2014 bgri briggs

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2014 bgri briggs

  1. 1. Alterations in host transcriptional activity to rust pathogens J. Briggs, J. Garbe, M. N. Rouse, J. Kurle University of Minnesota, USDA-ARS Cereal Disease Laboratory #bgri2014
  2. 2. Plant-Pathogen Interactions § Plants are exposed to a wide variety of potential pathogens § Relatively few infect and elicit disease symptoms § Successful colonization of a host requires: § Surpassing passive defenses § Suppressing or delaying resistance responses § Obtaining nutrients from the host
  3. 3. Disease   •  Affected  host  genes  are  dubbed  suscep4bility  genes  (S-­‐genes)   •  They  create  a  more  conducive  environment  for  the  pathogen   Pathogen  effectors   Differen0ally  expressed  host   proteins-­‐suscep0ble  response   Effector Triggered Susceptibility
  4. 4. S-genes § Why should we find them? § Mutated S-genes can confer resistance, sometimes separate from standard pathways § pmr6 mutant in Arabidopsis thaliana § Mutant S-gene derived resistance can be non- race-specific, durable, and involved conserved interactions § mlo mutants in Hordeum vulgare pmr6 in A. thaliana Vogel et al. 2002 § Are there conserved pathways which are altered during rust infection? § Are S-genes present we can manipulate/mutate for resistance? § Specifically, we want to identify putative S-genes, mutate and assess them for resistant/susceptible phenotypes.
  5. 5. Plant and Pathogen Materials § Utilized three compatible host-rust systems § Barley, soybean, and maize § Shifts focus from host specific to broad interactions
  6. 6. Experimental Design § 6, 12, 24, 72 hours post inoculation Mock Inoculated (control) Inoculated
  7. 7. Transcript Analysis Workflow FastQC TopHat2 TopHat2- Corrected Interval Cufflinks Cuffmerge- All Time Points CuffDiff
  8. 8. Comparing Differential Expression Homologous DEGs Barley DEGs Soybean DEGs Maize DEGs S-Genes
  9. 9. Soybean § Differential expression characterized by early up-regulation § No differential expression at 24 hours 0 50 100 150 200 250 6 12 24 72 TotalDEGs Hours Post Inoculation Soybean: Up vs. Down-regulation Up-regulated Down-regulated 40896 78385 Soybean Differential Expression Similar Expression Differential Expression Differential Expression- Homologous to Barley
  10. 10. Soybean Genes Mapped § Location of soybean DEGs homologous to barley DEGs
  11. 11. Barley § Differential expression follows a biphasic pattern § No differential expression at 6 hpi § Significantly more up-regulation 40963 1410 84 Barley Differential Expression Similar Expression Differential Expression Differential Expression- Homologous to Soy 0 200 400 600 800 1000 1200 6 12 24 72 TotalDEGs Hours Post Inoculation Barley: Up vs. Down-regulation Up-regulated Down-regulated
  12. 12. Barley genes mapped § Partially mapped, about 30 unmapped genes § Some have a general location §  8 on 1H §  5 on 3HL, 2 on 3HS §  3 on 4HL §  1 on 5HL §  2 on 6HL §  6 on 7HL §  3 on unknown contigs
  13. 13. Overrepresented GO categories § Regulation of Transcription § Sequence specific DNA binding § Protein Phosphorylation § Serine-threonine kinase activity § Carbohydrate Metabolism § Hydrolases § Oxidation Reduction § Metal ion binding § Peptidase Inhibition § Trypsin-, serine-, aspartic-type
  14. 14. Heat Shock Transcription Factors § Some associated with defense responses, results vary by study -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 3 3.5 4 6 12 24 72 Log2FoldChange Hours Post Inoculation Regulation of Transcription Response to heat Barley 1 Soybean 1
  15. 15. Glutathione S-Transferase § Typically up-regulated in resistant responses § Mediates stress tolerance, detoxification -4 -3.5 -3 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 6 12 24 72 Log2FoldChange Hours Post Inoculation Glutathione S-Transferases Barley 2 Barley 3 Soybean 2
  16. 16. Transmembrane Nitrate Transporters § Knockdown mutant alleles in poplar are associated with earlier activation salicylic acid stimulated defenses and rust resistant -1 -0.5 0 0.5 1 1.5 2 2.5 6 12 24 72 Log2FoldChange Hours Post Inoculation Nitrate Transmembrane Transporters Barley 4 Soybean 3
  17. 17. Serine Peptidase Inhibitors § Shown to be up-regulated in response to biotrophic and necrotrophic fungi § Overexpression shown to increase fungal resistance -8 -6 -4 -2 0 2 4 6 6 12 24 72 Log2foldchange Hours after Inoculation Soybean Peptidase Inhibitors Soybean 4 Soybean 5 Soybean 6 -2.5 -2 -1.5 -1 -0.5 0 0.5 6 12 24 72 Log2FoldChange Hours after Inoculation Barley Peptidase Inhibitors Barley 5 Barley 6 Barley 7
  18. 18. Changes in Regulation § If multiple genes exhibit similar changes in expression it is possible they are regulated via a similar mechanism -8 -6 -4 -2 0 2 4 6 6 12 24 72 Log2foldchange Hours after Inoculation Soybean Peptidase Inhibitors Soybean 4 Soybean 5 Soybean 6 -2.5 -2 -1.5 -1 -0.5 0 0.5 6 12 24 72 Log2FoldChange Hours after Inoculation Barley Peptidase Inhibitors Barley 5 Barley 6 Barley 7 -1.5 -1 -0.5 0 0.5 1 1.5 2 6 12 24 72 Log2FoldChange Hours after Inoculation Soybean Transcription Factor Soybean 7 -1 -0.5 0 0.5 1 1.5 2 2.5 6 12 24 72 Log2FoldChange Hours after Inoculation Barley Transcription Factors Barley 8 Barley 9
  19. 19. Conclusion § Conserved patterns of differential gene expression are being identified § Candidate S-genes also identified § Their involvement in the infection process and ability to alter the host-rust interaction is yet to be determined
  20. 20. Acknowledgements § Rouse Lab USDA-ARS §  Matthew Rouse §  Jayaveeramuthu Nirmala §  Sam Stoxen § Kurle Lab U of Minnesota §  James Kurle §  Grace Anderson § U of Minnesota Supercomputing Institute §  John Garbe § Monsanto graduate fellowship
  21. 21. Questions or Comments?

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