Azido-Coronatine: A Useful Platform for “Click Chemistry”-Mediated Probe Synthesis for Bioorganic Studies

We report on the development of azide-coronatine as a useful platform for azide alkyne cycloaddition (“click chemistry”)-mediated synthesis of molecular probes. (+)-Azido-coronatine was synthesized in 10 steps with 11% yield using improved synthesis of coronafacic acid, in which the highly exo-selective Diels-Alder reaction (endo:exo > 1:25) is the key step. Azido coronatine was as effective as the original coronatine in a stomatal opening assay, and was easily modified to a fluorescein isothiocyanate (FITC)-labeled probe with high yield.     

Priming for enhanced defence responses by specific inhibition of the Arabidopsis response to coronatine

The priming agent β-aminobutyric acid (BABA) is known to enhance Arabidopsis resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000 by potentiating salicylic acid (SA) defence signalling, notably PR1 expression. The molecular mechanisms underlying this phenomenon remain unknown. A genome-wide microarray analysis of BABA priming during Pst DC3000 infection revealed direct and primed up-regulation of genes that are responsive to SA, the SA analogue benzothiadiazole and pathogens. In addition, BABA was found to inhibit the Arabidopsis response to the bacterial effector coronatine (COR). COR is known to promote bacterial virulence by inducing the jasmonic acid (JA) response to antagonize SA signalling activation. BABA specifically repressed the JA response induced by COR without affecting other plant JA responses. This repression was largely SA-independent, suggesting that it is not caused by negative cross-talk between SA and JA signalling cascades. Treatment with relatively high concentrations of purified COR counteracted BABA inhibition. Under these conditions, BABA failed to protect Arabidopsis against Pst DC3000. BABA did not induce priming and resistance in plants inoculated with a COR-deficient strain of Pst DC3000 or in the COR-insensitive mutant coi1-16. In addition, BABA blocked the COR-dependent re-opening of stomata during Pst DC3000 infection. Our data suggest that BABA primes for enhanced resistance to Pst DC3000 by interfering with the bacterial suppression of Arabidopsis SA-dependent defences. This study also suggests the existence of a signalling node that distinguishes COR from other JA responses.     

Norcoronatine and N-coronafacoyl-L-valine,phytotoxic analogues of coronatine produced by a strain of Pseudomonas syringae pv. glycinea

Compounds in liquid cultures of the phytopathogenic bacterium Pseudomonas syringae pv. glycinea that cause chlorosis after application to young bean leaves have been investigated. Known compounds that were isolated and identified were coronatine, the major component, and N-coronafacoyl-L-valine, which are both biologically active, and coronafacic acid which is inactive. In addition a new minor component was isolated and purified. Mass spectrometry indicated that this was an amide of coronafacic acid, bearing one less methylene group than coronatine. Mass spectral and NMR data, together with a study of the products from acid hydrolysis of the new compound, established its structure to be norcoronatine (i.e. a methyl substituent in place of the 2-ethyl substituent on the cyclopropyl moiety of coronatine). The probable biosynthetic derivation of norcoronatine is discussed.     

干旱胁迫下冠菌素对玉米幼苗光合参数和内源激素含量的影响

采用沙培称重法研究冠菌素对玉米幼苗光合参数及内源激素系统影响的结果表明,干旱胁迫下,0.01μmol·L-1冠菌素显著增加玉米幼苗茎基部粗、根长、干鲜重、光合速率、蒸腾速率和气孔导度,降低胞间二氧化碳浓度,提高幼苗的ABA和1-氨基环丙烷-1-羧酸(ACC)含量。叶片与根中ABA/GA3大时耐旱性增大。     

Site-directed mutagenesis of the temperature-sensing histidine protein kinase CorS from Pseudomonas syringae

Several plant pathogenic bacteria belonging to the species Pseudomonas syringae produce the phytotoxin coronatine to enhance their virulence. Pseudomonas syringae pv. glycinea PG4180 synthesizes coronatine at the virulence-promoting temperature of 18 degrees C, but not at 28 degrees C, its optimal growth temperature. In contrast, temperature has virtually no effect on coronatine synthesis in P. syringae pv. tomato strain DC3000. A modified two-component system controlling coronatine synthesis and consisting of the histidine protein kinase (HPK), CorS, the response regulator, CorR, and a third essential component, CorP, had been identified previously in both strains. CorS had been identified previously as a potential thermo-sensor. Comparison of the amino acid sequences of the HPKs from the two organisms revealed distinct differences. Site-directed mutagenesis of CorS from PG4180 was used to identify amino acyl residues potentially important for temperature signal perception. Point mutations and combinations of these were introduced into corS of PG4180 to generate corS variants with increased similarities to the respective allele from strain DC3000. These mutations resulted in either loss of activity, increase of thermoresponsiveness, or had no effect on CorS activity. Although none of the introduced mutations resulted in a clear conversion of CorS activity from thermo-responsive to temperature-independent, amino acyl residues important for temperature-dependent CorS activity and coronatine biosynthesis were identified.     

