Il-Pyung Ahn

Vitamin B1 functions as an activator of plant disease resistance

Vitamin B1 (thiamine) is an essential nutrient for humans. Vitamin B1 deficiency causes beriberi, which disturbs the central nervous and circulatory systems. In countries in which rice (Oryza sativa) is a major food, thiamine deficiency is prevalent because polishing of rice removes most of the thiamine in the grain. We demonstrate here that thiamine, in addition to its nutritional value, induces systemic acquired resistance (SAR) in plants. Thiamine-treated rice, Arabidopsis (Arabidopsis thaliana), and vegetable crop plants showed resistance to fungal, bacterial, and viral infections. Thiamine treatment induces the transient expression of pathogenesis-related (PR) genes in rice and other plants. In addition, thiamine treatment potentiates stronger and more rapid PR gene expression and the up-regulation of protein kinase C activity. The effects of thiamine on disease resistance and defense-related gene expression mobilize systemically throughout the plant and last for more than 15 d after treatment. Treatment of Arabidopsis ecotype Columbia-0 plants with thiamine resulted in the activation of PR-1 but not PDF1.2. Furthermore, thiamine prevented bacterial infection in Arabidopsis mutants insensitive to jasmonic acid or ethylene but not in mutants impaired in the SAR transduction pathway. These results clearly demonstrate that thiamine induces SAR in plants through the salicylic acid and Ca2+-related signaling pathways. The findings provide a novel paradigm for developing alternative strategies for the control of plant diseases.

Vitamin B1-Induced Priming Is Dependent on Hydrogen Peroxide and the NPR1 Gene in Arabidopsis

Thiamine confers systemic acquired resistance (SAR) on susceptible plants through priming, leading to rapid counterattack against pathogen invasion and perturbation of disease progress. Priming reduces the metabolic cost required for constitutive expression of acquired resistance. To investigate the effects of priming by thiamine on defense-related responses, Arabidopsis (Arabidopsis thaliana) was treated with thiamine and effects of pathogen challenge on the production of active oxygen species, callose deposition, hypersensitive cell death, and pathogenesis-related 1 (PR1)/Phe ammonia-lyase 1 (PAL1) gene expression was analyzed. Thiamine did not induce cellular and molecular defense responses except for transient expression of PR1 per se; however, subsequent Pseudomonas syringae pv tomato challenge triggered pronounced cellular defense responses and advanced activation of PR1/PAL1 gene transcription. Thiamine treatment and subsequent pathogen invasion triggered hydrogen peroxide accumulation, callose induction, and PR1/PAL1 transcription activation in Arabidopsis mutants insensitive to jasmonic acid (jar1), ethylene (etr1), or abscisic acid (abi3-3), but not in plants expressing bacterial NahG and lacking regulation of SAR (npr1 [nonexpressor of PR genes 1]). Moreover, removal of hydrogen peroxide by catalase almost completely nullified cellular and molecular defense responses as well as SAR abolishing bacterial propagation within plants. Our results indicated that priming is an important cellular mechanism in SAR by thiamine and requires hydrogen peroxide and intact NPR1.

Co-expression of a modified maize ribosome-inactivating protein and a rice basic chitinase gene in transgenic rice plants confers enhanced resistance to sheath blight

Chitinases, β-1,3-glucanases, and ribosome-inactivating proteins are reported to have antifungal activity in plants. With the aim of producing fungus-resistant transgenic plants, we co-expressed a modified maize ribosome-inactivating protein gene, MOD1, and a rice basic chitinase gene, RCH10, in transgenic rice plants. A construct containing MOD1 and RCH10 under the control of the rice rbcS and Act1 promoters, respectively, was co-transformed with a plasmid containing the herbicide-resistance gene bar as a selection marker into rice by particle bombardment. Several transformants analyzed by genomic Southern-blot hybridization demonstrated integration of multiple copies of the foreign gene into rice chromosomes. Immunoblot experiments showed that MOD1 formed approximately 0.5% of the total soluble protein in transgenic leaves. RCH10 expression was examined using the native polyacrylamide-overlay gel method, and high RCH10 activity was observed in leaf tissues where endogenous RCH10 is not expressed. R1 plants were analyzed in a similar way, and the Southern-blot patterns and levels of transgene expression remained the same as in the parental line. Analysis of the response of R2 plants to three fungal pathogens of rice, Rhizoctonia solani, Bipolaris oryzae, and Magnaporthe grisea, indicated statistically significant symptom reduction only in the case of R. solani (sheath blight). The increased resistance co-segregated with herbicide tolerance, reflecting a correlation between the resistance phenotype and transgene expression.

