Xianming Chen, USDA-ARS

1. Towards an understanding of
the molecular mechanisms
of durable and non-durable
resistance to stripe rust
Xianming Chen
USDA-ARS, Wheat Genetics, Quality, Physiology, and Disease
Research Unit, Pullman, WA and
Department of Plant Pathology, Washington State University

2. Stripe rust
damage can be
HUGE
Average 2010
3 86
0 10 20 30 40 50 60 70 80 90 100

3. Distribution of Stripe Rust in the US in 2010
Grain Yield Loss in the US
2003: 89 M. Bu (2.42 M. MT)
2005: 73 M. Bu (1.99 M. MT)
2010: 87 M. Bu (2.38 M. MT)

4. Damage can be prevented

5. Yield Loss by Stripe Rust and Increase by Fungicide on Winter Wheat Cultivars, Pullman, WA 2010
Area in 2010 Stripe rust AUDPC Yield (BU/A) YL (%) YI (%) TW (Lb/Bu)
Cultivar Acres % Check Fung. Check Fung. Dif. by rust by fung. Check Fung.
PS 279 0 0.00 2937.50 1300.75 *** 29.88 65.19 35.31 *** 54.17 118.19 55.44 58.08 **
Declo 10,900 0.62 2752.50 566.88 *** 42.15 75.27 33.12 *** 44.00 78.58 54.56 56.67 *
Lambert 13,150 0.75 2373.75 409.50 *** 64.43 96.65 32.22 ** 33.34 50.01 56.56 58.04 *
Xerpha 159,766 9.13 1624.38 495.75 ** 70.49 95.44 24.95 *** 26.15 35.40 57.83 58.43
Tubbs 06 16,200 0.93 2036.25 487.00 *** 78.75 102.32 23.57 *** 23.03 29.93 55.65 56.32
Eddy 34,000 1.94 1915.75 330.13 *** 71.37 92.53 21.16 ** 22.87 29.65 60.69 61.92 **
Bauermeister 29,500 1.69 1053.75 392.50 *** 67.28 84.75 17.47 * 20.61 25.96 57.97 58.43
Paladin 13,000 0.74 2103.13 713.00 *** 63.72 80.03 16.31 * 20.38 25.60 58.29 59.56 **
ORCF-103 19,900 1.14 1342.50 450.63 *** 83.07 97.35 14.28 ** 14.67 17.19 56.81 56.78
Masami 23,000 1.31 630.75 285.50 *** 86.10 100.64 14.53 ** 14.44 16.88 56.21 56.81
Stephens 27,100 1.55 1202.50 196.25 *** 102.07 116.44 14.37 ** 12.34 14.08 58.15 58.61
ORCF-102 169,000 9.66 580.88 184.63 ** 90.04 102.57 12.53 * 12.22 13.92 57.48 58.19
Brundage 96 39,400 2.25 397.50 82.63 ** 98.37 110.98 12.61 ** 11.36 12.82 57.83 58.61 **
Cashup 15,800 0.90 1117.63 291.25 ** 78.78 88.11 9.33 ** 10.59 11.84 59.17 59.14
Eltan 309,850 17.71 728.25 265.25 * 85.99 96.05 10.07 10.48 11.71 56.60 56.74
Westbred 528 108,100 6.18 513.63 135.00 * 97.44 108.21 10.77 ** 9.95 11.05 59.35 59.95
Chuckar 17,800 1.02 60.50 27.00 106.62 114.81 8.19 * 7.14 7.68 59.21 59.56
Bruehl 168,900 9.65 78.75 36.00 *** 95.27 102.05 6.78 6.65 7.12 57.13 56.78
Finley 42,500 2.43 297.25 71.38 * 61.70 64.98 3.28 5.04 5.31 62.17 62.73
Farnum 10,900 0.62 67.25 9.00 * 66.43 67.93 1.51 2.22 2.27 59.49 59.77
Madsen 95,100 5.43 39.88 9.00 110.12 111.30 1.18 1.06 1.07 59.59 59.59
AP700CL 48,000 2.74 171.38 36.00 * 116.76 115.12 -1.64 -1.43 -1.41 58.75 59.10
Rod 56,883 3.25 654.50 242.75 ** 104.87 103.19 -1.69 -1.63 -1.61 57.52 57.41
Buchanan 25,700 1.47 345.75 230.38 ** 93.32 89.06 -4.25 ** -4.77 -4.56 57.87 58.19
Mean 1042.74 302.01 *** 81.88 95.04 13.17 *** 14.79 21.61 57.93 58.56 ***
f
Mean (Excl. PS 279) (83.11 960.36 258.58 *** 84.14 96.34 12.20 *** 13.07(9.36 ) 17.41 58.04 58.58 ***

