The AvrB_AvrC domain of AvrXccC of Xanthomonas campestris pv. campestris is required to elicit plant defense responses and manipulate ABA homeostasis.

Plant disease induced by Xanthomonas campestris pv. campestris depends on type III effectors but the molecular basis is poorly understood. Here, AvrXccC8004 was characterized, and it was found that the AvrB_AvrC domain was essential and sufficient to elicit defense responses in an Arabidopsis-resistant ecotype (Col-0). An upregulation of genes in responding to the AvrB_AvrC domain of AvrXccC8004 was shown in a profile of host gene expression. The molecular changes were correlated with morphological changes observed in phenotypic and ultrastructural characterizations. Interestingly, the abscisic acid (ABA)-signaling pathway was also a prominent target for the AvrB_AvrC domain of AvrXccC8004. The highly elicited NCED5, encoding a key enzyme of ABA biosynthesis, was increased in parallel with ABA levels in AvrXccC8004 transgenic plants. Consistently, the X. campestris pv. campestris 8004 ΔavrXccC mutant was severely impaired in the ability to manipulate the accumulation of ABA and induction of ABA-related genes in challenged leaves. Moreover, exogenous application of ABA also enhanced the susceptibility of Arabidopsis to the X. campestris pv. campestris strains. These results indicate that the AvrB_AvrC domain of AvrXccC8004 alone has the activity to manipulate ABA homeostasis, which plays an important role in regulating the interactions of X. campestris pv. campestris and Arabidopsis.

A Plant Endophytic Bacterium Priestia megaterium StrainBP-R2 Isolated from the Halophyte Bolboschoenus planiculmis Enhances Plant Growth under Salt and Drought Stresses.

Global warming and climate change have contributed to the rise of weather extremes. Severe drought and soil salinization increase because of rising temperatures. Economically important crop production and plant growth and development are hindered when facing various abiotic stresses. Plant endophytic bacteria live inside host plants without causing visible harm and can be isolated from surface-sterilized plant tissues. Using plant endophytic bacteria to stimulate plant growth and increase environmental stress tolerance has become an alternative approach besides using the traditional breeding and genetically modifying approaches to select or create new crop types resistant to different environmental stresses. The plant endophytic bacterium, Priestia megaterium (previously known as Bacillus megaterium) strain BP-R2, was isolated from the surface-sterilized root tissues of the salt marsh halophyte Bolboschoenus planiculmis. The bacteria strain BP-R2 showed high tolerance to different sodium chloride (NaCl) concentrations and produced the auxin plant hormone, indole acetic acid (IAA), under various tested growth conditions. Inoculation of Arabidopsis and pak choi (Brassica rapa L. R. Chinensis Group) plants with the strain BP-R2 greatly enhanced different growth parameters of the host plants under normal and salt and drought stress conditions compared to that of the mock-inoculated plants. Furthermore, the hydrogen peroxide (H2O2) content, electrolyte leakage (EL), and malondialdehyde (MDA) concentration accumulated less in the BP-R2-inoculated plants than in the mock-inoculated control plants under salt and drought stresses. In summary, the plant endophytic bacterium strain BP-R2 increased host plant growth and stress tolerance to salt and drought conditions.

Genomic and biochemical characterization of antifungal compounds produced by Bacillus subtilis PMB102 against Alternaria brassicicola.

Bacillus subtilis is ubiquitous and capable of producing various metabolites, which make the bacterium a good candidate as a biocontrol agent for managing plant diseases. In this study, a phyllosphere bacterium B. subtilis PMB102 isolated from tomato leaf was found to inhibit the growth of Alternaria brassicicola ABA-31 on PDA and suppress Alternaria leaf spot on Chinese cabbage (Brassica rapa). The genome of PMB102 (Accession no. CP047645) was completely sequenced by Nanopore and Illumina technology to generate a circular chromosome of 4,103,088 bp encoding several gene clusters for synthesizing bioactive compounds. PMB102 and the other B. subtilis strains from different sources were compared in pangenome analysis to identify a suite of conserved genes involved in biocontrol and habitat adaptation. Two predicted gene products, surfactin and fengycin, were extracted from PMB102 culture filtrates and verified by LC-MS/MS. The antifungal activity of fengycin was tested on A. brassicicola ABA-31 in bioautography to inhibit hyphae growth, and in co-culturing assays to elicit the formation of swollen hyphae. Our data revealed that B. subtilis PMB102 suppresses Alternaria leaf spot by the production of antifungal metabolites, and fengycin plays an important role to inhibit the vegetative growth of A. brassicicola ABA-31.

