Towards an understanding of the molecular mechanisms of durable and non-durable resistance to stripe rust

Xianming Chen

Towards an understanding of the molecular mechanisms of durable and non-durable

resistance to stripe rust

USDA-ARS, Wheat Genetics, Quality, Physiology, and Disease Research Unit, Pullman, WA and

Department of Plant Pathology, Washington State University

Stripe rustdamage can be

HUGE

0 20 40 50 60 70 80 90 10010 303 862010Average

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)

Damage can be prevented

YL (%) YI (%)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 58.43Tubbs 06 16,200 0.93 2036.25 487.00 *** 78.75 102.32 23.57 *** 23.03 29.93 55.65 56.32Eddy 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 58.43Paladin 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 56.78Masami 23,000 1.31 630.75 285.50 *** 86.10 100.64 14.53 ** 14.44 16.88 56.21 56.81Stephens 27,100 1.55 1202.50 196.25 *** 102.07 116.44 14.37 ** 12.34 14.08 58.15 58.61ORCF-102 169,000 9.66 580.88 184.63 ** 90.04 102.57 12.53 * 12.22 13.92 57.48 58.19Brundage 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 59.14Eltan 309,850 17.71 728.25 265.25 * 85.99 96.05 10.07 10.48 11.71 56.60 56.74Westbred 528 108,100 6.18 513.63 135.00 * 97.44 108.21 10.77 ** 9.95 11.05 59.35 59.95Chuckar 17,800 1.02 60.50 27.00 106.62 114.81 8.19 * 7.14 7.68 59.21 59.56Bruehl 168,900 9.65 78.75 36.00 *** 95.27 102.05 6.78 6.65 7.12 57.13 56.78Finley 42,500 2.43 297.25 71.38 * 61.70 64.98 3.28 5.04 5.31 62.17 62.73Farnum 10,900 0.62 67.25 9.00 * 66.43 67.93 1.51 2.22 2.27 59.49 59.77Madsen 95,100 5.43 39.88 9.00 110.12 111.30 1.18 1.06 1.07 59.59 59.59AP700CL 48,000 2.74 171.38 36.00 * 116.76 115.12 -1.64 -1.43 -1.41 58.75 59.10Rod 56,883 3.25 654.50 242.75 ** 104.87 103.19 -1.69 -1.63 -1.61 57.52 57.41Buchanan 25,700 1.47 345.75 230.38 ** 93.32 89.06 -4.25 ** -4.77 -4.56 57.87 58.19Mean 1042.74 302.01 *** 81.88 95.04 13.17 *** 14.79 21.61 57.93 58.56 ***Mean (Excl. PS 279) (83.11 960.36 258.58 *** 84.14 96.34 12.20 *** 13.07(9.36f) 17.41 58.04 58.58 ***

Stripe rust AUDPC Yield (BU/A) TW (Lb/Bu)Area in 2010

Yield Loss by Stripe Rust and Increase by Fungicide on Winter Wheat Cultivars, Pullman, WA 2010

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.

All-Stage (Seedling) Resistance:

Effective in all growth stagesHigh-level resistance

Can be detected in seedling stage

Easy to incorporate into cultivars

Not durable if a singlegene is involved

Yr5 AVS

http://cahnrsnews.wsu.edu/reportertools/news/2007/vogel-building-2007-09.htmlPhoto source:

Dr. Orville A. Vogel was the first to develop wheat cultivars with partial resistance to stripe rust

GainesNugainesLuke

196019651970

Brevor 1949

5 505 402 10

3 30Cultivar Release Flow.

8 608 403 15

5 30S. elong

5 603 403 5

2 20Head.

Pullman Mt. VernonIT and % in 2008

Omar 1955 8 90 8 60 8 100

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-plantstage.