6. In Washington State in 2010:
•The various levels of resistance including HTAP
resistance were estimated to reduce yield loss from
potentially more than 60% to about 9%.
• The application of fungicides in more than 60% of winter
and spring wheat acreage in Washington State was
estimated to reduce yield loss further to about 3% (about
4.5 million bushels) in average.
• The total resistance in all wheat cultivars collectively was
able to save 73 million bushes ($512 million) and the
fungicide application further saved 13.7 million bushels
($96 million) at the cost of estimated over $27 million.

7. All-Stage (Seedling) Resistance:
Can be detected in seedling stage
Effective in all growth stages Yr5 AVS
High-level resistance
Easy to incorporate
into cultivars
Not durable if a single
gene is involved

8. Dr. Orville A. Vogel was the first to develop wheat
cultivars with partial resistance to stripe rust
IT and % in 2008
Pullman Mt. Vernon
Cultivar Release Flow. S. elong Head.
Brevor 1949 3 30 5 30 2 20
Omar 1955 8 90 8 60 8 100
Gaines 1960 5 50 8 60 5 60
Nugaines 1965 5 40 8 40 3 40
Luke 1970 2 10 3 15 3 5
Photo source: http://cahnrsnews.wsu.edu/reportertools/news/2007/vogel-building-2007-09.html

9. Dr. Roland F. Line
characterized high-temperature
adult-plant (HTAP) resistance as
the type of resistance expressed
at high post-inoculation
temperatures and at adult-plant
stage.

10. High-Temperature Adult-Plant Resistance
Expresses when weather is warm and
plants grow old
Low to high-level resistance
Durable
Conferred by quantitative trait loci
Relatively difficult to detect and difficult to
incorporate into cultivars
May not be adequate

11. Year
release Wheat cultivars and their possible source of HTAP resistance
1949 Brevor
Nord
1960 Gaines Desprez
1965 Nugaines
1970 Luke
1971 Hyslop
1972 Sprague
1976 Raeder McDermid Daws
1977 Stephens
1979 Walladay
1982 Lewjain
1983 Hill 81
1984 Basin John
1985 Malcolm Dusty Batum
1986 Oveson
1987 Frontana Spillman Wakanz
1988 Madsen
1990 Eltan Kmor
1991 Express Bonneville
1992 Macvicar Rod
1993 Rohde
1994 Lambert Alpowa Wawawai
1997 Boundary
1998 Weatherford Coda Hiller
2000 Edwin Cappelle Desprez Hubbard
2001 Bruehl Brundage 96 Chukar Finch Gary
2002 Tubbs
2004 Masami
2005 MDM Bauermeister Otis Louise
2006 Darwin
2007 Xerpha

12. Why is race-specific all-stage resistance
not durable and nonrace-specific HTAP
resistance durable?

13. Wheat GeneChip
 55,052 probe sets
 Derived from the public content
of the T. aestivum UniGene Build
#38
 Probe sets consist of pairs of 11
perfect match (PM) and
mismatch (MM) 25-mer
oligonucleotides

14. Yr5 Experimental Design
 Near isogenic lines (Isolines)
Yr5 AvS
 Yr5 (AvSYr5NIL: resistant)
 yr5 (AvS: susceptible)
 Mock- and P. s. tritici-inoculation
 Seedling stage (~10 days)
 PST-100
 Time-course sampling (6, 12, 24, 48 h)
 3 biological replicates
 RNA extraction, labeling and hybridization

15. Yr5 Data Analysis
120
Differentially expressed
100
80
transcripts
Yr5
Yr5
60
yr5
40
20
yr5
0
6 12 24 48
Time-point (hpi)

16. Transcript Separation
Yr5 yr5
Biotrophic
HR-specific interaction
transcripts 61 54 19 specific
transcripts
Basal defense
transcripts

17. Transcript Annotation
• HR-specific transcripts
–Defense signaling via protein kinases
–Oxidative burst and HR
–PR proteins and phenylpropanoids

