Song Mi Cho

2R,3R-butanediol, a bacterial volatile produced by Pseudomonas chlororaphis O6, is involved in induction of systemic tolerance to drought in Arabidopsis thaliana.

Root colonization of plants with certain rhizobacteria, such as Pseudomonas chlororaphis O6, induces tolerance to biotic and abiotic stresses. Tolerance to drought was correlated with reduced water loss in P. chlororaphis O6-colonized plants and with stomatal closure, indicated by size of stomatal aperture and percentage of closed stomata. Stomatal closure and drought resistance were mediated by production of 2R,3R-butanediol, a volatile metabolite of P. chlororaphis O6. Root colonization with bacteria deficient in 2R,3R-butanediol production showed no induction of drought tolerance. Studies with Arabidopsis mutant lines indicated that induced drought tolerance required the salicylic acid (SA)-, ethylene-, and jasmonic acid-signaling pathways. Both induced drought tolerance and stomatal closure were dependent on Aba-1 and OST-1 kinase. Increases in free SA after drought stress of P. chlororaphis O6-colonized plants and after 2R,3R-butanediol treatment suggested a primary role for SA signaling in induced drought tolerance. We conclude that the bacterial volatile 2R,3R-butanediol was a major determinant in inducing resistance to drought in Arabidopsis through an SA-dependent mechanism.

Galactinol is a signaling component of the induced systemic resistance caused by Pseudomonas chlororaphis O6 root colonization.

Root colonization by Pseudomonas chlororaphis O6 in cucumber elicited an induced systemic resistance (ISR) against Corynespora cassiicola. In order to gain insight into O6-mediated ISR, a suppressive subtractive hybridization technique was applied and resulted in the isolation of a cucumber galactinol synthase (CsGolS1) gene. The transcriptional level of CsGolS1 and the resultant galactinol content showed an increase several hours earlier under O6 treatment than in the water control plants following C. cassiicola challenge, whereas no difference was detected in the plants without a pathogen challenge. The CsGolS1-overexpressing transgenic tobacco plants demonstrated constitutive resistance against the pathogens Botrytis cinerea and Erwinia carotovora, and they also showed an increased accumulation in galactinol content. Pharmaceutical application of galactinol enhanced the resistance against pathogen infection and stimulated the accumulation of defense-related gene transcripts such as PR1a, PR1b, and NtACS1 in wild-type tobacco plants. Both the CsGolS1-overexpressing transgenic plants and the galactinol-treated wild-type tobacco plants also demonstrated an increased tolerance to drought and high salinity stresses.

Tobacco cultivars vary in induction of systemic resistance against Cucumber mosaic virus and growth promotion by Pseudomonas chlororaphis O6 and its gacS mutant

The colonization of plant roots with certain rhizosphere bacteria promotes plant growth and induces long lasting systemic protection against a broad spectrum of plant pathogens. The role of the global regulator, GacS, in the rhizosphere colonist Pseudomonas chlororaphis O6 in stimulating growth promotion and induced resistance against Cucumber mosaic virus was examined in tobacco. Responses were compared in tobacco cvs Samsun and GX3. Root colonization of Samsun with wild-type O6 and the gacS complemented mutant-elicited reduced viral symptoms and viral titre. On GX3, there was little affect on symptoms when roots were colonized by the wild-type, gacS mutant or complemented mutant but colonization by both the wild-type and the gacS mutant lowered viral titre. Wild-type O6 and the gacS mutant caused plant growth to be maintained in both tobacco cultivars after viral infection, although the affect was stronger with GX3 than Samsun. In contrast, although a chemical inducer, benzothiadiazole, reduced symptoms and viral titre in both cultivars, plant growth was suppressed. Our results indicate rhizobacteria-elicited induced viral resistance without a negative impact on growth but there was a differential response between cultivars. Detailed knowledge regarding the mechanisms inherent to these differences between cultivars requires further investigation.

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.

Induced Systemic Drought and Salt Tolerance by Pseudomonas chlororaphis O6 Root Colonization is Mediated by ABA-independent Stomatal Closure