A Plant Endophytic Bacterium, Burkholderia seminalis Strain 869T2, Promotes Plant Growth in Arabidopsis, Pak Choi, Chinese Amaranth, Lettuces, and Other Vegetables.

Plant endophytic bacteria live inside host plants, can be isolated from surface-sterilized plant tissues, and are non-pathogenic. These bacteria can assist host plants in obtaining more nutrients and can improve plant growth via multiple mechanisms. Certain Gram-negative Burkholderia species, including rhizobacteria, bioremediators, and biocontrol strains, have been recognized for their plant-growth-promoting abilities, while other isolates have been identified as opportunistic plant or human pathogens. In this study, we observed the auxin production, siderophore synthesis, and phosphate solubilization abilities of B. seminalis strain 869T2. Our results demonstrated that strain 869T2 promoted growth in Arabidopsis, ching chiang pak choi, pak choi, loose-leaf lettuce, romaine lettuce, red leaf lettuce, and Chinese amaranth. Leafy vegetables inoculated with strain 869T2 were larger, heavier, and had more and larger leaves and longer and heavier roots than mock-inoculated plants. Furthermore, inoculations of strain 869T2 into hot pepper caused increased flower and fruit production, and a higher percentage of fruits turned red. Inoculation of strain 869T2 into okra plants resulted in earlier flowering and increased fruit weight. In conclusion, the plant endophytic bacterium Burkholderia seminalis 869T2 exerted positive effects on growth and production in several plant species.

Effects of galU mutation on Pseudomonas syringae-plant interactions.

Bacterial galU coding for a uridine diphosphate-glucose pyrophosphorylase plays an important role in carbohydrates biosynthesis, including synthesis of lipopolysaccharides (LPS), membrane-derived oligosaccharides, and capsular polysaccharides. In this study, we characterized the galU mutant of Pseudomonas syringae pv. syringae 61 (Psy61), a necrotizing plant pathogen whose pathogenicity depends on a functional type III secretion system (T3SS), and showed that the Psy61 galU mutant had reduced biofilm formation ability, was nonmotile, and had an assembled T3SS structure but failed to elicit hypersensitive response in resistant plants and necrotic lesions in susceptible plants. Moreover, the defective LPS and other pathogen-associated molecular patterns (PAMPs) on the surface of the Psy61 galU mutant were capable of inducing PAMP-triggered immunity, which severely compromised the ability of the Psy61 galU mutant to survive in planta. Our results demonstrated that the complete LPS protected plant-pathogenic bacteria from host innate immunity, similar to what was found in animal pathogens, prior to the translocation of T3S effectors and bacterial multiplication.

Phenylacetic Acid and Methylphenyl Acetate From the Biocontrol Bacterium Bacillus mycoides BM02 Suppress Spore Germination in Fusarium oxysporum f. sp. lycopersici.

Bacillus mycoides strain BM02 originally isolated from the tomato rhizosphere was found to have beneficial functions in tomato by promoting plant growth and reducing the severity of Fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici (Fol). Cytological experiments demonstrated B. mycoides BM02 reduced Fol invasion by reducing spore attachment and increasing hyphal deformation in hydroponics-grown tomato root tissues. Two volatile antifungal compounds, phenylacetic acid (PAA) and methylphenyl acetate (MPA), were identified from the culture filtrates of B. mycoides BM02 by GC-MS analysis. Chemically synthesized PAA, and to a lower extent MPA, suppressed spore germination but have no effects on the hyphal growth of Fol. Our results indicated that the biocontrol agent B. mycoides BM02 produced an array of bioactive compounds including PAA and MPA to suppress plant diseases caused by Fol and other pathogenic microorganisms.

[Postoperative acute urinary retention in anorectal diseases treated with acupuncture combined with grain-moxibustion: a randomized controlled trial].

