Expression profiles of putative defence-related proteins in oil palm (Elaeis guineensis) colonized by Ganoderma boninense.

Basal stem rot (BSR) is a major disease of oil palm caused by a pathogenic fungus, Ganoderma boninense. However, the interaction between the host plant and its pathogen is not well characterized. To better understand the response of oil palm to G. boninense, transcript profiles of eleven putative defence-related genes from oil palm were measured by quantitative reverse-transcription (qRT)-PCR in the roots of oil palms treated with G. boninense from 3 to 12 weeks post infection (wpi). These transcripts encode putative Bowman-Birk serine protease inhibitors (EgBBI1 and 2), defensin (EgDFS), dehydrin (EgDHN), early methionine-labeled polypeptides (EgEMLP1 and 2), glycine-rich RNA binding protein (EgGRRBP), isoflavone reductase (EgIFR), metallothionein-like protein (EgMT), pathogenesis-related-1 protein (EgPRP), and type 2 ribosome-inactivating protein (EgT2RIP). The transcript abundance of EgBBI2 increased in G. boninense-treated roots at 3 and 6. wpi compared to those of controls; while the transcript abundance of EgBBI1, EgDFS, EgEMLP1, EgMT, and EgT2RIP increased in G. boninense-treated roots at 6 or 12. wpi. Meanwhile, the gene expression of EgDHN was up-regulated at all three time points in G. boninense-treated roots. The expression profiles of the eleven transcripts were also studied in leaf samples upon inoculation of G. boninense and Trichoderma harzianum to identify potential biomarkers for early detection of BSR. Two candidate genes (EgEMLP1 and EgMT) that have different profiles in G. boninense-treated leaves compared to those infected by T. harzianum may have the potential to be developed as biomarkers for early detection of G. boninense infection

Transcriptome of oil palm (Elaeis guineensis Jacq.) roots treated with Ganoderma boninense

Basal stem rot (BSR) is the most devastating disease of oil palm. In this study, we examined the transcriptional responses of oil palm roots treated with a causal agent of BSR, Ganoderma boninense using a cDNA microarray approach. A total of 61 from 3,748 transcripts examined were found to be significantly up- or down-regulated in oil palm roots infected with G. boninense at 3 and 6 weeks post inoculation compared to those from uninfected roots. The differentially expressed genes identified in the artificially infected oil palm roots included genes encoding isoflavone reductase, Em protein H2, SPX domain-containing protein 1, pathogenesis-related protein 1, vicilin-like antimicrobial peptide. The gene expression of isoflavone reductase, which is involved in the production of phytoalexin and three related genes in the phenylpropanoid biosynthetic pathway was also profiled in the treated oil palms using real-time quantitative reverse transcription PCR. This information has contributed to our understanding of the defense mechanisms of oil palm in response to G. boninense, the future development of molecular markers for marker assisted breeding and screening of oil palms that are tolerant to G. boninense

Oil palm defensin: a thermal stable peptide that restricts the mycelial growth of Ganoderma boninense

Plant defensins are plant defence peptides that have many different biological activities, including antifungal, antimicrobial, and insecticidal activities. A cDNA (EgDFS) encoding defensin was isolated from Elaeis guineensis. The open reading frame of EgDFS contained 231 nucleotides encoding a 71-amino acid protein with a predicted molecular weight at 8.69 kDa, and a potential signal peptide. The eight highly conserved cysteine sites in plant defensins were also conserved in EgDFS. The EgDFS sequence lacking 30 amino acid residues at its N-terminus (EgDFSm) was cloned into Escherichia coli BL21 (DE3) pLysS and successfully expressed as a soluble recombinant protein. The recombinant EgDFSm was found to be a thermal stable peptide which demonstrated inhibitory activity against the growth of G. boninense possibly by inhibiting starch assimilation. The role of EgDFSm in oil palm defence system against the infection of pathogen G. boninense was discussed

