Probenazole Research Articles

Chemical induction of leaf senescence and powdery mildew resistance involves ethylene-mediated chlorophyll degradation and ROS metabolism in cucumber.

Timely initiation of leaf senescence is an integral part of plant development and, importantly, an adaptive strategy by which plants cope with various stresses, e.g. to limit the spread of pathogens. Powdery mildew is a major cucumber disease that promotes the initiation/progression of leaf senescence and reduces leaf photosynthesis, resulting in severe losses of yield and quality. However, how powdery mildew induces leaf senescence and how cucumber plants respond to enhance their resistance remain unclear. Here, with established agrochemical induction and pathogen inoculation systems, we demonstrate that both probenazole (PBZ) and powdery mildew activate ethylene (ET) biosynthesis and signal transduction, consequently promoting leaf senescence and enhancing plant resistance to powdery mildew through CsEIN3 to directly upregulate the expression of CsCCGs and CsRBOHs. Our analysis convincingly suggests that the regulation of leaf senescence and powdery mildew resistance is interconnected and mediated mainly by ET in cucumber.

The NPR1-WRKY46-WRKY6 signaling cascade mediates probenazole/salicylic acid-elicited leaf senescence in Arabidopsis thaliana.

Endogenous salicylic acid (SA) regulates leaf senescence, but the underlying mechanism remains largely unexplored. The exogenous application of SA to living plants is not efficient for inducing leaf senescence. By taking advantage of probenazole (PBZ)-induced biosynthesis of endogenous SA, we previously established a chemical inducible leaf senescence system that depends on SA biosynthesis and its core signaling receptor NPR1 in Arabidopsis thaliana. Here, using this system, we identified WRKY46 and WRKY6 as key components of the transcriptional machinery downstream of NPR1 signaling. Upon PBZ treatment, the wrky46 mutant exhibited significantly delayed leaf senescence. We demonstrate that NPR1 is essential for PBZ/SA-induced WRKY46 activation, whereas WRKY46 in turn enhances NPR1 expression. WRKY46 interacts with NPR1 in the nucleus, binding to the W-box of the WRKY6 promoter to induce its expression in response to SA signaling. Dysfunction of WRKY6 abolished PBZ-induced leaf senescence, while overexpression of WRKY6 was sufficient to accelerate leaf senescence even under normal growth conditions, suggesting that WRKY6 may serve as an integration node of multiple leaf senescence signaling pathways. Taken together, these findings reveal that the NPR1-WRKY46-WRKY6 signaling cascade plays a critical role in PBZ/SA-mediated leaf senescence in Arabidopsis.

Acibenzolar-S-methyl and probenazole activate stomatal-based defense at different times to control bacterial blight of cabbage

In efforts to control bacterial blight caused by Pseudomonas cannabina pv. alisalensis (Pcal) on cabbage using plant defense activators acibenzolar-S-methyl (ASM) and probenazole (PBZ), a soil drench with ASM and PBZ effectively suppressed Pcal lesion formation and reduced bacterial populations compared with a water drench. Although ASM and PBZ activated stomatal-based defense against Pcal resulting in lower bacterial populations and less-severe symptoms, ASM, but not PBZ, induced stomatal-based defense within 4 h after the soil drench. Thus, ASM and PBZ activate a stomatal-based defense in cabbage against Pcal but differ the timing of induction.

The plant activator saccharin induces resistance to wheat powdery mildew by activating multiple defense-related genes

Saccharin and its parental compound probenazole (PBZ) are plant activators of effective defense responses to (hemi) biotrophic pathogens. Here, we demonstrate that pretreatment of wheat seedlings with saccharin or PBZ results in a significant reduction of powdery mildew caused by Blumeria graminis f. sp. tritici. Transcriptional analysis revealed the expression of 15 defense-related genes including PR genes, WCI genes, LOX, AOS, NPR1, PAL and WRKY genes in wheat seedlings exposed to either saccharin or PBZ. Moreover, the saccharin- and PBZ-enhanced expression of those genes during fungal infection further proved a close correlation with increased resistance in wheat.

