NEW MEXICO AMP ALLIANCE FOR MINORITY PARTICIPATION Identifying

NEW MEXICO AMP ALLIANCE FOR MINORITY PARTICIPATION Identifying Genes Required for Vegetative Incompatibility of Cryphonectria parasitica Desa Daniel and Rong Mu Dr. Dawe Assistant Professor in Biology New Mexico Alliance For Minority Participation New Mexico State University

Out. Line Background Interest Experiments Future Work Acknowledgements

American Chestnut Tree Predominate hardwood tree during the 1800’s Known for their nuts and resistant decaying wood. 1900’s effected by Cryphonectria parasitica (Chestnut Blight) Chestnut Blight is an fungus introduced from Asia Trees die before bearing fruit Now only found in the mid-west Transplanted

Chestnut blight Cryphonectria parasitica chestnut blight dead chestnut

Vegetative Incompatibility Saupe, 2000.

Vegetative Incompatibility • Manifestation Compartmentation and death of fusion hyphae, which usually lead to the formation of barrage on solid medium Growth inhibition Failure to form stable heterokaryons with continued growth • Significance Limit horizontal transfer of infectious elements Possible use of virulence attenuating viruses as a biological control

Vegetative hyphal fusion in Filamentous Fungi EU 5 EU 1 EP 155 A) Hyphal fusion test between EP 155 and Eu 1 and Eu 5 strains in day 5. EU 5 EP 155 EU 1 EP 155 B) Hyphal fusion test between EP 155 and Eu 1 and Eu 5 strains in day 15.

Factors may involved in vegetative incompatibility in C. parasitica 6 vegetative incompatibility (vic) loci allelic incompatibility nonallelic incompatibility Vib 1(vegetative incompatibility blocked)-like gene Firstly found in Neuraspora crassa Participate in allelic incompatibility system Functions: Suppresses conidiation during het-c vegetative incompatibility Regulates hyphal compartmentation and death rate Mod. A (modifiers of vegetative incompatibility)-like gene Firstly found in Podospora anserina Participate in nonallelic incompatibility system Functions: Controls growth arrest in incompatible interaction Involves in female sterility

Experiments Method of making deletion construct—PCR-based method Yu et al. , 2004

Results of PCR-based method • Summary Kb Kb 1 4 3 Kb 1 1) 5’flank and 3’fank. 3) Final product. 2) Hphcassette. Successfully amplified 3 fragments Failed in the last step. Factors that may effect the results include: Taq polymerase, the length of overlapping sequences in primers and molar ratio of 3 fragments.

Future Work Alternative strategies for generation of a construct Yeast Based Method Over Expression

Yeast Based Method Preparato in 3 fragments Gapped vector (PRS 426) Liquid culture of yeast(FY 834 strain) Selective medium (-URA) Yeasttransformato in Alkali-Cation yeast transformation kit

Acknowledgments Rong Mu Dr. Angus Dawe The Dawe Lab New Mexico Alliance for Minority Participation NSF HRD 083171

References Glass and Kaneko, Fatal Attraction: Nonself Recognition and Heterokaryon 2003 p 1 -8 Vol 2, No 1 Incompatibility in Fliamentous Fungi Lucarre, Paoletti, and Clave, Cell Death by Incompatibility in the Fungus Podospora 2007 p 101 -111 Nuss, Hypovirulence: Mycoviruses at the Fungal-plant Interface 2005 Vol 3 p 632 – 639 Powell, G. H. 1898. The European and Japanese chestnuts in the Eastern United States. Delaware College Agricultural Experiment Station bulletin 42, Newark, DE, 16 pp Questions?