΔbluf2 grown in NB medium for 6 h under white light, blue light, and darkness. Cells were harvested by centrifugation at 5000 rpm for 30 min at 4 °C. Bacteria were washed and resuspended in 400 μL of ice-cold 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM Phenylmethylsulfonyl fluoride (PMSF), and then disrupted by intermittent sonication. Suspensions were clarified by centrifugation at 12,000g for 20 min at 4 °C. Protein concentrations in soluble cell extracts were determined by the Sedmak and Grossberg method [56] with bovine serum albumin as standard. For the evaluation of catalase activity in gels, aliquots of cell extracts containing 30 μg of soluble protein were electrophoresed on 8% (w/v) non-denaturing polyacrylamide gels and stained for catalase activity as described by Scandalios [57]. Briefly, following the electrophoretic separation the gel was first soaked for 1 min in a 0.5% hydrogen peroxide solution, washed twice in running distilled water, and then immersed for 2 min in a 1.0% solution of potassium iodide acidified with glacial acetic acid. The peroxide releases iodine which stains the starch gel dark green except in the areas where the catalase activity has destroyed the peroxide. The experiment was repeated three times, with similar results in all cases.

Plant material and inoculation
Citrus sinensis cv. Valencia orange plants were kindly provided by Catalina Anderson and Gastón Alanis (INTA Concordia, Argentina). Plants were grown in a greenhouse with a photoperiod of 16 h light (150 μE/m 2 s 1 ) and 8 h dark at a temperature of 25 °C and 80% humidity. Bacterial suspensions of Xcc WT, XccΔbluf2 and cXccΔbluf2 strains adjusted to 10 6 CFU/mL with 10 mM MgCl 2 were inoculated by cotton swab or pressure infiltration on the abaxial phase of orange leaves. MgCl 2 was used as a control for non-infected leaves. Inoculated plants were kept in a growth cabinet with normal photoperiod. Symptom development and disease progression were phenotypically monitored and registered. Canker lesions were quantified using Image J software (v1.41; National Institutes of Health, Bethesda, MD, USA).
To perform growth curves in planta, 0.8 cm diameter leaf discs from the infiltrated zones were taken at different days 1 3 after bacterial infiltrations. The discs were ground in 100 μL of 10 mM MgCl 2 followed by serial dilution and plating onto SB agar plates. Colonies were counted after 48 h of inoculation at 28 °C. All plant bioassays were conducted in three independent experiments using different plants, in each independent experiment three leaves were infiltrated.

Statistical analysis
Descriptive statistical analyses were performed with at least three independent biological samples. In each case, the plotted data correspond to the average of these independent determinations, with the corresponding standard error indicated by the error bars. Data were subjected to multifactorial ANOVA and Fisher's multiple comparison tests (see S1 Appendix).

Plant-pathogen Xcc possess two bluf photoreceptors
The Xcc genome has two genes previously annotated as hypothetical, which present a BLUF domain XAC2120 (bluf1) and XAC3278 (bluf2). To understand the conservation degree between these genes a pairwise alignment between the bluf1 (158 aa) and the bluf2 (147 aa) sequences was performed, followed by the confirmation of the domain's presence using Pfam and Interpro [35,36] (Fig. 1a, b). The nucleotide alignment had 65% identity, whereas the amino acid alignment had 69% identity. The largest variation was observed in the C-terminal region. The similarity between the two proteins was also observed at the predicted 3D structural level, where the BLUF motif is evident according to phyre2 prediction [37] (Fig. 1a, b). This is demonstrated by the confidence values (100%), by the coverage in the alignment of the residues in the region of the Bluf domain (104), and by the degree of identity, of 59% in Bluf1 and 32% in Bluf2 when compared to the reference structure in the PDB (AppA-2bun from R. sphaeroides). Despite this identity variation, the xyz values (in Angstroms) that denote the position of protein atoms in the Cartesian plane (topology) are very similar. This can be confirmed by the high degree of superposition of amino acid residues in proteins (93% of the structural composition), when evaluated using the DALI tool [38]. Although BLUF1 and BLUF2 are highly conserved at the structural level, analysis of these paralogous genes indicates that bluf2 was acquired by lateral transfer. The bluf2 gene (CDS coordinates 3,858,688-3,859,131 minus strand) is found within a genome island of 48.874 bp (Fig. 1c), according to IslandViewer [58] and three other methods (SIGI-HMM, IslandPath-DIMOB and Islander). Additionally, this island shows in its composition some insertion signatures, such as two upstream transposases and one phage integrase downstream, inserted at the 3′-position of a glycine tRNA (Fig. 1d).
Inside this putative HGT region (putatively acquired evolutionarily by mechanisms other than vertical inheritance), proteins associated with plasmids and other transposition elements were found, together with a high number of hypothetical genes (69.5%). Unlike bluf2, bluf1 (CDS coordinates 2,477,224-2,477,700 plus strand) is not in a genome island (that has evidence of horizontal origins), although it is very close to the replication endpoint. A phylogenetic analysis also suggests that the evolutionary histories of bluf1 and bluf2 are different (Fig. 1f). The reconstructed tree shows that bluf1 and bluf2 belong to two separate and well-supported clades, providing additional evidence for the hypothesis that bluf2 was acquired through lateral transfer. In contrast, bluf1 is more in line with the presumed Xanthomonas citri species tree [59,60]. We searched the orthologue neighborhood of bluf2 (Fig. 1e). All putative genes in this island were found in other Xanthomonas citri subsp. citri genomes, except in strain Aw12879. In other citrus infecting Xanthomonas species, such as X. axonopodis pv. citrumelo strain F1 and Xanthomonas fuscans pv. aurantifolii strain 10,535, only a few genes located in the island were found, with bluf2 found only in X. fuscans. In X. campestris pv. vesicatoria, X. oryzae pv. oryzae, and X. campestris pv. campestris bluf2 was not found, but curiously in a few genomes of X. arboricola this gene was found in regions that appear to be unrelated to the described island.

