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Madan K. BhattacharyyaAgronomy Department
Iowa State University
Novel Approaches inFighting Sudden
Death Syndrome inSoybean
http://soysdsresistance.ag.iastate.edu/index.html
Dr
Why to enhance sudden death syndrome(SDS) resistance in soybean?
http://soysdsresistance.ag.iastate.edu/index.html
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Why to enhance sudden death syndrome(SDS) resistance in soybean?
v The estimated soybean yield suppression fromSDS in 2010 was 2.1% of total yield, which wasvalued at $0.82 billion.
v Growing SDS resistant cultivars is the majormethod of controlling this disease.
v Unfortunately, SDS resistance encoded by a largenumber QTL; and breeding SDS resistance ischallenging.
v We need novel single-genes encoding SDSresistance.
http://soysdsresistance.ag.iastate.edu/index.html
Two Novel Approaches to EnhanceSDS Resistance in Soybean
1) Understand how the pathogen causesSDS and apply the knowledge to createSDS resistant soybean lines.
2) Identify and express plant genes toenhance SDS resistance.
3/16/16 4
http://soysdsresistance.ag.iastate.edu/index.html
Lead PI: Madan K. Bhattacharyya (ISU)Co-Pis: Liang Dong (ISU)
Jing Yong Ye (UTSA)
SDS symptoms – (i) root necrosis and (ii) foliar SDS
Small chlorotic spotson leaves
Dead interveinal tissue falls off
Leaf drop –followed by flower and pod drop
Young seedlings Mature plants
Pathogen remains in infected roots.
http://soysdsresistance.ag.iastate.edu/index.html
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v In North America, Fusarium virguliforme causes SDS.v In South America, F. tucumaniae is the major causal
agent of SDS.v Both are haploid organisms.v They produce sickle-shaped conidia for spread of the
disease.v They overwinter as chlamydospores.
v
http://soysdsresistance.ag.iastate.edu/index.html
Express plant genes
Microfluidic Assays
Dong Lab, Iowa State University
http://soysdsresistance.ag.iastate.edu/index.html
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How quickly conidium germinate?
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Conidia development
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Structure of the MAT locus
Hughes et al. 2013: Mycologia 106:686–697.
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Frequency distributions of alleles at the Mat locusamong the Fusarium spp.
Hughes et al. 2013: Mycologia 106:686–697.
A maximum-likelihood tree of Fusarium species (based on genome-wide SNP data)
BRR
BRR (bean root rot)
SDS
SDS
SDS
SDS
BRR
Huang et al. 2016: PloS One, Under Revision Huang et al. 2016: PloS One, Under Revision
BRR
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Phylogenetic tree of F. virguliforme isolates.
13
http://soysdsresistance.ag.iastate.edu/index.html
http://soysdsresistance.ag.iastate.edu/index.html
Phylogenetic tree of F. tucumaniae isolates.
14
http://soysdsresistance.ag.iastate.edu/index.html
Two Approaches to EnhanceSDS Resistance in Soybean
1) Understand how the pathogen causesSDS and apply the knowledge to createSDS resistance.
2) Express plant genes to enhance SDSresistance.
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FvTox1 – acidic proteincauses foliar SDS
3/16/16 16Brar et al. (2011) MPMI 24: 1179-1188
Pudake et al. (2013) Current Genetics, 59:107-117
http://soysdsresistance.ag.iastate.edu/index.html
Lead PI: Madan K. Bhattacharyya (ISU)Co-PIs: Liang Dong (ISU)
Jing Yong Ye (UTSA)
Brar and Bhattacharyya 2012: MPMI 25:817–824
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Work flow for identifying peptides fromphage display peptides libraries
3/16/16 18Wang et al. (2015) PLoS OneDOI:10.1371/journal.pone.0145156
http://soysdsresistance.ag.iastate.edu/index.html
http://soysdsresistance.ag.iastate.edu/index.html
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Four classes of M13 phage clonesdisplayed FvTox1-interacting peptides.