Antagonism between SA- and JA-signaling conditioned by saccharin in Arabidopsis thaliana renders resistance to a specific pathogen

Saccharin is generated from probenazole (PBZ) in plants and acts as a plant defense activator. Our study of the mechanism underlying saccharin-induced systemic acquired resistance in Arabidopsis thaliana suggests an antagonistic interaction between salicylic acid (SA)- and jasmonic acid (JA)-signaling as revealed through gene expression analyses. In wild-type plants (Col-0) exposed to saccharin, there was a consistent increase in callose deposition and in expression of SA-marker genes, PR1 and PR2, which coincided with a decrease in expression of JA-marker genes such as VSP2, LOX2 and PDF1.2. Actually, pretreatment of Col-0 with saccharin or PBZ conferred resistance to Pseudomonas syringae pv. tomato DC3000, but not to Pectobacterium carotovorum subsp. carotovorum, Botrytis cinerea, or Colletotrichum higginsianum. Enhanced expression of SA- and JA-marker genes and the augmented deposition of callose were evident after a challenge with virulent DC3000 in saccharin-pretreated plants. Consistently, pretreatment of saccharin and PBZ with SA- and JA-defective mutants led to diminished resistance in NahG-transgenic and npr1 mutant plants, but not in jar1 mutant plants, suggesting that saccharin and PBZ induce resistance in A. thaliana against Pto DC3000 mainly via activation of SA-signaling, leading to suppression of JA/ET-signaling and vice versa. Collectively, an antagonism between SA- and JA-signaling conditioned by saccharin renders resistance to a specific pathogen in Arabidopsis.

Novel bacterial control agent tolprocarb enhances systemic acquired resistance in Arabidopsis and rice as a second mode of action

The fungicide tolprocarb (TPC) is a melanin biosynthesis inhibitor, but it may also have another mode of action. Here in tests of TPC for inducing plant systemic acquired resistance (SAR), TPC induced promoter activity of the tobacco pathogenesis-related gene PR-1a in Arabidopsis thaliana and genes for PBZ1, β-1,3-glucanase, and chitinase 1 in the defense-related salicylic acid (SA) signaling pathway in rice, but not genes for the jasmonate signaling pathway. Probenazole (PBZ), a commercially used plant defense activator, induced genes in both signaling pathways. The antibacterial activity of TPC was equivalent to that of PBZ. Irrigation with 200 μM TPC prevented growth by Pseudomonas syringae pv. maculicola in A. thaliana, and 30 μM TPC inhibited Xanthomonas oryzae pv. oryzae growth in rice. The results of this study suggest that TPC functions not only as a melanin biosynthesis inhibitor but also as an SAR inducer and is applicable as a novel bacterial control agent that induces SAR activity in both A. thaliana and rice.

Control of Fusarium wilt of melon by combined treatment with biocontrol, plant-activating, and soil-alkalizing agents

Fusarium wilt of melon is one of the destructive diseases of melon worldwide. To develop a method to control the soilborne disease Fusarium wilt of melon, we applied various combinations of a crop-protective microbe, a plant activator, and a soil pH increaser during seedling cultivation in preliminary greenhouse tests and chose the crop-protective Fusarium oxysporum strain SL0037, plant activator probenazole (PBZ), and soil-alkalizer slag fertilizer (SFR) for further tests in the field. From 2012 to 2014, the effects of treatment combinations with SL0037, PBZ, and SFR were tested in a field that was amended with the melon wilt pathogen F. oxysporum f. sp. melonis. Although disease incidence varied among the 3 years of the study, the main stem of melon plants was longer than that of the untreated control plants or those treated with one or two of the three agents. Results suggest a combination of SL0037, PBZ, and SFR treatments can be used to control Fusarium wilt of melon and improve growth of melon plants.

Overexpression of a rice heme activator protein gene (OsHAP2E) confers resistance to pathogens, salinity and drought, and increases photosynthesis and tiller number.

Heme activator protein (HAP), also known as nuclear factor Y or CCAAT binding factor (HAP/NF-Y/CBF), has important functions in regulating plant growth, development and stress responses. The expression of rice HAP gene (OsHAP2E) was induced by probenazole (PBZ), a chemical inducer of disease resistance. To characterize the gene, the chimeric gene (OsHAP2E::GUS) engineered to carry the structural gene encoding β-glucuronidase (GUS) driven by the promoter from OsHAP2E was introduced into rice. The transgenic lines of OsHAP2Ein::GUS with the intron showed high GUS activity in the wounds and surrounding tissues. When treated by salicylic acid (SA), isonicotinic acid (INA), abscisic acid (ABA) and hydrogen peroxide (H2 O2 ), the lines showed GUS activity exclusively in vascular tissues and mesophyll cells. This activity was enhanced after inoculation with Magnaporthe oryzae or Xanthomonas oryzae pv. oryzae. The OsHAP2E expression level was also induced after inoculation of rice with M. oryzae and X. oryzae pv. oryzae and after treatment with SA, INA, ABA and H2 O2, respectively. We further produced transgenic rice overexpressing OsHAP2E. These lines conferred resistance to M. oryzae or X. oryzae pv. oryzae and to salinity and drought. Furthermore, they showed a higher photosynthetic rate and an increased number of tillers. Microarray analysis showed up-regulation of defence-related genes. These results suggest that this gene could contribute to conferring biotic and abiotic resistances and increasing photosynthesis and tiller numbers.

