광 인지에 의한 식물병원균 Pseudomonas cichorii JBC1의 병원성 및 생활사 적응 조절 = Light sensing regulates virulence and lifestyle adaptation of the phytopathogen Pseudomonas cichorii JBC1

다국어 초록 (Multilingual Abstract)

Pseudomonas cichorii JBC1 (PcJBC1) is a non-photosynthetic phytopathogenic bacterium responsible for leaf spot and blight diseases in economically important crops and vegetables. Throughout its life cycle, PcJBC1 inhabits highly dynamic environmental niches, ranging from the phyllosphere to internal plant tissues. In these habitats, the bacterium encounters rapidly fluctuating environmental cues, including changes in oxygen availability, redox status, host-derived metabolites, and exposure to light and ultraviolet (UV) radiation. Among these factors, light represents a particularly complex environmental signal, acting not only as a source of sensory information but also as a potent cellular stressor, especially under high-intensity blue light (BL) and UV exposure. Consequently, phytopathogenic bacteria must deploy precise light-sensing and signal-transduction mechanisms to ensure survival, stress tolerance, and successful infection of their hosts. Despite its ecological and agricultural relevance, the molecular basis by which PcJBC1 perceives and integrates light signals across the UV-to-visible spectrum to regulate its pathogenic lifestyle has remained largely unexplored.
In this study, a comprehensive and integrative strategy combining computational biology, molecular genetics, and biophysical analyses was employed to systematically characterize the light-sensing machinery of PcJBC1. Bacterial signal transduction is frequently mediated by the Per–Arnt–Sim (PAS) domain, a versatile and widespread sensory module capable of detecting diverse environmental and intracellular signals, including gases, metabolites, redox states, and light. A genome-wide in silico survey identified 41 PAS domain–containing proteins in PcJBC1, accounting for approximately 0.82% of the predicted proteome. Sequence and domain architecture analyses revealed conserved ligand-binding motifs characteristic of light-, oxygen-, and redox-responsive PAS systems. Notably, these PAS domains were predominantly associated with canonical output modules, such as histidine kinases (HKs)/hybrid HKs and GGDEF or GGDEF–EAL domains, indicating a strong linkage to two-component signaling and cyclic di-GMP regulatory networks. From this dataset, three candidate light-responsive proteins were prioritized for functional characterization: a putative LOV-domain protein (Pc-LOV1; locus tag PCH70_11150), a bacteriophytochrome (Pc-BphP; PCH70_14470), and a cryptochrome/photolyase (Pc-Phr; PCH70_09020).
The putative BL photoreceptor Pc-LOV1, a hybrid PAS–HK–response regulator protein, was cloned and biochemically characterized. Spectroscopic analyses confirmed that Pc-LOV1 exhibits canonical LOV photochemistry, binding flavin mononucleotide (FMN) as a chromophore and undergoing reversible light-induced cysteinyl–flavin adduct formation. The protein displayed a characteristic dark-state absorption peak at 448 nm and a relatively long adduct-state recovery lifetime (τrec = 67.03 ± 4.34 min at 25 °C). Functional analyses using a lov1 deletion mutant (JBC1Δlov1) demonstrated that Pc-LOV1 functions as a BL–dependent negative regulator of pathogenicity. BL exposure markedly suppressed disease severity in plants inoculated with the wild-type strain, whereas this suppression was abolished in JBC1∆lov1, which exhibited enhanced virulence regardless of illumination conditions. Pc-LOV1–mediated repression extended to multiple virulence-associated traits, including swarming motility, exopolysaccharide (EPS) production, and transcription of key pathogenicity determinants, such as the type III secretion system (T3SS) genes hrpA and hrpL, as well as the cichofactin lipopeptide biosynthesis genes cifA and cifB. These results indicate that Pc-LOV1 transduces BL signals to attenuate virulence, potentially optimizing energy expenditure and minimizing photo-induced stress under high-irradiance conditions.
The putative bacteriophytochrome Pc-BphP, a PAS–GAF–PHY–HK hybrid protein, was identified as a sensor for longer-wavelength light. Spectroscopic characterization confirmed Pc-BphP as a prototypical phytochrome, exhibiting a red-light–absorbing ground state (Pr; λmax675 nm) that photoconverts to a far-red–absorbing active state (Pfr; λmax705 nm) upon red light (RL) illumination. In addition, Pc-BphP also showed responsiveness to green light (GL), with a secondary absorbance peak around 520 nm. Both RL and GL induced efficient Pr–Pfr photoconversion, followed by thermal reversion to the Pr state in darkness. Functional analyses revealed that Pc-BphP plays a central role in mediating RL- and GL-dependent regulation of PcJBC1 physiology and pathogenicity. Under dark conditions, the JBC1ΔbphP mutant displayed hypervirulence and enhanced colonization of plant tissues. In contrast, RL and GL significantly suppressed these phenotypes in the wild-type and complemented strains, but not in the mutant. Importantly, Pc-BphP mediated distinct wavelength-specific regulatory outputs. RL predominantly repressed acute virulence traits, including swarming motility and expression of T3SS effector genes (avrE1, hopA1). In contrast, GL selectively downregulated type VI secretion system (T6SS) genes (ppkA, rhsB), which are associated with interbacterial competition and host interactions, while simultaneously enhancing motility. These results suggest that Pc-BphP enables PcJBC1 to fine-tune its pathogenic strategy, promoting virulence suppression or dispersal depending on the prevailing light environment, such as open leaf surfaces versus shaded tissues.
