Blue-light perception by epiphytic Pseudomonas syringae drives chemoreceptor expression, enabling efficient plant infection.

Adaptation and efficient colonization of the phyllosphere are essential processes for the switch to an epiphytic stage in foliar bacterial pathogens. Here, we explore the interplay among light perception and global transcriptomic alterations in epiphytic populations of the hemibiotrophic pathogen Pseudomonas syringae pv. tomato DC3000 (PsPto) following contact with tomato leaves. We found that blue-light perception by PsPto on leaf surfaces is required for optimal colonization. Blue light triggers the activation of metabolic activity and increases the transcript levels of five chemoreceptors through the function of light oxygen voltage and BphP1 photoreceptors. The inactivation of PSPTO_1008 and PSPTO_2526 chemoreceptors causes a reduction in virulence. Our results indicate that during PsPto interaction with tomato plants, light perception, chemotaxis, and virulence are highly interwoven processes.

A Method for Investigating the Pseudomonas syringae-Arabidopsis thaliana Pathosystem Under Various Light Environments.

Arabidopsis thaliana and Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) comprise an effective model pathosystem for resolving mechanisms behind numerous aspects of plant innate immunity. Following the characterization of key molecular components over the past decades, we may begin investigating defense signaling under various environmental conditions to gain a more holistic understanding of the underlying processes. As a critical regulator of growth and development, exploration into the influence of light on pathogenesis is a logical step toward a systems-level understanding of innate immunity. Based on methods described previously, here we describe a method for investigating plant immune responses under various light environments.

Pseudomonas syringae pv. tomato exploits light signals to optimize virulence and colonization of leaves.

Light is pervasive in the leaf environment, creating opportunities for both plants and pathogens to cue into light as a signal to regulate plant-microbe interactions. Light enhances plant defences and regulates opening of stomata, an entry point for foliar bacterial pathogens such as Pseudomonas syringae pv. tomato DC3000 (PsPto). The effect of light perception on gene expression and virulence was investigated in PsPto. Light induced genetic reprogramming in PsPto that entailed significant changes in stress tolerance and virulence. Blue light-mediated up-regulation of type three secretion system genes and red light-mediated down-regulation of coronatine biosynthesis genes. Cells exposed to white light, blue light or darkness before inoculation were more virulent when inoculated at dawn than dusk probably due to an enhanced entry through open stomata. Exposure to red light repressed coronatine biosynthesis genes which could lead to a reduced stomatal re-opening and PsPto entry. Photoreceptor were required for the greater virulence of light-treated and dark-treated PsPto inoculated at dawn as compared to dusk, indicating that these proteins sense the absence of light and contribute to priming of virulence in the dark. These results support a model in which PsPto exploits light changes to maximize survival, entry and virulence on plants.

Light regulates motility, attachment and virulence in the plant pathogen Pseudomonas syringae pv tomato DC3000.

Pseudomonas syringae pv tomato DC3000 (Pto) is the causal agent of the bacterial speck of tomato, which leads to significant economic losses in this crop. Pto inhabits the tomato phyllosphere, where the pathogen is highly exposed to light, among other environmental factors. Light represents a stressful condition and acts as a source of information associated with different plant defence levels. Here, we analysed the presence of both blue and red light photoreceptors in a group of Pseudomonas. In addition, we studied the effect of white, blue and red light on Pto features related to epiphytic fitness. While white and blue light inhibit motility, bacterial attachment to plant leaves is promoted. Moreover, these phenotypes are altered in a blue-light receptor mutant. These light-controlled changes during the epiphytic stage cause a reduction in virulence, highlighting the relevance of motility during the entry process to the plant apoplast. This study demonstrated the key role of light perception in the Pto phenotype switching and its effect on virulence.

Replication arrest is a major threat to growth at low temperature in Antarctic Pseudomonas syringae†Lz4W.

Chromosomal damage was detected previously in the recBCD mutants of the Antarctic bacterium Pseudomonas syringae Lz4W, which accumulated linear chromosomal DNA leading to cell death and growth inhibition at 4°C. RecBCD protein generally repairs DNA double-strand breaks by RecA-dependent homologous recombination pathway. Here we show that ΔrecA mutant of P. syringae is not cold-sensitive. Significantly, inactivation of additional DNA repair genes ruvAB rescued the cold-sensitive phenotype of ΔrecBCD mutant. The ΔrecA and ΔruvAB mutants were UV-sensitive as expected. We propose that, at low temperature DNA replication encounters barriers leading to frequent replication fork (RF) arrest and fork reversal. RuvAB binds to the reversed RFs (RRFs) having Holliday junction-like structures and resolves them upon association with RuvC nuclease to cause linearization of the chromosome, a threat to cell survival. RecBCD prevents this by degrading the RRFs, and facilitates replication re-initiation. This model is consistent with our observation that low temperature-induced DNA lesions do not evoke SOS response in P. syringae. Additional studies show that two other repair genes, radA (encoding a RecA paralogue) and recF are not involved in providing cold resistance to the Antarctic bacterium.

