Ju Yeon Park

Inactivation of pqq genes of Enterobacter intermedium 60‐2G reduces antifungal activity and induction of systemic resistance

Enterobacter intermedium 60-2G, a phosphate solubilizing bacterium, has the ability to induce systemic resistance in plants against soft rot pathogen Erwinia carotovora. Glucose dehydrogenase, an enzyme that utilizes pyrroloquinoline quinone (PQQ) as a cofactor, is required for the synthesis of gluconic acid by E. intermedium 60-2G. Here, we report that the pqqA and pqqB genes are required for phosphate solubilization and induced systemic resistance against a soft rot pathogen in tobacco. Mutations in either the pqqA or pqqB gene abolished the production of 2-ketogluconic acid and eliminated the ability of E. intermedium to solubilize hydroxyapatite. Addition of gluconic acid to the growth media restored the ability of the pqqA mutant to produce 2-ketogluconic acid. Interestingly, both pqqA and pqqB mutants of E. intermedium lost their ability to inhibit the growth of the rice pathogen Magnaporthe grisea KI-409. Additionally, induced systemic resistance against the soft rot pathogen was attenuated in the pqq mutants. These functions were restored by complementation with the wild-type pqq gene cluster. Our findings suggest that PQQ plays an important function in beneficial traits including phosphate solubilization, antifungal activity, and induced systemic resistance of E. intermedium, possibly by acting as a cofactor for several enzymes including glucose dehydrogenase.

Draft Genome Sequence of the Biocontrol Bacterium Pseudomonas putida B001, an Oligotrophic Bacterium That Induces Systemic Resistance to Plant Diseases

Pseudomonas putida B001 is a rhizobacterium that was isolated on the basis of its abilities to grow under low-nutrient conditions and induce systemic resistance against bacterial, fungal, and viral diseases of plants. Here we report the draft genome sequence and automatic annotation of strain B001. Comparison of this sequence to the sequenced genome of P. putida KT2440 points to a subset of gene functions that may be related to the defense-inducing functions of B001.

Antiviral peptide from Pseudomonas chlororaphis O6 against tobacco mosaic virus (TMV)

Although Pseudomonas chlororaphis O6 (O6) is known to be a rhizobacterium capable of inducing systemic resistance against plant virus, its antiviral products from O6 remain unknown. In the present study, an antiviral cyclic peptide was identified from the cell-free supernatant of O6. O6 cultures grown on Luria Bertani medium were centrifuged, and the resulting supernatant was extracted with organic solvent, followed by a series of column chromatography and preparative high performance liquid chromatography (HPLC). Bioassay-guided fractionations were involved in the isolation of antiviral products against tobacco mosaic virus (TMV). Time of flight mass spectrometry (TOF-MS) analysis of the isolated product detected (M+H)+ peak at m/z 887.4242 that generated m/z 756.3859, 657.3180, 556.2724, 459.2208, 345.1873, and 171.1130 as the main fragment ions. NMR analyses characterized all protons and carbons of the isolated product. Based on the data, the isolated antiviral product was determined to be a cyclic peptide with molecular formula C39H67N9O12S consisting of seven different amino acids. The antiviral peptide exhibited more than 95% disease suppression of TMV at 1,000 μg/mL. O6 may play a role in promoting plant growth.

Identification and Transcriptional Analysis of Priming Genes in Arabidopsis thaliana Induced by Root Colonization with Pseudomonas chlororaphis O6

