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Plants elicit defense responses when exposed to pathogens, which partly contribute to the resistance of plants to Agrobacterium tumefaciens–mediated transformation. Some pathogenic bacteria have sophisticated mechanisms to counteract these defense responses by injecting Type III effectors (T3Es) through the Type III secretion system (T3SS). By engineering A. tumefaciens to express T3SS to deliver T3Es, we suppressed plant defense and enhanced plant genetic transformation. Here, we describe the optimized protocols for mobilization of T3SS-expressing plasmid to engineer A. tumefaciens to deliver proteins through T3SS and fractionation of cultures to study proteins from pellet and supernatants to determine protein secretion from engineered A. tumefaciens. 

Abstract Agrobacterium-mediated plant transformation (AMT) is the basis of modern-day plant biotechnology. One major drawback of this technology is the recalcitrance of many plant species/varieties toAgrobacteriuminfection, most likely caused by elicitation of plant defense responses. Here, we develop a strategy to increase AMT by engineeringAgrobacterium tumefaciensto express a type III secretion system (T3SS) fromPseudomonas syringaeand individually deliver theP. syringaeeffectors AvrPto, AvrPtoB, or HopAO1 to suppress host defense responses. Using the engineeredAgrobacterium, we demonstrate increase in AMT of wheat, alfalfa and switchgrass by ~250%–400%. We also show that engineeredA. tumefaciensexpressing a T3SS can deliver a plant protein, histone H2A-1, to enhance AMT. This strategy is of great significance to both basic research and agricultural biotechnology for transient and stable transformation of recalcitrant plant species/varieties and to deliver proteins into plant cells in a non-transgenic manner.