Rhizobacteria-Induced Priming in Arabidopsis Is Dependent on Ethylene, Jasmonic Acid, and NPR1

A nonpathogenic rhizobacterium, Pseudomonas putida LSW17S, elicited systemic protection against Fusarium wilt and pith necrosis caused by Fusarium oxysporum f. sp. lycopersici and P. corrugata in tomato (Lycopersicon esculentum L.). LSW17S also confers disease resistance against P. syringae pv. tomato DC3000 (DC3000) on Arabidopsis ecotype Col-0. To investigate mechanisms underlying disease protection, expression patterns of defense-related genes PR1, PR2, PR5, and PDF1.2 and cellular defense responses such as hydrogen peroxide accumulation and callose deposition were investigated. LSW17S treatment exhibited the typical phenomena of priming. Strong and faster transcription of defense-related genes was induced and hydrogen peroxide or callose were accumulated in Arabidopsis treated with LSW17S and infected with DC3000. In contrast, individual actions of LSW17S and DC3000 did not elicit rapid molecular and cellular defense responses. Priming by LSW17S was translocated systemically and retained for more than 10 days. Treatment with LSW17S reduced pathogen proliferation in Arabidopsis ecotype Col-0 expressing bacterial NahG; however, npr1, etr1, and jar1 mutations impaired inhibition of pathogen growth. Cellular and molecular priming responses support these results. In sum, LSW17S primes Arabidopsis for NPR1-, ethylene-, and jasmonic acid-dependent disease resistance, and efficient molecular and cellular defense responses.

Rice defense mechanisms against Cochliobolus miyabeanus and Magnaporthe grisea are distinct

ABSTRACT Responses of rice to Magnaporthe grisea and Cochliobolus miyabeanus were compared. In Tetep, a rice cultivar resistant to both fungi, pathogen inoculation rapidly triggered the hypersensitive reaction (HR), resulting in microscopic cell death. In rice cv. Nakdong, susceptible to both pathogens, M. grisea did not cause HR, whereas C. miyabeanus caused rapid cell death similar to that associated with HR, which appeared similar to that observed in cv. Tetep, yet failed to block fungal ramification. Treatment with conidial germination fluid (CGF) from C. miyabeanus induced rapid cell death in both cultivars, suggesting the presence of phytotoxins in CGF. Pretreatment of cv. Nakdong with CGF significantly increased resistance to M. grisea, while the same treatment was ineffective against C. miyabeanus. Similarly, in cv. Nakdong, benzothiadiazole (BTH) significantly increased resistance to M. grisea, but was ineffective against C. miyabeanus. Methyl jasmonate (MeJA) treatment appeared to be ineffective against either fungus. Increased resistance of cv. Nakdong to M. grisea by BTH or CCF treatment was correlated with more rapid induction of three monitored PR genes. Application of MeJA resulted in the expression of JAmyb in cv. Nakdong being induced faster than in untreated plants in response to M. grisea infection. In contrast, the expression pattern of the PR and JAmyb genes in response to C. miyabeanus was nearly identical between cvs. Nakdong and Tetep, and neither BTH nor MeJA treatment significantly modified their expression patterns in response to C. miyabeanus infection. Our results suggest that rice employs distinct mechanisms for its defense against M. grisea versus C. miyabeanus.

Glufosinate Ammonium-Induced Pathogen Inhibition and Defense Responses Culminate in Disease Protection in bar -Transgenic Rice

Glufosinate ammonium diminished developments of rice (Oryza sativa) blast and brown leaf spot in 35S:bar-transgenic rice. Pre- and postinoculation treatments of this herbicide reduced disease development. Glufosinate ammonium specifically impeded appressorium formation of the pathogens Magnaporthe grisea and Cochliobolus miyabeanus on hydrophobic surface and on transgenic rice. In contrast, conidial germination remained unaffected. Glufosinate ammonium diminished mycelial growth of two pathogens; however, this inhibitory effect was attenuated in malnutrition conditions. Glufosinate ammonium caused slight chlorosis and diminished chlorophyll content; however, these alterations were almost completely restored in transgenic rice within 7 d. Glufosinate ammonium triggered transcriptions of PATHOGENESIS-RELATED (PR) genes and hydrogen peroxide accumulation in transgenic rice and PR1 transcription in Arabidopsis (Arabidopsis thaliana) wild-type ecotype Columbia harboring 35S:bar construct. All transgenic Arabidopsis showed robust hydrogen peroxide accumulation by glufosinate ammonium. This herbicide also induced PR1 transcription in etr1 and jar1 expressing bar; however, no expression was observed in NahG and npr1. Fungal infection did not alter transcriptions of PR genes and hydrogen peroxide accumulation induced by glufosinate ammonium. Infiltration of glufosinate ammonium did not affect appressorium formation of M. grisea in vivo but inhibited blast disease development. Hydrogen peroxide scavengers nullified blast protection and transcriptions of PR genes by glufosinate ammonium; however, they did not affect brown leaf spot progression. In sum, both direct inhibition of pathogen infection and activation of defense systems were responsible for disease protection in bar-transgenic rice.

