Semester

Spring

Date of Graduation

2019

Document Type

Dissertation

Degree Type

PhD

College

Davis College of Agriculture, Natural Resources and Design

Department

Not Listed

Committee Chair

Nikola Kovinich

Committee Co-Chair

Daniel G. Panaccione

Committee Member

Daniel G. Panaccione

Committee Member

Vagner A. Benedito

Committee Member

Stephen P. DiFazio

Committee Member

Jonathan R. Cumming

Abstract

Glyceollin phytoalexins are the pathogen-elicited defense metabolites that belong to the isoflavonoid family of molecules of soybean (Glycine max L. Merr). Phytophthora root and stem rot of soybean caused by Phytophthora sojae is a destructive disease throughout the soybean-growing regions worldwide causing devastating economic damages (globally $1-2 billion and over $250 million in the USA) every year. Engineering soybean plants that produce higher levels of glyceollins could confer resistance against this pathogen. Glyceollins also show anticancer and neuroprotective activities in mammals, therefore they are important for agriculture and medical research. Firstly, we were interested in understanding how biotic and chemical elicitors regulate glyceollin biosynthesis differently. We found that combining the biotic elicitor WGE (wall glucan elicitor) extracted for P. sojae and the chemical elicitor silver nitrate had an additive effect on the elicitation of glyceollins I. They elicited glyceollins by separate biosynthetic mechanisms. We treated soybean seedlings with a panel of abiotic stress conditions and found that acidic medium elicited the highest amount of glyceollins in root tissues of 1700µg gt-1 (gram tissue, Fresh Weight). Then we focused on the identification of transcription factors (TFs) that regulate the biosynthesis of glyceollins in response to abiotic and biotic stress. Based on a comparative transcriptomics approach using RNA-seq, we chose to functionally characterize the NAC (NAM/ATAF1/CUC2) family TF GmNAC42-1, a homolog of the camalexin regulator ANAC042 from Arabidopsis and an R2R3-MYB (myeloblastosis)-type TF GmMYB29A2, the homolog of the stilbene regulator VvMYB14 from grapevine. Overexpressing and RNAi silencing of GmNAC42-1 and GmMYB29A2 in WGE-elicited hairy roots resulted in significantly increased and decreased accumulation of glyceollin metabolites and biosynthesis gene transcripts such as glycinol 4-dimethylallyl transferase (G4DT) and isoflavone synthase2 (IFS2). The GmNAC42–1 and GmMYB29A2 proteins directly bound the promoters of glyceollin biosynthesis genes IFS2 and G4DT in the yeast one-hybrid (Y1H) system and localized to the nucleus when expressed with an N-terminal green fluorescent protein (GFP) tag. EMSA (Electrophoretic Mobility Shift Assay) confirmed that MYB29A2 protein binds glyceollin gene promoters. Overall, our findings indicate that GmNAC42–1 and GmMYB29A2 are essential components of a glyceollin gene regulatory network (GRN) and could be used for the large-scale, economic bioproduction of glyceollins for therapeutic use and to enhance resistance against P. sojae.

Embargo Reason

Publication Pending

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