Abstract: To establish the gene knockout technical system for Pseudomonas amygdali pv. tabaci (Pta), Pta strain D151 (race 0) was used as material and plasmid pEX18Gm was selected as the starting vector to construct the pEX18Gm-HopF1-KO suicide plasmid. After conjugation transfer and several rounds of resistance screening, the effector HopF1 was knocked out in the Pta strain (race 0). The results showed that five positive mutant strains were obtained by PCR identification of the bacterial solution, and the positive transformation rate was 50%. Sequencing results further confirmed that the HopF1 gene knockout strains were successfully constructed. This study showed that the technical system of Pta gene knockout was successfully established, which lay the foundation for the cultivation of Pta-race-specialized resistant tobacco varieties and the cloning of Pta-race-specialized resistant genes in tobacco.