Identification of defense-related genes during early interaction of oil palm (Elaeis guineensis) and Ganoderma boninense for biomarker development

Ganoderma boninense is a phytopathogen that causes basal stem rot (BSR) disease leading to a significant decline in oil palm yield. By practicing a hemibiotrophic feeding lifestyle, the fungus could strategically manipulate and overwhelm their host defense mechanism by switching from biotrophic to necrotrophic mode of infection. Furthermore, early phase of G. boninense infection is not detectable and the first emergence of physical symptom denotes extensive spread of infection and cellular damage. Hence, identifying the early phase of BSR infection in oil palm is of utmost important to tackle or attenuate the progress of infection. This study aims to characterize molecular defense response and identify differentially-expressed genes (DEGs) of oil palm during early phase of G. boninense infection which have potentials to be genetic biomarker. The study also aims to develop biomarker-specific aptamerbased biosensor targeting the biomarker for on-site early detection of BSR in oil palm. Oil palm seedlings were artificially infected with G. boninense inoculums and root samples were collected following a time-course of 0, 3, 7, and 11 days-post-inoculation (dpi) for ribonucleic acid (RNA) sequencing (RNA-Seq) and identification of DEGs. The host counter-attack was evidenced based on fungal hyphae colonisation observed at 3 dpi which became significantly reduced at 7 and 11 dpi. Analysis of DEGs revealed the upregulation of many defense-related genes such as pathogenesis-relatedprotein1 (EgPR1), protease inhibitors (PIs) (EgBGIA), pattern-recognition receptor (PRR) proteins (EgLYK3), chitinases (EgCht), and expansins (EgEXPB18) at 3 and 7 dpi, which showed a decrease at 11 dpi. Conversely, later stage of the infection (11 dpi) witnessed upregulation of genes involved in biosynthesis of cuticular wax and suberins, defense-related transcription factors like EgERF113 and EgMYC2 and iron deficiency response genes. Reactive oxygen species (ROS) elicitors such as peroxidases (EgPER) and NADPH oxidases (EgRBOH) were upregulated and maintained throughout the treatment period. Suppression of auxin signaling and iron uptake genes likely led to compromised growth and altered nutrient distribution. Selected DEGs were assessed for expression across various BSR stages in oil palm plantations. These stages include symptomless (no signs of BSR infection), mild (showing fruiting bodies at the base but no foliar symptoms or stem rot, with continued yield production), and severe (featuring fruiting bodies at the base, unopened leaves, mid frond collapse, no yield, and severe stem rot), as well as BSR-free farm via multiplex end-point polymerase chain reaction (mPCR). Results showed that auxin efflux carrier component 5-like (EgPIN5) was expressed in symptomless group but was not expressed in mild, severe or BSR-free groups, supporting the potential of the gene as biomarker for early stage of BSR infection. EgPIN5-specific aptamer was developed through 10 rounds of Systematic Evolution of Ligands by EXponential enrichment (SELEX). Twenty-six aptamer candidates were discovered bearing ‘CCAT CATG ATC’ consensus sequence that base-complement with a region of EgPIN5. Additionally, the aptamers were proposed to bind to the RNA target through a DNA-RNA stem-loop kissing complex, determined through simulations to achieve the highest possible number of base pairings. Next, a sandwich-type aptamer-based lateral flow assay (LFA) strip was devised utilizing the versatile deoxyribonucleic acid (DNA)-functionalized gold nanoparticles (AuNPs) for colorimetric detection of EgPIN5 RNA. The LFA strip demonstrated visible colorimetric signal of EgPIN5 RNA at 12.5 ng/μL and the limit of detection (LOD) was recorded at 2.45 ng/μL. This study was performed as a proof-of-concept which utilizes LFA strip for rapid point-of-care (POC) EgPIN5 RNA detection, a biomarker of early phase BSR infection in oil palm.

Text IPTSM 2023 2 - IR.pdf Download (1MB) Official URL or Download Paper: https://ethesis.upm.edu.my/id/eprint/18687

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