Physiological characterization of a Xanthomonas citri subsp. citri bluf2 mutant strain
In order to understand the biological role of Xcc-BLUF2 protein we constructed a bluf2 gene deletion mutant called

Xcc-bluf2 gene expression
The expression levels of bluf2 gene under different light conditions were analyzed. RNA was extracted from Xcc WT, XccΔbluf2 and cXccΔbluf2 strains grown under white light, blue light, and darkness for 6 h in XVM2 medium.
RT-PCR reactions showed that bluf2 gene had the same level of expression in all light treatments (see Supplementary Information Fig. S3). The XccΔbluf2 did not show bluf2 gene expression, confirming the knock-out mutation. Nevertheless, the complemented strain presented a higher bluf2 level of gene expression than Xcc WT in all light conditions. The statistical analysis can be found in Supplementary Information Appendix S1.

Blue light is implicated in the regulation of swimming motility
Bacterial motility is a very important feature since it allows bacteria to colonize different surfaces and host tissues. Swimming motility is an individual flagella-dependent movement that occurs in a liquid medium [61]. We studied swimming motility from different strains under different light conditions using 0.3% agar plates. After 48 h of incubation at 28 °C, Xcc WT migrated further than the mutant strain under blue light and in darkness (p ≤ 0.05). On the other hand, the motility of the mutant strain was greater under blue light than in any other light conditions. Under white light the swimming motility of all the strains were similar (Fig. 2a). The complemented strain does not recover the WT phenotype in any case. The diameter measurements of the migration zones confirmed the observed phenotypes (Fig. 2b). The statistical analysis can be found in Supplementary Information Appendix S1.
To determine if the differences in swimming motility are due to variations in flagella synthesis, we collected samples from the migration zones generated by Xcc WT and XccΔbluf2 incubated under different light conditions and we analyzed them by transmission electron microscopy (TEM). No differences between strains or light conditions were observed. All strains conserve the characteristic bacillary form and show a single polar flagellum similar in length and thickness (see Supplementary Materials Fig. S4).

The absence of the bluf2 gene modifies Xcc adhesion
Adhesion on the plant surface is a requirement for successful plant colonization by a bacterial phytopathogen [9]. We studied the ability of Xcc strains to adhere to biotic surfaces. The adhesion ability of the Xcc WT strain on the orange leaf surface under light treatments was higher than in darkness. Furthermore, the attachment capability of XccΔbluf2 strain was higher than the Xcc WT strain in all light conditions assayed (Fig. 3).

