Phytophthora sojae Transformation Based on the CRISPR/Cas9 System

Area of Science:

• Plant Pathology
• Oomycete Genetics
• Molecular Biology

Background:

• Phytophthora sojae serves as a crucial model organism for studying plant pathogenic oomycetes.
• Traditional gene function studies in Phytophthora relied heavily on gene silencing techniques.
• CRISPR/Cas9 genome editing has recently been established and widely adopted in oomycete research.

Purpose of the Study:

• To establish a detailed protocol for CRISPR/Cas9-based genome editing in Phytophthora sojae.
• To describe the process of PEG-mediated stable transformation of P. sojae protoplasts.
• To enable precise gene knockout for functional analysis in oomycetes.

Main Methods:

• Co-transformation of two plasmids into P. sojae protoplasts: one expressing Cas9 and single-guide RNA, and a homologous replacement vector.
• Utilizing CRISPR/Cas9 technology for targeted genome modification.
• Employing polyethylene glycol (PEG)-mediated transformation for stable integration.

Main Results:

• Successful implementation of CRISPR/Cas9 technology in P. sojae.
• Precise knockout of candidate genes by replacing their open reading frames (ORFs) with the hygromycin B phosphotransferase (HPH) gene.
• Demonstrated efficiency of PEG-mediated stable transformation.

Conclusions:

• The developed protocol provides a robust method for CRISPR/Cas9-mediated gene knockout in P. sojae.
• This advancement facilitates detailed functional genomics studies in oomycetes.
• The precise gene editing capability opens new avenues for understanding oomycete pathogenicity.