Among solar wavelengths, UV radiation poses a severe threat to bacterial survival due to its capacity to induce DNA damage. Pc-Phr, identified as a class I cyclobutane pyrimidine dimer (CPD) photolyase, was characterized for its role in counteracting UV-induced damage, a critical factor for phyllosphere survival. Spectroscopic analyses confirmed that Pc-Phr contains both the catalytic flavin adenine dinucleotide (FAD) cofactor and the antenna chromophore methenyltetrahydrofolate (MTHF), exhibiting characteristic BL-induced photoreduction from oxidized FAD (FADox; λmax445 nm) to the catalytically active FADH⁻ state (λmax360 nm). Functionally, Pc-Phr displayed robust BL-dependent photoreactivation activity, efficiently repairing UV-C–induced CPD lesions in vitro and restoring viability to UV-irradiated PcJBC1 cells in vivo. Expression of phr was strongly induced by UV-C exposure and further enhanced by BL, highlighting a coordinated regulation of DNA repair and light sensing. Deletion of phr significantly compromised bacterial survival under UV-C radiation, high-intensity blue light, and oxidative stress induced by hydrogen peroxide. Moreover, the phr deletion mutant (JBC1∆phr) exhibited significantly attenuated virulence, reduced in planta proliferation, and impaired initial attachment to leaf surfaces. These results demonstrate that photolyase activity contributes not only to genome maintenance but also to pathogenic fitness under light-exposed and stress-prone conditions encountered during plant infection, particularly on light-exposed phyllosphere surfaces.
Collectively, this work demonstrates that PcJBC1 possesses a sophisticated and multi-layered light-sensing network that integrates information across the UV-to-visible spectrum to regulate virulence, stress tolerance, ecological adaptation, and host interactions. Pc-LOV1 and Pc-BphP function as wavelength-specific signal transducers that modulate motility, secretion systems, effector gene expression, and host colonization in response to BL, RL, and GL. Pc-Phr operates as a BL–driven DNA repair enzyme that safeguards genomic integrity and supports bacterial survival and pathogenicity under UV and oxidative stress. Together, these systems reveal that light as a major ecological determinant shaping the infection strategies of PcJBC1. This study provides the first comprehensive molecular framework for understanding light sensing in P. cichorii, advances our understanding of environmental signal integration in plant-pathogenic bacteria, and lays the groundwork for future investigations into downstream signaling pathways linking photon perception to physiological outputs. This study also open potential for developing sustainable, light-based strategies to suppress bacterial virulence and mitigate plant disease in agricultural systems.

목차 (Table of Contents)

• CHAPTER 1 1
• Genome-wide identification and functional annotation of PAS domains signaling proteins in Pseudomonas cichorii JBC1 1
• ABSTRACT 2
• 1.1. INTRODUCTION 3
• 1.2. MATERIALS AND METHODS 5
• 1.2.1. Identification and analysis of PAS domain containing proteins in Pseudomonas cichorii JBC1 5
• 1.2.2. Prediction of subcellular localization of PAS protein 5
• 1.2.3. Ligand-binding prediction 6
• 1.2.4. Phylogenetic analysis of PAS domains 6
• 1.3. RESULTS 6
• 1.3.1. Overview of PAS domains in PcJBC1 genome 6
• 1.3.2. Domain architecture of PAS-containing proteins 11
• 1.3.3. Predicted subcellular localization of PAS proteins 13
• 1.3.4. Predicted ligand-binding capabilities 14
• 1.3.5. Phylogenetic analysis of PAS domains 14
• 1.4. DISCUSSION 17
• 1.5. LITERATURE CITED 21
• CHAPTER 2 29
• PAS blue light photosensor (Pc-LOV1) modulates virulence and lifestyles of Pseudomonas cichorii JBC1 29
