Priming of Immune System in Tomato by Treatment with Low Concentration of L-Methionine.

Various metabolites, including phytohormones, phytoalexins, and amino acids, take part in the plant immune system. Herein, we analyzed the effects of L-methionine (Met), a sulfur-containing amino acid, on the plant immune system in tomato. Treatment with low concentrations of Met enhanced the resistance of tomato to a broad range of diseases caused by the hemi-biotrophic bacterial pathogen Pseudomonas syringae pv. tomato (Pst) and the necrotrophic fungal pathogen Botrytis cinerea (Bc), although it did not induce the production of any antimicrobial substances against these pathogens in tomato leaf tissues. Analyses of gene expression and phytohormone accumulation indicated that Met treatment alone did not activate the defense signals mediated by salicylic acid, jasmonic acid, and ethylene. However, the salicylic acid-responsive defense gene and the jasmonic acid-responsive gene were induced more rapidly in Met-treated plants after infection with Pst and Bc, respectively. These findings suggest that low concentrations of Met have a priming effect on the phytohormone-mediated immune system in tomato.

Characterization of Disease Resistance Induced by a Pyrazolecarboxylic Acid Derivative in Arabidopsis thaliana.

Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is induced through the salicylic acid (SA)-mediated signaling pathway. Here, we characterized 3-chloro-1-methyl-1H-pyrazole-5-carboxylic acid (CMPA) as an effective SAR inducer in Arabidopsis. The soil drench application of CMPA enhanced a broad range of disease resistance against the bacterial pathogen Pseudomonas syringae and fungal pathogens Colletotrichum higginsianum and Botrytis cinerea in Arabidopsis, whereas CMPA did not show antibacterial activity. Foliar spraying with CMPA induced the expression of SA-responsible genes such as PR1, PR2 and PR5. The effects of CMPA on resistance against the bacterial pathogen and the expression of PR genes were observed in the SA biosynthesis mutant, however, while they were not observed in the SA-receptor-deficient npr1 mutant. Thus, these findings indicate that CMPA induces SAR by triggering the downstream signaling of SA biosynthesis in the SA-mediated signaling pathway.

Strigolactones Modulate Salicylic Acid-Mediated Disease Resistance in Arabidopsis thaliana.

Strigolactones are low-molecular-weight phytohormones that play several roles in plants, such as regulation of shoot branching and interactions with arbuscular mycorrhizal fungi and parasitic weeds. Recently, strigolactones have been shown to be involved in plant responses to abiotic and biotic stress conditions. Herein, we analyzed the effects of strigolactones on systemic acquired resistance induced through salicylic acid-mediated signaling. We observed that the systemic acquired resistance inducer enhanced disease resistance in strigolactone-signaling and biosynthesis-deficient mutants. However, the amount of endogenous salicylic acid and the expression levels of salicylic acid-responsive genes were lower in strigolactone signaling-deficient max2 mutants than in wildtype plants. In both the wildtype and strigolactone biosynthesis-deficient mutants, the strigolactone analog GR24 enhanced disease resistance, whereas treatment with a strigolactone biosynthesis inhibitor suppressed disease resistance in the wildtype. Before inoculation of wildtype plants with pathogenic bacteria, treatment with GR24 did not induce defense-related genes; however, salicylic acid-responsive defense genes were rapidly induced after pathogenic infection. These findings suggest that strigolactones have a priming effect on Arabidopsis thaliana by inducing salicylic acid-mediated disease resistance.

Induction of tocopherol biosynthesis through heat shock treatment in Arabidopsis.

Plants have developed various self-defense systems to survive many types of unfavorable conditions. Heat shock (HS) treatment, an abiotic stress, activates salicylic acid (SA) biosynthesis to enhance resistance to biotic stresses in some plant species. Since SA is produced from the shikimate pathway, other related metabolic pathways were expected to be upregulated by HS treatment. We speculated that tocopherol biosynthesis utilizing chorismic acid would be activated by HS treatment. In Arabidopsis, expression analysis of tocopherol biosynthetic genes, HPPD, VTE2, VTE3, VTE1, and VTE4, in combination with measurement of metabolites, indicated that HS treatment enhanced the biosynthesis and accumulation of tocopherols. Analyses using an SA biosynthesis-deficient mutant indicated that the upregulation of tocopherol biosynthesis was independent of the SA-mediated signaling pathway.

Arabidopsis MAPKKK δ-1 is required for full immunity against bacterial and fungal infection.