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Class (sequence) Clone Number Peptide Library
Class I (SYLPETIYEYRL)
1,2,4,5,6,7,8,10,11,12,13,14,15,16,17,18,19,20,23,28,29,30,36,38,39
Ph.D.-12
Class Ⅱ (VENKTRYHDREV)
3,9,22,25,27,37 Ph.D.-12
Class Ⅲ (HEGAWHNYARSV)
24 Ph.D.-12
ClassⅣ (SNGRVAD)
31 Ph.D.-7
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Nine synthetic genes encodingFvTox1-interacting peptides
20
http://soysdsresistance.ag.iastate.edu/index.html
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Transgenic soybean lines carrying FvTox1-interactingPep -1 showed enhanced foliar SDS resistance.
http://soysdsresistance.ag.iastate.edu/index.html
FvTox1-interacting peptides conferenhanced tolerance of roots to FvTox1.
A
B D
CR² = 0.51712
0
10
20
30
40
50
60
0 1 2 3 4 5 6
Roo
t Rot
Sev
erity
(%)
Foliar SDS Score
R² = 0.83681
0
10
20
30
40
50
0 1 2 3 4 5 6
Stem
Rot
Sev
erity
(%)
Foliar SDS Score
R²#=#0.83919#
0
20
40
60
80
100
120
140
0 1 2 3 4 5 6
Deve
lopm
ent o
f New
Ro
ot (
%)
Foliar SDS Score
A
C
B
22Williams 82 Williams 82 + Pep 1
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23
0 0.5
1 1.5
2 2.5
3 3.5
4
MN1
606
Pep
9C -
7
Pep
8C -
9
Pep
1C -
12
Pep
5C -
14
Pep
2C -
8
Pep
5C -
4
Pep
1 - 6
Pep
9 -
25
Pep
9C -
2
Pep
1 -
7
Pep
2C
- 14
Pep
8 -
14
Pep
8C
- 7
Pep
2C
- 6
Pep
5 -
15
Pep
1C
- 6
Pep
9 -
3
Spen
cer
Will
iam
s 82
Pep
8 -
17
Folia
r SDS
Sco
re
0 0.5
1 1.5
2 2.5
3 3.5
4 4.5
5
Folia
r SDS
Sco
re
Resistant Susceptible
02468
1 01 21 4
Num
ber o
f Pla
nts
R e s i s t an tS us c e p t ib l e
B
A
Transgenic soybean lines showed enhancedresistance to F. virguliforme infection.
B. Wang and M.K. Bhattacharyya, unpublished
http://soysdsresistance.ag.iastate.edu/index.html
Express plant genes
Summary
1. Demonstrated that FvTox1-interacting Pep-1enhances foliar SDS resistance.
2. FvTox1-interacting peptides conferringenhanced foliar SDS resistance also showedimproved tolerance of roots to FvTox1.
3. FvTox1 most likely causes suppression of rootgrowth and root necrosis in addition to foliarSDS.
24
http://soysdsresistance.ag.iastate.edu/index.html
Two Approaches to EnhanceSDS Resistance in Soybean
1) Understand how the pathogen causesSDS and apply the knowledge to createSDS resistance.
2) Identify and express plant genes toenhance SDS resistance.
1. Arabidopsis nonhost resistance genes2. Altered expression of soybean genes
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Phytophthora sojae susceptible (pss) mutants
pen1-1 pss1
Mutant Family Mutant Family Mutant Family
pss1 0.2B17I9 pss11 0.25B8C1 pss21 0.25B9D7
pss2 0.2B3D2 pss12 0.25 B9A1 pss22 0.25B9F1
pss3 0.3B6A7 pss13 0.25B8H5 pss23 0.2B20I2
pss4 0.2B21F1 pss14 0.3B6E8 pss24 0.2B9G4
pss5 0.2B15I6 pss15 0.3B6H8 pss25 0.2B8B7
pss6 0.2B4G5 pss16 0.25B7F8 pss26 0.3B1C7
pss7 0.2B21B9 pss17 0.25B7G4 pss27 0.3B8B8
pss8 0.2B4A5 pss18 0.25B9C3 pss28 0.25B9E3
pss9 0.2B21D6 pss19 0.3B6E4 pss29 0.2B1A9
pss10 0.25B7I5 pss20 0.3B6C8 pss30 0.25B7D2
Identified 30 pss mutants.
