Kasumi Takeuchi

Flagellin Glycosylation Island in Pseudomonas syringae pv. glycinea and Its Role in Host Specificity

The deduced amino acid sequences of the flagellins of Pseudomonas syringae pv. tabaci and P. syringae pv. glycinea are identical; however, their abilities to induce a hypersensitive reaction are clearly different. The reason for the difference seems to depend on the posttranslational modification of the flagellins. To investigate the role of this posttranslational modification in the interactions between plants and bacterial pathogens, we isolated genes that are potentially involved in the posttranslational modification of flagellin in P. syringae pv. glycinea (glycosylation island); then defective mutants with mutations in these genes were generated. There are three open reading frames in the glycosylation island, designated orf1, orf2, and orf3. orf1 and orf2 encode putative glycosyltransferases, and mutants with defects in these open reading frames, deltaorf1 and deltaorf2, secreted nonglycosylated and slightly glycosylated flagellins, respectively. Inoculation tests performed with these mutants and original nonhost tobacco leaves revealed that deltaorf1 and deltaorf2 could grow on tobacco leaves and caused symptom-like changes. In contrast, these mutants failed to cause symptoms on original host soybean leaves. These data indicate that putative glycosyltransferases encoded in the flagellin glycosylation island are strongly involved in recognition by plants and could be the specific determinants of compatibility between phytopathogenic bacteria and plant species.

Identification of glycosylation genes and glycosylated amino acids of flagellin in Pseudomonas syringae pv. tabaci

A glycosylation island is a genetic region required for glycosylation. The glycosylation island of flagellin in Pseudomonas syringae pv. tabaci 6605 consists of three orfs: orf1, orf2 and orf3. Orf1 and orf2 encode putative glycosyltransferases, and their deletion mutants, Δorf1 and Δorf2, exhibit deficient flagellin glycosylation or produce partially glycosylated flagellin respectively. Digestion of glycosylated flagellin from wild‐type bacteria and non‐glycosylated flagellin from Δorf1 mutant using aspartic N‐peptidase and subsequent HPLC analysis revealed candidate glycosylated amino acids. By generation of site‐directed Ser/Ala‐substituted mutants, all glycosylated amino acid residues were identified at positions 143, 164, 176, 183, 193 and 201. Matrix‐assisted laser desorption/ionization time of flight (MALDI‐TOF) mass spectrometry (MS) analysis revealed that each glycan was about 540 Da. While all glycosylation‐defective mutants retained swimming ability, swarming ability was reduced in the Δorf1, Δorf2 and Ser/Ala‐substituted mutants. All glycosylation mutants were also found to be impaired in the ability to adhere to a polystyrene surface and in the ability to cause disease in tobacco. Based on the predicted tertiary structure of flagellin, S176 and S183 are expected to be located on most external surface of the flagellum. Thus the effect of Ala‐substitution of these serines is stronger than that of other serines. These results suggest that glycosylation of flagellin in P. syringae pv. tabaci 6605 is required for bacterial virulence. It is also possible that glycosylation of flagellin may mask elicitor function of flagellin molecule.

A Homologue of the 3-Oxoacyl-(Acyl Carrier Protein) Synthase III Gene Located in the Glycosylation Island of Pseudomonas syringae pv. tabaci Regulates Virulence Factors via N-Acyl Homoserine Lactone and Fatty Acid Synthesis

Pseudomonas syringae pv. tabaci 6605 possesses a genetic region involved in flagellin glycosylation. This region is composed of three open reading frames: orf1, orf2, and orf3. Our previous study revealed that orf1 and orf2 encode glycosyltransferases; on the other hand, orf3 has no role in posttranslational modification of flagellin. Although the function of Orf3 remained unclear, an orf3 deletion mutant (Deltaorf3 mutant) had reduced virulence on tobacco plants. Orf3 shows significant homology to a 3-oxoacyl-(acyl carrier protein) synthase III in the fatty acid elongation cycle. The Deltaorf3 mutant had a significantly reduced ability to form acyl homoserine lactones (AHLs), which are quorum-sensing molecules, suggesting that Orf3 is required for AHL synthesis. In comparison with the wild-type strain, swarming motility, biosurfactant production, and tolerance to H2O2 and antibiotics were enhanced in the Deltaorf3 mutant. A scanning electron micrograph of inoculated bacteria on the tobacco leaf surface revealed that there is little extracellular polymeric substance matrix surrounding the cells in the Deltaorf3 mutant. The phenotypes of the Deltaorf3 mutant and an AHL synthesis (DeltapsyI) mutant were similar, although the mutant-specific characteristics were more extreme in the Deltaorf3 mutant. The swarming motility of the Deltaorf3 mutant was greater than that of the DeltapsyI mutant. This was attributed to the synergistic effects of the overproduction of biosurfactants and/or alternative fatty acid metabolism in the Deltaorf3 mutant. Furthermore, the amounts of iron and biosurfactant seem to be involved in biofilm development under quorum-sensing regulation in P. syringae pv. tabaci 6605.