OBJECTIVE: To observe the efficacy differences between acupuncture combined with grain-moxibustion and acupuncture on acute urinary retention after epidural anaesthesia for anorectal diseases. METHODS: A total of 60 patients were randomized into an acupuncture combined with grain-moxibustion group and an acupuncture group, 31 cases in each one. In the acupuncture group, acupuncture was applied at Zhongji (CV 3), Guanyuan (CV 4), Qihai (CV 6), Shuidao (ST 28), Pangguangshu (BL 28), Sanyinjiao (SP 6) and Yinlingquan (SP 9). In the acupuncture combined with grain-moxibustion group, grain-moxibustion was given at Zhongji (CV 3), Guanyuan (CV 4), Qihai (CV 6) and Shuidao (ST 28) on the basis of acupuncture. Those who failed to urinate 60 min after the first treatment received the second treatment. 30 min after the second treatment, the lower abdominal symptom scores before and after treatment as well as the bladder residual urine volume after the first urination after treatment were compared between the two groups. In addition, the clinical efficacy and security were evaluated. RESULTS: Compared before treatment, the symptom scores after treatment were reduced in the two groups (P<0.05), and the score in the acupuncture combined with grain-moxibustion group was lower than that in the acupuncture group after treatment (P<0.05). The bladder residual urine volume in the acupuncture combined with grain-moxibustion group was (26.71±17.01) mL, which was lower than (35.32±20.76) mL in the acupuncture group (P<0.05). The total effective rate was 93.5% (29/31) in the acupuncture combined with grain-moxibustion group, which was superior to 71.0% (22/31) in the acupuncture group (P<0.05). CONCLUSION: The efficacy of acupuncture combined with grain-moxibustion is superior to simple acupuncture on acute urinary retention after epidural anaesthesia for anorectal diseases, which is safe and reliable.

Presence of an Agrobacterium-Type Tumor-Inducing Plasmid in Neorhizobium sp. NCHU2750 and the Link to Phytopathogenicity.

The genus Agrobacterium contains a group of plant-pathogenic bacteria that have been developed into an important tool for genetic transformation of eukaryotes. To further improve this biotechnology application, a better understanding of the natural genetic variation is critical. During the process of isolation and characterization of wild-type strains, we found a novel strain (i.e., NCHU2750) that resembles Agrobacterium phenotypically but exhibits high sequence divergence in several marker genes. For more comprehensive characterization of this strain, we determined its complete genome sequence for comparative analysis and performed pathogenicity assays on plants. The results demonstrated that this strain is closely related to Neorhizobium in chromosomal organization, gene content, and molecular phylogeny. However, unlike the characterized species within Neorhizobium, which all form root nodules with legume hosts and are potentially nitrogen-fixing mutualists, NCHU2750 is a gall-forming pathogen capable of infecting plant hosts across multiple families. Intriguingly, this pathogenicity phenotype could be attributed to the presence of an Agrobacterium-type tumor-inducing plasmid in the genome of NCHU2750. These findings suggest that these different lineages within the family Rhizobiaceae are capable of transitioning between ecological niches by having novel combinations of replicons. In summary, this work expanded the genomic resources available within Rhizobiaceae and provided a strong foundation for future studies of this novel lineage. With an infectivity profile that is different from several representative Agrobacterium strains, this strain may be useful for comparative analysis to better investigate the genetic determinants of host range among these bacteria.

Differential expression of pectolytic enzyme genes in Xanthomonas citri subsp. citri and demonstration that pectate lyase Pel3 is required for the formation of citrus canker.

