1a. Objectives (from AD-416):
1. Generate transgenic wheat plants that overexpress TaBAK1. 2. Begin the evaluation of these transgenics for increased FHB resistance. 3. Conduct VIGS experiments to evaluate the possible functions of three other TaBAK1-related sequences in FHB resistance, TaBAK2, 3, 4 and 5. 4. Generate cDNA clones for transformation if any of the TaBAK1-related sequences prove to make significant contributions to FHB resistance. 5. Continue the characterization of transgenic wheat overexpressing ethylene-signaling genes.
1b. Approach (from AD-416):
Efforts to make significant improvement in the resistance of wheat to Fusarium head blight (FHB) require understanding the mechanism(s) of the naturally occurring FHB resistance pathways. Quantitative Trait Loci (QTL) conferring varying degrees of FHB resistance are known, and these are being used by breeders to generate useful FHB resistant wheat and barley varieties. However, none of the actual gene sequences that underlie these QTL and determine the mechanism of FHB resistance are known. Until the molecular mechanism of FHB resistance is better understood, efforts to engineer improved FHB resistance will be futile. The process of identifying the genes that are functionally essential to FHB resistance has been greatly hindered by the genetic complexities of wheat. In previous work funded by the USWBSI our group has developed a virus-induced gene silencing (VIGS) system that overcomes many of the obstacles for functional identification of genes involved in FHB resistance. Previous work has shown that wheat and barley plants that are resistant to FHB initiate complex defense responses when challenged by Fusarium graminearum. Understanding how these responses are initiated is a key question to address. Very recent results in our VIGS analyses have implicated a receptor-like protein, TaBAK1, as playing a key role in FHB resistance. In model plant system this protein functions in the perception of conserved pathogen-associated molecular patterns (PAMPs), leading to PAMP-triggered immunity (PTI). The discovery of a receptor-kinase protein that may play a key role in activating FHB resistance offers an excellent opportunity to engineer improved FHB resistance, and thereby directly serves the primary objective of the USWBSI Gene Discovery and Engineering Resistance research area.
3. Progress Report:
Identification of wheat genes involved in resistance to Fusarium head blight (FHB) has been very difficult for two major reasons. First, wheat has at least six copies of most genes so conventional mutagenesis cannot reveal the affect of the loss of gene function, because there are always other functional copies present that mask the mutation. Second, it is very difficult to transform wheat so T-DNA mutations or T-DNAs expressing RNAi constructs are not feasible. Our approach utilizes virus-induced gene silencing (VIGS) to simultaneously down-regulate the expression of all copies of chosen genes. As virus infection of wheat is very rapid, the results of VIGS experiments can be observed within one month of inoculating the plant with the VIGS construct. This project has led to the discovery that ethylene-signaling has a crucial role in resistance of wheat to FHB and that increasing ethylene signaling can improve resistance. In the past year several genes involved in ethylene signaling have been engineered for overexpression and then transformed into wheat. We are in the process of generating sufficient seed to begin to test the transgenics for increased resistance to FHB.