Title

Genetic Transformation of Tobacco with a Beta-Glucosidase Gene to Induce Constitutive Systemic Acquired Resistance against Tobacco Mosaic Virus

Date of Award

8-2004

Degree Name

Doctor of Philosophy

Department

Biological Sciences

First Advisor

Dr. Alexander Enyedi

Second Advisor

Dr. Todd Barkman

Third Advisor

Dr. Hector Quemada

Abstract

Salicylic acid (SA) is a signal molecule that can activate plant defense systems and provide plants with systemic acquired resistance. However, most SA is converted into an inactive form, SA 2-O-β-D-glucoside (GSA), and stored in the vacuole of plant cells. In this project, GSA was converted back into free SA in order to increase the endogenous SA level in plant cells and enhance plant disease resistance. A β-glucosidase gene cloned from the bacterium Butyrivibrio fibrisolvens H17c was inserted into the binary vector pGA482 under the control of the 35S Cauliflower Mosaic Virus promoter. Two vectors, pGLU100 plasmid (for cytosol expression) and pGLU200 plasmid (for vacuole targeted expression) were constructed. Agrobacterium tumefaciens containing either the pGLU100 or the pGLU200 plasmid was used to transform leaf discs of tobacco (Nicotiana tabacum cv. Xanthi-nc NN genotype). In pGLU100 transgenic tobacco plants, the β-glucosidase gene was designed to be expressed in the cytosol of cells while in pGLU200 transgenic tobacco plants, the β-glucosidase gene was designed to be expressed in the vacuole of plant cells. Vacuole expression was accomplished by adding an endoplasmic reticulum (ER)-leading sequence at the 5 end of the β-glucosidase gene and a vacuole-targeting sequence at the 3 end of β-glucosidase gene. Polymerase chain reaction (PCR) analysis of the genome showed that 53% of pGLU100 and 51% of pGLU200 plants of T0 generation contained the bglA gene. Reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated that the bglA gene was expressed in 71% of T 0 generation pGLU100 plants containing bglA gene and 67% of T0 generation pGLU200 tobacco plants containing bglA gene. The results of SA extraction from uninoculated tobacco plants of T0 and T1 generation plants showed that free SA levels increased significantly in the transgenic plants, and the GSA level had decreased significantly compared to the untransformed NN type tobacco plants. The resistance of the T1 generation plants to tobacco mosaic virus (TMV) was detected by measuring the hypersensitive reaction (HR) lesion area 72 hours after TMV inoculation. The area of TMV-induced lesions was reduced by 51% in pGLU100 plants and 60% in pGLU200 plants compared to the NN type tobacco plants. The inheritance of the β-glucosidase gene was determined by Southern blot analysis and confirmed that the bglA gene was successfully passed on to the T1 generation plants. The constitutive expression of the β-glucosidase gene does not seem to affect the phenotype of the plants, indicating that β-glucosidase could be used as a potential gene in engineered plants to enhance disease resistance.

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