Yoshiteru Noutoshi

RRS1 and RPS4 provide a dual Resistance-gene system against fungal and bacterial pathogens

Colletotrichum higginsianum is a fungal pathogen that infects a wide variety of cruciferous plants, causing important crop losses. We have used map-based cloning and natural variation analysis of 19 Arabidopsis ecotypes to identify a dominant resistance locus against C. higginsianum. This locus named RCH2 (for recognition of C. higginsianum) maps in an extensive cluster of disease-resistance loci known as MRC-J in the Arabidopsis ecotype Ws-0. By analyzing natural variations within the MRC-J region, we found that alleles of RRS1 (resistance to Ralstonia solanacearum 1) from susceptible ecotypes contain single nucleotide polymorphisms that may affect the encoded protein. Consistent with this finding, two susceptible mutants, rrs1-1 and rrs1-2, were identified by screening a T-DNA-tagged mutant library for the loss of resistance to C. higginsianum. The screening identified an additional susceptible mutant (rps4-21) that has a 5-bp deletion in the neighboring gene, RPS4-Ws, which is a well-characterized R gene that provides resistance to Pseudomonas syringae pv. tomato strain DC3000 expressing avrRps4 (Pst-avrRps4). The rps4-21/rrs1-1 double mutant exhibited similar levels of susceptibility to C. higginsianum as the single mutants. We also found that both RRS1 and RPS4 are required for resistance to R. solanacearum and Pst-avrRps4. Thus, RPS4-Ws and RRS1-Ws function as a dual resistance gene system that prevents infection by three distinct pathogens.

Novel Plant Immune-Priming Compounds Identified via High-Throughput Chemical Screening Target Salicylic Acid Glucosyltransferases in Arabidopsis

This study used high-throughput screening to identify five immune priming chemicals that potentiate but do not directly induce defense responses. These compounds inhibit salicylic acid (SA) glucosyltransferases and increase SA during pathogen infection. Thus, SA glucosylation can be a target for developing novel crop protectants. Plant activators are compounds, such as analogs of the defense hormone salicylic acid (SA), that protect plants from pathogens by activating the plant immune system. Although some plant activators have been widely used in agriculture, the molecular mechanisms of immune induction are largely unknown. Using a newly established high-throughput screening procedure that screens for compounds that specifically potentiate pathogen-activated cell death in Arabidopsis thaliana cultured suspension cells, we identified five compounds that prime the immune response. These compounds enhanced disease resistance against pathogenic Pseudomonas bacteria in Arabidopsis plants. Pretreatments increased the accumulation of endogenous SA, but reduced its metabolite, SA-O-β-d-glucoside. Inducing compounds inhibited two SA glucosyltransferases (SAGTs) in vitro. Double knockout plants that lack both SAGTs consistently exhibited enhanced disease resistance. Our results demonstrate that manipulation of the active free SA pool via SA-inactivating enzymes can be a useful strategy for fortifying plant disease resistance and may identify useful crop protectants.

Sulfonamides identified as plant immune-priming compounds in high-throughput chemical screening increase disease resistance in Arabidopsis thaliana

Plant activators are agrochemicals that protect crops from diseases by activating the plant immune system. To isolate lead compounds for use as practical plant activators, we screened two different chemical libraries composed of various bioactive substances by using an established screening procedure that can selectively identify immune-priming compounds. We identified and characterized a group of sulfonamide compounds – sulfameter, sulfamethoxypyridazine, sulfabenzamide, and sulfachloropyridazine – among the various isolated candidate molecules. These sulfonamide compounds enhanced the avirulent Pseudomonas-induced cell death of Arabidopsis suspension cell cultures and increased disease resistance in Arabidopsis plants against both avirulent and virulent strains of the bacterium. These compounds did not prevent the growth of pathogenic bacteria in minimal liquid media at 200 μM. They also did not induce the expression of defense-related genes in Arabidopsis seedlings, at least not at 24 and 48 h after treatment, suggesting that they do not act as salicylic acid analogs. In addition, although sulfonamides are known to be folate biosynthesis inhibitors, the application of folate did not restore the potentiation effects of the sulfonamides on pathogen-induced cell death. Our data suggest that sulfonamides potentiate Arabidopsis disease resistance by their novel chemical properties.