Exopolysaccharide production in Xcc is regulated by light
Bacteria produce a variety of extracellular polysaccharides. These can contribute to the epiphytic survival of pathogenic bacteria in the plant tissue and the development of disease symptoms in the host [9]. Extracellular polysaccharide (EPS) is also related to the pathogenicity process in some bacterial species [50,[62][63][64]. Xanthomonas species produce an EPS called xanthan, which confers characteristic mucoid appearance to the bacterial colonies [50]. To study possible modifications in EPS production, xanthan was quantified from the supernatants of three-days-old cultures of Xcc WT, XccΔbluf2 and cXccΔbluf2 grown under white light, blue light and darkness. Under blue light, all strains produce a statistically significant higher amount of EPS compared to white light and dark conditions (p ≤ 0.05). Xcc WT strain produced more EPS than XccΔbluf2 in darkness (p ≤ 0.05).
The complemented strain only partially recovered the WT phenotype (Fig. 4a). The statistical analysis can be found in Supplementary Information Appendix S1.

Blue light affects the bacterial biofilm production
Biofilms are groups of intertwined microorganisms that form a matrix of extracellular polymers which adhere to each other and to different surfaces. This allows its adaptation in a joint way to fluctuating environmental conditions such as environmental stress, low nutrients concentrations, etc. [65,66]. We assayed the biofilm production of Xcc WT, XccΔbluf2 and cXccΔbluf2 strains on glass tubes containing SB liquid medium after 3 days of static incubation under different light conditions. We observed that Xcc WT strain produced more highly flocculated aggregates in the air-liquid interface under blue light (Fig. 4b). On the other hand, biofilm development in XccΔbluf2 was not affected by different light treatments. The complemented strain did not recover the WT phenotype in any case. These macroscopic results were corroborated by spectroscopic quantification of the bound dye at 540 nm (p ≤ 0.05) (Fig. 4c). The statistical analysis can be found in Supplementary Information Appendix S1.

Xcc-BLUF2 is implicated in ROS detoxification and catalases activities
To analyze the role of BLUF2 photoreceptor in the ROS detoxification mechanisms, we assessed bacterial survival in the presence of hydrogen peroxide under different light conditions. XccΔbluf2 strain was more susceptible to hydrogen peroxide treatment compared to the Xcc WT strain in all lightning conditions (p ≤ 0.05). The complemented strain presented the highest level of ROS sensitivity (Fig. 5). The statistical analysis can be found in Supplementary Information Appendix S1. Catalases are the antioxidant enzymes involved in the detoxification pathways catalyzing the dismutation of H 2 O 2 to water and oxygen. Four genes encoding putative catalase enzymes have been identified in the Xcc genome: srpA (XAC3990), katE (XAC1211) and catB (XAC4029 and XAC4030) encode putative monofunctional catalases, while katG (XAC1301) encodes a bifunctional catalase-peroxidase [67]. To determine if BLUF2 photoreceptor has a role in the ROS detoxification modulating the catalase activity, we performed native gel electrophoresis with soluble protein extracts from Xcc WT, XccΔbluf2 and cXccΔbluf2 grown under different light conditions (Fig. 6a), the catalase activity was observed by differential staining. In the case of KatE, the highest activity was observed in Xcc WT cultures grown under white light. On the other hand, the KatE activity was practically undetectable in the other lighting conditions. Regarding KatG, Xcc WT presented higher activity than the mutant strain under white and blue light. Under dark conditions, Xcc WT and XccΔbluf2 presented low catalase activity levels. WT phenotype was restored partially by complemented strain (Fig. 6b, c). These results are consistent with the H 2 O 2 survival assay.

The deletion of the Xcc bluf2 modifies plant disease symptoms
We also evaluated the phenotype produced in planta by Xcc WT, XccΔbluf2 and cXccΔbluf2 strains using two different protocols of infection, swabbing and inoculation with a needleless syringe on the abaxial face of orange leaves. After 30 days of swabbing plan infection, different phenotypes symptoms were observed between XccΔbluf2, WT and complemented strains (p < 0.05) (Fig. 7a). The mutant strain rendered a greater amount of cankers on the inoculated area compared to the wild type and complemented strain. Furthermore, the complemented strain restored the WT phenotype (Fig. 7b). The statistical analysis can be found in Supplementary Information Appendix S1.  On the other hand, when the infections were carried out by infiltration of the abaxial face of the orange leaves, after 14 days it was observed that the mutant strain presented a lesion with a larger water-soaking area compared to the WT and complemented strains (Fig. 7c). The bacterial growth at different times after inoculation was analyzed. We observed that the growth rate was similar for Xcc WT, XccΔbluf2 and cXccΔbluf2 strains up to 14 days after inoculation (Fig. 7d). Therefore, using two different strategies of plant infection, it was observed that the deletion of the bluf gene yielded a bacterium that produces greater symptoms.