Bacterial canker, caused by Xanthomonas citri subsp. citri (Xcc), is one of the most destructive diseases of citrus. The pectolytic enzymes produced by phytobacteria are important virulence factors involved in tissue maceration, electrolyte loss and cell death of host plants. In this study, the promoter activity of the pectolytic enzyme genes pel1, pel2, pel3, pglA, and peh-1 were investigated in Xcc XW19 strain using the ß-glucuronidase (GUS) gene as a reporter. GUS activity expressed under the control of the pel1, pel3, pglA, and peh-1 gene promoters positively correlated with bacterial growth. These gene promoters displayed high GUS activity in the presence of sodium polypectate. In addition, the four genes were induced in XVM2 minimal medium. However, only pel1 was subjected to catabolite repression by glucose. GUS activity was significantly enhanced in the XW19-derived reporter strains after they were inoculated into the leaves of Mexican lime and grapefruit, suggesting the involvement of the pel1, pel3, pglA, and peh-1 genes in XW19 pathogenesis. The pel3 promoter produced the highest GUS activity under all test conditions, whereas no GUS activity was detected using the pel2 promoter in vitro and in planta. In comparison with wild type Xcc, a pel3 mutant generated from Xcc XW19 using unmarked mutagenesis displayed reduced growth and induced smaller canker lesions on the leaves of Mexican lime, demonstrating that Pel3 of Xcc strain XW19 is a virulence factor.

The pathogenicity factor HrpF interacts with HrpA and HrpG to modulate type III secretion system (T3SS) function and t3ss expression in Pseudomonas syringae pv. averrhoi.

To ensure the optimal infectivity on contact with host cells, pathogenic Pseudomonas syringae has evolved a complex mechanism to control the expression and construction of the functional type III secretion system (T3SS) that serves as a dominant pathogenicity factor. In this study, we showed that the hrpF gene of P. syringae pv. averrhoi, which is located upstream of hrpG, encodes a T3SS-dependent secreted/translocated protein. Mutation of hrpF leads to the loss of bacterial ability on elicitation of disease symptoms in the host and a hypersensitive response in non-host plants, and the secretion or translocation of the tested T3SS substrates into the bacterial milieu or plant cells. Moreover, overexpression of hrpF in the wild-type results in delayed HR and reduced t3ss expression. The results of protein-protein interactions demonstrate that HrpF interacts directly with HrpG and HrpA in vitro and in vivo, and protein stability assays reveal that HrpF assists HrpA stability in the bacterial cytoplasm, which is reduced by a single amino acid substitution at the 67th lysine residue of HrpF with alanine. Taken together, the data presented here suggest that HrpF has two roles in the assembly of a functional T3SS: one by acting as a negative regulator, possibly involved in the HrpSVG regulation circuit via binding to HrpG, and the other by stabilizing HrpA in the bacterial cytoplasm via HrpF-HrpA interaction prior to the secretion and formation of Hrp pilus on the bacterial surface.

Finished Genome Sequence of Collimonas arenae Cal35.

We announce the finished genome sequence of soil forest isolate Collimonas arenae Cal35, which comprises a 5.6-Mbp chromosome and 41-kb plasmid. The Cal35 genome is the second one published for the bacterial genus Collimonas and represents the first opportunity for high-resolution comparison of genome content and synteny among collimonads.

Arabidopsis HFR1 is a potential nuclear substrate regulated by the Xanthomonas type III effector XopD(Xcc8004).

XopDXcc8004, a type III effector of Xanthomonas campestris pv. campestris (Xcc) 8004, is considered a shorter version of the XopD, which lacks the N-terminal domain. To understand the functions of XopDXcc8004, in planta, a transgenic approach combined with inducible promoter to analyze the effects of XopDXcc8004 in Arabidopsis was done. Here, the expression of XopDXcc8004, in Arabidopsis elicited the accumulation of host defense-response genes. These molecular changes were dependent on salicylic acid and correlated with lesion-mimic phenotypes observed in XVE::XopDXcc8004 transgenic plants. Moreover, XopDXcc8004 was able to desumoylate HFR1, a basic helix-loop-helix transcription factor involved in photomorphogenesis, through SUMO protease activity. Interestingly, the hfr1-201 mutant increased the expression of host defense-response genes and displayed a resistance phenotype to Xcc8004. These data suggest that HFR1 is involved in plant innate immunity and is potentially regulated by XopDXcc8004.

Plant innate immunity induced by flagellin suppresses the hypersensitive response in non-host plants elicited by Pseudomonas syringae pv. averrhoi.