Defining essential processes in plant pathogenesis with Pseudomonas syringae pv. tomato DC3000 disarmed polymutants and a subset of key type III effectors

Pseudomonas syringae pv. tomato DC3000 and its derivatives cause disease in tomato, Arabidopsis and Nicotiana benthamiana. The primary virulence factors include a repertoire of 29 effector proteins injected into plant cells by the type III secretion system and the phytotoxin coronatine. The complete repertoire of effector genes and key coronatine biosynthesis genes have been progressively deleted and minimally reassembled to reconstitute basic pathogenic ability in N. benthamiana, and in Arabidopsis plants that have mutations in target genes that mimic effector actions. This approach and molecular studies of effector activities and plant immune system targets have highlighted a small subset of effectors that contribute to essential processes in pathogenesis. Most notably, HopM1 and AvrE1 redundantly promote an aqueous apoplastic environment, and AvrPtoB and AvrPto redundantly block early immune responses, two conditions that are sufficient for substantial bacterial growth in planta. In addition, disarmed DC3000 polymutants have been used to identify the individual effectors responsible for specific activities of the complete repertoire and to more effectively study effector domains, effector interplay and effector actions on host targets. Such work has revealed that AvrPtoB suppresses cell death elicitation in N. benthamiana that is triggered by another effector in the DC3000 repertoire, highlighting an important aspect of effector interplay in native repertoires. Disarmed DC3000 polymutants support the natural delivery of test effectors and infection readouts that more accurately reveal effector functions in key pathogenesis processes, and enable the identification of effectors with similar activities from a broad range of other pathogens that also defeat plants with cytoplasmic effectors.     

Coronatine biosynthesis: Incorporation of l-[U-14C]isoleucine and l-[U-14C] threonine into the 1-amido-1-carboxy-2-ethylcyclopropyl moiety

Addition of either l-[U-¹⁴C]threonine or l-[U-¹⁴C]isoleucine to 2.7-day-old shaking liquid cultures of Pseudomonas syringae pv. atropurpurea resulted in incorporation of radioactivity into coronatine, but not into N- coronafacoylvaline, another phytotoxin excreted by P.s. atropurpurea. In contrast, addition ofl-[U-¹⁴C]valine did not lead to incorporation of radioactivity into coronatine, but instead into coronafacoylvaline. Acid hydrolysis of the purified [¹⁴C] coronatine obtained after incorporation of either [¹⁴C]isoleucine or [¹⁴C]threonine demonstrated that > 94% of the radioactivity was present in the 1-amido-1-carboxy-2-ethylcyclopropyl moiety of coronatine, and < 6 % was in the coronafacoyl moiety. These findings are used to propose a biosynthetic pathway for coronatine.     

冠菌素对玉米幼苗耐干旱胁迫的诱导效应

以玉米幼苗为材料,在20%PEG模拟干旱条件下,研究了植物生长物质冠菌素0.001~1.0μmol?L-1处理时叶片水分状况及其脯氨酸、可溶性糖、MDA含量及抗氧化酶活性.实验表明,在干旱胁迫条件下,低浓度(0.01μmol?L-1)的冠菌素处理能显著降低干旱胁迫下叶片水势,并显著提高玉米幼苗叶片相对含水量、可溶性糖和游离脯氨酸的含量;同时能显著提高玉米叶片中POD、CAT和APX的活性(分别比对照高32.1%、13.8%和29.5%),而对SOD和GR的活性无显著影响;此时叶片中MDA的含量显著比对照降低11.6%,能够在一定程度上维持细胞膜的完整性.高浓度冠菌素处理(1.0μmol?L-1)下各指标的变化趋势则相反.结果证明,低浓度冠菌素(0.01μmol?L-1)能增强玉米幼苗耐干旱胁迫的能力.