Transciptome profiling at early infection of Elaeis guineensis by Ganoderma boninense provides novel insights on fungal transition from biotrophic to necrotrophic phase

Background: Basal stem rot (BSR) caused by hemibiotroph Ganoderma boninense is a devastating disease resulting in a major loss to the oil palm industry. Since there is no physical symptom in oil palm at the early stage of G. boninense infection, characterisation of molecular defense responses in oil palm during early interaction with the fungus is of the utmost importance. Oil palm (Elaeis guineensis) seedlings were artificially infected with G. boninense inoculums and root samples were obtained following a time-course of 0, 3, 7, and 11 days-post-inoculation (d.p.i) for RNA sequencing (RNA-seq) and identification of differentially expressed genes (DEGs). Results: The host counter-attack was evidenced based on fungal hyphae and Ganoderma DNA observed at 3 d.p.i which became significantly reduced at 7 and 11 d.p.i. DEGs revealed upregulation of multifaceted defense related genes such as PR-protein (EgPR-1), protease inhibitor (EgBGIA), PRR protein (EgLYK3) chitinase (EgCht) and expansin (EgEXPB18) at 3 d.p.i and 7 d.p.i which dropped at 11 d.p.i. Later stage involved highly expressed transcription factors EgERF113 and EgMYC2 as potential regulators of necrotrophic defense at 11 d.p.i. The reactive oxygen species (ROS) elicitor: peroxidase (EgPER) and NADPH oxidase (EgRBOH) were upregulated and maintained throughout the treatment period. Growth and nutrient distribution were probably compromised through suppression of auxin signalling and iron uptake genes. Conclusions: Based on the analysis of oil palm gene expression, it was deduced that the biotrophic phase of Ganoderma had possibly occurred at the early phase (3 until 7 d.p.i) before being challenged by the fungus via switching its lifestyle into the necrotrophic phase at later stage (11 d.p.i) and finally succumbed the host. Together, the findings suggest the dynamic defense process in oil palm and potential candidates that can serve as phase-specific biomarkers at the early stages of oil palm-G. boninense interaction

EgJUB1 and EgERF113 transcription factors as potential master regulators of defense response in Elaeis guineensis against the hemibiotrophic Ganoderma boninense

Background: Hemibiotrophic pathogen such as the fungal pathogen Ganoderma boninense that is destructive to oil palm, manipulates host defense mechanism by strategically switching from biotrophic to necrotrophic phase. Our previous study revealed two distinguishable expression profiles of oil palm genes that formed the basis in deducing biotrophic phase at early interaction which switched to necrotrophic phase at a later stage of infection. Results: The present report is a continuing study from our previous published transcriptomic profiling of oil palm seedlings against G. boninense. We focused on identifying differentially expressed genes (DEGs) encoding transcription factors (TFs) from the same RNA-seq data; resulting in 106 upregulated and 108 downregulated TFs being identified. The DEGs are involved in four established defense-related pathways responsible for cell wall modification, reactive oxygen species (ROS)-mediated signaling, programmed cell death (PCD) and plant innate immunity. We discovered upregulation of JUNGBRUNNEN 1 (EgJUB1) during the fungal biotrophic phase while Ethylene Responsive Factor 113 (EgERF113) demonstrated prominent upregulation when the palm switches to defense against necrotrophic phase. EgJUB1 was shown to have a binding activity to a 19 bp palindromic SNBE1 element, WNNYBTNNNNNNNAMGNHW found in the promoter region of co-expressing EgHSFC-2b. Further in silico analysis of promoter regions revealed co-expression of EgJUB1 with TFs containing SNBE1 element with single nucleotide change at either the 5th or 18th position. Meanwhile, EgERF113 binds to both GCC and DRE/CRT elements promoting plasticity in upregulating the downstream defense-related genes. Both TFs were proven to be nuclear-localized based on subcellular localization experiment using onion epidermal cells.Conclusion: Our findings demonstrated unprecedented transcriptional reprogramming of specific TFs potentially to enable regulation of a specific set of genes during different infection phases of this hemibiotrophic fungal pathogen. The results propose the intricacy of oil palm defense response in orchestrating EgJUB1 during biotrophic and EgERF113 during the subsequent transition to the necrotrophic phase. Binding of EgJUB1 to SNBE motif instead of NACBS while EgERF113 to GCC-box and DRE/CRT motifs is unconventional and not normally associated with pathogen infection. Identification of these phase-specific oil palm TFs is important in designing strategies to tackle or attenuate the progress of infection

