This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We were able to determine the optimal time of giving a light pulse. Greatest phase shifts can be observed 7 h into the dark phase of a 12 h light/12 h dark cycle. We have not optimized the duration of the light pulse yet. However, since two of the photoreceptors we are investigating for their possible involvement in circadian entrainment by light in our model organism absorb in the blue region of the light spectrum, we already tested blue light. We have preliminary evidence that blue light is indeed effective. This is contrary to previously reported findings that were based on a particular mutant of the organism and obtained under ambiguous conditions. Plant-like cryptochrome: We succeeded in identifying several of our transformants with the RNA interference construct for this particular photoreceptor as having indeed reduced amounts of the protein. They will now provide the basis for experiments that test, whether these strains also show reduced abilities to phase shift upon blue light pulses. Animal-like cryptochrome: We did not succeed in cloning the coding sequence for this protein, because we were not able to amplify it by PCR. We only succeeded in amplifying shorter fragments, most likely due to the high GC content of the DNA. Because of our difficulties, and because the protein is most likely a DNA photolyase rather than a photoreceptor as previously suggested, we have abandoned this part of the project. Chlamyopsin: Experiments are currently underway to clone a marker into an RNA interference construct received by a collaborator, so that strains with reduced amounts of this photoreceptor can be created. Phototropin: When testing RNA interference strains for this photoreceptor that we received from a colleague, we did not succeed in detecting the protein in western blots. There is some evidence that the problem arises from degradation problems with the antibodies during shipment and subsequent storage. Our collaborator has kindly agreed to send us another sample in a more secure fashion.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Exploratory Grants (P20)
Project #
5P20RR016481-11
Application #
8360111
Study Section
Special Emphasis Panel (ZRR1-RI-4 (01))
Project Start
2011-05-01
Project End
2012-04-30
Budget Start
2011-05-01
Budget End
2012-04-30
Support Year
11
Fiscal Year
2011
Total Cost
$63,877
Indirect Cost
Name
University of Louisville
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
057588857
City
Louisville
State
KY
Country
United States
Zip Code
40292
Green, Kimberly A; Becker, Yvonne; Fitzsimons, Helen L et al. (2016) An Epichloë festucae homologue of MOB3, a component of the STRIPAK complex, is required for the establishment of a mutualistic symbiotic interaction with Lolium perenne. Mol Plant Pathol 17:1480-1492
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Becker, Yvonne; Eaton, Carla J; Brasell, Emma et al. (2015) The Fungal Cell-Wall Integrity MAPK Cascade Is Crucial for Hyphal Network Formation and Maintenance of Restrictive Growth of Epichloë festucae in Symbiosis With Lolium perenne. Mol Plant Microbe Interact 28:69-85
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