Bacteria perceive light signals via photoreceptors and modulate many physiological and chemical processes that are essential for growth and survival[1, 4]. Although many studies have reported that bacterial LOV photoreceptors are involved in many physiological processes, the roles of LOV proteins vary between the reports and the function of LOV in P. cichorii has remained unexplored.
In this study, the existence of conserved photosensory domains within each ORF of the PcJBC1 genome was analyzed by accessing the CDD server and identifying the LOV protein (Pc-LOV1) that is composed of light-perceiving domains coupled with signaling domains. Pc-LOV1 is composed of an N-terminal LOV domain (aa 15–132), HK domain (aa 147–383), and C-terminal RR (aa 402–515). The domain structure was primarily similar to LOV proteins found in bacterial plant pathogens, such as PstDC3000, PaATCC11528, PssB728a, Xac306, and XccATCC33913, which have an N-terminal LOV domain, followed by a HK domain and a C-terminal RR domain[4, 7, 18]. PcJBC1 and all the analyzed bacterial strains contained a G*NCRFLQ motif with a conserved cysteine, which is known to be important for photochemistry and signaling[6, 19]. Multiple alignments of the amino acid sequence of Pc-LOV1 also showed high similarity to LOV proteins of bacterial plant pathogens, such as PstDC3000, PaATCC11528, and PssB728a. Altogether, the domain structure and multi-alignment analyses indicate that Pc-LOV1 is a member of the HKs subfamily of light-inducible, two-component signal transduction systems.
LOV proteins respond to BL through conformational changes that result from BL-mediated protein–flavin adduct formation at a conserved cysteine in the LOV domain[5]. LOV-HK of PstDC3000 is associated primarily with FMN and secondarily with FAD[7] and exhibits BL-mediated autophosphorylation followed by rapid transfer of phosphate to the RR domain[6, 7]. The four LOV-HKs of B. melitensi and two LOV of E. litoralis exhibited two absorption peaks at 450 and 370 nm[5]. The recombinant LOV protein of Xac306 showed absorption maxima at 375, 450, and 475 nm in the dark[1]. The aureochrom-LOV of the diatom Phaeodactylum tricornutum and the LOV of Methylobacterium radiotolerans[20] showed maximum absorption at 448 and 450 nm, respectively, in the dark[21]. In this study, dark-state Pc-LOV1 presented an absorption maximum peak at 448 nm and two shoulder peaks at 425 and 475 nm, which are typical features of oxidized flavin chromophores. BL illumination induced covalent adduct formation between the flavin chromophore and a conserved cysteine on a microsecond timescale, whereas the adduct-state decay kinetics vary from seconds to days or longer[5, 22–24]. In our study, BL illumination of Pc-LOV1 for 1 min completely eliminated the absorption peak of the protein, and the absorption peak was completely restored after 4 h in the dark, indicating the fully oxidized state of flavin chromophores. These results indicate that Pc-LOV1 forms a typical flavin adduct in response to BL.
Plant pathogens are exposed to various wavelengths and intensities of light in the environment, including on plant surfaces. BL inactivates microbes through cell membrane damage, inactivation of virulence factors, DNA oxidation, and the changes caused by reactive oxygen species (ROS)[25–27]. Therefore, we first assessed the susceptibility of PcJBC1 and Pc-LOV1-deficient mutant strains to various intensities of BL. The survival rates of PcJBC1, JBC1Δlov1, and JBC1Δlov1+plov1 significantly decreased when they were exposed to BL at intensities > 5 µmol/m2s. Notably, JBC1Δlov1 showed higher survival rates than PcJBC1 under high BL intensities of 10 and 20 µmol/m2s. These results indicate that the presence of Pc-LOV1 increases the susceptibility of PcJBC1 to BL, and consequently, leads to detrimental damage by BL. However, the underlying mechanisms of the increased susceptibility require further investigation.