The genome of Arabidopsis encodes more than 60 mitogen-activated protein kinase kinase (MAPKK) kinases (MAPKKKs); however, the functions of most MAPKKKs and their downstream MAPKKs are largely unknown. Here, MAPKKK δ-1 (MKD1), a novel Raf-like MAPKKK, was isolated from Arabidopsis as a subunit of a complex including the transcription factor AtNFXL1, which is involved in the trichothecene phytotoxin response and in disease resistance against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (PstDC3000). A MKD1-dependent cascade positively regulates disease resistance against PstDC3000 and the trichothecene mycotoxin-producing fungal pathogen Fusarium sporotrichioides. MKD1 expression was induced by trichothecenes derived from Fusarium species. MKD1 directly interacted with MKK1 and MKK5 in vivo, and phosphorylated MKK1 and MKK5 in vitro. Correspondingly, mkk1 mutants and MKK5RNAi transgenic plants showed enhanced susceptibility to F. sporotrichioides. MKD1 was required for full activation of two MAPKs (MPK3 and MPK6) by the T-2 toxin and flg22. Finally, quantitative phosphoproteomics suggested that an MKD1-dependent cascade controlled phosphorylation of a disease resistance protein, SUMO, and a mycotoxin-detoxifying enzyme. Our findings suggest that the MKD1-MKK1/MKK5-MPK3/MPK6-dependent signaling cascade is involved in the full immune responses against both bacterial and fungal infection.

Characterization of plant immunity-activating mechanism by a pyrazole derivative.

A newly identified chemical, 4-{3-[(3,5-dichloro-2-hydroxybenzylidene)amino]propyl}-4,5-dihydro-1H-pyrazol-5-one (BAPP) was characterized as a plant immunity activator. BAPP enhanced disease resistance in rice against rice blast disease and expression of a defense-related gene without growth inhibition. Moreover, BAPP was able to enhance disease resistance in dicotyledonous tomato and Arabidopsis plants against bacterial pathogen without growth inhibition, suggesting that BAPP could be a candidate as an effective plant activator. Analysis using Arabidopsis sid2-1 and npr1-2 mutants suggested that BAPP induced systemic acquired resistance (SAR) by stimulating between salicylic acid biosynthesis and NPR1, the SA receptor protein, in the SAR signaling pathway.

Involvement of ethylene signaling in Azospirillum sp. B510-induced disease resistance in rice.

A bacterial endophyte Azospirillum sp. B510 induces systemic disease resistance in the host without accompanying defense-related gene expression. To elucidate molecular mechanism of this induced systemic resistance (ISR), involvement of ethylene (ET) was examined using OsEIN2-knockdown mutant rice. Rice blast inoculation assay and gene expression analysis indicated that ET signaling is required for endophyte-mediated ISR in rice. ABBREVIATIONS: ACC: 1-aminocyclopropane-1-carboxylic acid; EIN2: ethylene-insensitive protein 2; ET: ethylene; ISR: induced systemic resistance; JA: jasmonic acid; RNAi: RNA interference; SA: salicylic acid; SAR: systemic acquired resistance.

Abscisic acid modulates salicylic acid biosynthesis for systemic acquired resistance in tomato.

Among the regulatory mechanisms of systemic acquired resistance (SAR) in tomato, antagonistic interaction between salicylic acid (SA) and abscisic acid (ABA) signaling pathways was investigated. Treatment with 1,2-benzisothiazol-3(2H)-one1,1-dioxide (BIT) induced SAR in tomato thorough SA biosynthesis. Pretreatment of ABA suppressed BIT-induced SAR including SA accumulation, suggesting that ABA suppressed SAR by inhibiting SA biosynthesis.

Effects of colonization of a bacterial endophyte, Azospirillum sp. B510, on disease resistance in tomato.

A plant growth-promoting bacteria, Azospirillum sp. B510, isolated from rice, can enhance growth and yield and induce disease resistance against various types of diseases in rice. Because little is known about the interaction between other plant species and this strain, we have investigated the effect of its colonization on disease resistance in tomato plants. Treatment with this strain by soil-drenching method established endophytic colonization in root tissues in tomato plant. The endophytic colonization with this strain-induced disease resistance in tomato plant against bacterial leaf spot caused by Pseudomonas syringae pv. tomato and gray mold caused by Botrytis cinerea. In Azospirillum-treated plants, neither the accumulation of SA nor the expression of defense-related genes was observed. These indicate that endophytic colonization with Azospirillum sp. B510 is able to activate the innate immune system also in tomato, which does not seem to be systemic acquired resistance.

