RESUMO
Plant immune responses can be triggered by chemicals, microbes, pathogens, insects, or abiotic stresses. In particular, induced systemic resistance (ISR) refers to the activation of the immune system due to a plant's interaction with beneficial microorganisms. The phenolic compound, 2,4-diacetylphloroglucinol (DAPG), which is produced by beneficial Pseudomonas spp., acts as an ISR elicitor, yet DAPG's mechanism in ISR remains unclear. In this study, transgenic Arabidopsis thaliana plants overexpressing the DAPG hydrolase gene (phlG) were generated to investigate the functioning of DAPG in ISR. DAPG was applied onto 3-week-old A. thaliana Col-0 and these primed plants showed resistance to the pathogens Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000. However, in the phlG transgenic A. thaliana, the ISR was not triggered against these pathogens. The DAPG-mediated ISR phenotype was impaired in transgenic A. thaliana plants overexpressing phlG, thus showing similar disease severity when compared to untreated control plants. Furthermore, the DAPG-treated A. thaliana Col-0 showed an increase in their gene expression levels of PDF1.2 and WRKY70 but this failed to occur in the phlG transgenic lines. Collectively, these experimental results indicate that jasmonic acid/ethylene signal-based defense system is effectively disabled in phlG transgenic A. thaliana lines.
RESUMO
The family of PhlG proteins catalyses the hydrolysis of carbon-carbon bonds and is widely distributed across diverse bacterial species. Two members of the PhlG family have been separately identified as 2,4-diacetylphloroglucinol (2,4-DAPG) hydrolase and phloretin hydrolase; however, the extent of functional divergence and catalytic substrates for most members of this family is still unknown. Here, using sequence similarity network and gene co-occurrence analysis, we categorized PhlG proteins into several subgroups and inferred that PhlG proteins from Mycobacterium abscessus (MaPhlG) are likely to be functionally equivalent to phloretin hydrolase. Indeed, we confirmed the hydrolytic activity of MaPhlG towards phloretin and its analog monoacetylphloroglucinol (MAPG), and the crystal structure of MaPhlG in complex with MAPG revealed the key residues involved in catalysis and substrate binding. Through mutagenesis and enzymatic assays, we demonstrated that H160, I162, A213 and Q266, which are substituted in 2,4-DAPG hydrolase, are essential for the activity towards phloretin. Based on the conservation of these residues, potential phloretin hydrolases were identified from Frankia, Colletotrichum tofieldiae and Magnaporthe grisea, which are rhizosphere inhabitants. These enzymes may be important for rhizosphere adaptation of the producing microbes by providing a carbon source through anaerobic degradation of flavonoids. Taken together, our results provided a framework for understanding the mechanism of functional divergence of PhlG proteins.
Assuntos
Hidrolases/química , Mycobacterium abscessus/enzimologia , Floretina/metabolismo , Cristalografia por Raios X , Hidrolases/genética , Hidrolases/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Mutagênese , Floroglucinol/análogos & derivados , Floroglucinol/química , Floroglucinol/metabolismo , Conformação Proteica , Rizosfera , Especificidade por SubstratoRESUMO
The PhlG protein from Mycobacterium abscessus 103 (mPhlG), which shares 30% sequence identity with phloretin hydrolase from Eubacterium ramulus and 38% sequence identity with 2,4-diacetylphloroglucinol hydrolase from Pseudomonas fluorescens Pf-5, is a putative carbon-carbon bond hydrolase. Here, the expression, purification and crystallization of mPhlG are reported. Crystals were obtained using a precipitant consisting of 100 mM citric acid pH 5.0, 1.0 M lithium chloride, 8%(w/v) polyethylene glycol 6000. The crystals diffracted to 1.87 Å resolution and belonged to space group P21, with unit-cell parameters a = 71.0, b = 63.4, c = 74.7 Å, α = 90.0, ß = 103.2, γ = 90.0°. Assuming the presence of two mPhlG molecules in the asymmetric unit, VM was calculated to be 2.5 Å(3) Da(-1), which corresponds to a solvent content of 50%.