discussion
Xanthomonas citri subsp. citri is the bacterium responsible for citrus canker type A [68]. Its genome has been completely sequenced, presenting two genes coding for BLUF domain proteins, one for a LOV domain protein and one that encodes for a bacteriophytochrome [12,34]. Xcc is the only bacterium that presents two BLUF receptors to date, and one of these copies (encoded by the gene XAC3278) is inserted in a large region (XAC3247-XAC3299) characterized as a lateral transfer island. Although phylogenetic analysis of the genes supports the hypothesis of distinct evolutionary origins, the proteins share a high degree of identity and structural topology (Fig. 1). Functionally, the presence of two genes coding for BLUF photoreceptors in the genome of Xcc suggests that the light perception is important in the pathogen life and could be involved in the colonization of the host tissues and the development of disease. Several authors have demonstrated that the virulence process in Xanthomonas species is mediated by sensing light through photoreceptor proteins [10,12,24].
The Xcc BLUF photoreceptors belong to the category of small BLUF proteins, similar to the BLUF protein of A. baumannii [16] and other Acinetobacter species [8,17]. The alignment of the Xcc BLUF photoreceptors with other BLUF proteins already characterized show that they possess conserved amino acids essential for the photochemistry of this family protein [15,17]. The role of the LOV protein in Xcc was previously described in our laboratory, demonstrating its functionality as a photoreceptor [10,11] and its participation in the virulence process [10,12]. These reports Fig. 6 Catalase activity. a Catalase activity was analyzed by native gel electrophoresis. Protein was extracted from Xcc WT (1), XccΔbluf2 (2) and cXccΔbluf2 (3) cultures grown 6 h in NB medium in different light conditions. Activity was measured by densitometric quantification of KatE (b) and KatG (c) bands intensities. Integrated optical density (IOD), arbitrary units (A.U) led us to the hypothesis that Xcc BLUF-type photoreceptors could also be involved in the light modulation of the virulence in this bacterium. We constructed the XccΔbluf2 mutant strain and cXccΔbluf2 complemented strain. The growth curves in the liquid medium showed that the bluf2 gene is not essential for Xcc viability. Furthermore, the viability of Xcc is not affected by the light treatment (see Supplementary Information Fig. S1).
We analyzed the expression of the bluf2 gene by RT-PCR in cultures grown under different lighting conditions. The bluf2 gene showed the same level of expression in all light treatments at 28 °C (see Supplementary Materials Fig.  S3). This result indicates that this gene is expressed in both light and dark conditions and that light does not induce its expression. Furthermore, Bitrian et al. reported that the 4 four genes encoding BLUF type photoreceptor are expressed in dark conditions in Acinetobacter baylyi ADP1. All four genes were expressed both in light and in darkness but three of these genes showed a decreased expression in the light compared to dark [69]. Moreover, the expression level of blsA, a blue light photoreceptor from A. baumannii was the same under light and darkness at 37 °C [19]. In concordance with these results, Bonomi et al. [70] reported that the protein levels of wild type LOV in Rhizobium leguminosarum also remains unchanged in light and dark conditions. Furthermore, the BLUF activation could be a consequence of post-translation modifications. Several BLUF proteins have shown the formation of oligomers upon the absorption of light, like FixD from Synechocystis sp. PCC6803 [71], AppA from R. sphaeroides [72] and YcgF from E. coli [73]. This conformational change that induces the aggregation would be important in the formation of the signaling state and for the interaction with effector proteins [73]. BLUF-type photoreceptors are involved in regulating several light-dependent physiological responses in bacteria, for example, the control of gene expression from photosynthesis components [74], the biofilm formation [5,17] and motility [5,8,16,69].
To study the role of BLUF photoreceptor in Xcc motility, we performed swimming assays with Xcc WT, XccΔbluf2 and cXccΔbluf2 in different light conditions. Swimming motility is a bacterial movement dependent on flagella [75]. Our results showed that Xcc WT migrated further than XccΔbluf2 under blue light and dark conditions in swimming plates (Fig. 2a). However, the evaluation of flagella by TEM revealed that this structure was similar in all strains and light treatments (see Supplementary Information Fig.  S4). Some authors reported that the motility in A. baumannii is modified in response to a variety of environmental conditions such as changes in temperature, nutrients, and light irradiance [16,76,77]. Mussi et al. reported that BlsA, a BLUF protein, is involved in this response integrating both, blue light and temperature signals in vivo [16]. The swimming motility results could be explained by the flagella post-translational modifications, such as was observed in the swimming of Pseudomonas syringae pv. tabaci [78]. Also, other authors have reported that the regulation of flagellar proteins is modulated by light [11,22,26,70]. Furthermore, Kraiselburd et al., reported that the blue light receptor LOV is a component of the Xcc motility regulation network [11].