Datura stramonium agglutinin: cloning, molecular characterization and recombinant production in Arabidopsis thaliana.

Datura stramonium seeds contain at least three chitin-binding isolectins [termed Datura stramonium agglutinin (DSA)] as homo- or heterodimers of A and B subunits. We isolated a cDNA encoding isolectin B (DSA-B) from an immature fruit cDNA library; this contained an open reading frame encoding 279 deduced amino acids, which was confirmed by partial sequencing of the native DSA-B peptide. The sequence consisted of: (i) a cysteine (Cys)-rich carbohydrate-binding domain composed of four conserved chitin-binding domains and (ii) an extensin-like domain of 37 residues containing four SerPro4-6 motifs that was inserted between the second and third chitin-binding domains (CBDs). Although each chitin-binding domain contained eight conserved Cys residues, only the second chitin-binding domain contained an extra Cys residue, which may participate in dimerization through inter-disulfide bridge formation. Using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, the molecular mass of homodimeric lectin composed of two B-subunits was determined as 68,821 Da. The molecular mass of the S-pyridilethylated B-subunit were found to be 37,748 Da and that of the de-glycosylated form was 26,491 Da, which correlated with the molecular weight estimated from the deduced sequence. Transgenic Arabidopsis plants overexpressing the dsa-b demonstrated hemagglutinating activity. Recombinant DSA-B was produced as a homodimeric glycoprotein with a similar molecular mass to that of the native form. Moreover, the N-terminus of the purified recombinant DSA-B protein was identical to that of the native DSA-B, confirming that the cloned cDNA encoded DSA-B.

Thermodynamics-based rational design of DNA block copolymers for quantitative detection of single-nucleotide polymorphisms by affinity capillary electrophoresis.

Diblock copolymers composed of allele-specific oligodeoxyribonucleotide (ODN) and poly(ethylene glycol) (PEG) are used as an affinity probe of free-solution capillary electrophoresis to quantitatively detect single-base substitutions in genetic samples. During electrophoresis, the probe binds strongly to a wild-type single-stranded DNA analyte (WT) through hybridization, while it binds weakly to its single-base-mutated DNA analyte (MT) due to a mismatch. Complex formation with the probe augments the hydrodynamic friction of either analyte, thereby retarding its migration. The difference in affinity strength leads to separation of the WT, MT, and contaminants, including the PCR primers used for sample preparation. The optimal sequence of the probe's ODN segment is rationally determined in such a way that the binding constant between the ODN segment and MT at the capillary temperature is on the order of 10(6) M(-1). The validity of this guideline is verified using various chemically synthesized DNA analytes, as well as those derived from a bacterial genome. The peak area ratio of MT agrees well with its feed ratio, suggesting the prospective use of the present method in SNP allele frequency estimation.

Induction of systemic acquired resistance by heat shock treatment in Arabidopsis.

Systemic acquired resistance (SAR) is a potent innate immunity system in plants and has been used in rice fields. Development of SAR, involving priming, is achieved by activation of salicylic acid (SA)-mediated pathway. To determine whether heat shock (HS) treatment can induce SAR, we analyzed the effects of HS on Arabidopsis. HS treatment induced disease resistance, expression of SAR marker genes, and SA accumulation in wild-type but not in SA-deficient sid2 and NahG plants, indicating induction of SAR. Time course analysis of the effects of HS indicated that SAR was activated transiently, differently from biological induction, with a peak at 2-3 d after HS, and that it ceased in several days. Production of reactive oxygen species was observed before SA biosynthesis, which might be a trigger for SAR activation. The data presented here suggest that HS can induce SAR, but there exist unknown regulation mechanisms for the maintenance of SAR.

Response of tomatoes primed by mycorrhizal colonization to virulent and avirulent bacterial pathogens.

Most plants interact with arbuscular mycorrhizal fungi, which enhance disease resistance in the host plant. Because the effects of resistance against bacterial pathogens are poorly understood, we investigated the effects of mycorrhizal colonization on virulent and avirulent pathogens using phytopathological and molecular biology techniques. Tomato plants colonized by Gigaspora margarita acquired resistance not only against the fungal pathogen, Botrytis cinerea, but also against a virulent bacterial pathogen, Pseudomonas syringae pv. tomato DC3000 (Pst). In G. margarita-colonized tomato, salicylic acid (SA)- and jasmonic acid (JA)-related defense genes were expressed more rapidly and strongly compared to those in the control plants when challenged by Pst, indicating that the plant immunity system was primed by mycorrhizal colonization. Gene expression analysis indicated that primed tomato plants responded to the avirulent pathogen, Pseudomonas syringae pv. oryzae, more rapidly and strongly compared to the control plant, where the effect on the JA-mediated signals was stronger than in the case with Pst. We found that the resistance induced by mycorrhizal colonization was effective against both fungal and bacterial pathogens including virulent and avirulent pathogens. Moreover, the activation of both SA- and JA-mediated signaling pathways can be enhanced in the primed plant by mycorrhizal colonization.