Light regulation of swarming motility in Pseudomonas syringae integrates signaling pathways mediated by a bacteriophytochrome and a LOV protein.

The biological and regulatory roles of photosensory proteins are poorly understood for nonphotosynthetic bacteria. The foliar bacterial pathogen Pseudomonas syringae has three photosensory protein-encoding genes that are predicted to encode the blue-light-sensing LOV (light, oxygen, or voltage) histidine kinase (LOV-HK) and two red/far-red-light-sensing bacteriophytochromes, BphP1 and BphP2. We provide evidence that LOV-HK and BphP1 form an integrated network that regulates swarming motility in response to multiple light wavelengths. The swarming motility of P. syringae B728a deletion mutants indicated that LOV-HK positively regulates swarming motility in response to blue light and BphP1 negatively regulates swarming motility in response to red and far-red light. BphP2 does not detectably regulate swarming motility. The histidine kinase activity of each LOV-HK and BphP1 is required for this regulation based on the loss of complementation upon mutation of residues key to their kinase activity. Surprisingly, mutants lacking both lov and bphP1 were similar in motility to a bphP1 single mutant in blue light, indicating that the loss of bphP1 is epistatic to the loss of lov and also that BphP1 unexpectedly responds to blue light. Moreover, whereas expression of bphP1 did not alter motility under blue light in a bphP1 mutant, it reduced motility in a mutant lacking lov and bphP1, demonstrating that LOV-HK positively regulates motility by suppressing negative regulation by BphP1. These results are the first to show cross talk between the LOV protein and phytochrome signaling pathways in bacteria, and the similarity of this regulatory network to that of photoreceptors in plants suggests a possible common ancestry. IMPORTANCE Photosensory proteins enable organisms to perceive and respond to light. The biological and ecological roles of these proteins in nonphotosynthetic bacteria are largely unknown. This study discovered that a blue-light-sensing LOV (light, oxygen, or voltage) protein and a red/far-red-light-sensing bacteriophytochrome both regulate swarming motility in the foliar pathogen Pseudomonas syringae. These proteins form an integrated signaling network in which the bacteriophytochrome represses swarming motility in response to red, far-red, and blue light, and LOV positively regulates swarming motility by suppressing bacteriophytochrome-mediated blue-light signaling. This is the first example of cross talk between LOV and phytochrome signaling pathways in bacteria, which shows unexpected similarity to photoreceptor signaling in plants.

Arabidopsis thylakoid formation 1 is a critical regulator for dynamics of PSII-LHCII complexes in leaf senescence and excess light.

In higher plants, photosystem II (PSII) is a large pigment-protein supramolecular complex composed of the PSII core complex and the plant-specific peripheral light-harvesting complexes (LHCII). PSII-LHCII complexes are highly dynamic in their quantity and macro-organization to various environmental conditions. In this study, we reported a critical factor, the Arabidopsis Thylakoid Formation 1 (THF1) protein, which controls PSII-LHCII dynamics during dark-induced senescence and light acclimation. Loss-of-function mutations in THF1 lead to a stay-green phenotype in pathogen-infected and senescent leaves. Both LHCII and PSII core subunits are retained in dark-induced senescent leaves of thf1, indicative of the presence of PSII-LHCII complexes. Blue native (BN)-polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis showed that, in dark- and high-light-treated thf1 leaves, a type of PSII-LHCII megacomplex is selectively retained while the stability of PSII-LHCII supercomplexes significantly decreased, suggesting a dual role of THF1 in dynamics of PSII-LHCII complexes. We showed further that THF1 interacts with Lhcb proteins in a pH-dependent manner and that the stay-green phenotype of thf1 relies on the presence of LHCII complexes. Taken together, the data suggest that THF1 is required for dynamics of PSII-LHCII supramolecular organization in higher plants.

Transcriptional profile of P. syringae pv. phaseolicola NPS3121 at low temperature: physiology of phytopathogenic bacteria.

