Efficient genome editing in Pseudomonas syringae pv. actinidiae using the CRISPR/FnCas12a system

Area of Science:

• Plant Pathology
• Microbial Genetics
• Molecular Biology

Background:

• CRISPR-based gene editing is underutilized in plant pathogens.
• Pseudomonas syringae pv. actinidiae (Psa) causes kiwifruit bacterial canker (BC).
• Psa biovar 3 (Psa3) is responsible for global BC pandemics.

Purpose of the Study:

• Establish a CRISPR/FnCas12a system for Psa gene editing.
• Investigate the function of hopH1 and hopZ5 effectors in Psa virulence.
• Explore effector-host interactions in the kiwifruit-Psa pathosystem.

Main Methods:

• Constructed the CRISPR/FnCas12a system in the pBBR1-MCS2 vector for Psa.
• Designed CRISPR RNAs (crRNAs) to target hopH1 and hopZ5 genes.
• Utilized PCR primers to screen for gene deletions and vector presence.

Main Results:

• Successfully established genome editing in Psa using CRISPR/FnCas12a.
• Demonstrated that crRNA targeting position affects gene deletion efficiency.
• The double mutant ΔhopZ5ΔhopH1 exhibited altered virulence on different kiwifruit cultivars, suggesting effector-host interactions.

Conclusions:

• The CRISPR/FnCas12a system is effective for gene editing in Psa.
• hopH1 and hopZ5 effectors play roles in Psa virulence and interact with kiwifruit resistance genes.
• This system facilitates pathogen gene function analysis and effector-target interaction studies.