Studies on regulation of plant physiology by pesticides.

Some agrochemicals have unique activities on plant, which modes of actions differ from those of herbicides and plant growth regulators. Because these induce useful and important phenotypic characteristics by activating physiological mechanisms in plant cell, understanding the underlying mechanism of their activities should be crucial for plant physiology and agriculture. As examples of such agrochemicals, studies on agrochemicals that activate the plant immune systems or root elongation, are described. Plant activators, inducers of systemic acquired resistance, were divided into two types, acting on upstream and downstream of salicylic acid (SA) biosynthesis, respectively. They have been useful research tools to clarify the regulation mechanism of SA-mediated disease resistance and to investigate another type of disease resistance mechanism mediated by brassinosteroids. By analyzing the roles of phytohormones in the isoprothiolane-induced root elongation indicated a positive effect of jasmonic acid and ethylene on primary root elongation. These types of research, categorized to one of chemical biology, would provide novel insight into plant physiology, which also contribute to control of crops.

Community- and genome-based views of plant-associated bacteria: plant-bacterial interactions in soybean and rice.

Diverse microorganisms are living as endophytes in plant tissues and as epiphytes on plant surfaces in nature. Questions about driving forces shaping the microbial community associated with plants remain unanswered. Because legumes developed systems to attain endosymbioses with rhizobia as well as mycorrhizae during their evolution, the above questions can be addressed using legume mutants relevant to genes for symbiosis. Analytical methods for the microbial community have recently been advanced by enrichment procedures of plant-associated microbes and culture-independent analyses targeting the small subunit of rRNA in microbial ecology. In this review, we first deal with interdisciplinary works on the global diversity of bacteria associated with field-grown soybeans with different nodulation genotypes and nitrogen application. A subpopulation of Proteobacteria in aerial parts of soybean shoots was likely to be regulated through both the autoregulation system for plant-rhizobium symbiosis and the nitrogen signaling pathway, suggesting that legumes accommodate a taxonomically characteristic microbial community through unknown plant-microbe communications. In addition to the community views, we then show multiphasic analysis of a beneficial rice endophyte for comparative bacterial genomics and plant responses. The significance and perspectives of community- and genome-based approaches are discussed to achieve a better understanding of plant-microbe interactions.

Acquired resistance to the rice blast in transgenic rice accumulating the antimicrobial peptide thanatin.

Thanatin is an antimicrobial peptide with a strong and wide-ranging antimicrobial spectrum, including certain species of fungi and Gram-negative and Gram-positive bacteria. To evaluate the application of thanatin to the generation of disease-resistant plants, we introduced a synthetic thanatin gene into rice. Several transformants that expressed the introduced gene showed significant level of antimicrobial activity. The substances showing antimicrobial activity were partially purified from these transformants and their properties were determined. The molecule with characteristics similar to those of native thanatin on the elution pattern in HPLC analysis had an identical molecular mass to that of native molecule. It should also be noted that the transformant acquired a sufficient level of resistance to the rice blast fungus, Magnaporthe oryzae, presumably due to the repressive activity of thanatin to its initial stage of infection. This result demonstrates that thanatin has antifungal activity for M. oryzae and that the introduction of the thanatin gene into rice is effective in generating a plant resistant to rice blast disease.

ABA hypersensitive germination2-1 causes the activation of both abscisic acid and salicylic acid responses in Arabidopsis.