A Chemical Biology Approach Reveals an Opposite Action between Thermospermine and Auxin in Xylem Development in Arabidopsis thaliana

Thermospermine, a structural isomer of spermine, is produced through the action of ACAULIS5 (ACL5) and suppresses xylem differentiation in Arabidopsis thaliana. To elucidate the molecular basis of the function of thermospermine, we screened chemical libraries for compounds that can modulate xylem differentiation in the acl5 mutant, which is deficient in thermospermine and shows a severe dwarf phenotype associated with excessive proliferation of xylem vessels. We found that the isooctyl ester of a synthetic auxin, 2,4-D, remarkably enhanced xylem vessel differentiation in acl5 seedlings. 2,4-D, 2,4-D analogs and IAA analogs, including 4-chloro IAA (4-Cl-IAA) and IAA ethyl ester, also enhanced xylem vessel formation, while IAA alone had little or no obvious effect on xylem differentiation. These effects of auxin analogs were observed only in the acl5 mutant but not in the wild type, and were suppressed by the anti-auxin, p-chlorophenoxyisobutyric acid (PCIB) and α-(phenyl ethyl-2-one)-IAA (PEO-IAA), and also by thermospermine. Furthermore, the suppressor of acaulis51-d (sac51-d) mutation, which causes SAC51 overexpression in the absence of thermospermine and suppresses the dwarf phenotype of acl5, also suppressed the effect of auxin analogs in acl5. These results suggest that the auxin signaling that promotes xylem differentiation is normally limited by SAC51-mediated thermospermine signaling but can be continually stimulated by exogenous auxin analogs in the absence of thermospermine. The opposite action between thermospermine and auxin may fine-tune the timing and spatial pattern of xylem differentiation.

ImprimatinC1, a novel plant immune-priming compound, functions as a partial agonist of salicylic acid

Plant activators are agrochemicals that protect crops from pathogens. They confer durable resistance to a broad range of diseases by activating intrinsic immune mechanisms in plants. To obtain leads regarding useful compounds, we have screened a chemical library using an established method that allows selective identification of immune-priming compounds. Here, we report the characterisation of one of the isolated chemicals, imprimatinC1, and its structural derivative imprimatinC2. ImprimatinC1 functions as a weak analogue of salicylic acid (SA) and activates the expression of defence-related genes. However, it lacks antagonistic activity toward jasmonic acid. Structure-activity relationship analysis suggests that imprimatinC1 and C2 can be metabolised to 4-chlorobenzoic acid and 3,4-chlorobenzoic acid, respectively, to function in Arabidopsis. We also found that imprimatinC1 and C2 and their potential functional metabolites acted as partial agonists of SA. Thus, imprimatinC compounds could be useful tools for dissecting SA-dependent signal transduction pathways.

Diuretics Prime Plant Immunity in Arabidopsis thaliana

Plant activators are agrochemicals that activate the plant immune system, thereby enhancing disease resistance. Due to their prophylactic and durable effects on a wide spectrum of diseases, plant activators can provide synergistic crop protection when used in combination with traditional pest controls. Although plant activators have achieved great success in wet-rice farming practices in Asia, their use is still limited. To isolate novel plant activators applicable to other crops, we screened a chemical library using a method that can selectively identify immune-priming compounds. Here, we report the isolation and characterization of three diuretics, bumetanide, bendroflumethiazide and clopamide, as immune-priming compounds. These drugs upregulate the immunity-related cell death of Arabidopsis suspension-cultured cells induced with an avirulent strain of Pseudomonas syringae pv. tomato in a concentration-dependent manner. The application of these compounds to Arabidopsis plants confers disease resistance to not only the avirulent but also a virulent strain of the pathogen. Unlike salicylic acid, an endogenous phytohormone that governs disease resistance in response to biotrophic pathogens, the three diuretic compounds analyzed here do not induce PR1 or inhibit plant growth, showing potential as lead compounds in a practical application.

