Aug 2026· Journal of Fungi· Vol 12· 0 citations· 125 references
Medicine
TL;DR
The first successful application of CRISPR/Cas9 genome editing in G. boninense is reported, establishing 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.
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.
A new protoplast-based platform enables transient transformation and proof-of-concept CRISPR/Cas12a-mediated genome editing in European beech, a key but recalcitrant forest tree species.
Virginia Zahn, Alice-Jeannine Sievers, B. Kersten et al.· Communications Biology· 0 citations
It is demonstrated that Avr4 does not explain the resistance of Calcutta 4, suggesting that resistance is instead triggered by the recognition of other hitherto unknown effectors.
Maikel B. F. Steentjes, Gregory Ashe, Patricia Schöppl et al.· bioRxiv· 0 citations
The results demonstrate the successful deployment of CRISPR/Cas9 for targeted genome engineering in sugarbeet and establish a reliable platform for future gene-editing efforts aimed at enhancing resistance to a wide range of pathogens and diseases affecting the crop.
Z. Khan, Tinley Hathaway, C. Chu et al.· Frontiers in Genome Editing· 0 citations
Ganoderma boninense, a soil-borne pathogenic fungus causing basal stem rot (BSR) disease in oil palm, is the major threat to cultivation in Southeast Asia, with the potential to reduce oil production by up to 80% in severely affected plantations. With a view to marker-assisted selection (MAS) of resistant planting material, we combined genetics and transcriptomics approaches to investigate the defense mechanisms of oil palm against infection by G. boninense. We first performed quantitative trait locus (QTL) mapping of BSR resistance using a multi-locus Bayesian variable selection approach applied to more than 10 years of data collected during pre-nursery tests of Deli and La Mé breeding populations. We found 6 and 5 BSR resistance loci segregating in Deli and La Mé respectively, with no overlap between them. Then we performed an RNA-sequencing approach on libraries of roots and bole of oil palm seedlings inoculated or not with G. boninense, focusing on Deli × La Mé crosses with contrasted BSR resistance. A Bayesian variable selection on predictors obtained by tracing the resistance loci haplotypes in profiled individuals identified 195 differentially expressed genes (DEGs) for the G. boninense inoculation × QTL interaction, among the 551 DEGs found for inoculation effects. The DEGs were located both in cis and trans positions compared to the segregating QTL intervals, enabling the prioritization of candidate genes and resistance mechanisms associated. Focusing on a strong effect QTL region on chromosome 4 in the Deli genetic background, we identified three candidate genes in cis positions that exhibited differential expression both in response to G. boninense inoculation and between resistant and susceptible haplotypes. Our study is the first combining QTL and RNA-seq approaches in oil palm based on genetically connected experimental setup, providing valuable information on the underlying mechanisms and paving the way for MAS of BSR resistant planting material.
Aurélie Daval, David Lopez, Teresa Cuéllar et al.· Microorganisms· 0 citations
Cotton leaf curl disease (CLCuD), caused by begomoviruses and their betasatellites, is a major threat to cotton production, especially in South Asia, where periodic viral outbreaks continue to affect cotton yields, quality, and livelihoods. The advent of CRISPR/Cas9 gene-editing technology has transformed plant biotechnology, offering efficient, accurate, and programmable methods for combating viral pathogens at the genetic level. Here, the antiviral efficacy of two most widely used CRISPR/Cas9 binary plant expression vectors, pKSE401 and pHSE401, was tested in Nicotiana benthamiana against Cotton leaf curl Kokhran virus (CLCuKoV) and Cotton leaf curl Multan betasatellite (CLCuMuB). The guide-RNAs (gRNAs) were designed to target viral genes that play significant roles in pathogenicity and replication, with pHSE401 encoding a single gRNA and pKSE401 a multiplex of two gRNAs. Agrobacterium-mediated transient transformation and viral inoculation experiments revealed that both CRISPR/Cas9 vectors effectively delayed symptom onset and reduced virus titers relative to infected controls. Remarkably, the multiplex pKSE401 system was more effective at suppressing viral infection, achieving about a 90% reduction in viral accumulation compared with a 75% reduction by the single gRNA pHSE401 construct. pKSE401-treated plants showed delayed symptom development, reduced severity, and partial recovery, demonstrating the improved efficiency of multiplex genome editing. The results demonstrate the cutting-edge potential of CRISPR/Cas9 multiplex approaches as next-generation methods for designing sustainable resistance to multifaceted plant virus diseases. This research not only contributes to our understanding of CRISPR-based antiviral response mechanisms but also provides a promising avenue for designing broad-spectrum, sustainable resistance against viral epidemics in cotton and other commercially valuable crops.
Farwa Yaqub, Sidra Ashraf, Ahmed Al‐Harrasi et al.· Plant Protection· 0 citations
Three endogenous promoters with high expression levels in the hypocotyls and callus are identified, to replace the 35S promoter of Cas9, and enhanced sgRNA (esgRNA) enhanced CRISPR/Cas9 editing efficiency by 52.1% compared to native sgRNA.