A secreted protein of 15 kDa plays an important role in Phytophthora palmivora development and pathogenicity.

Phytophthora palmivora is a destructive oomycete plant pathogen with a wide host range. So far, little is known about the factors governing its infection structure development and pathogenicity. From the culture filtrate of a P. palmivora strain isolated from papaya, we identified a secreted glycoprotein of 15 kDa, designated as Ppal15kDa, using liquid chromatography tandem mass spectrometry. Two gene variants, Ppal15kDaA and Ppal15kDaB were amplified from a P. palmivora papaya isolate. Transient expression of both variants in Nicotiana benthamiana by agroinfiltration enhanced P. palmivora infection. Six Ppal15kDa mutants with diverse mutations were generated via CRISPR/Cas9-mediated gene editing. All mutants were compromised in infectivity on N. benthamiana and papaya. Two mutants with all Ppal15kDa copies mutated almost completely lost pathogenicity. The pathogenicity of the other four containing at least one wild-type copy of Ppal15kDa was compromised at varying levels. The mutants were also affected in development as they produced smaller sporangia, shorter germ tubes, and fewer appressoria. The affected levels in development corresponded to the levels of reduction in pathogenicity, suggesting that Ppal15kDa plays an important role in normal development of P. palmivora infection structures. Consistent with its role in infection structure development and pathogenicity, Ppal15kDa was found to be highly induced during appressorium formation. In addition, Ppal15kDa homologs are broadly present in Phytophthora spp., but none were characterized. Altogether, this study identified a novel component involved in development and pathogenicity of P. palmivora and possibly other Phytophthora spp. known to contain a Ppal15kDa homolog.

Enhanced Resistance to Leaf Fall Disease Caused by Phytophthora palmivora in Rubber Tree Seedling by Sargassum polycystum Extract.

The brown seaweed (Sargassum polycystum C. Agardh-Sargassaceae) extract was examined for its bioelicitor properties in the rubber tree seedling (Hevea brasiliensis (Willd. ex A.Juss.) Müll.Arg. - Euphorbiaceae) and its application to reduce the leaf fall disease caused by Phytophthora palmivora (Edwin John) Butler, 1917 (Peronosporaceae). The major purpose of this study was to apply this seaweed extract (SWE) to improve the disease resistance in rubber tree seedling compared to a chemical fungicide (1% metalaxyl). After foliar spraying of SWE solution, two antioxidant enzymes, catalase (CAT) and peroxidase (POD) and systemic acquired resistance (SAR)-triggered enzyme, ß-1,3-glucanase (GLU), were analyzed. Both secondary metabolites, a phytoalexin scopoletin (Scp) and a signaling molecule salicylic acid (SA) were measured by high performance liquid chromatography (HPLC). Both SWE- and metalaxyl-treated plants had a close disease index (DI)-score which were 16.90 ± 1.93 and 15.54 ± 1.25, respectively, while the positive control sprayed with P. palmivora showed DI-score of 29.27 ± 1.89 which was much higher than those treated with SWE or fungicide. CAT, POD, and GLU were increased in rubber tree leaves treated with SWE solution. Furthermore, Scp and SA were significantly increased in SWE-treated leaves. Enhanced systemic acquired resistance induction, 2.09 folds of SA accumulation, was observed in the distal area comparing to the local area of SWE application. In conclusion, the positive effects of SWE elicitation from these studies revealed that SWE could be used as an alternative biocontrol agent for foliar spraying to enhance the defense responses in rubber tree seedling against P. palmivora.

Novel Cell Death-Inducing Elicitors from Phytophthora palmivora Promote Infection on Hevea brasiliensis.

Elicitors play an important role in plant and pathogen interactions. The discovery of new elicitors and their effects on plant defense responses is significant and challenging. In this study, we investigated novel elicitors from Phytophthora palmivora and their effects on plant defenses. A crude elicitor isolated by ethanol precipitation from culture filtrates of P. palmivora induced cell death in tobacco leaves. When tobacco leaves were infiltrated with this cell death-inducing elicitor, the accumulations of H2O2, salicylic acid (SA), scopoletin (Scp), and abscisic acid (ABA) were detected. Accumulations of SA, Scp, and ABA were also induced in rubber tree leaves. P. palmivora infection significantly increased in rubber tree leaves pretreated with the elicitor and cotreated with the elicitor and zoospores of P. palmivora. This elicitor can be described as compound elicitor because Fourier-transform infrared spectroscopy revealed that it consisted of both polysaccharide and protein. We also found that the cell death effect caused by this compound elicitor was completely neutralized by Proteinase K. The compound elicitor was composed of four fractions which were beta-glucan, high-molecular-weight glycoprotein, broad-molecular-weight glycoprotein and 42-kDa protein. Interestingly, the broad-molecular-weight glycoprotein caused the highest level of cell death in tobacco leaves, while the beta-glucan had no effect. The high-molecular-weight glycoprotein, broad-molecular-weight glycoprotein and 42-kDa protein fractions not only caused cell death in tobacco leaves but also induced high levels of SA accumulation. Furthermore, these three fractions clearly promoted P. palmivora infection of rubber tree leaves.

Sulphated Polysaccharide from Acanthophora spicifera Induced Hevea brasiliensis Defense Responses Against Phytophthora palmivora Infection.

