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 ( Psa 3) 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 Δ hopZ5 Δ hopH1 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.