19. Yr39 Experimental Design
• Yr39 (resistant) and yr39 (susceptible) RILs
from an AvS/Alpowa F7 population
• Mock- and P. s. tritici-inoculation
–Flag leaf stage (~35 days)
–Time-course sampling (12, 24, 48 h)
–3 biological replicates
• RNA extraction, labeling
and hybridization

20. Fungal Biomass
0.15
Accumulation of Pst beta-tubulin
mRNA transcripts (1/delta Ct)
yr39
0.10
Yr39
*
*
0.05
0.00
0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 102
Time (hpi)

21. Differential Expression
 In reference to mock-inoculated controls
 For Yr39 HTAP resistance interaction
 215 transcripts significant at 48 hpi
 207 induced and 8 repressed
 For yr39 compatible interaction
 Just 1 transcript induced at 48 hpi

22. HTAP Resistance-Specific Transcripts
• All 99 transcripts were induced
• 50.5% involved in pathogen defense and signal
transduction
• 10 R gene-like transcripts including;
– Yr10 all-stage stripe rust resistance protein
– Cf2/Cf5 disease resistance protein homolog
– Barley stem rust resistance protein Rpg1
– Maize NADPH-dependent HC-toxin reductase Hm1
homolog

23. HTAP Resistance-Specific Transcripts
• Other interesting induced transcripts;
– Several protein kinase signaling proteins
– Transcriptional regulatory transcripts
including a WRKY5 homolog
– Five beta-1,3-glucanase transcripts
– Wheat pathogen-induced WIR1A protein
– Six phenylalanine ammonia-lyase (PAL)
transcripts
• Quantitative RT-PCR confirmed results

24. Genes Regulated by Yr5 Race-Specific
and Yr39 Nonrace-Specific
Resistance to Stripe Rust
Race-specific Basal Nonrace-specific Basal
resistance defense resistance defense
yr39
Yr5 yr5 Yr39
61 19 99 1
HR 54
specific Biotrophic
91
interaction
specific

25. Comparison of transcripts regulated by the Yr5 race-specific all-
stage resistance and the Yr39 nonrace-specific HTAP resistance
Race-specific Nonrace-specific
resistance resistance
Yr5 Yr39
61 99
14

26. Functional category Yr5 race-specific Yr39 race non-
resistance specific
resistance
• Defense
• Resistance proteins
- Yr10 homolog - +
- Rpg1 homolog - +
- Cf2/Cf9 homolog - +
- Hm1 homolog - +
• Cell wall
- WIR1A protein + +
- Proline-rich protein + -
• Pathogenesis-related proteins
- Beta-1,3-glucanase + +
- PR protein 10 + -
- Thaumatin-like protein + -
- Chitinase + -
• Phenylpropanoid
- Phenylalanine-ammonia lyase + +
- UDP-glucosyl transferase - +
- Hydroxyanthranilate hydroxyl cinnamoyl - +
transferase

27. Functional category Yr5 race-specific Yr39 race non-
resistance specific
resistance
• Defense
• Oxidative stress
- Peroxidase + +
• Hypersensitive response
- S1/P1 nuclease + -
- Chromosome condensation factor + -
• Miscellaneous
- ABC transporter - +
- Putative disease resistance protein - +
- Latex protein allergen - +
• Signal transduction
- Protein kinase + +
- Receptor protein kinase - +
- Calmodulin protein + +
• Transcription
- WRKY5 homolog - +
- Myb transcription factor + -

28. Meta-analysis Design
• Custom oligonucleotide microarray
– 116 significant transcripts from Yr5 results
– 207 significant transcripts from Yr39 results
• Aim to identify common/unique gene expression
signatures involved in each resistance

29. Resistance Type Comparison
(More Genes of Races-Specific vs. Nonrace-specific)
• 8 wheat genotypes with race-specific resistance
– Yr1, Yr5, Yr7, Yr8, Yr9, Yr10, Yr15 and Yr17
• 4 genotypes with nonrace-specific resistance
– Yr18, Yr29, Yr36 and Yr39
• Mock and incompatible interaction
– Seedling and adult plant stage

30. Race-specific Resistance
• Seedling stage phenotype effect
– Combined genotype data
• 28 transcripts significant
– P <0.10 and fold change >2.0
• Compared to 116 transcripts in Yr5 response
– Meta-analysis narrowed the gene list

