Gene expression is regulated at multiple levels, from control of transcription to co-transcriptional processing steps to control of translation and decay. This multi-layered approach allows for sophisticated gene expression programs to be implemented. Previously, our lab engineered HyP5SM, a plant-derived alternatively spliced cassette exon which can be inserted directly and tracelessly into a variety of open reading frames to inducibly regulate protein expression in dicot plants. HyP5SM takes advantage of a gene regulation strategy common in nature—alternative splicing coupled to nonsense-mediated decay—to produce "default off, inducible on" gene expression. Here, we explored the benefits of combining two levels of regulation, a conditional promoter and an NMD-targeted cassette exon, and showed that we could obtain tight, inducible regulation of toxic genes. We demonstrate that addition of HyP5SM can regulate even the hypersensitive response (HR) phenotype, a defensive programmed cell death response initiated by disease-resistant plants upon detection of specific pathogen effector proteins. Effector-triggered immune pathways are important for disease-specific resistance to plant pathogens. We combine the dexamethasone inducible promoter and the HyP5SM cassette exon to regulate pathogen effector proteins. The inducible promoter alone results in leaky effector protein and HR, but HyP5SM renders the leaky transcript non-productive, thus eliminating leaky protein and the resulting auto-immune responses. Furthermore, plants inducibly recover both effector protein expression and the HR phenotype. We have tested this with Bs2/AvrBs2- and RPP1/ATR1Δ51-dependent hypersensitive response pathways in Nicotiana benthamiana and Nicotiana tabacum, respectively. We also show that Arabidopsis thaliana plants transgenic for these resistance/effector gene pairs are viable, healthy, and can complete their full life cycle (seed-to-seed) without inducing leaky HR phenotype from the pathogen effector transgene, unless the HR immune phenotype is specifically induced with dexamethasone. We also present progress identifying additional factors involved in the alternative splicing regulation of HyP5SM. The HyP5SM cassette can be generally applied to regulate other genes in dicot plants, and may be utilized with conditional, constitutive, or native promoters. Finally, we present progress toward engineering new variants of HyP5SM for gene regulation in mammalian cells.
|Advisor:||Hammond, Ming C.|
|Commitee:||Cate, Jamie, Staskawicz, Brian J., Vance, Russell|
|School:||University of California, Berkeley|
|Department:||Molecular & Cell Biology|
|School Location:||United States -- California|
|Source:||DAI-B 79/08(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Plant sciences|
|Keywords:||Alternative splicing, Engineered cassette exons, Gene regulation, Hypersensitive response, Inducible regulation, Leaky expression|
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