Department of Plant Pathology, The Ohio State University, OARDC, 1680 Madison Avenue, Wooster, Ohio 44691, USA(Received on May 11, 2011; Revised on July 21, 2011; Accepted on July 21, 2011)Root colonization of Arabidopsis thaliana with Pseudo-monas chlororaphis O6 induces systemic tolerance againstdiverse pathogens, as well as drought and salt stresses. Inthis study, we demonstrated that 11 genes in the leaveswere up-regulated, and 5 genes were down-regulated asthe result of three- to five-days root colonization by P.chlororaphis O6. The identified priming genes wereinvolved in cell signaling, transcription, protein synthesis,and degradation. In addition, expression of selectedpriming genes were induced in P. chlororaphis O6-colonized plants subjected to water withholding. Genesencoding defense proteins in signaling pathways re-gulated by jasmonic acid and ethylene, such as VSP1and PDF1.2, were additional genes with enhancedexpression in the P. chlororaphis O6-colonized plants.This study indicated that the expression of priminggenes, as well as genes involved in jasmonic acid- andethylene-regulated genes may play an important role inthe systemic induction of both abiotic and biotic stressdue to root colonization by P. chlororaphis O6. Keywords :disease defense, drought stress, induced systemicresistance, priming Induced resistance or tolerance against abiotic and bioticstresses is stimulated in plants by two systems. In systemicacquired resistance (SAR), plants develop a broad spectrumof systemic resistance against plant diseases upon pathogenattack. SAR involves elevated salicylic acid (SA) levels andboth local and systemic accumulation of pathogenesis-related (PR) proteins (Van Loon et al., 2006). Expression ofPR genes, PR-1 in particular, is used as a marker for SARinduction (Durrant and Dong, 2004) and a key regulatoryprotein is NPR1 (Pierterse and Van Loon, 2004). Aciben-zolar-S-methyl is a chemical that activates the SARpathway (Bostock, 2005).Colonization of roots by microbes also elicits systemicresistance against abiotic stresses and a broad spectrum ofdiseases caused by bacterial, fungal, and viral pathogens(Kim et al., 2011). Induced systemic resistance (ISR) causesthe plant to induce different array of genes than SAR. Onerhizobacterium, Pseudomonas chlororaphis O6, inducesISR for many plant diseases and enhanced drought tolerance(Han et al., 2006; Kim et al., 2004a; Ryu et al., 2007;Spencer et al., 2003). Three days of root colonization by P.chlororaphis O6 reduces opening of leaf stomata (Cho etal., 2008), thereby demonstrating a rapidly induced systemiceffect.Enhanced plant gene activation was observed in oilseedrape treated with the ISR-inducers P. thivervalensis MLG45and Bacillus amyloliquefaciens (Carieaux et al., 2003;Sarosh et al., 2009), in response to abiotic stress and afterchallenge by insects (Conrath et al., 2002). Differentialexpressed cucumber genes were induced in P. chlororaphisO6-colonized cucumber after challenge with the fungalpathogen Corynespora cassicola (Kim et al., 2004a). Oneinduced plant gene encoding galactinol synthase (CsGolS1),when over expressed in tobacco, increases galactinol levels,enhances disease resistance against Botrytis cinerea andPectobacterium carotovorum, and provides drought andsalinity tolerance (Kim et al., 2008). Another study ofplants inoculated with the ISR-inducer P. fluorescens WCS417r, showed 81 genes to increase in expression afterchallenge inoculation with the bacterial speck pathogen P.syringae pv. tomato (Verhagen et al., 2004). Few changes ingene expression were noted in plants colonized by P.fluorescens WCS 417r without pathogen challenge (VanWees et al., 1999; Verhagen et al., 2004). This enhancedgene activation in the colonized plants by ISR-inducingrhizobacteria is referred to as priming. However, the plant

The GacS-regulated sigma factor RpoS governs production of several factors involved in biocontrol activity of the rhizobacterium Pseudomonas chlororaphis O6.

Pseudomonas chlororaphis O6 possesses many beneficial traits involved in biocontrol of plant diseases. In this paper, we examined the effect of a mutation in rpoS encoding a stress-related alternative sigma factor to better understand the regulation of these traits. Biochemical studies indicated that production of acyl homoserine lactones was altered and phenazine was increased in the P. chlororaphis O6 rpoS mutant. The rpoS mutation reduced hydrogen cyanide levels, but the rpoS mutant still displayed a level of in vitro antifungal activity against Fusarium graminearum and Alternaria alternata. Tomato root colonization by the rpoS mutant was lower than that by the wild type at 5, 7, and 13 days after inoculation. The rpoS mutant was less effective than the wild type in induction of systemic resistance to two foliar pathogens after root inoculation of the tomato plants. Our findings demonstrate that the stationary-phase sigma factor RpoS regulates production of several key factors involved in the biocontrol potential of P. chlororaphis O6, some independently of the global regulator GacS.

Draft Genome Sequence of the Biocontrol Bacterium Chromobacterium sp. Strain C-61

Chromobacterium sp. strain C-61 is a plant-associated bacterium with proven capacities to suppress plant diseases. Here, we report the draft genome sequence and automatic annotation of strain C-61. A comparison of this sequence to the sequenced genome of Chromobacterium violaceum ATCC 12472 indicates the novelty of C-61 and a subset of gene functions that may be related to its biocontrol activities.

The RpoS Sigma Factor Negatively Regulates Production of IAA and Siderophore in a Biocontrol Rhizobacterium, Pseudomonas chlororaphis O6.