A new pathogen, Pseudomonas syringae pv. averrhoi (Pav), which causes bacterial spot disease on carambola was identified in Taiwan in 1997. Many strains of this pathovar have been isolated from different locations and several varieties of hosts. Some of these strains, such as HL1, are nonmotile and elicit a strong hypersensitive response (HR) in nonhost tobacco leaves, while other strains, such as PA5, are motile and elicit a weak HR. Based on the image from a transmission electron microscope, the results showed that HL1 is flagellum-deficient and PA5 has normal flagella. Here we cloned and analyzed the fliC gene and glycosylation island from Pav HL1 and PA5. The amino acid sequences of FliC from HL1 and PA5 are identical to P. s. pvs. tabaci (Pta), glycinea and phaseolicola and share very high similarity with other pathovars of P. syringae. In contrast to the flagellin mutant PtaΔfliC, PA5ΔfliC grows as well as wild type in the host plant, but it elicits stronger HR than wild type does in non-host plants. Furthermore, the purified Pav flagellin, but not the divergent flagellin from Agrobacterium tumefaciens, is able to impair the HR induced by PA5ΔfliC. PA5Δfgt1 possessing nonglycosylated flagella behaved as its wild type in both bacterial growth in host and HR elicitation. Flagellin was infiltrated into tobacco leaves either simultaneously with flagellum-deficient HL1 or prior to the inoculation of wild type HL1, and both treatments impaired the HR induced by HL1. Moreover, the HR elicited by PA5 and PA5ΔfliC was enhanced by the addition of cycloheximide, suggesting that the flagellin is one of the PAMPs (pathogen-associated molecular patterns) contributed to induce the PAMP-triggered immunity (PTI). Taken together, the results shown in this study reveal that flagellin in Pav is capable of suppressing HR via PTI induction during an incompatible interaction.

The Arabidopsis short-chain dehydrogenase/reductase 3, an abscisic acid deficient 2 homolog, is involved in plant defense responses but not in ABA biosynthesis.

ABSCISIC ACID DEFICIENT2 (ABA2) encodes a short-chain dehydrogenase/reductase1 (SDR1) that catalyzes the multi-step conversion of xanthoxin to abscisic aldehyde during abscisic acid (ABA) biosynthesis in Arabidopsis thaliana. In this study, AtSDR2 and AtSDR3, the two closest homologs to AtABA2, were investigated for their potential role in ABA biosynthesis. AtSDR2 showed undetectable transcription in plants grown under normal conditions or under stress. AtSDR3 and AtABA2 have different spatial and temporal expression patterns. Complementation testing demonstrated that the pABA2::SDR3 transgene failed to complement the aba2 mutant phenotype, and that transgenic plants showed the same levels of ABA as the aba2 mutants. These data suggest that AtSDR3 confers no functional redundancy to AtABA2 in ABA biosynthesis. Interestingly, microarray data derived from Genevestigator suggested that AtSDR3 might have a function that is related to plant defense. Pseudomonas syringae pv. tomato (Pst) DC3000 infection and systemic acquired resistance (SAR) activator application further demonstrated that AtSDR3 plays an important role in plant defense responses at least partially through the regulation of AtPR-1 gene expression.

Yiqi zhuyu decoction combined with FOLFOX-4 as first-line therapy in metastatic colorectal cancer.

OBJECTIVE: To evaluate the efficacy and safety of yiqi zhuyu decoction (YZD) combined with oxaliplatin plus 5-flurouracil/leucovorin (FOLFOX-4) in the patients with metastatic colorectal cancer (MCRC). METHODS: A total of 120 patients with MCRC were randomly divided into the experimental group (FOLFOX-4 plus YZD, 60 cases) and the control group (FOLFOX-4 plus placebo, 60 cases), according to the sequence of hospitalization from January 2005 to December 2007. The treatment was supposed to be continued until disease progression (PD) or for 48 weeks (i.e., up to 24 cycles of FOLFOX-4). Response rate (RR), progression-free survival (PFS), overall survival (OS) and adverse events (AEs) were observed. RESULTS: RR was 41.5% in the experimental group and 34.0% in the control group [odds ratio (OR): 1.18, 95% CI: 0.77 to 1.82, P=0.432]. Median PFS were 9.0 months and 8.0 months, respectively [hazard ratio (HR): 0.78, 95% CI: 0.53 to 1.15, P=0.215]. Median OS were 21.0 months and 18.0 months (HR: 0.65, 95% CI: 0.43 to 0.99, P=0.043) and grade 3/4 AEs were 56.6% and 76.7% (OR: 0.61, 95% CI: 0.18 to 0.87, P=0.020), respectively. CONCLUSIONS: YZD combined with FOLFOX-4 chemotherapy significantly improved OS in this first-line trial in the patients with MCRC and significantly decreased grade 3/4 AEs. However, RR was not improved, and PFS did not reach statistical significance by the addition of YZD. The treatment of YZD combined with FOLFOX-4 may be necessary in order to optimize efficacy and safety.