• ABSTRACT 30
• 2.1. INTRODUCTION 31
• 2.2. MATERIALS AND METHODS 33
• 2.2.1. Identification of LOV1 sequence and domain analysis 33
• 2.2.2. Deletion of lov1 using CRISPR-CAS9 33
• 2.2.3. Complementation of lov1-deficient mutant 34
• 2.2.4. Creation of construct for Pc-LOV1 overexpression 38
• 2.2.5. Isolation and purification of Pc-LOV1 38
• 2.2.6. UV-Vis spectrophotometry analysis 39
• 2.2.7. Exposure of BL to bacterial cells 39
• 2.2.8. Virulence assay using midrib of Kimchi cabbage 40
• 2.2.9. Swarming motility assay 41
• 2.2.10. Exopolysaccharide production assay 41
• 2.2.11. RNA isolation and qPCR for gene expression analysis 41
• 2.2.12. Statistical analysis 42
• 2.3. RESULTS 42
• 2.3.1. Domain architecture and UV-Vis absorption spectra of Pc-LOV1 42
• 2.3.2. Influence of Pc-LOV1 and BL on the survival 48
• 2.3.3. Effects of Pc-LOV1 and BL on virulence 50
• 2.3.4. Effects of Pc-LOV1 and BL on swarming motility 51
• 2.3.5. Effects of Pc-LOV1 and BL on exopolysaccharide 52
• 2.3.6. Pc-LOV1 mediates regulation of gene expression in response to BL 53
• 2.4. DISCUSSION 57
• 2.5. LITERATURE CITED 62
• CHAPTER 3 69
• Bacteriophytochrome of Pseudomonas cichorii modulates its virulence and lifestyles in response to red and green light 69
• ABSTRACT 70
• 3.1. INTRODUCTION 71
• 3.2. MATERIALS AND METHODS 73
• 3.2.1. Identification of BphP from PcJBC1 and its domain analysis 73
• 3.2.2. Overexpression of Pc-BphP protein 74
• 3.2.3. Purification of Pc-BphP protein 74
• 3.2.4. UV-Vis spectroscopic analysis 75
• 3.2.5. Deletion of bphP using CRISPR-CAS9 75
• 3.2.6. Complementation of bphP-deficient mutant 76
• 3.2.7. Virulence assay using midrib of Kimchi cabbage 78
• 3.2.8. Attachment and colonization assay 78
• 3.2.9. Swarming motility assay 79
• 3.2.10. RNA isolation and qPCR 79
• 3.2.11. Statistical analysis 80
• 3.3. RESULTS 80
• 3.3.1. Domain organization 80
• 3.3.2. Absorbance spectra of Pc-BphP by illumination of RL and GL 86
• 3.3.3. Influences of RL/GL and Pc-BphP on virulence 88
• 3.3.4. Influence of RL/GL and Pc-BphP on the attachment and colonization 90
• 3.3.5. Influence of RL/GL and Pc-BphP on the swarming motility 92
• 3.3.6. Pc-BphP modulates virulence-associated gene expression in response to RL/GL. 93
• 3.4. DISCUSSION 95
• 3.5. LITERATURE CITED 100
• CHAPTER 4 109
• Roles of photolyase in survival, stress resistance and virulence of Pseudomonas cichorii JBC1 109
• ABSTRACT 110
• 4.1. INTRODUCTION 111
• 4.2. MATERIALS AND METHODS 113
• 4.2.1. Phr gene identification and domain analysis 113
• 4.2.2. Overexpression of Pc-Phr 114
• 4.2.3. Purification of Pc-Phr 115
• 4.2.4. Spectroscopic absorption analysis 115
• 4.2.5. In vitro photorepairing activity of Pc-Phr 116
• 4.2.6. Deletion of phr using CRISPR-CAS9 116
• 4.2.7. Complementation of the phr-deficient mutant 117
• 4.2.8. UV-C and BL treatment to bacterial strains 119
• 4.2.9. In vivo photorepairing activity of Pc-Phr 119
• 4.2.10. RNA isolation and qPCR for phr expression level by UV and BL illumination 120
• 4.2.11. Virulence assay using midrib of Kimchi cabbage 120
• 4.2.12. H2O2 sensitivity assay 121
• 4.2.13. Leaf attachment assay 121
• 4.2.14. Statistical analysis 122
• 4.3. RESULTS 122
• 4.3.1. Domain architecture and phylogenetic classification 122
• 4.3.2. UVVis absorption properties 128
• 4.3.3. In vitro photorepair activity of Pc-Phr 130
• 4.3.4. Effects of Pc-Phr on UV-C and BL sensitivity in PcJBC1 131
• 4.3.5. In vivo photoreactivation of Pc-Phr 133
• 4.3.6. Expression of the phr gene in PcJBC1 134
• 4.3.7. Effects of Pc-Phr on virulence and in planta survival 136
• 4.3.8. H2O2 sensitivity 137
• 4.3.9. Leaf attachment 139
• 4.4. DISCUSSION 139
• 4.5. LITERATURE CITED 144
• 요약(국문초록) 151
• ACKNOWLEDGEMENT 155