Submitted to: International Wheat Scab Symposium Proceedings
Publication Type: Proceedings
Publication Acceptance Date: 11/25/2004
Publication Date: 12/10/2004
Citation: Tobias, D., Pritsch, C., Dahleen, L.S., Jha, A. Expression patterns of chitinase and thaumatin-like proteins in three events of barley (hordeum vulgare cv. conlon). Proceedings of the 2nd International Symposium on Fusarium Head Blight; incorporating the 8th European Fusarium Seminar; Dec. 11-15, 2004, Orlando, FL. Vol. 1. pg. 269. Interpretive Summary: Fusarium head blight (FHB), predominantly caused by the fungus Fusarium graminearum, is a devastating disease in cereal grain crops including barley. Currently, there are no barley genotypes that are highly resistant to FHB. Introducing a combination of antifungal or antitoxin genes into barley may provide greater resistance to the disease. We have transformed barley with two antifungal genes from rice. Transgenic plants were analyzed to confirm the presence of the two genes and their expression. Further analysis of progenies for their protein expression is underway to discriminate between rice proteins from those that could be endogenous to barley.
Technical Abstract: Fusarium head blight (FHB), predominantly caused by Fusarium graminearum, is a devastating disease in barley and other cereal grains. Currently, there are no reports of barley genotypes that are highly resistant to FHB. The barley malting cultivar Conlon was previously transformed with two antifungal genes, chi (chitinase) and tlp (thaumatin-like protein) by particle bombardment. T2 homozygous plants from three transformation events were obtained and analyzed for transgene integration and expression. In the the present report, T3 progenies from the three transformation events were further analyzed for chitinase (chi) and thaumatin-like protein (tlp) expression in both leaf and spike tissues. Southern hybridization confirmed that all transgenic lines tested from each event were positive for tlp but only one event (Event 2) showed stable integration of the chi transgene. Western blots showed a 26 kD rice chi in Event 2 leaves and spikes that was not detected in transgenic lines from the other two events. Aside from a 35-kD putative barley chi from all transgenic plants and the wildtype, other chitinase bands of 25 and 31 kD were detected in leaves. In all three events including the non-transgenic Conlon spikes, a chi protein close to 25 kD was present. The expected 23-kD protein of the rice tlp was highly expressed in Event 2 leaves but had lower levels in spikes of the other two events (Events 1 and 3). A native tlp which co-migrates with the rice tlp is expressed in the spikes of wildtype Conlon and transgenics making transgene expression levels difficult to determine. It is possible that the bands detected in leaves or spikes other than the 26 kD rice chi or 23 kD rice tlp could be either isoforms that are native to barley or could have been derived by proteolytic processing. To further analyze the differential protein expression and isoforms in leaf and spike tissues of transgenic and wildtype Conlon, isoelectric focusing gel electrophoresis is currently underway. Northern blot analyses are being used to confirm the mRNA expression level of chi and tlp using gene specific probes. RT-PCR analyses are being conducted using to discriminate rice chi and tlp transcripts from corresponding endogenous barley transcripts.