BACKGROUND: Low temperatures play key roles in the development of most plant diseases, mainly because of their influence on the expression of various virulence factors in phytopathogenic bacteria. Thus far, studies regarding this environmental parameter have focused on specific themes and little is known about phytopathogenic bacteria physiology under these conditions. To obtain a global view regarding phytopathogenic bacteria strategies in response to physiologically relevant temperature changes, we used DNA microarray technology to compare the gene expression profile of the model bacterial pathogen P. syringae pv. phaseolicola NPS3121 grown at 18°C and 28°C. RESULTS: A total of 236 differentially regulated genes were identified, of which 133 were up-regulated and 103 were down-regulated at 18°C compared to 28°C. The majority of these genes are involved in pathogenicity and virulence processes. In general, the results of this study suggest that the expression profile obtained may be related to the fact that low temperatures induce oxidative stress in bacterial cells, which in turn influences the expression of iron metabolism genes. The expression also appears to be correlated with the profile expression obtained in genes related to motility, biofilm production, and the type III secretion system. CONCLUSIONS: From the data obtained in this study, we can begin to understand the strategies used by this phytopathogen during low temperature growth, which can occur in host interactions and disease development.

Defence responses of Arabidopsis thaliana to infection by Pseudomonas syringae are regulated by the circadian clock.

The circadian clock allows plants to anticipate predictable daily changes in abiotic stimuli, such as light; however, whether the clock similarly allows plants to anticipate interactions with other organisms is unknown. Here we show that Arabidopsis thaliana (Arabidopsis) has circadian clock-mediated variation in resistance to the virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), with plants being least susceptible to infection in the subjective morning. We suggest that the increased resistance to Pst DC3000 observed in the morning in Col-0 plants results from clock-mediated modulation of pathogen associated molecular pattern (PAMP)-triggered immunity. Analysis of publicly available microarray data revealed that a large number of Arabidopsis defence-related genes showed both diurnal- and circadian-regulation, including genes involved in the perception of the PAMP flagellin which exhibit a peak in expression in the morning. Accordingly, we observed that PAMP-triggered callose deposition was significantly higher in wild-type plants inoculated with Pst DC3000 hrpA in the subjective morning than in the evening, while no such temporal difference was evident in arrhythmic plants. Our results suggest that PAMP-triggered immune responses are modulated by the circadian clock and that temporal regulation allows plants to anticipate and respond more effectively to pathogen challenges in the daytime.

Accumulation of chlorophyll catabolites photosensitizes the hypersensitive response elicited by Pseudomonas syringae in Arabidopsis.

• The staygreen (SGR) gene encodes a chloroplast-targeted protein which promotes chlorophyll degradation via disruption of light-harvesting complexes (LHCs). • Over-expression of SGR in Arabidopsis (SGR-OX) in a Columbia-0 (Col-0) background caused spontaneous necrotic flecking. To relate this to the hypersensitive response (HR), Col-0, SGR-OX and RNAi SGR (SGRi) lines were challenged with Pseudomonas syringae pv tomato (Pst) encoding the avirulence gene avrRpm1. Increased and decreased SGR expression, respectively, accelerated and suppressed the kinetics of HR-cell death. In Col-0, SGR transcript increased at 6 h after inoculation (hai) when tissue electrolyte leakage indicated the initiation of cell death. • Excitation of the chlorophyll catabolite pheophorbide (Pheide) leads to the formation of toxic singlet oxygen ((1)O(2)). Pheide was first detected at 6 hai with Pst avrRpm1 and was linked to (1)O(2) generation and correlated with reduced Pheide a oxygenase (PaO) protein concentrations. The maximum quantum efficiency of photosystem II (F(v)/F(m)), quantum yield of electron transfer at photosystem II (φPSII), and photochemical quenching (qP) decreased at 6 hai in Col-0 but not in SGRi. Disruption of photosynthetic electron flow will cause light-dependent H(2)O(2) generation at 6 hai. • We conclude that disruption of LHCs, possibly influenced by SGR, and absence of PaO produce phototoxic chlorophyll catabolites and oxidative stress leading to the HR.

62-kb plasmids harboring rulAB homologues confer UV-tolerance and epiphytic fitness to Pseudomonas syringae pv. syringae mango isolates.

The presence of genetic determinants homologous to rulAB genes for ultraviolet (UV) radiation resistance was determined in a collection of Pseudomonas syringae pv. syringae strains isolated from mango. The potential role of these plasmids in UV tolerance and ecological fitness in the mango phyllosphere was also evaluated. Nearly all of the 62-kb plasmids present in the P. syringae pv. syringae strains hybridized with a rulAB probe, but these 62-kb plasmids showed differences in restriction patterns. In vitro assays of tolerance to UV radiation of P. syringae pv. syringae strains showed a higher survival of the strains harboring the 62-kb plasmids compared to strains lacking plasmids when exposed to UVC or UVA+B fractions. Similar results were observed when transconjugants harboring the 62-kb plasmid were tested. Survival assays were carried out under field conditions, and a higher survival of P. syringae pv. syringae strains harboring 62-kb plasmids under direct solar radiation on the adaxial surface of leaves was also observed. When the assays were carried out in shady areas or on the abaxial surface of leaves, survival time was comparable for all the assayed strains, whether or not they contained a 62-kb plasmid hybridizing to rulAB. Our results indicate that P. syringae pv. syringae strains harboring 62-kb plasmids show an increase in ecological fitness when colonizing the mango phyllosphere.