Cell attachment is an important stage tied to virulence in bacterial pathogens. We studied the adhesion to biotic surfaces of Xcc WT and XccΔbluf2 strains in white light, blue light and darkness, employing XVM2 medium that simulates the apoplast leaf and promotes the expression of adhesins genes [79]. Xcc WT showed less adhesion in darkness, indicating a light dependence on the adhesion process (Fig. 3). This behavior was previously observed in Xcc by Kraiselburd et al. [11]. XccΔbluf2 presented more attachment than Xcc WT in all light conditions, suggesting that BLUF2 photoreceptor negatively regulates the mechanism of bacterial adhesion through light absorption. Taking into account that lov gene regulates this feature in a positive way and bluf2 gene in a negative one we concluded that the adhesion is regulated through these receptors in a light dependent way.
The biofilm is a microbial community where bacteria are immersed in EPS matrix linked to a biotic or abiotic surface, which provides a microenvironment where they are protected from the host plant defense system [80,81]. The EPS production is an important feature in Xcc pathogenicity, and it is involved in biofilm formation [55,63,79,82]. We found that all strains of Xcc produced more EPS under blue light (Fig. 4a). These results are consistent with the results obtained in biofilm formation assays. The WT strain produced a major biofilm under blue light. In the mutant strain we observed a minor biofilm formation, suggesting that BLUF2 receptor is involved in the development of this structure under blue light and dark conditions (Fig. 4b).
Altogether these results indicated that blue light is involved in biofilm production through EPS production. In addition, BLUF2 photoreceptor would act as a positive regulator of these features under blue light and darkness.
Regulation of biofilm formation by blue light and specifically through BLUF proteins has been demonstrated in a several non-phototrophic organisms such as A. baylii, A. baumannii, E. coli [14], Caulobacter crescentus [83] and Idiomarina loihiensis [1]. In Rhodopseudomonas palustris, an anoxygenic phototrophic soil bacterium, biofilm formation is negatively regulated by a BLUF domain protein. This bacterium can control biofilm formation via a blue lightdependent modulation of its c-di-GMP level by the BLUF domain protein, PapB [84]. Several reports demonstrated that light and photoreceptors proteins are implicated in the networks of attachment process in some phytopathogenic bacteria [10,26,29,30,85]. Bonomi et al. reported that X. campestris WT produces significantly more EPS and biofilm in the dark than in the white light treatment [24]. On the other hand, Kraiselburd et al., reported non differences in EPS production between light and dark treatment in Xcc WT [11]. It is important to highlight that the quality, duration, and intensity of light treatments are critical to the phenotype regulation [26]. Also, white light contains in addition to blue components, red components, probably exciting other photoreceptors such as LOV, Bluf1 and bacteriophytochrome also present in Xcc and signaling through another pathway to the same biological processes [31,32]. This could explain some differences observed between white and blue light treatments for some of the phenotypes tested. Under certain conditions, it could be assumed that more than one photoreceptor is probably involved in the regulation of this feature, as was observed for swarming motility in Pseudomonas syringae, which is mediated by a bacteriophytochrome and LOV proteins [31].
Xcc employs different detoxifying enzymes, catalases and superoxide dismutases, to face the presence of ROS when is exposed to biotic and abiotic stress. There are four catalaseencoding genes in Xcc genome, katE, katG, catB and srpA. Monofunctional KatE and bifunctional KatG catalases contribute to Xcc virulence, at different stages of the pathogenic process. KatG is required for the epiphytic survival of Xcc on host leaves prior to colonization, promoting virulence as the epiphytic population size of the pathogen determines the probability of disease occurrence. Once inside the apoplast environment KatE induction provides protection against plant-produced hydrogen peroxide, thus favoring the successful establishment of Xcc in the host plant [67,86]. KatG activity was higher in Xcc WT than XccΔbluf2 under white and blue light. On the other hand, KatE activity is only observed in WT strain under white light but is practically null in the other strains and light treatment (Fig. 6). In agreement with these results, when XccΔbluf2 was exposed to H 2 O 2 , it presented a lower survival capability than Xcc WT (Fig. 5), suggesting a potential protective role of bluf2 gene upon oxidative stress. The catalase activity was lower in Xcc WT cultures grown in darkness than in white and blue light. These