Thermospermine suppresses auxin-inducible xylem differentiation in Arabidopsis thaliana

Thermospermine, a structural isomer of spermine, is synthesized by a thermospermine synthase designated ACAULIS5 (ACL5). Thermospermine-deficient acl5 mutant of Arabidopsis thaliana shows severe dwarfism and excessive xylem differentiation. By screening for compounds that affect xylem differentiation in the acl5 mutant, we identified auxin analogs that remarkably enhanced xylem vessel differentiation in the acl5 mutant but not in the wild type. The xylem-inducing effect of auxin analogs was clearly suppressed by thermospermine, indicating that auxin-inducible xylem differentiation is normally limited by thermospermine. Here, we further characterized xylem-inducing effect of auxin analogs in various organs. Auxin analogs promoted protoxylem differentiation in roots and cotyledons in the acl5 mutant. Our results indicate that the opposite action between thermospermine and auxin in xylem differentiation is common in different organs and also suggest that thermospermine might be required for the suppression of protoxylem differentiation.

Isolation and characterization of the plant immune-priming compounds Imprimatin B3 and -B4, potentiators of disease resistance in Arabidopsis thaliana

Plant activators are chemical crop protectants that fortify the immune system in plants. Unlike pesticides that target pathogens, plant activators provide durable effects against a broad spectrum of diseases, which have not been overcome by pathogenic microbes. Plant activators are not only useful agrochemicals, but can also help to elucidate the details of the plant immune system. Using an established high-throughput screening procedure, we previously identified 5 compounds, designated as Imprimatins, which prime plant immune response. These compounds increased disease resistance against pathogenic Pseudomonas bacteria in Arabidopsis plants by inhibiting 2 salicylic acid (SA) glucosyltransferases (SAGTs), resulting in accumulation of the phytohormone SA. Here, we report the isolation of 2 additional Imprimatins, B3 and B4, which are structurally similar to Imprimatin B1 and B2. Because these compounds did not have strong inhibitory effects on SAGTs in vitro, they may exert their function after metabolic conversion in vivo.

Synthesis and properties of peptide dendrimers containing fluorescent and branched amino acids

In this report, we describe dendritic peptides possessing central fluorescent amino acids with adjacent branched amino acids. These fluorescent-peptide dendrimers were prepared using (9-fluorenyl)methoxycarbonyl (Fmoc)-based solid-phase peptide synthesis and Fmoc-derivative fluorescent and branched amino acids. The branched amino acids featured multiple carboxylic acids in their side chains, making the fluorescent-peptide dendrimers highly water-soluble compared with the analogous peptides containing the fluorescent amino acids only. The branched amino acid units also improved the fluorescence intensity of the dendrimers. Based on high-pressure liquid chromatography and fluorescence spectroscopy results, we determined that the fluorescent groups were located in the core and that the carboxylic acids were located on the surface of the dendrimers. Fluorescence resonance energy transfer was achieved among the three proximal fluorescent groups in one of the fabricated fluorescent-peptide dendrimers.

Characterization of quorum sensing-controlled transcriptional regulator MarR and Rieske (2Fe-2S) cluster-containing protein (Orf5), which are involved in resistance to environmental stresses in Pseudomonas syringae pv. tabaci 6605

Pseudomonas syringae pv. tabaci 6605 (Pta6605) produces acyl homoserine lactones (AHLs), quorum sensing (QS) molecules that are indispensable for virulence in host tobacco infection. Genome-wide transcriptional profiling of several QS-defective mutants revealed that the expression of the genes encoding the MarR family transcriptional regulator (MarR) and a Rieske 2Fe-2S cluster-containing protein (Orf5) located adjacent to psyI, a gene encoding AHL synthetase, are significantly repressed. Exogenous application of AHL recovered the expression of both marR and orf5 genes in the ΔpsyI mutant, indicating that AHL positively regulates the expression of these genes. To investigate the role of these genes in the virulence of Pta6605, ΔmarR and Δorf5 mutants were generated. Both mutants showed decreased swimming and swarming motilities, decreased survival ability under oxidative and nitrosative stresses and, consequently, reduced virulence on host tobacco plants. Transmission electron micrographs showed that the structure of the cell membranes of ΔmarR and Δorf5 mutants was severely damaged. Furthermore, not only the ratio of dead cells, but also the amount of flagella, extracellular DNA and protein released into the culture supernatant, was significantly increased in both mutants, indicating that the disruption of marR and orf5 genes might induce structural changes in the membrane and cell lysis. Because both mutants showed partly similar expression profiles, both gene products might be involved in the same regulatory cascades that are required for QS-dependent survival under environmentally stressed conditions.