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Fourteen pss mutants are alsosusceptible to F. virguliforme.
pss12
pss15
pss16
pss1
pss2
pss5
pss3
pss10
pss18
pss19
pss20
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Field evaluation of transgenic soybean plantscarrying Arabidopsis nonhost resistance Pss1 gene
0 20 40 60 80
100
SDS
Res
ista
nt P
lant
s (%
)
30-Sep 15-Oct
29B. Wang et al. unpublished
http://soysdsresistance.ag.iastate.edu/index.html
Dr
30
0
20
40
60
80
100
SDS
Resi
stan
t Pla
nts
(%)
Field evaluation of transgenic soybean plantscarrying Arabidopsis nonhost resistance
Pss30 gene193-16.2 35S-Pss30-16.2 232-2 Prom2-Pss30-2
232-5 Prom2-Pss30-5 Williams 82
A
C
B
D
193-16.2 35S-Pss30-16.2 232-2 Prom2-Pss30-2
232-5 Prom2-Pss30-5 Williams 82
A
C
B
D
S. Kambakam et al., unpublished
http://soysdsresistance.ag.iastate.edu/index.html
Two Approaches to EnhanceSDS Resistance in Soybean
1) Understand how the pathogen causesSDS and apply the knowledge to createSDS resistance.
2) Identify and express plant genes toenhance SDS resistance.
1. Arabidopsis nonhost resistance genes2. Altered expression of soybean genes
3/16/16 31
http://soysdsresistance.ag.iastate.edu/index.html
Soybean genes suppressed duringF. virguliforme infection.
32
http://soysdsresistance.ag.iastate.edu/index.html
http://soysdsresistance.ag.iastate.edu/index.html
Expression of GmSAMT1 enhances SDS resistancein transgenic soybean plants under field conditions
33
http://soysdsresistance.ag.iastate.edu/index.html
http://soysdsresistance.ag.iastate.edu/index.html
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Expression of GmDS1 enhances SDS resistance intransgenic soybean plants under field conditions
http://soysdsresistance.ag.iastate.edu/index.html
Current Status
Gene Name Growth Chamber Field EvaluationPss1 Resistant ResistantPss30 Resistant ResistantPss6 Resistant Not YetPss21 Resistant Not YetPss25 Partially Resistant Not YetGmSAMT1 Resistant ResistantGmDS1 Resistant ResistantGmARP1 Resistant ResistantGmSEQ1 Resistant Not Yet
3/16/16 35
Acknowledgements
Bhattacharyya Lab
Sekhar Kambakam, Ph.D.
Micheline Ngaki, Ph.D.
Bing Wang, Ph.D.
Siva Swaminathan, Ph.D.
Jill Heinrich
Previous Scientists
Hargeet Brar
Ramesh Pudake, Ph.D.
Devinder Sandhu, Ph.D.
Min Xu, Ph.D.
Binod Sahu, Ph.D.
Rishi Sumit, M.S.
Prashant Singh, Ph.D.
Program Grant no. 2013-68004-20374 11:25:52 AM 36
Dong LabXuan Qiao
Huang LabAnindya Das
Ye Lab (UT San Antonio)Bailin Zhang, Ph.D.
Teresa J. HughesKerry O’Donnell
USDA-ARS
ISU Plant Transformation FacilityIowa Soybean Association
CPBRUnited Soybean Board
3/16/16 37
Slide 1Slide 2Slide 3Two Novel Approaches to Enhance SDS Resistance in SoybeanSlide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Two Approaches to Enhance SDS Resistance in SoybeanFvTox1 – acidic protein causes foliar SDSSlide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Two Approaches to Enhance SDS Resistance in SoybeanSlide 26Slide 27Slide 28Slide 29Slide 30Two Approaches to Enhance SDS Resistance in SoybeanSlide 32Slide 33Slide 34Current StatusAcknowledgementsSlide 37