31. Transcript Annotation
Functional category Putative function Fold change p value
Defense Putative disease resistance protein 2.45 0.017
Defense Putative disease resistance protein 2.36 0.017
Defense - alkaloid 28 transcripts annotated
• 21 of the Reticuline oxidase 2.01 0.078
Defense - cell wall Pathogen induced WIR1A protein 2.15 0.000
– 15 (71%) involved in defense/signaling
Defense - oxidative stress Blue copper-binding protein 4.11 0.000
Defense - oxidative stress Blue copper-binding protein 2.35 0.012
Defense - oxidative stress Peroxidase 2.54 0.022
Defense - oxidative stress Peroxidase 2.71 0.089
Defense - phenylpropanoid Phenylalanine ammonia-lyase 2.13 0.004
Defense - phenylpropanoid Phenylalanine ammonia-lyase 5.43 0.001
Defense - PR protein Beta-1,3-glucanase 2.04 0.087
Defense - PR protein PR protein 10 2.01 0.003
Defense - R protein NB-ARC domain containing protein 2.66 0.024
Signal transduction Calmodulin-binding protein 2.82 0.055
Signal transduction LRR-containing extracellular glycoprotein 2.57 0.001
Transcription Transcription factor 2.40 0.000

32. Nonrace-specific resistance
• Only detectable at adult-stage
• Zero significant transcripts for nonrace-
specific resistance phenotype effect
• Directly compared race-specific resistance to
nonrace-specific resistance
– 5 transcripts significant for race-specific
resistance
– 1 transcript significant for nonrace-specific
resistance

33. Race-specific resistance
Functional Category Putative Function Fold change p value
Defense - cell wall Hydroxyproline-rich glycoprotein 10.78 0.000
Defense - R protein NB-ARC domain containing protein 2.22 0.006
Signal transduction Protein kinase 5.50 0.000
Unknown No homology 4.38 0.000
Unknown No homology 2.43 0.000
Nonrace-specific resistance
Functional category Putative function Fold change p value
Transport Nonclathrin coat protein 2.16 0.000

34. Relationships of Yr genes based on common and unique
transcripts in response to stripe rust infection
Yr1
Yr5
Yr17
Yr15
Yr8
Yr10
Yr18
Yr39
Yr9
Yr29

35. Conclusions and Perspectives
 In comparison with race-specific all-stage resistance, nonrace-
specific HTAP resistance is contributed by a relatively great
number of defense-related genes, which may explain the durability
of HTAP resistance.
 Genes contributing to all-stage resistance are induced fast and
their transcription levels increased dramatically in the infection
process, while those contributing to HTAP resistance are induced
more slowly and their transcription changes are less dramatic.
 All-stage resistances mediated by different R genes tend to share
many common defense genes, while HTAP resistances-mediated
by different genes do not have many defense genes in common.
 Transcription factors identified in these studies may play key roles
in the networks of plant defense. Further characterization of these
genes may enhance our understanding of molecular mechanisms
of different types of resistance.

36. Cloning a Pleiotropic Drug Resistance/
ABC Transporter Gene from Alpowa
Ta.6990.1.S1_at
1.29*
Mean log2 fold change
(Pst inoculated – mock
1.4
1.2
inoculated) 1
0.8 * Significant
0.6
0.25 0.27
0.4
0.02
0.2
0
24hai 48hai
HTAP Resistance (Yr39)
Susceptible (yr39)

37. Genes with similar sequence to Ta.6990.1.S1
Ta.6990.1.S1_at is likely a PDR
[Pleiotropic Drug Resistance]-type ABC
[ATP Binding Cassette] transporter

38. ABC Gene
associated with
Ta.6990.1.S1_at
Yr18/Lr34
Ta.6990.1.S1_at Yr18/Lr34
Size (kb) 7.4 11.8
Introns 18 24
Chromosome 7A* 7D*
• Lr34-A, a homolog of Lr34, is located on 7A, but its sequence
is dissimilar from the Ta.6990.1 associated gene.
The gene associated with the Ta.6990.1.S1 transcript is
substantially different from Yr18/Lr34 (47% similar) even
though both are PDR- type ABC transporters.

39. Thank You
Tristan Coram
Xueling Huang
Meinan Wang
Andrea Dolezal