ABA and salicylic acid (SA) are believed to act antagonistically. We previously reported that an ABA-hypersensitive mutant ahg2-1, which had reduced expression of poly(A)-specific ribonuclease (PARN), exhibited pleiotropic phenotypes including unique enhanced ABA- and SA-sensitive phenotypes. In this study, we characterized the increased SA-sensitive phenotype of this mutant in detail and addressed its relationship with ABA-related and dwarf phenotypes. We found that the ahg2-1 mutant had a high endogenous SA level and an elevated resistance to bacterial pathogens. Double mutant analyses showed that Arabidopsis plants defective in the SA signaling pathway (npr1 and pad4 mutants and nahG transgenic plants) could suppress neither the ABA hypersensitivity nor the dwarf phenotypes. These results indicate that ABA-related, SA-related and dwarf phenotypes of the ahg2-1 mutant are independent of each other. To obtain more insight into the molecular basis of the effect of ahg2-1, microarray analyses were conducted not only for ahg2-1 but also for ahg2sid2 or ahg2abi1 so as to reduce the secondary effects of SA or ABA. The resulting data indicate that ahg2-1 has a unique gene expression profile, consistent with the novel phenotype of this mutant. Detailed comparison of the expression profiles of up- or down-regulated genes implied that ahg2-1 somehow affects mitochondrial function. Our data suggest that a partial loss of PARN activity affects ABA, SA and mitochondrial function independently, and that the regulation of mRNA levels is deeply implicated in diverse cellular functions.

Production of short-chain-length/medium-chain-length polyhydroxyalkanoate (PHA) copolymer in the plastid of Arabidopsis thaliana using an engineered 3-ketoacyl-acyl carrier protein synthase III.

Short-chain-length/medium-chain-length (SCL/MCL) polyhydroxyalkanoate (PHA) was produced in the plastids of Arabidopsis thaliana. Phe87Thr (F87T) mutated 3-ketoacyl-acyl carrier protein (ACP) synthase III (FabH) from Escherichia coli , and Ser325Thr/Gln481Lys (ST/QK) mutated polyhydroxyalkanoate (PHA) synthase (PhaC1) from Pseudomonas sp. 61-3, along with the beta-ketothiolase (PhaA) and acetoacetyl-CoA reductase (PhaB) from Ralstonia eutropha (Cupriavidus necator) genes were introduced into Arabidopsis. The transgenic Arabidopsis produced PHA copolymers composed of monomers consisting of 4-14 carbons. The introduction of the engineered PHA synthase resulted in a 10-fold increase in PHA content compared to plants expressing the wild-type PHA synthase. In addition, expression of the engineered fabH gene in the plastid led to an increase in the amount of the SCL monomer, 3-hydroxybutyrate, incorporated into PHA, and contributed to supply of MCL monomers for PHA production.

Effects of colonization of a bacterial endophyte, Azospirillum sp. B510, on disease resistance in rice.

Agriculturally important grasses contain numerous diazotrophic bacteria, the interactions of which are speculated to have some other benefits to the host plants. In this study, we analyzed the effects of a bacterial endophyte, Azospirillum sp. B510, on disease resistance in host rice plants. Rice plants (Oryza sativa cv. Nipponbare) were inoculated with B510 exhibited enhanced resistance against diseases caused by the virulent rice blast fungus Magnaporthe oryzae and by the virulent bacterial pathogen Xanthomonas oryzae. In the rice plants, neither salicylic acid (SA) accumulation nor expression of pathogenesis-related (PR) genes was induced by interaction with this bacterium, except for slight induction of PBZ1. These results indicate the possibility that strain B510 is able to induce disease resistance in rice by activating a novel type of resistance mechanism independent of SA-mediated defense signaling.

Regulation of biosynthesis, perception, and functions of strigolactones for promoting arbuscular mycorrhizal symbiosis and managing root parasitic weeds.

Strigolactones (SLs) are carotenoid-derived plant secondary metabolites that play important roles in various aspects of plant growth and development as plant hormones, and in rhizosphere communications with symbiotic microbes and also root parasitic weeds. Therefore, sophisticated regulation of the biosynthesis, perception and functions of SLs is expected to promote symbiosis of beneficial microbes including arbuscular mycorrhizal (AM) fungi and also to retard parasitism by devastating root parasitic weeds. We have developed SL mimics with different skeletons, SL biosynthesis inhibitors acting at different biosynthetic steps, SL perception inhibitors that covalently bind to the SL receptor D14, and SL function inhibitors that bind to the serine residue at the catalytic site. In greenhouse pot tests, TIS108, an azole-type SL biosynthesis inhibitor effectively reduced numbers of attached root parasites Orobanche minor and Striga hermonthica without affecting their host plants; tomato and rice, respectively. AM colonization resulted in weak but distinctly enhanced plant resistance to pathogens. SL mimics can be used to promote AM symbiosis and to reduce the application rate of systemic-acquired resistance inducers which are generally phytotoxic to horticultural crops. © 2019 Society of Chemical Industry.