1. Turfgrass and Genetics Because plants have diseases, too! By Jessica Kaczmarek

2. Why are turfgrasses important? Used in: – Lawns of buildings – Roadsides – Athletic fields – Golf courses Benefits – Reduce soil erosion – Trap dust and pollutants – Moderate temperature – Beautify the environment $$ Multibillion dollar business annually! $$

3. Creeping Bentgrass Gets Sick Meet…the DOLLAR SPOT and BROWN PATCH ! Two fungal pathogens!

4. What can be done? Alternative I Fungicide Pathogen strains become resistant Negative impact on human health Alternative II Transgenic plants More sustainable resistance to pathogens Safer for human health

5. Atlantic White Shrimp, Litopenaeus setiferus Antimicrobial peptide Penaeidin4-1 (Pen4-1)

6. ANTIMICROBIAL PEPTIDES (AMP’S) Short sequence peptides with fewer than 50 amino acid residues, most of which have antimicrobial activity against many pathogens.

7. Expression of a Novel Antimicrobial Peptide Penaeidin4-1 in Creeping Bentgrass (Agrostis stolonifera L.) Enhances Plant Fungal Disease Resistance Dr. Chen, et al., at Clemson University Published September 12, 2011

8. Promising Conclusions Expressing Pen4-1 in Creeping Bentgrass DEFENDS Against Fungal Pathogens.

9. DNA Construct

10. Pen 4-1 Nucleotide Sequence from Shrimp Altered for Plant-Optimized Codon Usage

11. How to Make Transgenic Plants Technique: Agrobacterium-mediated Transformation 1.Cut grass leaves in small pieces (1cmx1cm). 2.Soak in Agrobacterium fluid for 10 minutes. 3.Cells along cut will be transformed by Agrobacterium. DNA insertion occurs. 4.Under right conditions, plants will regrow.

12. Wait, wouldn’t transformation disrupt the genes or regulatory sequences at the transgene integration site? Highly unlikely. Transgenic plants show similar phenotypes and have increased resistance to pathogens.

13. In vitro plant leaf inoculation assay: R. solani (brown patch)

14. In vivo plant inoculation bioassay: R. solani

15. Second wave of inoculation: R. solani

16. In vitro plant leaf inoculation assay: S. homoeocarpa (dollar spot)

17. In vivo plant inoculation: S. homeocarpa

18. Future Stable transmission of Pen4-1 into next generations using the original transgenic plants (large-scale application)

19. Ask A Question Time

20. What makes Pen4-1 a good candidate for genetic engineering of enhanced disease resistance in plants? Inhibits many plant pathogenic fungal species, including multi-drug resistant species

21. How did the experimenters know the DNA construct was taken up during the Agrobacterium- mediated transformation? Extract genomic DNA and conduct PCR amplification; increased pathogen resistance is evident

22. Are transgenic plants different from normal plants? No significant differences in general plant morphology, root and shoot development was observed

23. References 1.“Expression of a Novel Antimicrobial Peptide Penaeidin4-1 in Creeping Bentgrass (Agrostic stolonifera L.) Enhances Plant Fungal Disease Resistance.” Man Zhou, Qian Hu, Zhigang Li, Dayong Li, Chin- Fu Chen, Hong Luo. Published September 12. 2011. 2.Google Images for all images.