Response of an aspartic protease gene OsAP77 to fungal, bacterial and viral infections in rice

BackgroundAspartic protease (APs) plays important roles in plant growth, development and biotic and abiotic stresses. We previously reported that the expression of a rice AP gene (OsAP77, Os10g0537800) was induced by probenazole (PBZ), a chemical inducer of disease resistance. In this study we examined some characteristics of this gene in response to fungal, bacterial and viral pathogens.ResultsTo elucidate the spatial and temporal expression of OsAP77, the chimeric gene was constructed carrying the structural gene encoding β-glucuronidase (GUS) driven by the OsAP77 promoter. This construct was introduced into rice and the transgenic lines were tested to analyze gene expression by fungal, bacterial and viral infections. Inoculation with Magnaporthe oryzae or Xanthomonas oryzae pv. oryzae revealed the enhanced GUS activities in vascular tissues surrounding the symptom sites by each pathogen. Moreover, GUS activity also increased after inoculation with Cucumber mosaic virus (CMV). Transgenic plants immersed in a solution containing salicylic acid (SA), isonicotinic acid (INA), hydrogen peroxide (H2O2) or abscisic acid (ABA) showed an increased level of GUS activity exclusively in vascular tissues. RT-PCR analysis showed that OsAP77 was induced not only by infection with these pathogens, but also after treatment with SA, INA, H2O2 or ABA. A knockout mutant line of OsAP77 by the insertion of Tos17 after inoculation with M. oryzae, X. oryzae pv. oryzae or CMV showed an enhanced susceptibility compared to wild type.ConclusionThese results suggest that the expression of OsAP77 is induced by pathogen infection and defense related signaling molecules in a vascular tissue specific manner and that this gene has a positive role of defense response against fungal, bacterial and viral infections.Electronic supplementary materialThe online version of this article (doi:10.1186/s12284-014-0009-2) contains supplementary material, which is available to authorized users.

Identification of Regulatory cis-elements Upstream of AtNPR1 that are Responsive to Probenazole Treatment in Transgenic Tobacco Plants

Probenazole (PBZ) is a highly effective chemical inducer of systemic acquired resistance (SAR). We found that the transcript level of NPR1, a key regulator of SAR, was significantly up-regulated upon PBZ treatment in Arabidopsis. To identify cis-elements involved in this response, a series of 5′ deleted fragments in the upstream region of NPR1 were fused to the GUS gene, and the resultant constructs were then introduced into tobacco plants. We have shown that even the shortest of these fragments was able to drive the expression of GUS at a similar level to that of the largest fragment after PBZ treatment. Further mutation analysis within the fragment showed only when both of the two W-boxes present at -128 and -123 were mutated could the responsiveness to PBZ treatment be completely abolished. These results suggest that these two W-boxes are necessary for the full responsiveness of AtNPR1 to PBZ treatment in tobacco plants. This requirement implies that one or more of WRKY transcription factors may play a key role in the positive regulation of PBZ-induced SAR, mediated by AtNPR1. Moreover, the characteristic cross-species responsiveness of the AtNPR1 upstream region to PBZ treatment demonstrates that a conserved regulatory mechanism of PBZ-induced SAR may exist in diverse plant species.

Proteomic analysis of rice defense response induced by probenazole

Here, we report the first proteomic analysis of rice defense response induced by probenazole (PBZ), an agricultural chemical that has been widely used to protect rice plants from rice blast and the bacterial blight pathogen. Two-dimensional gel electrophoresis (2-DE) was utilized to identify a total of 40 protein spots including 9 protein spots that are up-regulated by PBZ and 31 abundant protein spots. A total of 11 unique proteins from these 9 spots were identified by LC–MS/MS, and the majority of them were classified and/or possessed orthologs in defense-related functions. Five protein spots with only one protein species identified in each spot appear to be PBZ-regulated proteins. They are a putative glutathione S-transferase GSTU17, a putative phenylalanine ammonia-lyase (PAL, XP_466843), a putative caffeic acid 3- O-methyltransferase (COMT), a putative NADH-ubiquinone oxidoreductase, and a putative glucose-1-phosphate adenyltransferase. However, the other six protein species identified from the remaining four protein spots could not be conclusively described as PBZ-regulated proteins due to either the co-migration of two protein species in one spot or the presence of one protein species in two spots. Through real-time reverse transcription polymerase chain reaction (RT-PCR), it was determined that PAL (XP_466843) is likely regulated at the protein level, whereas GSTU17 and COMT were regulated at the mRNA level after PBZ application. Interestingly, the mRNA transcripts of two PAL paralogs were found to be up-regulated by PBZ. We propose that PAL, COMT, and GSTU17 are likely to confer PBZ-induced disease resistance via such functions as biosynthesis and transport of flavonoid-type phytoalexin and/or lignin biogenesis.

