Identification, characterisation and functional analysis of elicitor proteins from Ganoderma boninense

Ganoderma boninense, the causal agent of Basal Stem Rot (BSR) disease, threatens oil palm production in Southeast Asia where there is no effective control method currently available. The fungus, like various phytopathogenic fungi, is anticipated to secrete elicitor proteins that modify plant immune responses to promote infection during its early interaction with oil palm. Understanding the roles of G. boninense elicitor proteins in oil palm root penetration and pathogenicity is imperative for developing effective BSR disease management strategies. Therefore, this study focused on (i) the isolation and identification of candidate elicitor proteins with hypersensitive response (HR)-inducing activity from the culture filtrate of G. boninense, (ii) the isolation, characterisation and expression analysis of genes encoding two cerato-platanin proteins in G. boninense-oil palm pathosystem, and (iii) the elucidation of the elicitation of early defence responses and the improvement of disease resistance by extracellular protein sample from G. boninense in oil palm. In this study, peptide matches to candidate elicitor proteins such as cellulases, chitinase, glucanases, pectate lyases, xylanases, cerato-platanin (CP) proteins, fungalysin and carboxypeptidases were identified in two active fractions obtained from a bioassay-guided fractionation of the 14-day-old fungal culture filtrate using anion exchange chromatography (AIEX), followed by a nano-LC-MS/MS analysis of the active AIEX fractions against the UniProtKB database of G. sinense ZZ0214-1. GbCP2 and GbCP6 were identified as potential elicitor proteins based on their effector properties in a bioinformatic investigation of GbCP1 through 9, the G. boninense CP gene models. The coding regions of 528 bp and 450 bp of GbCP2 and GbCP6, respectively, were successfully isolated using polymerase chain reaction (PCR) method. The deduced amino acid sequences of GbCP2 and GbCP6 resembled elicitor CP proteins from several ascomycetes and basidiomycetes. The quantitative real-time PCR analysis of GbCP2 and GbCP6 in oil palm root-infecting mycelia at 3, 7 and 11 d after inoculation (DAI) revealed that GbCP6 was significantly up-regulated by 16.3-fold at 11 DAI. GbCP2, on the other hand, was not differentially expressed at any sampling point, indicating that the protein may have a different function. Besides, the extracellular protein sample induced HR-associated cell death and reactive oxygen species (ROS) generation in oil palm leaf sections at 24-h post-infiltration. In addition, the activities of guaiacol peroxidase and superoxide dismutase enzymes were significantly higher in extracellular protein sample-treated oil palm roots at 12, 24, 48, and 72 h post-treatment (hpt) compared to the control. Significant induction of RbohB, a gene encoding respiratory burst oxidase homolog B, by 2.5-fold at 12 hpt further confirmed ROS accumulation and the HR-inducing potential of the extracellular protein sample. BSR disease incidence of 45% and disease reduction of 71.43% at 9 months after inoculation showed the potential of the extracellular protein sample from G. boninense strain PER71 as a plant defence activator. In light of all findings, it can be concluded that G. boninense secretes elicitor proteins that elicit events involved in early defence during G. boninense-oil palm interaction.

Text 122838.pdf Download (1MB) Official URL or Download Paper: https://ethesis.upm.edu.my/id/eprint/18683

Actions (login required)

View Item