Inhibition of prostaglandin E2 production by synthetic minor prenylated chalcones and flavonoids: synthesis, biological activity, crystal structure, and in silico evaluation

The discovery of potent inhibitors of prostaglandin E2 (PGE2) synthesis in recent years has been proven to be an important game changer in pharmaceutical industry. It is known that excessive production of PGE2 triggers a vast array of biological signals and physiological events that contributes to inflammatory diseases such as rheumatoid arthritis, atherosclerosis, cancer, and pain. In this Letter, we report the synthesis of a series of minor prenylated chalcones and flavonoids which was found to be significantly active in suppressing the PGE2 production secreted by lipopolysaccharide-induced mouse macrophage cells (RAW 264.7). Among the compounds tested, 14b showed a dose-response inhibition of PGE2 production with an IC50 value of 2.1 μM. The suppression upon PGE2 secretion was not due to cell death since 14b did not reduce the cell viability in close proximity to the PGE2 inhibition concentration. The obtained atomic coordinates for the single-crystal XRD of 14b was then applied in the docking simulation to determine the potential important binding interactions with murine COX-2 and mPGES-1 putative binding sites

Evaluation of curcumin derivatives as new cyclooxygenase-2 inhibitors via in silico and in vitro analyses

Prostaglandin E2 (PGE2) is one of the lipid mediators of inflammation. Chronic inflammation drives overproduction of PGE2 that leads to development of chronic inflammatory diseases. PGE2 is synthesized by cyclooxygenase (COX) enzyme that exists in isoforms of COX-1, which is constitutively expressed; and COX-2, which is expressed upon induction. Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit COXs to control excessive production of PGE2 during inflammation but, most of commercialized NSAIDs selectively inhibit COX-1 or being non-selective which compensate for limitations and detrimental side effects of the medicine. Hence,deciphering the mechanisms of selectively inhibiting COX-2 is of great interest. Curcumin was known as remedy to treat the inflammatory-related diseases, but suffers from poor bioavailability and instability. Synthesis of curcumin derivatives was carried out to overcome the limitations. Thus, the objectives of this study are to investigate the effects of 43 curcumin derivatives towards activated cellular PGE2 production and COX’s activity, as well as to understand its mechanism of actions in silico and in vitro.In this study, effects of curcumin derivatives on PGE2 production in murine macrophage (RAW264.7) cells which was stimulated by combination of interferongamma (IFN-) and lipopolysaccharide (LPS), were evaluated using immunoassay procedures. Quantitative structure-activity relationship (QSAR) analysis was performed to correlate between the structure and PGE2 inhibition activity of curcumin derivatives.Enzymatic assay and molecular docking analysis were performed to decipher the mechanism of inhibition on COX activity by curcumin derivatives. Effects of active curcumin derivatives on gene expression of COX-1 and COX-2 were also determined.Results demonstrated that 3 out of 43 compounds significantly inhibited PGE2 production in IFN-γ/LPS-stimulated RAW264.7 cells dose-dependently which were 2,6-bis(2-fluorobenzylidene)cyclohexanone (compound 25), 2,6-bis(4-fluorobenzylidene)cyclohexanone (compound 27), and 2,5-bis(3,4,5-trimethoxybenzylidene)cyclopentanone (compound 43) with IC50 values of 6.15 ± 0.48 M, 5.78 ± 1.67 M and 12.15 ± 1.88 M respectively which were higher than that of curcumin. Furthermore, these three compounds were not toxic to the cells (cytotoxicity IC50>500 μM). The PGE2 inhibitory effect was contributed by the suppression of the IFN-γ/LPS-stimulated COX-2 gene expression, without affecting the phorbol myristate acetate (PMA)-stimulated COX-1 gene expression in RAW264.7 cells by these three compounds. Arene substitution patterns and substituents of electron withdrawing groups may contribute to the PGE2 inhibition activity of the compounds. Besides,QSAR study recommended that positive contribution of lipophilicity and numbers of rotatable bonds, and negative contribution of kappa_2 descriptor of the compounds were crucial for their anti-inflammatory properties. The enzymatic assay showed that most curcumin derivatives tested selectively inhibited COX-1 activity rather than COX-2. However, compounds 25 and 43 selectively inhibited COX-2, unlike compound 27 which favours towards COX-1 activity. Moreover, docking study revealed that compounds 25 and 43 interacted with COX’s active site receptors that favour towards COX-2 inhibition. Arg120, His90, Phe518 and Arg513 are important receptors involved in COX-2 inhibition, while Arg120 and Ser530 are important receptors in COX-1 inhibition. In conclusion, the experimental data have provided mechanistic insights into properties of compounds 25, 27, and 43 as COX-itors.Compounds 25 and 43 could be potential lead compounds for development of new COX-2 selective inhibitors