Disease symptoms that developed in tomato and A. thaliana plants, as well as bacterial populations in plant tissues, were significantly reduced when bacteria were grown under BL conditions[13]. In our previous studies, PcJBC1-caused disease incidence was significantly affected by light quality and quantity[14, 15]. In this study, BL significantly reduced the PcJBC1-led disease severity in Kimchi cabbage while the JBC1Δlov1-led disease severity was not significantly suppressed by BL. In addition, the virulence of strains containing Pc-LOV1, i.e., PcJBC1 and JBC1Δlov1+plov1, was higher in darkness than under BL illumination. Kraiselburd et al.[1] reported that the LOV-HK deficient Xac306 (Xac∆lov) were more virulent to orange leaves under light conditions and resulted in marked differences in symptom development compared to the WT. Río-Álvarez et al.[13] reported that the LOV-HK of PstDC3000 repressed type 3 secretion system (T3SS) and effectors, which probably results in reduced virulence in A. thaliana plants under white light (WL) and BL conditions. In this study, the virulence of Pc-LOV1-deficient mutant was significantly higher than that of the PcJBC1. Furthermore, the gene expressions of hrpL and hrpA in JBC1Δlov1 were higher than those in PcJBC1, which corresponded to the disease severity observed in cabbage midrib. Pauwelyn et al.[17] reported that the cifAB deletion mutant, which is responsible for the biosynthesis of cichofactin, a phytotoxic lipopeptide, exhibited reduced virulence in lettuce and swarming motility, but enhanced biofilm formation compared to the WT. In our study, the expression levels of cifA and cifB in PcJBC1 decreased under BL compared to under dark condition but not in PcJBC1Δlov1, indicating the suppression of cifA and cifB expression by BL and consequent reduction in the virulence of PcJBC1. Overall, our results indicate that the presence of Pc-LOV1 and BL illumination reduce the virulence of PcJBC1, suggesting a negative role for Pc-LOV1 in the pathogenic process of PcJBC1. Disease incidence and gene expression assay results suggest that the virulence of PcJBC1 is delicately tuned by light through the participation of several other light-sensing signals and factors, which require further study.
The motility of plant pathogenic bacteria contributes to their infection potential by enabling them to reach favorable sites, such as stomata and wounds. Oberpichler et al.[28] reported that the production of FlaA and FlaB proteins in Agrobacterium tumefaciens was significantly increased after light treatment. The swarming motility of Xac∆lov increased irrespective of light[1]. The swarming motility of PstDC3000∆lov decreased under BL but not in the dark[4]. In another study, Río-Álvarez et al.[13] reported that the swarming motility of PstDC3000 was truncated when grown under WL (70 µE/m2s) or BL (20 µE/m2s). The reduced motility was caused by the downregulation of gene expression involved in the synthesis of bacterial flagella and regulation of flagellar functions, which are mediated by BL via LOV[9, 13]. In this study, the swarming motility of PcJBC1 was significantly reduced by BL and the motility of JBC1Δlov1 was suppressed under both BL-illuminated and dark conditions compared to PcJBC1. Furthermore, the expression of genes involved in flagella formation in PcJBC1 was significantly decreased by BL illumination, and Pc-LOV1 deficiency suppressed gene expression under both BL-illuminated and dark conditions. Our results indicate that gene expression for flagella synthesis is negatively regulated by BL, which supports changes in swarming motility.
Plant pathogenic bacteria must effectively colonize plant surfaces and maintain a sufficient mass of cells to cause disease in host plants. The adhesion of Xac∆lov to abiotic and biotic surfaces was significantly diminished compared to that of WT, and light increased the adhesion of Xac∆lov and WT compared to dark conditions, which indicates the essential role of LOV-HK for the adhesion of Xac306[1]. The biocontrol agents Bacillus amyloliquefaciens and Pseudomonas chlororaphis showed significantly higher biofilm formation when exposed to BL[29, 30]. Biofilm formation and attachment of PstDC3000 to the leaves of Arabidopsis are favored under BL, suggesting that BL provides a signal for the bacterial switch to an attached lifestyle[13]. In this study, biofilm formation and attachment to plant surface of PcJBC1 was promoted by BL and those of JBC1Δlov1 were significantly decreased in comparison to PcJBC1 irrespective of light conditions. Collectively, our results indicate that biofilm formation and epiphytic attachment of PcJBC1 are positively regulated by Pc-LOV1 via the BL. Although the attachment and biofilm formation of JBC1Δlov1 was decreased, BL still influenced these abilities, suggesting the participation of other BL-sensing signals.