Probenazole-Induced Accumulation of Salicylic Acid Confers Resistance to Magnaporthe grisea in Adult Rice Plants

Probenazole (PBZ) is the active ingredient of Oryzemate, an agrochemical which is used for the protection of rice plants from Magnaporthe grisea (blast fungus). While PBZ was reported to function upstream of salicylic acid (SA) in Arabidopsis, little is known about the mechanism of PBZ-induced resistance in rice. The role of SA in blast fungus resistance is also unclear. The recommended application period for Oryzemate is just before the Japanese rainy season, at which time rice plants in the field have reached the 8-leaf stage with adult traits. Thus, the involvement of SA in PBZ-induced resistance was studied in compatible and incompatible blast fungus-rice interactions at two developmentally different leaf morphology stages. Pre-treatment of inoculated fourth leaves of young wild-type rice plants at the 4-leaf stage with PBZ did not influence the development of whitish expanding lesions (ELs) in the susceptible interaction without the accumulation of SA and pathogenesis-related (PR) proteins. However, PBZ pre-treatment increased accumulation of SA and PR proteins in the eighth leaves of adult plants at the 8-leaf stage, resulting in the formation of hypersensitive reaction (HR) lesions (HRLs). Exogenous SA induced resistance in adult but not young plants. SA concentrations in blast fungus-inoculated young leaves were essentially the same in compatible and incompatible interactions, suggesting that PBZ-induced resistance in rice is age-dependently regulated via SA accumulation.

Role of the α subunit of heterotrimeric G-protein in probenazole-inducing defense signaling in rice

Certain cellular responses to stresses and stimuli are regulated by a G-protein-mediated signaling pathway. A rice dwarf mutant that is defective in the α subunit of the heterotrimeric G-protein was found to be fully protected from blast fungus by the plant activator probenazole (PBZ) despite the 1-day delay in induction of the PR-10 gene PBZ1 by PBZ. These results suggest that the signaling pathway for protection by PBZ is not via the G-protein, although G-protein is involved in the induction of PBZ1 by PBZ.

Probenazole induces systemic acquired resistance in tobacco through salicylic acid accumulation

Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is effective against a broad range of pathogens. SAR development in dicotyledonous plant such as tobacco and Arabidopsis is mediated by salicylic acid (SA). The signaling pathway upstream of SA accumulation is partially clarified in Arabidopsis but little is known in other plant species. We investigated the SAR signaling pathway in tobacco using probenazole (PBZ) and its active metabolite, 1,2-benzisothiazole-1,1-dioxide (BIT), which induce SAR in Arabidopsis through SA accumulation. Wild-type tobacco treated with PBZ or BIT exhibited increased expression of several pathogenesis related (PR) genes, increased levels of free and total SA, and enhanced resistance to the viral pathogen tobacco mosaic virus (TMV), the bacterial pathogen Pseudomonas syringae pv. tabaci ( Pst), and the fungal pathogen Oidium sp. The role of SA in activating resistance following PBZ or BIT treatment was analysed using NahG transgenic plants. PBZ or BIT treatment did not induce disease resistance and PR-1 expression in NahG transgenic plants, which indicated that these compounds induce SAR by triggering signaling at a point upstream of SA accumulation. These results suggested that similar signaling pathways upstream of SA function in dicotyledonous plants species.

Molecular cloning and characterization of a rice phosphoinositide-specific phospholipase C gene, OsPI-PLC1, that is activated in systemic acquired resistance

