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We developed this protocol to assay the extent of proliferation of Pseudomonas syringae pv. glycinea in soybean leaves. This method specifically enables accurate pathogenesis assays of soybean plants at V2/V3 (2nd/3rd trifoliate) or higher stages of growth. The leaves of soybean plants at these growth stages are not amenable to bacterial infiltration using routine needleless syringe infiltration due to the high number of trichomes on these mature leaves. This method enables efficient infiltration of bacteria into the epidermal cells of mature leaves using a pressure pump.