Thesis
Using BSMV-VIGS to identify candidate rust resistance genes to be employed as silencing targets in stably transformed wheat through the development of an Agrobacterium tumefaciens transformation protocol
Washington State University
Master of Science (MS), Washington State University
2013
Handle:
https://hdl.handle.net/2376/101772
Abstract
Wheat is one of the most important agronomic crops in the world and is threatened annually by a multitude of biotic and abiotic pests. The use of resistant cultivars is the most economical, effective, and environmentally sound method of protecting against losses from Puccinia rust diseases of wheat. Because resistance genes are often circumvented by pathogen mutations, finding genes that confer more durable resistance and that can be more rapidly deployed is important for developing new cultivars with resistance to disease, especially rust caused by Puccinia species in wheat. Genes from the rust pathogens could be useful for engineering new resistance. RNA sequence experiments have revealed a number of genes with heightened expression in haustoria, which may be important for the production and dispersal of effectors and other essential proteins for Puccinia establishment. Four genes that have high haustorial expression, few gene family members, probable signal peptide or transmenbrane domains, and homology to genes across the three species of Puccinia that infect wheat were selected for silencing experiments. Transient silencing of the four genes using virus induced gene silencing (VIGS) with a barley stripe mosaic virus based vector tested their effectiveness as potential rust resistance genes. VIGS was observed on plants approximately 10 days after infection, after which they were inoculated with P.graminis or P.striiformis. Rust infection was evaluated from 10-22 days after infection; depending on the rust being studied. One gene, a predicted amino acid permease, showed a reduction in disease severity in P.graminis studies. Quantitative real-time PCR confirmed that silencing was occurring in the virus infected leaf tissue. Stable transformation using Agrobacterium tumefaciens was attempted to test the level of rust resistance provided by inserting a hairpin RNA construct of the P.graminis genes previously tested by BSMV-VIGS into the plant genome. Protocols were developed for both wheat and Brachypodium. Brachypodium, which has a more efficient and rapid method of transformation and regeneration than wheat, provides a basis for the level of resistance the selected genes may have on rust. To obtain a true indication of the resistance capabilities, stable transformants of wheat are required.
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Details
- Title
- Using BSMV-VIGS to identify candidate rust resistance genes to be employed as silencing targets in stably transformed wheat through the development of an Agrobacterium tumefaciens transformation protocol
- Creators
- Samantha Ilene Downey
- Contributors
- Scot Hulbert (Degree Supervisor)
- Awarding Institution
- Washington State University
- Academic Unit
- Crop and Soil Sciences, Department of
- Theses and Dissertations
- Master of Science (MS), Washington State University
- Publisher
- Washington State University; [Pullman, Washington] :
- Identifiers
- 99900525292001842
- Language
- English
- Resource Type
- Thesis