Phospholipid signaling is an important component in eukaryotic signal transduction pathways; in plants, it plays a key role in growth and development as well as in responses to environmental stress. We cloned and characterized a gene from rice encoding phosphoinositide-specific phospholipase C ( OsPI-PLC1, Oryza sativa L. phosphoinositide-specific phospholipase C1). OsPI-PLC1 encodes a 599 amino acid protein containing the catalytic X and Y domains, as well as a C2-like domain, characteristics of this class of enzymes. Expression of OsPI-PLC1 was induced by various chemical and biological inducers of plant defence pathways, including benzothiadiazole (BTH), salicylic acid (SA), dichloroisonicotinic acid, probenazole (PB), jasmonic acid (JA) and its methyl ester, Pseudomonas syringae pv. syringae, and wounding. All of these treatments were shown to induce resistance in rice against blast disease caused by Magnaporthe grisea. OsPI-PLC1 was activated within 6 h after inoculation with the blast fungus in BTH-treated rice seedlings and in the incompatible interaction between a resistant genotype of rice and M. grisea , whereas the expression in the BTH-untreated seedlings and in the compatible interaction was only induced to a relatively low level at later time points (30 h) after inoculation, indicating that expression of OsPI-PLC1 is associated with the resistance response and/or the incompatible interaction. In addition, BTH treatment also induced systemic expression of OsPI-PLC1. These results suggest that OsPI-PLC1 may be involved in the signaling pathways leading to disease resistance in rice.

Molecular cloning and characterization of a rice phosphoinositide-specific phospholipase C gene, OsPI-PLC1, that is activated in systemic acquired resistance,

Phospholipid signaling is an important component in eukaryotic signal transduction pathways; in plants, it plays a key role in growth and development as well as in responses to environmental stress. We cloned and characterized a gene from rice encoding phosphoinositide-specific phospholipase C ( OsPI-PLC1 , Oryza sativa L. phosphoinositide-specific phospholipase C1). OsPI-PLC1 encodes a 599 amino acid protein containing the catalytic X and Y domains, as well as a C2-like domain, characteristics of this class of enzymes. Expression of OsPI-PLC1 was induced by various chemical and biological inducers of plant defence pathways, including benzothiadiazole (BTH), salicylic acid (SA), dichloroisonicotinic acid, probenazole (PB), jasmonic acid (JA) and its methyl ester, Pseudomonas syringae pv. syringae , and wounding. All of these treatments were shown to induce resistance in rice against blast disease caused by Magnaporthe grisea . OsPI-PLC1 was activated within 6 h after inoculation with the blast fungus in BTH-treated rice seedlings and in the incompatible interaction between a resistant genotype of rice and M. grisea , whereas the expression in the BTH-untreated seedlings and in the compatible interaction was only induced to a relatively low level at later time points (30 h) after inoculation, indicating that expression of OsPI-PLC1 is associated with the resistance response and/or the incompatible interaction. In addition, BTH treatment also induced systemic expression of OsPI-PLC1 . These results suggest that OsPI-PLC1 may be involved in the signaling pathways leading to disease resistance in rice.

Protein phosphatase inhibitors activate defense responses in rice (Oryza sativa) leaves

The protein phosphatase inhibitor cantharidin activates defense responses in rice leaves when applied exogenously at concentrations ranging from 100 to 500 μM. Responses include the accumulation of the major rice phenolic phytoalexin sakuranetin and the lactone phytoalexin momilactone A. Accumulation of sakuranetin was preceded by an induction of phenylalanine ammonia lyase (PAL) activity and an increase in the activity of naringenin 7‐O‐methyltransferase (NOMT), the key enzyme in sakuranetin biosynthesis. Cantharidin also strongly induced accumulation of the probenazole (PBZ)‐inducible protein (PBZ1) and two novel, related proteins named PBZ2 and PBZ3. Endothall, a herbicide and potent protein phosphatase inhibitor, but not its inactive analog (1,4‐dimethylendothall) also induced sakuranetin accumulation, increased activity of NOMT and accumulation of the 3 PBZ proteins. In contrast, two other protein phosphatase inhibitors, calyculin A and microcystin LR, did not activate these defense responses. Induction of NOMT and PAL activity, and sakuranetin accumulation, was completely blocked by cycloheximide. Leaf segments treated with cantharidin and endothall showed brownish and orange colored lesions, respectively, similar to the lesion mimic mutants of rice. These results indicate a direct role for protein phosphorylation/dephosphorylation events in the activation of defense responses in rice, in particular on the accumulation of antifungal phytoalexins and the PBZ proteins.

Characterization of PBZ1, a probenazole-inducible gene, in suspension-cultured rice cells.

Probenazole (PBZ) induces non-race specific resistance in rice plants against rice blast fungus and PBZ1 was identified as a PBZ-inducible gene from rice. The induction of PBZ1 expression in suspension-cultured rice cells was investigated. Northern blot analysis indicated that PBZ1 was induced by PBZ in a dose-dependent manner. Enzyme-linked immunosorbent assay (ELISA) showed a dose and time-dependent accumulation of PBZ1 protein. Both mRNA and protein analysis showed that PBZ1 was not induced by salicylic acid or an active metabolite, 1,2-benzisothiazole-1,1-dioxide.