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

3. 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

4. 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

5. 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.

6. Plant and Pathogen Materials
§ Utilized three compatible host-rust systems
§ Barley, soybean, and maize
§ Shifts focus from host specific to broad interactions

7. Experimental Design
§ 6, 12, 24, 72 hours post
inoculation
Mock Inoculated (control) Inoculated

8. Transcript Analysis Workflow
FastQC
TopHat2
TopHat2-
Corrected
Interval
Cufflinks
Cuffmerge-
All Time
Points
CuffDiff

9. Comparing Differential Expression
Homologous
DEGs
Barley DEGs
Soybean DEGs
Maize DEGs
S-Genes

10. 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

11. Soybean Genes Mapped
§ Location of soybean DEGs homologous to barley DEGs

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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

18. 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

19. 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

20. 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

21. 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

22. Questions or Comments?