Submitted to: Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/28/2006
Publication Date: 9/1/2006
Citation: Federico, M.L., Iniguez-Luy, F.L., Skadsen, R.W., Kaeppler, H.F. Spatial and temporal divergence of expression in duplicate barley germin-like protein-encoding genes. Genetics. 174:179-190.
Interpretive Summary: The fungal pathogen Fusarium graminearum is one of the most destructive diseases affecting U.S. agriculture. It infects developing seeds in barley and wheat, causing Fusarium head blight. This reduces seed yields and deposits mycotoxins that make the harvest unfit for consumption. We are undertaking molecular biology approaches to stopping the fungus by identifying barley proteins that kill the fungus. The genes that encode these proteins will be attached to barley promoter DNA sequences that cause the proteins to be produced in the path of the fungus. A screening process was conducted to locate genes that are turned on only in tissues of developing barley seeds that are infected by Fusarium. This produced the gene GerB, which is turned on almost exclusively in the epicarp tissue surrounding the seed. GerB encodes a germin-like protein. We identified, cloned and analyzed the control region that turns on this gene. This DNA sequence (the promoter) was attached to a gene that encodes a green fluorescent protein. When this combination was introduced into barley, epicarp cells glowed green, while leaf and other cells did not. This research also identified an almost identical gene, GerF, that is expressed in different tissues than GerB. This occurs despite the fact that their promoter sequences are very similar. This discovery gives us an insight into how genes become duplicated and then slowly change independlently from one another. This research will benefit barley farmers, malting companies and breweries by leading to barley resistant to Fusarium.
Technical Abstract: Subfunctionalization is the process by which a pair of duplicate genes experiences a reduction of individual expression patterns while maintaining the complete expression pattern of the ancestral gene. Two germin-like protein (GLP) encoding genes, GerB and GerF, belong to a small gene family in barley (H. vulgare). Both share high nucleotide sequence homology in coding and non-coding regions. They also encode identical apoplastic proteins. The Ka/Ks ratio (=0.00182 /0.01409=0.129) indicates that they are undergoing purifying selection, probably due to function constraint on protein evolution. The use of RNA gel blots, coupled with single stranded conformation polymorphism (SSCP) analysis of RT-PCR products, elucidated the developmental and tissue-specific expression patterns of each gene. This provided evidence of both overlapping redundancy and early subfunctionalization. GerB is predominantly expressed in developing shoots; while GerF is predominantly expressed in seedling roots, developing spikes and pericarp/testa. Promoter deletion studies located a GerF region (-356/-97) responsible for high promoter activity and showed the ability of GerB and GerF upstream regions to drive gfp (green fluorescent protein gene) expression in coleoptiles, epicarps, and lemma/palea of developing spikes. This ability and the inducible response pattern exhibited by these duplicate genes during Fusarium graminearum infection make these promoters useful candidates for targeting transgene-mediated resistance in barley. In addition, GerB and GerF expression patterns are consistent with proposed roles in plant development and defense mechanisms for this gene family. These roles may explain why redundancy has been selectively maintained in this duplicate gene pair.