Effects of Glycosylation on Swimming Ability and Flagellar Polymorphic Transformation in Pseudomonas syringae pv. tabaci 6605

The role of flagellin glycosylation on motility was investigated in Pseudomonas syringae pv. tabaci. The swimming activity of glycosylation-defective mutants was prominently decreased in a highly viscous medium. The mutants showed differences in polymorphic transitions and in the bundle formation of flagella, indicating that glycosylation stabilizes the filament structure and lubricates the rotation of the bundle.

Flagellin Glycans from Two Pathovars of Pseudomonas syringae Contain Rhamnose in d and l Configurations in Different Ratios and Modified 4-Amino-4,6-Dideoxyglucose

Flagellins from Pseudomonas syringae pv. glycinea race 4 and Pseudomonas syringae pv. tabaci 6605 have been found to be glycosylated. Glycosylation of flagellin is essential for bacterial virulence and is also involved in the determination of host specificity. Flagellin glycans from both pathovars were characterized, and common sites of glycosylation were identified on six serine residues (positions 143, 164, 176, 183, 193, and 201). The structure of the glycan at serine 201 (S201) of flagellin from each pathovar was determined by sugar composition analysis, mass spectrometry, and (1)H and (13)C nuclear magnetic resonance spectroscopy. These analyses showed that the S201 glycans from both pathovars were composed of a common unique trisaccharide consisting of two rhamnosyl (Rha) residues and one modified 4-amino-4,6-dideoxyglucosyl (Qui4N) residue, beta-D-Quip4N(3-hydroxy-1-oxobutyl)2Me-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap. Furthermore, mass analysis suggests that the glycans on each of the six serine residues are composed of similar trisaccharide units. Determination of the enantiomeric ratio of Rha from the flagellin proteins showed that flagellin from P. syringae pv. tabaci 6605 consisted solely of L-Rha, whereas P. syringae pv. glycinea race 4 flagellin contained both L-Rha and D-Rha at a molar ratio of about 4:1. Taking these findings together with those from our previous study, we conclude that these flagellin glycan structures may be important for the virulence and host specificity of P. syringae.

Defense responses of Arabidopsis thaliana inoculated with Pseudomonas syringae pv. tabaci wild type and defective mutants for flagellin (ΔfliC) and flagellin-glycosylation (Δorf1)

Flagellin, an essential component of the bacterial flagellar filament, is capable of inducing a hypersensitive response (HR), including cell death, in a nonhost plant. A flagellin-defective mutant (ΔfliC) of Pseudomonas syringae pv. tabaci lacks both the flagellar filament and motility, whereas a flagellin-glycosylation-defective mutant (Δorf1) retains the flagellar filament but lacks the glycosyl modification of flagellin protein. To investigate the role of flagellin protein and its glycosylation in the interaction with its nonhost Arabidopsis thaliana, we analyzed plant responses after inoculation with these bacteria. Inoculation with wild-type P. syringae pv. tabaci induced HR, with the generation of reactive oxygen species and cell death. In contrast, inoculation with either ΔfliC or Δorf1 mutant induced a low level of HR, and inoculated leaves developed a disease-like yellowing. These mutant bacteria multiplied better than the wild-type bacteria in A. thaliana. These results indicate that A. thaliana expresses a defense reaction in response to the bacterial flagellin with its glycosyl structure.

A novel pathosystem to study the interactions between Lotus japonicus and Fusarium solani

A wilt disease of the model legume Lotus japonicus was observed in a greenhouse in Tokyo, Japan in May 2004. Roots of diseased plants were rotted and dark brown with lesions spreading to lower stems and leaves, resulting in rapid plant death. The causal agent was identified as Fusarium solani based on the morphology. Sequence analysis of rDNA supported the identification. Inoculation of roots of healthy plants with conidia reproduced characteristic disease symptoms, and F. solani was reisolated from lesions, satisfying Koch’s postulates. The isolate also caused chlorotic to necrotic lesions on leaves of healthy plants after wound-inoculation. Infection by F. solani of leaves of L. japonicus was confirmed histologically. Mycelia were observed in the intercellular spaces of parenchymatous tissues in the lesion area and the surrounding tissues. This is the first report of fungal disease on L. japonicus satisfying Koch’s postulates. We named it “Fusarium root rot of L. japonicus” as a new disease. The compatibility of L. japonicus and F. solani is expected to form a novel pathosystem for studying interactions between legumes and fungal pathogens.