Expression of selected groups of pathogenesis-related (PR) proteins at early stage of Ganoderma boninense infection of oil palm (Elaeis guineensis Jacq.) seedlings

Basal stem rot (BSR) is a major fungal disease of the oil palm (Elaeis guineensis Jacq.) caused by the pathogenic fungi of the Ganoderma spp. which degrade plant cell wall components. The information on molecular response of oil palm to these pathogens are still limited, even though it is crucial to develop a better strategy in handling and eliminating BSR. The most prevalent and virulent species associated with BSR is Ganoderma boninense. However, poor understanding of the defence response in oil palm towards this necrotrophic fungus has complicated the resolving measures. Early detection of Ganoderma attacks in oil palm where physical symptoms have not yet appeared can offer opportunities to prevent the spread of this necrotrophic fungi. Hence, characterization of defence-related molecular changes and production of anti-fungal agents during early interaction with G. boninense is of utmost important. In order to have a better understanding of the defence mechanisms deployed by oil palm against G. boninense, we compared the pathogenesis related proteins production of uninoculated with G. boninense inoculated oil palm seedlings. Oil palm (Elaeis guineensis) seedlings were artificially infected with G. boninense inoculums and RNA samples were taken from root tissues at different stages within less than 2 weeks post infection (0, 3, 7, 11 days after infection) and used for RNA-Seq analysis. Differential gene expression (DEGs) analyses displayed induced expression of genes encoding various types of PR proteins including a few classes of PR proteins that have not been previously reported, which can potentially be an important component of the complex defence machinery deployed by oil palm against G. boninense

Transcriptional regulatory mechanism of early defense response of oil palm against Ganoderma boninense

Basal stem rot (BSR) caused by Ganoderma boninense, a white-rot fungus is one of the major threats in oil palm industry that accounted for huge economic losses. Furthermore, the defense mechanism of oil palm against G. boninense infection is poorly understood. Elucidation of the molecular changes in defense response in oil palm at early stages of the fungal colonization where symptoms have not yet appeared will promise further insights for prevention measures. Whole transcriptome RNA sequencing (RNA-Seq) was performed on G. boninense-infected oil palm seedlings at 3, 7, and 11 days post inoculation (d.p.i.). Analysis on differentially expressed genes (DEGs) revealed strengthening of the plant cell wall as well as degradation of fungal cell wall through chitinase activity. Flavonoids were suggested to be synthesized at later stage (7 and 11 d.p.i). Besides, 1-aminocyclopropane-1-carboxylate synthase (involves in ethylene biosynthesis) and MYC2 transcription factor genes were observed significantly induced at from 3 – 11 d.p.i. Both of the genes presumably work in concert at early interactions between oil palm and G. boninense. This study further clarifies the roles of defense response genes in oil palm at early stages of interaction with G. boninense

Melanogenic inhibition and toxicity assessment of flavokawain A and B on B16/F10 melanoma cells and zebrafish (Danio rerio)

Excessive production of melanin implicates hyperpigmentation disorders. Flavokawain A (FLA) and flavokawain B (FLB) have been reported with anti-melanogenic activity, but their melanogenic inhibition and toxicity effects on the vertebrate model of zebrafish are still unknown. In the present study, cytotoxic as well as melanogenic effects of FLA and FLB on cellular melanin content and tyrosinase activity were evaluated in α-MSH-induced B16/F10 cells. Master regulator of microphthalmia-associated transcription factor (Mitf) and the other downstream melanogenic-related genes were verified via quantitative real time PCR (qPCR). Toxicity assessment and melanogenesis inhibition on zebrafish model was further observed. FLA and FLB significantly reduced the specific cellular melanin content by 4.3-fold and 9.6-fold decrement, respectively in α-MSH-induced B16/F10 cells. Concomitantly, FLA significantly reduced the specific cellular tyrosinase activity by 7-fold whilst FLB by 9-fold. The decrement of melanin production and tyrosinase activity were correlated with the mRNA suppression of Mitf which in turn down-regulate Tyr, Trp-1 and Trp-2. FLA and FLB exhibited non-toxic effects on the zebrafish model at 25 and 6.25 µM, respectively. Further experiments on the zebrafish model demonstrated successful phenotype-based depigmenting activity of FLA and FLB under induced melanogenesis. To sum up, our findings provide an important first key step for both of the chalcone derivatives to be further studied and developed as potent depigmenting agents