Institute of Environmentally-Friendly Agriculture, Department of Plant Biotechnology, Chonnam National University, Gwangju500-757, Korea (Received on November 13, 2011; Revised on January 5, 2012; Accepted on January 11, 2012)Root colonization by the rhizobacterium Pseudomonaschlororaphis O6 in Arabidopsis thaliana Col-0 plantsresulted in induced tolerance to drought and salinitycaused by halide salt-generated ionic stress but not byosmotic stress caused by sorbitol. Stomatal aperturesdecreased following root colonization by P. chlororaphisO6 in both wild-type and ABA-insensitive Arabidopsismutant plants. These results suggest that an ABA-independent stomatal closure mechanism in the guardcells of P. chlororaphis O6-colonized plants could be akey phenotype for induced systemic tolerance todrought and salt stress. Keywords : abiotic stress, induced tolerance to environ-mental stress, priming, stomatal closurePlants possess various survival systems against abioticstresses, such as cold, drought, and salinity (Zhu, 2001).Under environmental stresses, the level of the planthormone, abscisic acid (ABA) increases triggering adaptiveresponses essential for survival (Zhu, 2001). Duringdrought and salt stresses, ABA induces stomatal closure tominimize water loss through transpiration (Leung andGiraudat, 1998). Consequently, ABA-biosynthesis mutants(aba mutants) and some of the ABA-response plantmutants (i.e. the ABA-insensitive abi mutants) aresusceptible to drought stress, due to problem of stomatalaperture regulation (Leung and Giraudat, 1998; Schroederet al., 2001). However, another plant growth regulator,jasmonate (Evans, 2003; Suhita et al., 2004), also stimu-lates stomatal closure under drought conditions (Creelmanand Mullet, 1997).Root colonization of certain plant-associated microbeselicits physiological and biochemical change to enhancesystemic resistance against various biotic and abioticstresses in plants (Kim et al., 2011; Yang et al., 2010),termed to as “induced systemic resistance (ISR)” or“induced systemic tolerance (IST)”. IST against drought orsalt stresses in plants can be induced with systemicapplication of certain rhizobacteria including Gram-positive Bacillus strains (Ryu et al., 2004; Timmusk andWagner 1999; Zhang et al., 2010), an endophytic fungalisolate, Trichoderma harziarum (Bae et al., 2010), andcertain Gram-negative bacterial isolates, such as ACC-deaminase producing bacteria (Mayak et al., 2004). Rootcolonization by Pseudomonas chlororaphis O6 inducedsystemic resistance against a broad spectrum of plantdiseases caused by viral, bacterial, and fungal pathogens invarious plants by jasmonic acid-ethylene related pathways(Cho et al., 2008; Kim et al., 2004; Ryu et al., 2007;Spencer et al., 2003). Additionally, root colonization by P.chlororaphis O6 induced systemic tol erance against drought,a process accompanied by stomatal closure. Applying2R,3R-butanediol, a volatile produced by P. chlororaphisO6, resulted in induced tolerance through a salicylic acid(SA), jasmonic acid (JA) and ethylene-dependent mech-anism (Cho et al., 2008). However, mechanisms involvedin microbe-mediated IST against drought have not beencharacterized or elucidated.In this study, we tested the hypothesis that P. chloro-raphis O6 induces tolerance to other abiotic stresses, suchas salinity, osmotic pressure, cold and heat. We used addi-tional Arabidopsis mutants altered in ABA signalingpathways to identify the role of ABA in induced abioticstress tolerance. Parental A. thaliana ecotypes Columbia(Col-0) or Landsberg erecta (Ler), and mutant and trans-genic lines were obtained from the Ohio State University

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

Cladosporium sp. is the Major Causal Agent in the Microbial Complex Associated with the Skin Sooty Dapple Disease of the Asian Pear in Korea

Skin sooty dapple disease, a fungal disease that lowers Asian pear fruit quality, has emerged recently in Korea but has not yet been thoroughly characterized. This disease affects the surface of fruit, leaves, and young shoots of the Asian pear, typically appearing as a dark or pale black dapple on the fruit surface. The disease initiates on the fruit with small circular lesions that become bigger, eventually spreading to form large circular or indefinite lesions. Sparse dark or flourishing white-greyish aerial mycelia and appearance of a dark or pale black dapple on the fruit surface are typical signs of this disease. The disease was severe during cold storage of the Niitaka and Chuhwangbae varieties, but more limited on the Gamcheonbae and Hwangkeumbae varieties. To identify causal pathogens, 123 fungal isolates were obtained from lesions. The fungi that caused typical skin sooty dapple disease symptoms in our bioassay were identified. Based on their morphological characteristics, 74% of the isolates were Cladosporium sp. and 5-7 % of the isolates were Leptosphaerulina sp., Tripospermum sp., or Tilletiopsis sp. None of the isolates caused severe soft rot by injection to a wound plug, but some of the Cladosporium sp. isolates caused mild maceration. Therefore this microbiol complex cannot account for the soft rot also observed in stored fruits. The high frequency of isolation of Cladosporium sp. from disease tissues and bioassay on pear fruit surface suggest that Cladosporium sp. could be a major pathogen in the microbial complex associated with skin sooty dapple disease of the Asian pear in Korea.

Extracellular Polymeric Substances of Pseudomonas chlororaphis O6 Induce Systemic Drought Tolerance in Plants

Pseudomonas chlororaphis O6 induces systemic tolerance in plants against drought stress. A volatile, 2R, 3R-butanediol, produced by the bacterium causes partial stomatal closure, thus, limiting water loss from the plant. In this study, we report that applications of extracellular polymeric substances (EPS) from P. chlororaphis O6 to epidermal peels of leaves of Arabidopsis thaliana also reduce the size of stomatal openings. Growth of A. thaliana seedlings with applications of the EPS from P. chlororaphis O6 reduced the extent of wilting when water was withheld from the plants. Fluorescence measurements showed photosystem II was protected in the A. thaliana leaves in the water stressed EPS-exposed plants. These findings indicate that P. chlororaphis O6 has redundancy in traits associated with induction of mechanisms to limit water stress in plants.