Role of Flagella and Flagellin in Plant — Pseudomonas syringae Interactions

The role of flagella and monomer flagellin of Pseudomonas syringae pv. tabaci in plant-bacteria interactions was investigated by using non-polar fliC or fliD mutants. These mutants deleted the open reading frames for fliC and fliD, respectively, and both mutants lost all flagella and motility. The ΔfliC mutant did not produce flagellin, whereas ΔfliD mutant, that lost HAP2 protein, secreted a large amount of monomer flagellin in the culture medium. Inoculation of tomato leaves with wild type and ΔfliD mutant of P. s. pv. tabaci induced HR, whereas the ΔfliC mutant caused symptom-like change and propagated as P. s. pv. tomato. In tomato suspension cultured cells, wild type P. s. pv. tabaci induced visible HR-like changes. The ΔfliC mutant did not induce the HR, but the response was activated by the ΔfliD mutant. The expression of typical defence genes such as PAL and hsr203J was rapidly and strongly induced by inoculation with the ΔfliD mutant compared to inoculation with wild type P. s. pv. tabaci. On the other hand, both fliC and fliD mutants were reduced in virulence when inoculated into host tobacco leaves. Furthermore, complementation of fliC gene in ΔfliC mutant restored motility and HR-inducing ability in tomato, and virulence in tobacco. These results suggest that the monomer flagellin of P. s. pv. tabaci is an essential factor in the elicitation of HR in non host tomato cells, and flagella are required for complete virulence in host tobacco cells.

Studies on flagellin glycosylation and pathogenicity of Pseudomonas syringae

Isolates of the phytopathogenic bacterium Pseudomonas syringae can be classified into more than 50 pathovars on the basis of virulence for host plant species. Our previous studies have shown that P. syringae flagellin elicits hypersensitive reactions (HR) in nonhost plants. Although the deduced amino acid sequences of the flagellin proteins of P. syringae pv. tabaci 6605 (Pta6605) and P. syringae pv. glycinea race 4 (Pgl4) are identical, the flagellin protein of Pgl4 causes cell death in nonhost tobacco cells, whereas that of Pta6605 does not. Further, the flagellin proteins of these two pathovars are glycosylated. In this study, we describe genetic and structural analyses of the glycosylation of flagellin in Pgl4 and Pta6605.

Role of Flagellin Glycosylation in Bacterial Virulence

Pseudomonas syringae pv. tabaci 6605 (Pta6605) is a causal agent for wildfire disease in tobacco. Recently, we found that flagellin, a major constituent in the flagella filament of this pathogen, is a potent elicitor of hypersensitive reaction in non-host plant species. We also found that flagellin is required for virulence against the host plant. In this study, we investigated the biochemical features of the glycosyl moiety of flagellin and the phytopathological role of flagellin glycosylation. DNA sequence analysis of the flagellum gene cluster revealed that two genes (orf1 and orf2) encoding a putative glycosyltransferase were located upstream of the fliC gene. To investigate the role of flagellin glycosylation, we generated deletion mutants for orf1 (∆orf1) and orf2 (∆orf2) in Pta6605 and pv. glycinea race 4 (Pgl4). The mutants, ∆orf1 and ∆orf2, of both pathovars produced nonglycosylated or par- tially glycosylated flagellins, respectively. Inoculation of host plants with these mutant strains confirmed that ∆orf1 and ∆orf2 had reduced ability to cause disease. Biochemical and genetic approaches revealed that a total of six serine residues of FliC were glycosylated in Pta6605. The serine residues were replaced individually with Ala by site-directed mutagenesis. All glycosylation-defective mutants includ- ing ∆orf1 and ∆orf2 and the six Ser/Ala-substituted mutants of pv. tabaci retained swimming ability but their swarming ability was reduced. The abilities to adhere to a polystyrene surface and to cause disease in host tobacco plants were also impaired in all Ser/Ala-substituted mutants of Pta6605. When tobacco leaves were inoculated with Pta6605 wild-type, bacteria were embedded and formed a biofilm-like structure in the matrix on the tobacco leaf surface. In contrast, mucoid material was rarely detected in the area surrounding the ∆orf1 mutant. These results suggest that glyco- sylation of flagellin in Pta6605 is required for swarming motility, adhesion, biofilm