towards an understanding of the molecular mechanisms of durable and non-durable resistance to stripe...
DESCRIPTION
Xianming Chen, USDA-ARSTRANSCRIPT
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