Inhibition of NPR1 Leads to Shoot Growth Improvement under Low-Calcium Conditions in Arabidopsis.

Under low-Ca conditions, plants accumulate salicylic acid (SA) and induce SA-responsive genes. However, the relationship between SA and low-Ca tolerance remains unclear. Here, we demonstrated that the inhibition or suppression of nonexpressor of pathogenesis-related 1 (NPR1) activity, a major regulator of the SA signaling pathway in the defense response, improves shoot growth under low-Ca conditions. Furthermore, mutations in phytoalexin-deficient 4 (PAD4) or enhanced disease susceptibility 1 (EDS1), which are upstream regulators of NPR1, improved shoot growth under low-Ca conditions, suggesting that NPR1 suppressed growth under low-Ca conditions. In contrast, growth of SA induction-deficient 2-2 (sid2-2), which is an SA-deficient mutant, was sensitive to low Ca levels, suggesting that SA accumulation by SID2 was not related to growth inhibition under low-Ca conditions. Additionally, npr1-1 showed low-Ca tolerance, and the application of tenoxicam-an inhibitor of the NPR1-mediated activation of gene expression-also improved shoot growth under low Ca conditions. The low-Ca tolerance of double mutants pad4-1, npr1-1 and eds1-22 npr1-1 was similar to that of the single mutants, suggesting that PAD4 and EDS1 are involved in the same genetic pathway in suppressing growth under low-Ca conditions as NPR1. Cell death and low-Ca tolerance did not correlate among the mutants, suggesting that growth improvement in the mutants was not due to cell death inhibition. In conclusion, we revealed that NPR1 suppresses plant growth under low-Ca conditions and that the other SA-related genes influence plant growth and cell death.

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.

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.

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.

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.

Studies on the mechanism of agricultural chemicals focused on plant hormone signals.

In recent years, it has become clear that the crosstalk of various plant hormones controls plant growth and disease resistance. Plant hormone signals may also be involved in the actions of a variety of pesticides and disease control techniques used for crop protection. From this point of view, we have focused on plant hormones to analyze the mode of action of pesticides that function in plants. Disease resistance inducers are pesticides that induce systemic acquired resistance (SAR) by activating the salicylic acid (SA)-mediated signaling pathway. However, when under unfavorable climate conditions, such as cold and cloudy weather, the resistance inducers are not sufficiently effective. Since the environmental stress response mediated by abscisic acid (ABA) may affect disease resistance, extensive studies of tobacco and tomato plants were performed, which clarified that SAR induction was suppressed by ABA. On the other hand, it was shown that transient high temperature treatment enhanced disease resistance via SA biosynthesis. These results suggest that changes in temperature due to climate change have an impact on disease resistance. The mode of action of a plant-growth regulator was analyzed by focusing on plant hormones. Isoprothiolane (IPT), an active ingredient of Fuji-one, is used as a plant-growth regulator and a fungicide. In Arabidopsis thaliana, we demonstrated that jasmonic acid and ethylene are required for the root elongation-promoting effect of IPT. As shown above, mode-of-action studies on pesticides in relation to plant hormones will lead to the development of new techniques for the better cultivation and protection of crops.

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.

Activation of cell proliferation in Arabidopsis root meristem by isoprothiolane.

A plant growth regulating agent "Fuji-one" has been used to control non-parasitic damping-off (Murenae disease) of rice seedlings. Its active ingredient, isoprothiolane (diisopropyl 1,3-dithiolan-2-ylidenemalonate, IPT), enhances root elongation of rice and Arabidopsis. To understand the mechanisms of IPT's effect on root development, its effect on Arabidopsis root cells was investigated histologically. IPT at a lower concentration (12.5 µg/mL) had no effect on root cell elongation, whereas it enhanced cell division in the root meristem. Histological analysis using phytohormone-related mutants indicated that jasmonic acid and ethylene were involved in the enhanced cell division. In contrast, IPT at a higher concentration (75 µg/mL) suppressed both cell elongation and cell division, in which jasmonic acid and ethylene were not involved. In addition, root hair formation was suppressed by treatment with IPT. These analyses demonstrated that IPT (12.5 µg/mL) enhanced root elongation by activating cell division in a jasmonic acid- and ethylene-dependent manner.

Involvement of phytohormones in root elongation activity of isoprothiolane in Arabidopsis.

Isoprothiolane (diisopropyl 1,3-dithiolan-2-ylidenemalonate, IPT), an active ingredient of "Fuji-one," has been used as a plant growth regulating agent to control non-parasitic damping-off (MURENAE disease) of rice seedlings. To understant plant growth regulating activity of IPT, its effect on root development was investigated in Arabidopsis. IPT enhanced root elongation at a lower concentration (12.5 µg/mL) but suppressed it at a higher concentration (75 µg/mL). Analysis using phytohormone-related mutants and chemical inhibitors revealed that the enhancement of root elongation by IPT required auxin, jasmonic acid, and ethylene signal transduction. Activation of the signal transduction mediated by these three phytohormones was confirmed by gene expression analysis. More detailed mechanisms of IPT's effect on root development were demonstrated via investigation using Arabidopsis and chemical inhibitors.

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.

Sulfur deficiency-induced repressor proteins optimize glucosinolate biosynthesis in plants.

Glucosinolates (GSLs) in the plant order of the Brassicales are sulfur-rich secondary metabolites that harbor antipathogenic and antiherbivory plant-protective functions and have medicinal properties, such as carcinopreventive and antibiotic activities. Plants repress GSL biosynthesis upon sulfur deficiency (-S); hence, field performance and medicinal quality are impaired by inadequate sulfate supply. The molecular mechanism that links -S to GSL biosynthesis has remained understudied. We report here the identification of the -S marker genes sulfur deficiency induced 1 (SDI1) and SDI2 acting as major repressors controlling GSL biosynthesis in Arabidopsis under -S condition. SDI1 and SDI2 expression negatively correlated with GSL biosynthesis in both transcript and metabolite levels. Principal components analysis of transcriptome data indicated that SDI1 regulates aliphatic GSL biosynthesis as part of -S response. SDI1 was localized to the nucleus and interacted with MYB28, a major transcription factor that promotes aliphatic GSL biosynthesis, in both yeast and plant cells. SDI1 inhibited the transcription of aliphatic GSL biosynthetic genes by maintaining the DNA binding composition in the form of an SDI1-MYB28 complex, leading to down-regulation of GSL biosynthesis and prioritization of sulfate usage for primary metabolites under sulfur-deprived conditions.

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.