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

CRISPR-based gene editing has rarely been studied in plant pathogens. In this report, the CRISPR/FnCas12a system was successfully established for gene editing in Pseudomonas syringae pv. actinidiae (Psa), which causes bacterial canker (BC) of kiwifruit. The system was constructed in a Psa-suitable vector pBBR1-MCS2 to edit hopH1 or/and hopZ5, which encode effectors only present in Psa biovar 3 (Psa3) responsible for BC pandemic in kiwifruit worldwide. Two different CRISPR RNAs (crRNA) were designed to edit either hopH1 or/and hopZ5, and two different sets of PCR primers were used to screen deletions of the target genes and the presence of the vector in Psa. The deletion in Psa was impacted by the position of the DNAs targeted by the crRNAs. The vector-eliminated mutant could receive the editing system iteratively. Interestingly, the double mutant ΔhopZ5ΔhopH1 showed significantly stronger virulence than the wild-type Psa on Actinidia eriantha cv. White (resistant to BC), but weak virulence on A. chinensis cv. Hongyang (highly susceptible to BC), respectively, suggesting that hopH1 or/and hopZ5 potentially matches an unknown resistance gene in White. In summary, we have established the CRISPR/FnCas12a genome-editing system to probe gene function in the pathogen and to explore effector-target interactions in kiwifruit-Psa-pathosyetem.