Establishment of CRISPR/Cas9 Genome Editing in Ganoderma boninense and Functional Validation of Hydrophobin-2 as a Virulence Determinant

Abstract

Oil palm is a major commodity crop in Southeast Asia, particularly in Malaysia and Indonesia, but its productivity is severely threatened by basal stem rot (BSR) and upper stem rot (USR) caused by the white-rot fungus Ganoderma boninense. Infected palms can lose up to 80% yield and die within 6–24 months (young) or 2–3 years (mature). Despite extensive field management efforts, disease incidence continues to rise, especially after replanting. Understanding infection mechanisms and validating fungal virulence factors are crucial for effective control, yet functional genomics in G. boninense has been limited. Previous RNAi-based gene silencing provided initial insights but was constrained by off-target effects and transient activity. Here, we report the first successful application of CRISPR/Cas9 genome editing in G. boninense for functional gene studies. Two genes were targeted: pyrG, essential in uridine monophosphate (UMP) biosynthesis; and hyd-2, encoding a hydrophobin, a potential virulence determinant implicated in host invasion. The disruption of pyrG produced a uracil auxotroph, and the knockout was validated by screening on 5-FOA and validated our system as a functional molecular tool. Disruption of hyd-2 reduced infection capability of the fungus by ~72% to ~91% in vitro. Mutations in both gene disruptions, including insertions, deletions, and substitutions, were confirmed by sequencing. Sequencing analysis also revealed incomplete editing events, as wild-type gene sequences were detected alongside edited alleles in the mutants. Future enhancements should focus on improving editing efficiency of the system. This work establishes a robust platform for functional genetic analysis and dissecting pathogenicity in G. boninense, ultimately advancing strategies to mitigate basal stem rot disease in oil palm.