High-Temperature Adult-Plant Resistance

Expresses when weather is warm and plants grow old

Low to high-level resistance

Conferred by quantitative trait lociDurable

Relatively difficult to detect and difficult to incorporate into cultivars

May not be adequate

Brevor

Nugaines

Luke

1949

196019651970

1977 Stephens

Hyslop1971

1976 Raeder Daws

Lewjain1982

McDermid

Hill 811983

Dusty1985

Sprague1972

1984

Gaines

JohnBatumMalcolm

1988 Madsen

1986 Oveson

Eltan1990 Kmor

MacvicarBonneville1991

1992 Rod1993 Rohde1994 Lambert

1998 HillerCodaWeatherford1997 Boundary

2001 Bruehl

Basin

FinchChukar

Cappelle Desprez

GaryBrundage 962002 Tubbs

2000 Hubbard

2004 Masami

Edwin

2005 MDM Bauermeister

2007 Xerpha

Nord Desprez

2006 Darwin

Alpowa

1979 Walladay

Wawawai

Express

Frontana

Louise

1987 WakanzSpillman

Otis

Yearrelease Wheat cultivars and their possible source of HTAP resistance

BrevorBrevor

NugainesNugaines

Luke

1949

196019651970

1977 StephensStephens

HyslopHyslop1971

1976 RaederRaeder DawsDaws

LewjainLewjain1982

McDermidMcDermid

Hill 81Hill 811983

DustyDusty1985

SpragueSprague1972

1984

GainesGaines

JohnJohnBatumBatumMalcolmMalcolm

1988 MadsenMadsen

1986 OvesonOveson

EltanEltan1990 KmorKmor

MacvicarMacvicarBonnevilleBonneville1991

1992 RodRod1993 RohdeRohde1994 LambertLambert

1998 HillerHillerCodaCodaWeatherfordWeatherford1997 Boundary

2001 BruehlBruehl

BasinBasin

FinchFinchChukarChukar

Cappelle DesprezCappelle Desprez

GaryGaryBrundage 96Brundage 962002 TubbsTubbs

2000 HubbardHubbard

2004 MasamiMasami

EdwinEdwin

2005 MDMMDM BauermeisterBauermeister

2007 XerphaXerpha

Nord DesprezNord Desprez

2006 DarwinDarwin

AlpowaAlpowa

1979 WalladayWalladay

WawawaiWawawai

ExpressExpress

FrontanaFrontana

LouiseLouise

1987 WakanzWakanzSpillmanSpillman

OtisOtis

Yearrelease Wheat cultivars and their possible source of HTAP resistance

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

resistance durable?

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

Yr5 Experimental Design Near isogenic lines (Isolines)

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

Yr5 AvS

Yr5 Data Analysis

yr5

Yr5

0

20

40

60

80

100

120

6 12 24 48

Time-point (hpi)

Diff

eren

tially

exp

ress

ed

tran

scri

pts

Yr5yr5

Transcript SeparationYr5 yr5

Basal defensetranscripts

HR-specifictranscripts

Biotrophicinteraction

specifictranscripts

54 1961

Transcript Annotation

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

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, labelingand hybridization

0.00

0.05

0.10

0.15

0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 102

Time (hpi)

Acc

umul

atio

n of

Pst

bet

a-tu

bulin

m

RN

A tr

ansc

ripts

(1/d

elta

Ct)

* *

Fungal Biomass

Yr39

yr39

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

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

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

Genes Regulated by Yr5 Race-Specific and Yr39 Nonrace-Specific Resistance to Stripe Rust

9991

1

Nonrace-specific Basal

Yr39yr39

resistance defenseRace-specificresistance

Yr5

Basaldefense

yr561

5419

HRspecific Biotrophic

interactionspecific

Comparison of transcripts regulated by the Yr5 race-specific all-stage resistance and the Yr39 nonrace-specific HTAP resistance

61 9914

Nonrace-specific

Yr39resistance

Race-specific

Yr5resistance

++++

+-

+---

+++

----

++

++++

+--

• 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

Yr39 race non-specific resistance

Yr5 race-specific resistance

Functional category

Functional category Yr5 race-specific resistance

Yr39 race non-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

+

++

---

+-+

-+

+

--

+++

+++

+-

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

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

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

Transcript Annotation

• 21 of the 28 transcripts annotated– 15 (71%) involved in defense/signaling

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 Reticuline oxidase 2.01 0.078 Defense - cell wall Pathogen induced WIR1A protein 2.15 0.000 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

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

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

Functional category Putative function Fold change p value Transport Nonclathrin coat protein 2.16 0.000

Race-specific resistance

Nonrace-specific resistance

Yr1Yr5

Yr8

Yr9

Yr10

Yr15Yr17

Yr18

Yr29

Yr39

Yr1Yr5

Yr8

Yr9

Yr10

Yr15Yr17

Yr18

Yr29

Yr39

Relationships of Yr genes based on common and uniquetranscripts in response to stripe rust infection

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 sharemany common defense genes, while HTAP resistances-mediatedby different genes do not have many defense genes in common.

Transcription factors identified in these studies may play key rolesin the networks of plant defense. Further characterization of thesegenes may enhance our understanding of molecular mechanismsof different types of resistance.

Cloning a Pleiotropic Drug Resistance/ABC Transporter Gene from Alpowa

00.20.40.60.8

11.21.4

24hai 48hai

0.25

1.29

0.020.27

HTAP Resistance (Yr39)Susceptible (yr39)

Mea

n lo

g 2fo

ld c

hang

e(P

stin

ocul

ated

–m

ock

inoc

ulat

ed)

*

* Significant

Ta.6990.1.S1_at

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

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.

Yr18/Lr34

ABC Gene associated withTa.6990.1.S1_at

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.

Thank YouTristan CoramXueling HuangMeinan WangAndrea Dolezal