Progress Toward Regulating the avrBs2 Gene to Study Plant Immunity Response

Bacterial spot disease caused by Xanthomonas campestris pv. vesicatoria is an agricultural problem that reduces yield and quality of tomatoes in the United States. Plants resistant to bacterial spot disease carry the Bs2 gene and are able to contain the infection by triggering a defensive "hypersensitive response" (HR), i.e. localized cell death. Previous research has found that Bs2-expressing resistant plants recognize bacterial protein AvrBs2. However, the downstream signaling events of the Bs2/AvrBs2 interaction that lead to HR are unknown. Studying the Bs2/AvrBs2 interaction is difficult because background expression of AvrBs2 from leaky inducible constructs is enough to trigger HR in plants with the Bs2 gene. In order to shed light on plant resistance to bacterial spot disease, I am designing a low background, inducible avrBs2 gene. Our lab has developed an alternatively-spliced cassette, Plant 5S Mimic (P5SM), which we have previously shown can control expression of enhanced Green Fluorescent Protein (eGFP) and other genes. The P5SM cassette exon contains a premature termination codon which results in nonsense-mediated decay of pre-mRNA. When we induce skipping of the P5SM exon, we recover gene expression. I am currently introducing P5SM into avrBs2. I expect to present results testing this construct with Agrobacterium-mediated transient transformations of Nicotiana benthamiana, a model plant in the tomato family. The addition of RNA-level gene control to an inducible promoter construct is a novel combination. This gene control system may provide a general solution for other plant biotechnology applications that do not tolerate leaky basal protein expression.