EPS generally contribute to the virulence of bacterial plant pathogens. The Xac∆lov produced significantly high amounts of EPS compared to WT[1]. BL-upregulated genes of PstDC3000 involved in alginate biosynthesis, an EPS that increases the attachment and biofilm formation of the strain in Arabidopsis leaves[13]. In our study, EPS production of PcJBC1 was slightly decreased by BL, whereas JBC1Δlov1 produced significantly higher amounts of EPS than PcJBC1 under both BL-illuminated and dark conditions, which correspond to the results of Xac306. Negative regulation of EPS production by Pc-LOV1 may influence PcJBC1 virulence
Bioavailable iron is an essential element for all living organisms and a critical factor in the pathogenesis of plant pathogenic bacteria. A defect in siderophore formation in Dickeya dadantii restricted symptom development in the inoculated leaves[31]. The virulence of pyoverdine-deficient mutants of Pseudomonas syringae pv. tabaci in tobacco plant was reduced[32]. In this study, siderophore productions by PcJBC1 and JBC1Δlov1 were suppressed by BL and JBC1Δlov1 produced significantly lower amount of siderophore compared to PcJBC1, irrespective of light. Our results indicate that siderophore production by PcJBC1 is negatively regulated by BL and suppressed by the deletion of Pc-LOV1, although it is still influenced by BL signals. Changes in EPS and siderophore production suggest that BL influences the secretion or production of metabolites in PcJBC1 via Pc-LOV1. Notably, in our swarming motility assays, the colors of the colonies differed with or without BL-illumination and Pc-LOV1. These results indicate that pigment production is also influenced by Pc-LOV1, which requires further investigation.
Bonomi et al.[11] reported that LOV protein levels in R. leguminosarum remain unchanged under light and dark conditions. In the present study, PcJBC1 showed the same level of lov1 expression with or without BL. This result indicates that lov1 is expressed at similar levels under both light and dark conditions and that BL does not induce its own expression.
Upon sensing light signals, the input domain of the photoreceptors binds to a chromophore and initiates a change in the photoreceptor protein, which binds to DNA or proteins and triggers a signal transduction cascade. HKs autophosphorylate upon activation, facilitating the subsequent transfer of the phosphoryl group to the response regulators to induce downstream effects[18, 33]. In this study, we analyzed the proteins that interact with Pc-LOV1 to understand the downstream networks regulated by photoreceptors. Pc-LOV1 was predicted to interact with specific functional proteins, such as flagellar formation proteins (FlhA, FlhB, FlhF, FliA, FliD, FliM, and FliG) and type IV pilus biogenesis proteins (PilH and PilJ), which influence bacterial motility and twitching. The biofilm dispersion protein (BdlA) and alginate biosynthesis transcriptional regulatory protein (AlgB), which influence biofilm formation and EPS production, were predicted to interact. Furthermore, Pc-LOV1 was predicted to interact with response regulators containing GAF, GGDEF, and EAL domains, as well as BphP. In many bacteria, the GGDEF domain is linked to a regulatory domain, such as a phosphorylation receiver or an oxygen-sensing domain, and functions as an intracellular signaling molecule[34, 35]. The EAL domain, found in diverse bacterial signaling proteins, may function as a diguanylate phosphodiesterase[36]. These proteins are involved in the regulation of physiological characteristics, such as virulence, motility, adhesion, biofilm formation, exopolysaccharide synthesis, and cell differentiation. Interaction analysis suggested that Pc-LOV1 regulates various processes through interactions with specific functional proteins and response regulators in coordination with other photoreceptors in response to BL from the environment. Further experimental studies on the interactions between Pc-LOV1 and candidate proteins are required.
In this study, we investigated the roles of Pc-LOV1 and BL in virulence and the ecological behaviors and physiologies associated with the pathogenicity of PcJBC1. Pc-LOV1 modulates motility, attachment to plant surfaces, metabolite production, and virulence in response to BL. To understand the modulation of these features, the expression of genes such as T3SS, cichofactin, flagella, and metabolite production were explored. The variable responses of these features to BL indicate that several other photosensory proteins participate in light sensing and downstream signaling. The signaling pathways, networks, and roles of Pc-LOV1 in the dark remain to be explored.