Transcriptional response of oil palm (Elaeis guineensis Jacq.) inoculated simultaneously with both Ganoderma boninense and Trichoderma harzianum

Application of beneficial microbes offers an environmentally friendly alternative for mitigation of basal stem rot (BSR) disease in oil palm. However, the biocontrol mechanisms of Trichoderma against the pathogenic Ganoderma spp. which cause BSR are largely unknown at the molecular level. To identify the transcripts involved during induced systemic resistance (ISR), we analyzed the root transcriptomes of oil palm seedlings inoculated simultaneously with both G. boninense and T. harzianum, and un-inoculated oil palm seedlings, as well as those that were inoculated with either pathogenic or beneficial fungi. Our analyses revealed that the biocontrol mechanisms of T. harzianum against G. boninense involve modulation of genes related to biosynthesis of phytohormones (ethylene, MeJA and MeSA), antioxidant (l-ascorbate and myo-inositol) and unique secondary metabolites such as momilactone, cell wall metabolisms, and detoxification of phytotoxic compounds; in addition to its role as a biofertilizer which improves nutritional status of host plant. The outcomes of this study have fueled our understanding on the biocontrol mechanisms involving T. harizianum against G. boninense infection in oil palm roots

Cloning and expression of oil palm (Elaeis guineensis Jacq.) type 2 ribosome inactivating protein in Escherichia coli

EgT2RIP is a type 2 ribosome-inactivating protein isolated from oil palm (Elaeis guineensis Jacq.). Its transcript abundance was reported to be up-regulated in oil palm roots upon inoculation of pathogenic fungus Ganoderma boninense in a recent study. This study aims to produce an active recombinant EgT2RIP protein for biological studies. The DNA fragments encoding Chain A (CA) and Chain B (CB) of EgT2RIP were cloned individually in an expression vector. Soluble CA and partially soluble CB were expressed in Escherichia coli Rosetta-gami 2 (DE3). Purified recombinant CA and CB were associated in a cysteine/cystine reduced/oxidized system, yielding a heterodimer protein (AB). The AB protein showed growth inhibitory activity against breast cancer cell lines (MCF-7) as well as non-tumorigenic breast epithelial cell line (MCF-10A) at IC50 = 1.4 and 10.9 μg mL−1, respectively. The active protein produced from this study may have the potential to be used for treatment in medical and agricultural fields

Leaf transcriptome of oil palm (Elaeis guineensis Jacq.) infected by Ganoderma boninense

Oil palm is susceptible to Ganoderma infection which causes basal stem rot (BSR). Induced defense gene profiles in oil palm leaves will assist the identification of markers for detection of infected oil palms. In this study, we have sequenced the mRNA samples from the leaves of G. boninense infected oil palm seedlings (LG) and in control treatment (LC). Differential gene expression analysis showed 711 and 482 genes that were up-and down-regulated more than fourfold in LG, respectively, compared to the LC. Differential gene expression analyses revealed the modulation of oil palm genes involved in defense response such as chitinases, glucanases, and thaumatin-like proteins that showed up-regulation in LG. In addition, genes for enzymes related to the biosynthesis of flavonoids, alkaloids, and terpenes were up-regulated, while many genes involved in photosynthesis were found to be suppressed in LG. Our findings provided information on how infected oil palm leaves diverting their resources into defense at the cost of other biological processes, contributing towards identification of candidate markers for the detection of infected oil palms

Expression profiling and functional characterization of selected oil palm genes in host-microbial interaction