The stationary-phase sigma factor, RpoS, influences the expression of factors important in survival of Pseudomonas chlororaphis O6 in the rhizosphere. A partial proteomic profile of a rpoS mutant in P. chlororaphis O6 was conducted to identify proteins under RpoS regulation. Five of 14 differentially regulated proteins had unknown roles. Changes in levels of proteins in P. chlororaphis O6 rpoS mutant were associated with iron metabolism, and protection against oxidative stress. The P. chlororaphis O6 rpoS mutant showed increased production of a pyoverdine-like siderophore, indole acetic acid, and altered isozyme patterns for peroxidase, catalase and superoxide dismutase. Consequently, sensitivity to hydrogen peroxide exposure increased in the P. chlororaphis O6 rpoS mutant, compared with the wild type. Taken together, RpoS exerted regulatory control over factors important for the habitat of P. chlororaphis O6 in soil and on root surfaces. The properties of several of the proteins in the RpoS regulon are currently unknown.

The global regulator GacS regulates biofilm formation in Pseudomonas chlororaphis O6 differently with carbon source.

An aggressive root colonizer, Pseudomonas chlororaphis O6 produces various secondary metabolites that impact plant health. The sensor kinase GacS is a key regulator of the expression of biocontrol-related traits. Biofilm formation is one such trait because of its role in root surface colonization. This paper focuses on the effects of carbon source on biofilm formation. In comparison with the wild type, a gacS mutant formed biofilms at a reduced level with sucrose as the major carbon source but at much higher level with mannitol in the defined medium. Biofilm formation by the gacS mutant occurred without phenazine production and in the absence of normal levels of acyl homoserine lactones, which promote biofilms with other pseudomonads. Colonization of tomato roots was similar for the wild type and gacS mutant, showing that any differences in biofilm formation in the rhizosphere were not of consequence under the tested conditions. The reduced ability of the gacS mutant to induce systemic resistance against tomato leaf mold and tomato gray mold was consistent with a lack of production of effectors, such as phenazines. These results demonstrated plasticity in biofilm formation and root colonization in the rhizosphere by a beneficial pseudomonad.

Isolation and Characterization of Oligotrophic Bacteria Possessing Induced Systemic Disease Resistance against Plant Pathogens

Biocontrol microbes have mainly been screened among large collections of microorganisms via. nutrient-rich in vitro assays to identify novel and effective isolates. However, thus far, isolates from only a few genera, mainly spore-forming bacilli, have been commercially developed. In order to isolate field-effective biocontrol microbes, we screened for more than 200 oligotrophic bacterial strains, isolated from rhizospheres of various soil samples in Korea, which induced systemic resistance against the soft-rot disease caused by Pectobacterium carotovorum SCC1; we subsequently conducted in planta bioassay screening. Two oligotrophic bacterial strains were selected for induced systemic disease resistance against the Tobacco Mosaic Virus and the gray mold disease caused by Botrytis cinerea. The oligotrophic bacterial strains were identified as Pseudomonas manteilii B001 and Bacillus cereus C003 by biochemical analysis and the phylogenetic analysis of the 16S rRNA sequence. These bacterial strains did not exhibit any antifungal activities against plant pathogenic fungi but evidenced several other beneficial biocontrol traits, including phosphate solubilization and gelatin utilization. Collectively, our results indicate that the isolated oligotrophic bacterial strains possessing induced systemic disease resistance could provide useful tools as effective biopesticides and might be successfully used as cost-effective and preventive biocontrol agents in the field.

Polyamine is a critical determinant of Pseudomonas chlororaphis O6 for GacS-dependent bacterial cell growth and biocontrol capacity

The Gac/Rsm network regulates, at the transcriptional level, many beneficial traits in biocontrol-active pseudomonads. In this study, we used Phenotype MicroArrays, followed by specific growth studies and mutational analysis, to understand how catabolism is regulated by this sensor kinase system in the biocontrol isolate Pseudomonas chlororaphis O6. The growth of a gacS mutant was decreased significantly relative to that of the wild-type on ornithine and arginine, and on the precursor of these amino acids, N-acetyl-l-glutamic acid. The gacS mutant also showed reduced production of polyamines. Expression of the genes encoding arginine decarboxylase (speA) and ornithine decarboxylases (speC) was controlled at the transcriptional level by the GacS sensor of P. chlororaphis O6. Polyamine production was reduced in the speC mutant, and was eliminated in the speAspeC mutant. The addition of exogenous polyamines to the speAspeC mutant restored the in vitro growth inhibition of two fungal pathogens, as well as the secretion of three biological control-related factors: pyrrolnitrin, protease and siderophore. These results extend our knowledge of the regulation by the Gac/Rsm network in a biocontrol pseudomonad to include polyamine synthesis. Collectively, our studies demonstrate that bacterial polyamines act as important regulators of bacterial cell growth and biocontrol potential.