Clinical study of external application of Qiyu oil gauze for promoting post-operational healing in patients with anal fistula.

OBJECTIVE: To observe the effects of the external application of Qiyu oil gauze (QYOG) for promoting post-operational healing in patients with anal fistula and to explore its mechanism of action so as to provide a beneficial scientific basis for its wide use. METHODS: Sixty patients with anal fistula scheduled to receive simple low anal fistulectomy were equally assigned, according to the sequence of hospitalization, to the tested group and the control group, and their wounds were classified according to longitudinal diameter into three grades (Grade I with a diameter below 2 cm; Grade II, 2-5 cm; and Grade III, over 5 cm). After the operation was completed and the operational wound was sterilized with benzalkonium bromide, the wound substratum was packed with QYOG in the test group and with vaseline gauze in the control group. The packing gauze was changed every day till the wound was healed. The healing time of the patients was observed, and the number of capillaries and positive cell percentages of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) in wound granulation tissues were counted on the 5th day after the operation. RESULTS: The wound healing time was 17.80+/-5.46 days in the test group, which was significantly shorter than that in the control group (21.90+/-6.32 days, P<0.01). The number of capillaries and positive cell percentages of VEGF and bFGF in wound granulation tissues on the 5th day in the tested group were higher than those in the control group (P<0.01), though the difference in EGF between the two groups was insignificant (P>0.05). CONCLUSION: QYOG could shorten the wound healing time after anal fistulectomy, which suggests that it participates in the stimulation of wound granulation tissues to produce VEGF and bFGF, and thus promotes capillary genesis and improves blood circulation in wounds so as to promote wound healing.

Identification of Pseudomonas syringae pv. syringae 61 type III secretion system Hrp proteins that can travel the type III pathway and contribute to the translocation of effector proteins into plant cells.

Pseudomonas syringae translocates effector proteins into plant cells via an Hrp1 type III secretion system (T3SS). T3SS components HrpB, HrpD, HrpF, and HrpP were shown to be pathway substrates and to contribute to elicitation of the plant hypersensitive response and to translocation and secretion of the model effector AvrPto1.

A chaperone-like HrpG protein acts as a suppressor of HrpV in regulation of the Pseudomonas syringae pv. syringae type III secretion system.

The cloned hrp/hrc cluster of Pseudomonas syringae pv. syringae 61 (Pss61) contains 28 proteins, and many of those are assembled into a type III secretion system (TTSS) that is responsible for eliciting the hypersensitive response (HR) in non-host plants and causing diseases on host plants (Huang et al., 1995). hrpG, the second gene in the hrpC operon, encodes a 15.4 kDa cytoplasmic protein whose predicted structure is similar to SicP (E-value: 0.19), a TTSS chaperone of Salmonella typhimurium. Two non-polar hrpG mutants, Pss61-N826 and Pss61-N674, were produced to investigate the biological function of hrpG gene. Pss61-N826, generated by replacing the coding sequence of hrpG with an nptII gene lacking both the promoter and the terminator, was found to be capable of eliciting the wild-type HR; whereas Pss61-N674 generated by replacement of a terminatorless nptII gene in the hrpG coding sequence showed the delayed HR phenotype. Northern and Western blotting analyses showed that the expression of hrpZ, hrcJ and hrcQb genes residing on two different operons in Pss61-N674 was reduced due to the nptII promoter-driven constitutive expression of hrpV that codes for a negative regulator. Interestingly, a plasmid-borne hrpG can derepress the hrp expression in Pss61-N674 and in Pss61 overexpressing HrpV without decreasing the hrpV transcript. Moreover, results of yeast two-hybrid assay, pull-down assay and far Western analysis show that HrpG and HrpV interact with each other in vivo and in vitro. Additionally, HrpV interacts with a positive regulator HrpS according to analysis of a yeast two-hybrid system. Based on the results presented in this study, we propose that HrpG acts as a suppressor of the negative regulator HrpV mediated via protein-protein interaction, leading to modulation of hrp/hrc expression subsequently freeing HrpS to promote the activation of other downstream hrp/hrc genes.

