1. Work presentation Gaurav Moghe Feb 4 th, 2008 – March 17 th, 2008

2. Projects Divergence between DDF1 and DDF2 Expression differences between A.thaliana and A.lyrata under JA-induced stress Novel coding regions in A. thaliana Amanitus-related toxins in other fungal genomes

3. 1. Divergence between DDF1 and DDF2 Objective: To find protein-level differences between DDF1 and DDF2 using translational fusions Subobjective: Verify transformants for GUS marker -- GUS Staining -- Homo/Heterozygosity using GUS-specific primers

4. Strategy: Translational fusions DDF1 DDF2 GUS Endogenous promoter Spatial and temporal differentiation of expression in Arabidopsis But, Existing lines from older A. thaliana strains. Moving everything to the newer Col0 strains. 10 lines 1 line

5. GUS Staining results Tried GUS staining from leaves, flowers, roots and first inter-node No GUS activity detected Progeny of transformants not differentiable by Hyg resistance phenotype

6. Future plans 1) New Col0 strains (DDF1-GUS) put on soil 2) Perform GUS staining with new Col0 strains under salt-stress from roots and inter-node 3) Verify homo / heterozygosity using GUS- specific primers

7. ChIP-Sequencing part 35S-cMyc-DDF1/2 construct (1 probable plant) 35S-DDF1/2-GFP construct Transformant should show over-expression phenotype, but that was not observed for GFP construct. So, will have to be re-done.

8. 2. JA-induced stress expression A. thalianaA. lyrata JA treated22 Control22 Extract RNA using RNAeasy Solexa sequencingqRT-PCR Analysis

9. RNAEasy (Qiagen) SampleYield260/280260/230 AT JAFrom 200- 600 ng/micL Above 2.0 (low/none protein contamination) AT JA AT Control AL JA AL Control

10. DNA contamination The extracted RNA samples (200ng) were PCR-amplified using GoTaq with: 1) actin_6 (flanks an intron) 2) DDF2 primers for 40X and 32X cycles. Both the reactions showed significant presence of DNA (~20ng after amplification)

11. DNA contamination: options The SuperArray RT2 First strand kit (Problem: Two-step RTPCR. No kit mentioned for Eppendorf Realplex machine) Design primers flanking the longest introns If possible, keep a smaller extension time(?) (Problem: Can affect RNA amplification) Keep RT(-) control during qRT-PCR (Problem: What is the best way to subtract effect of DNA?) Re-do the extraction

12. 3. Novel coding regions in A.thaliana Two directions of study Gene predictorsRNA genes 1.Augustus 2.GlimmerM 3.GeneID 4.TwinScan 5.EuGene 6.CONTRAST 7.SNAP Whole genome alignments BioPerl installation issue For some reason, not accepting Chr4 and Chr 5

13. Predicting existence of yet un-annotated RNA genes Question: Are there any regions in the genome that: 1) Are not known proteins/RNA? 2) Conserved across phylogeny? Are such regions: 1) RNA? 2) Protein?

14. RNA genes: Strategy Download A.thaliana PUTs from PlantGDB (best alternative to FLcDNA) BLASTx against UniProt (UniRef90) (1) RFAM database of known/predicted RNA molecules BLASTx against UniProt (UniRef90) (2) PUTs not known to be proteins Filter (1) using (2) distribution Discard significant matches Retain non-matching and not significantly matching PUTs

15. RNA genes: Strategy PUTs not known to be proteins BLASTn against RFAM (1) 10000 randomly generated PUTs for pre-selected size classes of the original PUTs BLASTn against RFAM (2) PUTs not known to be proteins or RNA Filter (1) using (2) distribution Discard significant matches Retain non-matching and not significantly matching PUTs

16. RNA genes: Strategy PUTs not known to be proteins or RNA BLAST against PUTs from different plants strategically placed in phylogeny Find conserved PUTs RNA prediction softwares 1)RNAZ 2)QRNA 3)ddbRNA 4)EvoFold 5)MSARI Conserved regions not yet annotated Are these PUTs mapped to A. thaliana genome? Yes

17. 4. Amanita toxin project (Walton lab) Multiple sequence alignment of several toxin genes from two fungal genera – Amanita and Galerina Sequenced fungal genomes tBLASTn with relaxed parameters does not give any significant hits

18. Use of Hidden Markov Model (HMM) to generate a PWM for MSA of toxins MARLP PCE HMM Scan 6-frame translated fungal genomes Hits of very low e-value (100-1000), but some degree of relevant sequence similarity Ama-like toxins MSA

19. Thank you