2014 bgri briggs

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

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

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

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.

Plant and Pathogen Materials § Utilized three compatible host-rust systems § Barley, soybean, and maize

§ Shifts focus from host specific to broad interactions

Experimental Design

§ 6, 12, 24, 72 hours post inoculation

Mock Inoculated (control) Inoculated

Transcript Analysis Workflow

FastQC

TopHat2

TopHat2-Corrected Interval

Cufflinks

Cuffmerge-All Time Points

CuffDiff

Comparing Differential Expression

Homologous DEGs

Barley DEGs

Soybean DEGs

Maize DEGs

S-Genes

Soybean § Differential expression characterized by early up-regulation § No differential expression at 24 hours

0

50

100

150

200

250

6 12 24 72

Tota

l DEG

s

Hours Post Inoculation

Soybean: Up vs. Down-regulation

Up-regulated Down-regulated

40896

783 85

Soybean Differential Expression

Similar Expression

Differential Expression

Differential Expression-Homologous to Barley

Soybean Genes Mapped § Location of soybean DEGs homologous to barley DEGs

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

Tota

l DEG

s

Hours Post Inoculation

Barley: Up vs. Down-regulation

Up-regulated Down-regulated

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

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

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

Log2

Fol

d C

hang

e

Hours Post Inoculation

Regulation of Transcription Response to heat

Barley 1 Soybean 1

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

Log2

Fol

d C

hang

e

Hours Post Inoculation

Glutathione S-Transferases

Barley 2 Barley 3 Soybean 2

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 Log2

Fol

d C

hang

e

Hours Post Inoculation

Nitrate Transmembrane Transporters

Barley 4

Soybean 3

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

Log2

fold

cha

nge

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

Log2

Fol

d C

hang

e

Hours after Inoculation

Barley Peptidase Inhibitors

Barley 5 Barley 6 Barley 7

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

Log2

fold

cha

nge

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

Log2

Fol

d C

hang

e

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

Log2

Fol

d C

hang

e

Hours after Inoculation

Soybean Transcription Factor

Soybean 7

-1

-0.5

0

0.5

1

1.5

2

2.5

6 12 24 72 Log2

Fol

d C

hang

e

Hours after Inoculation

Barley Transcription Factors

Barley 8 Barley 9

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

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

Questions or Comments?