HecA, a member of a class of adhesins produced by diverse pathogenic bacteria, contributes to the attachment, aggregation, epidermal cell killing, and virulence phenotypes of Erwinia chrysanthemi EC16 on Nicotiana clevelandii seedlings.

Erwinia chrysanthemi is representative of a broad class of bacterial pathogens that are capable of inducing necrosis in plants. The E. chrysanthemi EC16 hecA gene predicts a 3,850-aa member of the Bordetella pertussis filamentous hemagglutinin family of adhesins. A hecATn7 mutant was reduced in virulence on Nicotiana clevelandii seedlings after inoculation without wounding. Epifluorescence and confocal laser-scanning microscopy observations of hecA and wild-type cells expressing the green fluorescent protein revealed that the mutant is reduced in its ability to attach and then form aggregates on leaves and to cause an aggregate-associated killing of epidermal cells. Cell killing also depended on production of the major pectate lyase isozymes and the type II, but not the type III, secretion pathway in E. chrysanthemi. HecA homologs were found in bacterial pathogens of plants and animals and appear to be unique to pathogens and universal in necrogenic plant pathogens. Phylogenetic comparison of the conserved two-partner secretion domains in the proteins and the 16S rRNA sequences in respective bacteria revealed the two datasets to be fundamentally incongruent, suggesting horizontal acquisition of these genes. Furthermore, hecA and its two homologs in Yersinia pestis had a G+C content that was 10% higher than that of their genomes and similar to that of plant pathogenic Ralstonia, Xylella, and Pseudomonas spp. Our data suggest that filamentous hemagglutinin-like adhesins are broadly important virulence factors in both plant and animal pathogens.

Pseudomonas syringae exchangeable effector loci: sequence diversity in representative pathovars and virulence function in P. syringae pv. syringae B728a.

Pseudomonas syringae is a plant pathogen whose pathogenicity and host specificity are thought to be determined by Hop/Avr effector proteins injected into plant cells by a type III secretion system. P. syringae pv. syringae B728a, which causes brown spot of bean, is a particularly well-studied strain. The type III secretion system in P. syringae is encoded by hrp (hypersensitive response and pathogenicity) and hrc (hrp conserved) genes, which are clustered in a pathogenicity island with a tripartite structure such that the hrp/hrc genes are flanked by a conserved effector locus and an exchangeable effector locus (EEL). The EELs of P. syringae pv. syringae B728a, P. syringae strain 61, and P. syringae pv. tomato DC3000 differ in size and effector gene composition; the EEL of P. syringae pv. syringae B728a is the largest and most complex. The three putative effector proteins encoded by the P. syringae pv. syringae B728a EEL--HopPsyC, HopPsyE, and HopPsyV--were demonstrated to be secreted in an Hrp-dependent manner in culture. Heterologous expression of hopPsyC, hopPsyE, and hopPsyV in P. syringae pv. tabaci induced the hypersensitive response in tobacco leaves, demonstrating avirulence activity in a nonhost plant. Deletion of the P. syringae pv. syringae B728a EEL strongly reduced virulence in host bean leaves. EELs from nine additional strains representing nine P. syringae pathovars were isolated and sequenced. Homologs of avrPphE (e.g., hopPsyE) and hopPsyA were particularly common. Comparative analyses of these effector genes and hrpK (which flanks the EEL) suggest that the EEL effector genes were acquired by horizontal transfer after the acquisition of the hrp/hrc gene cluster but before the divergence of modern pathovars and that some EELs underwent transpositions yielding effector exchanges or point mutations producing effector pseudogenes after their acquisition.