Calcium/Calmodulin-Dependent Signaling for Prepenetration Development in Colletotrichum gloeosporioides

ABSTRACT Colletotrichum gloeosporioides forms a specialized infection structure, an appressorium, for host infection. Contacting hard surface induces appressorium formation in C. gloeosporioides, whereas hydrophobicity of the contact surface does not affect this infection-related differentiation. To determine if the calcium/calmodulin-dependent signaling system is involved in prepenetration morphogenesis in C. gloeosporioides pathogenic on red pepper, effects of calcium chelator (EGTA), phospholipase C inhibitor (neomycin), intracellular calcium modulators (TMB-8 and methoxy verampamil), and calmodulin antagonists (chloroproma-zine, phenoxy benzamine, and W-7) were tested on conidial germination and appressorium formation. Exogenous addition of Ca(2+), regardless of concentration, augmented conidial germination, while appressorial differentiation decreased at higher concentrations. Inhibition of appressorium formation by EGTA was partly restored by the addition of calcium ionophore A23187 or CaCl(2). Calcium channel blockers and calmodulin antagonists specifically reduced appressorium formation at micromolar levels. These results suggest that biochemical processes controlled by the calcium/calmodulin signaling system are involved in the induction of prepenetration morphogenesis in C. gloeosporioides pathogenic on red pepper.

Priming by Rhizobacterium Protects Tomato Plants from Biotrophic and Necrotrophic Pathogen Infections through Multiple Defense Mechanisms

A selected strain of rhizobacterium, Pseudomonas putida strain LSW17S (LSW17S), protects tomato plants (Lycopersicon esculentum L. cv. Seokwang) from bacterial speck by biotrophic Pseudomonas syringae pv. tomato strain DC3000 (DC3000) and bacterial wilt by necrotrophic Ralstonia solanacearum KACC 10703 (Rs10703). To investigate defense mechanisms induced by LSW17S in tomato plants, transcription patterns of pathogenesis-related (PR) genes and H2O2 production were analyzed in plants treated with LSW17S and subsequent pathogen inoculation. LSW17S alone did not induce transcriptions of employed PR genes in leaves and roots. DC3000 challenge following LSW17S triggered rapid transcriptions of PR genes and H2O2 production in leaves and roots. Catalase infiltration with DC3000 attenuated defense-related responses and resistance against DC3000 infection. Despite depriving H2O2 production and PR1b transcription by the same treatment, resistance against Rs10703 infection was not deterred significantly. H2O2 is indispensable for defense signaling and/or mechanisms primed by LSW17S and inhibition of bacterial speck, however, it is not involved in resistance against bacterial wilt.

Over-expression of rice leucine-rich repeat protein results in activation of defense response, thereby enhancing resistance to bacterial soft rot in Chinese cabbage

Pectobacterium carotovorum subsp. carotovorum causes soft rot disease in various plants, including Chinese cabbage. The simple extracellular leucine-rich repeat (eLRR) domain proteins have been implicated in disease resistance. Rice leucine-rich repeat protein (OsLRP), a rice simple eLRR domain protein, is induced by pathogens, phytohormones, and salt. To see whether OsLRP enhances disease resistance to bacterial soft rot, OsLRP was introduced into Chinese cabbage by Agrobacterium-mediated transformation. Two independent transgenic lines over-expressing OsLRP were generated and further analyzed. Transgenic lines over-expressing OsLRP showed enhanced disease resistance to bacterial soft rot compared to non-transgenic control. Bacterial growth was retarded in transgenic lines over-expressing OsLRP compared to non-transgenic controls. We propose that OsLRP confers enhanced resistance to bacterial soft rot. Monitoring expression of defense-associated genes in transgenic lines over-expressing OsLRP, two different glucanases and Brassica rapa polygalacturonase inhibiting protein 2, PDF1 were constitutively activated in transgenic lines compared to non-transgenic control. Taken together, heterologous expression of OsLRP results in the activation of defense response and enhanced resistance to bacterial soft rot.

Calcium/calmodulin-dependent signaling for prepenetration development in Cochliobolus miyabeanus infecting rice

Cochliobolus miyabeanus forms a specialized infection structure, an appressorium, to infect rice. Contacting a hard surface induces appressorium formation in C. miyabeanus, while the hydrophobicity of the substratum does not affect this morphogenic infection event. To determine whether the calcium/calmodulin-dependent signaling system is involved in prepenetration morphogenesis in C. miyabeanus, the effects of a calcium chelator (ethylene glycol tetraacetic acid; EGTA), phospholipase C inhibitor (neomycin), intracellular calcium channel blocker (TMB-8), calmodulin antagonists (chlorpromazine, phenoxybenzamine, and W-7), and calcineurin inhibitor (cyclosporin A) on morphogenesis and infection were examined. Addition of Ca2+ and the calcium ionophore A23187 did not affect conidial germination, while the number of appressoria decreased with higher concentrations. EGTA inhibited conidial germination and appressorium formation. The calcium channel blocker did not affect appressorium formation at any concentration; however, calmodulin antagonists and the calcineurin inhibitor specifically reduced appressorium formation at the micromolar level. One of the calmodulin antagonists, W-7, also inhibited accumulation of mRNA of the calmodulin gene within germinating conidia and/or appressorium-forming germ tubes. Thus, biochemical processes controlled by the calcium/calmodulin signaling system seem to be involved in the induction of prepenetration morphogenesis on rice.