Target Selection, Homokaryotic Isolation, and Screening Methods for Gene Editing in the Destructive Global Pathogen, <i>Phytophthora cinnamomi</i>
<i>Phytophthora cinnamomi</i> is a major plant pathogen that affects economically important crops and natural ecosystems, posing a threat to global biodiversity. While gene editing has emerged as a powerful tool for functional genomics in various <i>Phytophthora</i> species,...
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| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2025-02-01
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| Series: | International Journal of Plant Biology |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2037-0164/16/1/22 |
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| Summary: | <i>Phytophthora cinnamomi</i> is a major plant pathogen that affects economically important crops and natural ecosystems, posing a threat to global biodiversity. While gene editing has emerged as a powerful tool for functional genomics in various <i>Phytophthora</i> species, its application in <i>P. cinnamomi</i> remains underexplored. To address this gap, our study investigated the challenges of implementing CRISPR/Cas9-mediated gene editing in <i>P. cinnamomi</i>, with the insights gained applicable to other gene editing platforms. We designed guide RNAs (gRNAs) targeting <i>β-cinnamomin</i>, a highly basic elicitin expressed by the pathogen during early infection stages, known for its role in sterol recruitment. Using an “all-in-one” plasmid containing the gRNA, Cas9, and an antibiotic resistance gene as a selectable marker, we transformed <i>P. cinnamomi</i> protoplasts via PEG/CaCl<sub>2</sub>-mediated methods. The successful integration of the <i>nptII</i> gene, which confers geneticin (G418) resistance, was confirmed in heterokaryotic regenerants. To isolate pure mutants and eliminate wild-type dominance, we derived homokaryotic colonies from <i>nptII</i>-positive transformants. Mutation screening was performed using mismatch detection assays, T7 endonuclease 1 (T7E1), and restriction fragment length polymorphism (RFLP), followed by Sanger sequencing. Despite the integration of the <i>nptII</i> gene, the <i>β-cinnamomin</i> sequence in the transformants remained identical to the wild-type sequence, indicating challenges in detecting and achieving targeted gene disruption. This study identifies critical steps for optimising mutagenesis in <i>P. cinnamomi</i>, highlighting the importance of thorough preliminary screening, effective separation of heterokaryotic populations, and the isolation of homokaryotic colonies. |
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| ISSN: | 2037-0164 |