A blue light inducible two-component signal transduction system in the plant pathogen Pseudomonas syringae pv. tomato.

The open reading frame PSPTO2896 from the plant pathogen Pseudomonas syringae pv. tomato encodes a protein of 534 amino acids showing all salient features of a blue light-driven two-component system. The N-terminal LOV (light, oxygen, voltage) domain, potentially binding a flavin chromophore, is followed by a histidine kinase (HK) motif and a response regulator (RR). The full-length protein (PST-LOV) and, separately, the RR and the LOV+HK part (PST-LOV(DeltaRR)) were heterologously expressed and functionally characterized. The two LOV proteins showed typical LOV-like spectra and photochemical reactions, with the blue light-driven, reversible formation of a covalent flavin-cysteine bond. The fluorescence changes in the lit state of full-length PST-LOV, but not in PST-LOV(DeltaRR), indicating a direct interaction between the LOV core and the RR module. Experiments performed with radioactive ATP uncover the light-driven kinase activity. For both PST-LOV and PST-LOV(DeltaRR), much more radioactivity is incorporated when the protein is in the lit state. Furthermore, addition of the RR domain to the fully phosphorylated PST-LOV(DeltaRR) leads to a very fast transfer of radioactivity, indicating a highly efficient HK activity and a tight interaction between PST-LOV(DeltaRR) and RR, possibly facilitated by the LOV core itself.

Role of nucleotide excision repair and photoreactivation in the solar UVB radiation survival of Pseudomonas syringae pv. syringae B728a.

AIMS: To assess the role of DNA repair and photoreactivation in the solar radiation survival of the plant pathogen and leaf surface epiphyte Pseudomonas syringae pv. syringae (Pss). METHODS AND RESULTS: Mutants of Pss B728a, with insertional mutations within the nucleotide excision repair gene uvrA, photolyase gene phr, or uvrA phr double mutants, were constructed to examine the importance of individual repair mechanisms in solar UV radiation (UVR) survival. The survival of either the uvrA mutant or the phr mutant was reduced by approx. 10(2)-fold following exposure to a dose of 4.5 kJ m(-2) solar UVB (290-320 nm wavelengths) while the uvrA phr double mutant was reduced >10(6)-fold by the same dose. We constructed a transcriptional fusion between the Pss recA promoter and gfp to examine the induction of the SOS response in wild-type and mutant strains. Initiation of the recA mediated SOS response was more rapid and peaked at higher levels in mutant strains suggesting both increased DNA damage in mutant strains and also that photoreactivation and nucleotide excision repair remove DNA damage as it is incurred which is reflected in a delay of recA expression. Visualization of expression of B728a cells containing the recA::gfp reporter on UVB-irradiated bean leaves highlighted the movement of cells to intercellular spaces over time and that SOS induction was detectable when leaves were irradiated 48 h following leaf inoculation. CONCLUSIONS: This study indicated that solar UVB is detrimental to Pss B728a, DNA repair mechanisms play an important role in strain survival and expression of the SOS regulon on leaf surfaces contributes to survival of UVR-exposed cells during plant colonization. SIGNIFICANCE AND IMPACT OF THE STUDY: This work links previous laboratory-based UVR analyses with solar UVB dose-response analyses and highlights the role of photoreactivation in delaying induction of the SOS response following solar irradiation. Knowledge of population dynamics following direct solar irradiation will enhance our understanding of the biology of Pss in the phyllosphere.

Long-term effect of mutagenic DNA repair on accumulation of mutations in Pseudomonas syringae B86-17.

Forty replicate lineages of Pseudomonas syringae B86-17 cells expressing the rulAB mutagenic DNA repair (MDR) determinant or the rulB::Km MDR-deficient mutant GWS242 were passaged through single-cell bottlenecks (60 cycles), with a UV radiation (UVR) exposure given to half of the lineages at the beginning of each cycle. After every 10th bottleneck cycle, single-colony isolates from all 80 lineages were subjected to 39 phenotypic screens, with newly arising mutations detected in 60 and 0% of UVR-exposed or non-UVR-exposed B86-17 lineages, respectively, by the 60th cycle. Cellular fitness, measured as growth rate in a minimal medium, of UVR-exposed lineages of both B86-17 and GWS242 after 60 cycles was not significantly different from that of the ancestral strains. Although UVR exposure and MDR activity increased the occurrence of mutations in cells, a significant reduction in overall fitness was not observed.