RESUMO
Induced resistance by elicitors is considered to be an eco-friendly strategy to stimulate plant defense against pathogen attack. In this study, we elucidated the effect of salicylic acid (SA) on induced resistance in rubber tree against Phytophthora palmivora and evaluated the possible defense mechanisms that were involved. For SA pretreatment, rubber tree exhibited a significant reduction in disease severity by 41%. Consistent with the occurrence of induced resistance, the pronounced increase in H2O2 level, catalase (CAT) and peroxidase (POD) activities were observed. For defense reactions, exogenous SA promoted the increases of H2O2, CAT, POD and phenylalanine ammonia lyase (PAL) activities, including lignin, endogenous SA and scopoletin (Scp) contents. However, SA had different effects on the activity of each CAT isoform in the particular rubber tree organs. Besides, three partial cDNAs encoding CAT (HbCAT1, HbCAT2 and HbCAT3) and a partial cDNA encoding PAL (HbPAL) were isolated from rubber tree. Moreover, the expressions of HbCAT1, HbPAL and HbPR1 were induced by SA. Our findings suggested that, upon SA priming, the elevated H2O2, CAT, POD and PAL activities, lignin, endogenous SA and Scp contents, including the up-regulated HbCAT1, HbPAL and HbPR1 expressions could potentiate the resistance in rubber tree against P. palmivora.
Assuntos
Hevea/microbiologia , Hevea/fisiologia , Phytophthora/fisiologia , Ácido Salicílico/farmacologia , Árvores/microbiologia , Árvores/fisiologia , 3,3'-Diaminobenzidina/metabolismo , Sequência de Aminoácidos , Catalase/metabolismo , Clonagem Molecular , DNA Complementar/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Hevea/efeitos dos fármacos , Hevea/genética , Peróxido de Hidrogênio/metabolismo , Cinética , Lignina/metabolismo , Peroxidase/metabolismo , Fenóis/metabolismo , Fenilalanina Amônia-Liase/química , Fenilalanina Amônia-Liase/metabolismo , Phytophthora/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/microbiologia , Folhas de Planta/fisiologia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Escopoletina/metabolismo , Análise de Sequência de DNA , Árvores/efeitos dos fármacosRESUMO
Rubber tree (Hevea brasiliensis Muell. Arg) is an important economic crop in Thailand. Leaf fall and black stripe diseases caused by the aggressive oomycete pathogen Phytophthora palmivora, cause deleterious damage on rubber tree growth leading to decrease of latex production. To gain insights into the molecular function of H. brasiliensis subtilisin-like serine proteases, the HbSPA, HbSPB, and HbSPC genes were transiently expressed in Nicotiana benthamiana via agroinfiltration. A functional protease encoded by HbSPA was successfully expressed in the apoplast of N. benthamiana leaves. Transient expression of HbSPA in N. benthamiana leaves enhanced resistance to P. palmivora, suggesting that HbSPA plays an important role in plant defense. P. palmivora Kazal-like extracellular protease inhibitor 10 (PpEPI10), an apoplastic effector, has been implicated in pathogenicity through the suppression of H. brasiliensis protease. Semi-quantitative RT-PCR revealed that the PpEPI10 gene was significantly up-regulated during colonization of rubber tree by P. palmivora. Concurrently, the HbSPA gene was highly expressed during infection. To investigate a possible interaction between HbSPA and PpEPI10, the recombinant PpEPI10 protein (rPpEPI10) was expressed in Escherichia coli and purified using affinity chromatography. In-gel zymogram and co-immunoprecipitation (co-IP) assays demonstrated that rPpEPI10 specifically inhibited and interacted with HbSPA. The targeting of HbSPA by PpEPI10 revealed a defense-counterdefense mechanism, which is mediated by plant protease and pathogen protease inhibitor, in H. brasiliensis-P. palmivora interactions.
Assuntos
Hevea/enzimologia , Interações Hospedeiro-Patógeno , Phytophthora/metabolismo , Proteínas de Plantas/metabolismo , Inibidores de Proteases/metabolismo , Serina Proteases/metabolismo , Cromatografia de Afinidade , Resistência à Doença/fisiologia , Escherichia coli , Técnicas de Transferência de Genes , Hevea/genética , Hevea/parasitologia , Imunoprecipitação , Organismos Geneticamente Modificados , Phytophthora/genética , Proteínas de Plantas/genética , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Proteases/genética , Nicotiana/genética , Nicotiana/metabolismoRESUMO
This is the first report to present a full-length cDNA (designated HbPR-1) encoding a putative basic HbPR-1 protein from rubber tree (Hevea brasiliensis) treated with salicylic acid. It was characterized and also expressed in Nicotiana benthamiana using Agrobacterium-mediated transient gene expression system in order to investigate the role of HbPR-1 gene in rubber tree against its oomycete pathogen Phytopthora palmivora and to produce recombinant HbPR-1 protein for microbial inhibition test. The HbPR-1 cDNA was 647 bp long and contained an open reading frame of 492 nucleotides encoding 163 amino acid residues with a predicted molecular mass of 17,681 Da and an isoelectric point (pI) of 8.56, demonstrating that HbPR-1 protein belongs to the basic PR-1 type. The predicted 3D structure of HbPR-1 was composed of four α-helices, three ß-sheets, seven strands, and one junction loop. Expression and purification of recombinant HbPR-1 protein were successful using Agrobacterium-mediated transient expression and one-step of affinity chromatography. Heterologous expression of HbPR-1 in N. benthamiana reduced necrosis areas which were inoculated with P. palmivora zoospores, indicating that the expressed HbPR-1 protein played an important role in plant resistance to pathogens. The purified recombinant HbPR-1 protein was found to inhibit 64% of P. palmivora zoospore germination on a water agar plate compared with control, suggesting that it was an antimicrobial protein against P. palmivora.