Basal stem rot is a major disease of oil palm caused by a pathogenic fungus,Ganoderma boninense. It reduces the oil palm yield and causes severe economic loss to the oil palm industry. To better understand oil palm defence system during the host-pathogenic interactions, the gene expression profiles of eleven defence-related cDNAs in oil palm treated with G. boninense, Trichoderma harzianum, or mycorrhizas were studied. These cDNAs encode putative bowman-birk serine protease inhibitors (EgBBI1 and 2), defensin (EgDFS), dehydrin (EgDHN), early methionine labelled polypeptides (EgEMLP1 and 2), glycine rich RNA binding protein (EgGRRBP), isoflavone reductase (EgIFR), metallothionein-like protein (EgMT), pathogenesis-related protein-1 (EgPRP), and type 2 ribosome inactivating protein (EgT2RIP). These cDNAs were chosen because they are related to plant defence and were differentially expressed in oil palm upon inoculation by mycorrhizas or G. boninensen in a previous study. In this study, the transcript abundance of EgIFR and EgBBI2 increased in G. boninense-treated roots at 3 and/or 6 weeks post inoculation (wpi). While the gene expression of EgT2RIP, EgBBI1, and EgDFS increased in G. boninense-treated roots at 6 and/or 12 wpi. Meanwhile, EgDHN was up-regulated at all three time points. These reveal that these genes could have different roles at different stages during the infection. Transcript profiles in leaves showed two candidate genes encoding EgEMLP1 and EgMT with different profiles in G. boninense-treated leaves compared to that of T. harzianum. They may have the potential to be developed as biomarkers for early detection of G. boninense infection. Comparison of the transcripts expression profiles in the roots inoculated by G.boninense, T. harzianum, and mycorrhizas showed that some of the transcripts were increased by specific fungi (EgBBI1 and EgMT were up-regulated by G.boninense while EgPRP was up-regulated by mycorrhizas). However, EgDFS and EgT2RIP were up-regulated by all three fungi probably as a result of plant general defence mechanism. The putative functions of these cDNAs were identified by sequence analyses with other homologous proteins. SignalP predicted that EgBBI1 and 2 , EgDFS, EgPRP, and EgT2RIP are secretory proteins. The complete open reading frames (ORFs) of EgBBI1, EgDFS, EgIFR, EgPRP, and EgT2RIP were cloned for recombinant protein production. EgDFSm, EgT2RIPm-CA, and EgT2RIPm-CB were sub-cloned after removal of signal peptide and linker peptide sequences. Soluble recombinant proteins were obtained for EgDFSm, EgIFR,EgT2RIPm-CA while partially soluble protein was obtained for EgT2RIPm-CB. The recombinant EgDFSm managed to inhibit the growth of G. boninense mycelium by inhibiting the assimilation of starch, possibly by acting on α-amylase or calcium channel of the fungus. The butanol fraction of oil palm root extract treated with EgIFR showed some differences in its chemical profiles when analysed using a reverse-phase-high performance liquid chromatography (RP-HPLC). Lastly, the recombinant EgT2RIPm-CA and CB that formed EgT2RIPm-AB was found to be toxic to both mammalian cell lines, MCF-7 and MCF-10. In conclusion, the findings of this study have provided insights on the molecular events that happened during the plant-microbe interaction as well as functional roles of some proteins encoded by these cDNAs

Molecular defense response of oil palm to Ganoderma infection

Basal stem rot (BSR) of oil palm roots is due to the invasion of fungal mycelia of Ganoderma species which spreads to the bole of the stem. In addition to root contact, BSR can also spread by airborne basidiospores. These fungi are able to break down cell wall components including lignin. BSR not only decreases oil yield, it also causes the stands to collapse thus causing severe economic loss to the oil palm industry. The transmission and mode of action of Ganoderma, its interactions with oil palm as a hemibiotroph, and the molecular defence responses of oil palm to the infection of Ganoderma boninense in BSR are reviewed, based on the transcript profiles of infected oil palms. The knowledge gaps that need to be filled in oil palm–Ganoderma molecular interactions i.e. the associations of hypersensitive reaction (HR)-induced cell death and reactive oxygen species (ROS) kinetics to the susceptibility of oil palm to Ganoderma spp., the interactions of phytohormones (salicylate, jasmonate and ethylene) at early and late stages of BSR, and cell wall strengthening through increased production of guaiacyl (G)-type lignin, are also discussed