1. An EST library from Puccinia graminis f. sp. tritici reveals genes potentially involved in fungal differentiation Katja Broeker, Frank Bernard & Bruno M. Moerschbacher FEMS Microbiol Lett. 2006 Mar;256(2):273-81.. Advisor : Cing-Long Lan Student : Shun-Yang Cheng Date : 12/07/10

2. Introduction

3. Puccinia graminis f. sp. tritici The rust fungus. It’s a obligately biotrophic pathogen. n wheat plants Wheat is host to rust fungi. It cause serious losses in wheat harvests.

4. Kingdom Fungi Phylum Basidiomycota Class Teliomycetes Order Uredinales Family Pucciiaceae Genus Puccinia Species gramiais

5. http://4.bp.blogspot.com/_WomxvpSA0cQ/TIQ2aucbZOI/AAAAAAAAA Vg/qZVVmoGtY5s/s1600/PgLifecycle.jpg

6. http://www.gardeningblog.net/pictures/barberry.jpg

7. It’s causing black or stem, brown or leaf. Severe epidemics of black rust in Northern America in the 1950s Little is known about this fungus at the molecular level

8. This is the only rust fungus that can be grown in axenic culture so that it can be examined in pure culture apart from its host organism.

9. Materials and Methods

10. Wheat plants Triticum aestivum L. http://www.henriettesherbal.com/pictures/p14/pages/triticum-spelta- 4.htm

11. Plant growth and inoculation

12. Eight-day-old plants were inoculated with uredospores of Puccinia graminis PERS. f. sp. Tritici ERIKSS. & HENN. suspended in Freon- 113 (1,1,2-trichloro-1,2,2-trifluoroethane), or treated with Freon alone http://encyclopedia.airliquide.com/images_encyclopedie/molecules/Chlorotrifluoromethane.gif

13. inoculated or mock-inoculated plants were kept at high humidity in the dark for 18 h followed by the regular day–night rhythm in the growth chamber. Primary leaves were harvested at different days postinoculation (dpi) and directly frozen in liquid nitrogen.

14. Axenic culture of the rust fungus

15. cDNA library construction Suppression subtraction Hybridization PCR PCR-Select cDNA Subtraction kit from Clontech (Palo Alto,CA)

16. Suppression subtractive hybridization technique. FASEB J 9: A1421–A1421.

19. Total RNA was extracted from infected wheat leaves harvested at 5, 9,and 12 dpi. The driver cDNAs were derived from noninoculated wheat leaves.

20. The resulting cDNA library was ligated in pCR2.1 Topo vector. competent TOP10F’ Escherichia coli cells were heat shock-transformed with the ligation mix

21. The SSH-cDNA inserts of the plasmids from 454 clones were sequenced by MWG (Ebersberg, Germany).

22. Sequence analysis and bioinformatics The cDNA sequences were trimmed by removing vector and adaptor A contig analysis was performed using the Cap EST assembler. The contigs were compared to the protein database at NCBI using the BlastX program.

23. Cap EST assembler

24. BlastX Program http://blast.ncbi.nlm.nih.gov

27. http://140.136.180.180/xms/index.php

30. Semiquantitative RT-PCR polyT18 as a primer M-MLV reverse transcriptase RNAse H minus Point Mutant (Promega,Madison, WI).

31. templates for RTPCR cDNAs derived from 1. infected wheat leaves harvested 1, 3,5, 7, 9, and 11 days after inoculation (dpi) 2.Axenic mycelium growing as germ tubes, infection structures, nonsporulating mycelium, and sporulating mycelium.

34. Results

36. The EST-sequences were submitted to dbEST at NCBI and can be retrieved under the GenBank accession numbers CV191576– CV191778

40. Constitutively expressed genes

41. Genes expressed most strongly in mature colonies

42. Genes expressed most strongly in young colonies

43. Plant-induced genes

44. Genes with divergent expression patterns

45. Conclusion

46. Clones such as PGT_633 which appears to be involved in the differentiation of uredospores, Clones such as PGT_693 or PGT_659 which might be involved in plant-fungus interaction

47. Disscussion

48. In recent years, research was intensified to elucidate the infection processes of rust fungi. The focus was on genes specifically expressed in planta, since such genes are regarded as being potentially involved in the infection process Therefore, these sequences are the most abundant rust fungal sequences in the databases today

49. The two ESTs which were most frequently retrieved, namely PGT_728 (59 times) and PGT_731 (16 times) are judged putative infection-related ESTs by their similarities to plant-induced genes from Uromyces spec.

50. Thank You For Your Attention