results, together with the H 2 O 2 survival assay, indicated that the ROS detoxification mechanisms are regulated by light. The ROS assays with the complemented strain in Fig. 5 suggests that the expression level of bluf2 gene is important for the regulation of catalase activity. Probably the BLUF2 high expression in the complemented strain, rendered cells more susceptible to H2O2 compared to WT and knock-out mutant. In these scenarios, our hypothesis is that low expression levels of bluf2 induced the catalase rendering a better ROS detoxification. Recently, Müller et al. observed that light upregulates the expression of catalase genes and the catalase activity in A. baumannii. The A. baumannii BLUF defective mutant strain also presented lower levels of catalase activity [20]. The plant immune responses include a rapid accumulation of high amounts of ROS in a light dependent process [86]. Another environmental factor that affects ROS balance is ultraviolet radiation (UV-R). In this context, epiphytic bacteria sense the adverse environmental biological effects of UV radiation that is usually associated with oxidative stress [86]. Thus, phytopathogenic bacteria must overcome an oxidative stress in order to survive and successfully colonize the host plant [87]. A higher catalase activity in Xcc exposed to light agrees with the higher oxidative stress, as a consequence of the environment and the host plant defense system, which the bacteria suffer during the day.
The fact that no complementation in physiological assays (swarming, biofilm and ROS assay) was observed could be a consequence of the lack of fine-tuning in the expression control of bluf2 gene in the complemented strain. The complementation was carried out in trans by cloning the bluf2 gene under the control of the plasmid promoter sequence in a low copy vector. We observed that the expression levels of bluf2 gene in the complemented strain are 'overexpressed' under all light treatments (see Supplementary Materials Fig.  S2). As reported for other bacterial photoreceptor proteins, some of physiological features require a more precisely controlled level of these regulator proteins [10,24,30].
Finally, the role of the Xcc BLUF2 photoreceptor in the interaction with the host plants, sweet orange, was evaluated. In this case different infection protocols were used. The leaves were infected by swabbing or by inoculation with a needleless syringe. The leaves swabbing with XccΔbluf2 strain developed lesions phenotypically different from the lesions caused by the wild type strain. The infection with the mutant strain produced a greater number of cankers as well as the formation of an area of necrosis not observed in the WT and complemented strains, which produced similar phenotypes (Fig. 7a, b). Furthermore, when leaves were inoculated with the infiltration method, the development of symptoms was different in the leaves with the mutant strain compared to the WT and complemented strain. Specifically, a greater lesion area was observed with the mutant strain (Fig. 7c). However, the CFU counting showed no differences between the strains (Fig. 7d). The results obtained with the XccΔbluf2 strain were similar to those reported for the mutant strain in the Xcc LOV photoreceptor [11].
Similarly, to what was observed for the LOV protein in Pseudomonas syringae pv. tomato [25] and Xcc [11], the BLUF2 photoreceptor could be involved in the control of the damage produced by Xcc in the host plant tissue, acting as a negative regulator of bacterial virulence. Since this bacterium is a hemibiotrophic pathogen, its establishment and survival depend on maintaining the integrity of the host tissues. The modulation of damage in the host tissue is a previously characterized mechanism in this plant pathogen, which has been shown to be important for the permanence of the bacterium in the plant tissue long enough to develop the disease and spread to neighboring tissues [79]. Thus, the control of virulence by light and blue light photoreceptors would allow Xcc to proliferate in the host, making more likely the bacteria multiplication for the subsequent dissemination and colonization of new tissues [11,12].
In this study we described for the first time that the BLUF2 protein, a blue light photoreceptor, is functional in plant pathogenic bacteria modulating bacterial physiological features relevant for the pathogenicity process. Finally, the most relevant finding in this work is that the Xcc BLUF2 protein participated in the control of host plant infection. In Fig. 8 we present a model that summarizes the features modulated by light and BLUF2 receptor of Xcc. Further investigation respecting the role of phytopathogen photoreceptors on bacterial physiology and pathogenicity will yield a deeper understanding of the complexity and versatility of these proteins.  Fig. 8 Summary of the Xcc physiological features that are affected by BLUF2 photoreceptor and the different lighting conditions. BLUF2 is involved in the regulation of motility, adhesion capability, EPS and biofilm production, ROS detoxification processes, physiological characteristics that are related to pathogenicity