Elicitors from seaweeds are considered an alternative stimulant of plant defenses against pathogenic infection. Finding new sources of elicitors and exploring their effects on plant defenses is a significant undertaking. In this study, we extracted crude polysaccharide (CPS) from Acanthophora spicifera (a red alga) and tested the effects of the compound on rubber tree (Hevea brasiliensis) defense responses. Accumulations of salicylic acid (SA) and scopoletin (Scp) were measured by HPLC. The expression of SA- and Jasmonic acid (JA)-responsive genes was analyzed by semi-qRT-PCR. Strong anion exchange chromatography and Fourier-transform infrared (FTIR) spectroscopy were used for purification and functional characterization of CPS, respectively. The extracted CPS enhanced rubber tree defenses against Phytophthora palmivora infection. It induced SA and Scp accumulations and SA-responsive gene expression, but suppressed JA-responsive gene expression. We successfully separated the non-sulphated polysaccharide (F1) from the sulphated polysaccharides (SPS). Both peaks of SPS (F2 and F3) were identified as lambda (λ)-carrageenan. The F3 fraction showed greater elicitor activity on tobacco leaves. It induced SA and Scp accumulations and peroxidase activity but suppressed catalase activity. Furthermore, the purified λ-carrageenan did not cause cell death in tobacco or rubber tree leaves. Therefore, the elicitor from A. spicifera could be an alternative plant stimulant.

Salicylic Acid Induces Resistance in Rubber Tree against Phytophthora palmivora.

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.

The plant defense and pathogen counterdefense mediated by Hevea brasiliensis serine protease HbSPA and Phytophthora palmivora extracellular protease inhibitor PpEPI10.

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.

Molecular Cloning of HbPR-1 Gene from Rubber Tree, Expression of HbPR-1 Gene in Nicotiana benthamiana and Its Inhibition of Phytophthora palmivora.

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.

Molecular cloning and characterization of a novel bi-functional α-amylase/subtilisin inhibitor from Hevea brasiliensis.

A novel cDNA encoding a bi-functional α-amylase/subtilisin inhibitor (HbASI) was isolated from rubber (Hevea brasiliensis) leaves cultivar RRIM600. The HbASI had strong homology with the soybean trypsin inhibitor (Kunitz) family of protease inhibitors. Its putative amino acid sequence was similar to that of the α-amylase/subtilisin inhibitor from Ricinus communis (72% identity). Genomic sequencing indicated that the HbASI gene contained no introns. The messenger RNA of HbASI was detected in leaf, hypocotyl and root. The recombinant HbASI expressed extracellularly in Pichia pastoris exhibited inhibitory activity against α-amylase from Aspergillus oryzae, trypsin and subtilisin A. The HbASI gene was induced in the rubber leaves infected with a rubber tree pathogen, Phytophthora palmivora. It was also enhanced by salicylic acid (SA) treatment and mechanical wounding. In addition, the biological activity of the HbASI protein involving in the plant defence responses was also investigated. The HbASI at a concentration of 0.16 mg mL(-1) could inhibit the mycelium growth of P. palmivora. These data suggested that the HbASI protein might play a crucial role in defence against pathogen of rubber trees.

Hevea brasiliensis cell suspension peroxidase: purification, characterization and application for dye decolorization.

Peroxidases are oxidoreductase enzymes produced by most organisms. In this study, a peroxidase was purified from Hevea brasiliensis cell suspension by using anion exchange chromatography (DEAE-Sepharose), affinity chromatography (Con A-agarose) and preparative SDS-PAGE. The obtained enzyme appeared as a single band on SDS-PAGE with molecular mass of 70 kDa. Surprisingly, this purified peroxidase also had polyphenol oxidase activity. However, the biochemical characteristics were only studied in term of peroxidase because similar experiments in term of polyphenol oxidase have been reported in our pervious publication. The optimal pH of the purified peroxidase was 5.0 and its activity was retained at pH values between 5.0-10.0. The enzyme was heat stable over a wide range of temperatures (0-60°C), and less than 50% of its activity was lost at 70°C after incubation for 30 min. The enzyme was completely inhibited by ß-mercaptoethanol and strongly inhibited by NaN3; in addition, its properties indicated that it was a heme containing glycoprotein. This peroxidase could decolorize many dyes; aniline blue, bromocresol purple, brilliant green, crystal violet, fuchsin, malachite green, methyl green, methyl violet and water blue. The stability against high temperature and extreme pH supported that the enzyme could be a potential peroxidase source for special industrial applications.

Defense-related polyphenol oxidase from Hevea brasiliensis cell suspension: purification and characterization.

Polyphenol oxidase (PPO) was examined from the extract of leaf, seed, and cell suspension of Hevea brasiliensis, a rubber plant. The defense-related isozyme from Hevea cell suspension induced by culture filtrate of Phytophthora palmivora or by agitation stress was isolated through anion exchange and affinity chromatography, respectively. A 104-purification fold, migrated as a single band of 70 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of PPO, was obtained after further purified by the preparative gel electrophoresis. Based on reaction with catechol and dopamine but not with p-cresol and guaiacol, it is a diphenol-type PPO. The values of V(max)/K(m) ratio indicated that catechol was the most specific substrate. The optimal activity of the purified PPO was observed at pH 6.0. The PPO activity was retained at pH 4.0-10.0 and temperature 10-60 °C. The inhibitors which completely inhibited the activity were ascorbic acid, dithiothreitol, and ß-mercaptoethanol while sodium azide was a poor inhibitor. The PPO obtained from Hevea cell suspension possesses high specific activity and is stable at wide range of pH and temperature. It is therefore suitable for extreme condition uses and may lead to an alternative source of PPO in various industrial applications.