Ethylene Signaling in Wheat is Essential for Type I and II Resistance to Fusarium
graminearum and Tolerance to Deoxynivalenol
Megan Gillespie
Scofield Lab
Virus-Induced Gene Silencing (VIGS)
VIGS is a form of RNA-mediated gene silencing.
Replication of RNA viruses causes large amounts of dsRNA to accumulate which activates the RNA silencing mechanism.
The silencing mechanism targets the sequences represented in the dsRNA for homology-dependent degradation.
If the virus carries sequences homologous to plant genes, transcripts of the plant gene are also degraded.
Because the mechanism is homology-dependent, VIGS can silence homeologous genes present in polyploids.
Viral infection is easy, doesn’t require transformation.
RNA virus engineered to carry a fragment of plant
gene X
RNA silencing mechanism targets viral RNA for degradation Transcripts of plant gene X are also targeted for destruction
Plant gene X
General mechanism of RNA-induced gene silencing
Dicer AAAA RDRP
siRNAs miRNAs
AGO
AAAA AAAA
RNA Interference Silencing Complex (RISC)
DNA/Histone Methylation mRNA Degradation Translation Block TGS VIGS, PTGS, RNAi Developmental Processes
dsRNA dsRNA
RNAi Cascade
Genetic analysis of recessive mutations in diploids and hexaploids
Y y
X Y Y Y y y y
1/4 2/4 1/4
Y y Y Y Y Y
1A 1B 1D
y y Y Y Y Y
1A 1B 1D
X
The yy phenotype is always masked by Y’s on 1B and 1D
Diploid Hexaploid
No Virus BSMV:00 (control) BSMV:SAMs
Resistant (‘Ning’ 7840) plants become susceptible when SAMs is Silenced
Single floret inoculation point. The fungus has not moved past the boundary between the floret and the rachis indicating Type II resistance.
Fugus has moved past inoculation point and in this individual, down into the stem. All florets have been colonized by the fungus.
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%In
fectedFlorets
Treatment
ResistantPlantsBecomeSuscep blewhenSAMsisSilenced
P = 4.03 x 10-9 P = 0.03 P = 8.00 x 10-3 P = 9.00 x 10-3 P = 0.75
Ning Ning Ning Ning Len
Ethylene Biosynthesis: Methionine SAM ACC ET
SAMs ACS ACO
ETO (negative regulator of ET biosynthesis) Ethylene Signaling:
ET
ETR (Ethylene Receptors)
CTR (Negative Regulator of MAPK cascade)
MAPK
EIN2 (Positively regulates EIN3 by removing SCFEBF which is inhibiting EIN3)
The presence of ethylene inhibits the receptors which are positively regulating CTR which in turn is inhibiting a MAPKK. Once the negative regulation of CTR is relieved, the MAPK cascade proceeds to activate EIN2 which interacts with the SCFEBF complex. EIN3 is now released to activate various ethylene responsive transcription factors including the ERF’s.
EBF 1/2
EIN3/EIL Trancription Factor Activation (ERFs, etc.)
BSMV:ERF BSMV:00(Control)
Resistant (‘Ning’ 7840) plants become susceptible when ERF is Silenced
Single floret inoculation point. The fungus, again, has not moved into the rachis.
The fungus has moved past the point of inoculation and into the rachis. Within days, the fungus had completely colonized the florets and had moved into the stem
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Ning Ning Ning Ning Len
1-methylcyclopropene (1-MCP) binds irreversibly to the copper ion in the ethylene receptor, preventing ethylene from binding,
and thus blocking signal transduction.
ET ET
ET
ET
ET
1-MCP
1-MCP
1-MCP 1-MCP
1-MCP ETR ETR
ETR
CTR1
CTR1
CTR1
MAPK
1-MCP
Delayed Senescence in 1-MCP Treated Plants Indicates that 1-MCP is Effective in Disrupting Ethylene Signaling
Control Group Displaying Normal Senescence 1-MCP Treated Plants Displaying Delayed Senescence
Fusarium moves into the 1-MCP treated plants within 5 days of initial infection
Control: No movement of Fusarium into the rachis (5 DPI)
1-MCP Treated: Fusarium has moved into the rachis and is proceeding down the head into the stem (5 DPI)
After 2 weeks, Ning plants treated with 1-MCP become fully infected
Fusarium remains restricted to the infected floret
Fusarium has moved out of infected floret and infected the entire head
1-MCP Treated Control
Bobwhite plants treated with 1-MCP become even more susceptible
Fusarium has moved past the infected floret and colonized ~50% of the head.
Fusarium has moved rapidly out of the infected floret and colonized most of the head
Control 1-MCP Treated
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fectedFlorets
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Both Resistant and Suceptible Plants Treated with 1-MCP Become More Susceptible
Ning plants treated with 1-MCP become more sensitive to DON
No 1-MCP, ETOH No 1-MCP, DON 1-MCP, ETOH 1-MCP, DON
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%Necrosis
NingPlantsBecomeMoreSensi vetoDONWhenTreatedwith1-MCP
No 1-MCP, ETOH No 1-MCP, DON 1-MCP, ETOH 1-MCP, DON
‘Bobwhite’ Plants Treated with 1mM ACC become more resistant to Fusarium
Control (Water Treated)
100 μM ACC Treated
Fusarium has spread from the infected floret into the rest of the head.
Fusarium infection is visible only in the glume of the infected floret.
P-value = 1.06 x 10-4 Thus, we are 99% confident (α = .01) that ACC has an effect on Fusarium resistance.
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BobwhiteControl Bobwhite1mMACC
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BobwhitePlantsSprayedwith1mMACCandPointInoculatedwithFusariumBecomeMoreResistant(TypeIIAssay)
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Control ACC Control ACC Control ACC Control ACC Control ACC
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Ning (resistant genotype) Bobwhite (susceptible genotype)
Bobwhite and Ning Plants Sprayed with 1mM ACC and Sprayed with Fusarium Become More Resistant (Type I Assay)
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WaterTreated 1mMACCTreated
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veExpression
Relative Expression of ERF Increases in Bobwhite When Treated with 1mM ACC
Bobwhite plants sprayed with 1mM ACC have increased lignin content in the rachis
There appears to be less lignin in the rachis of the control plant.
Lignin appears abundant throughout the 1mM ACC treated plant.
Ning plants sprayed with 1mM ACC also appear to have more lignin
Control (Water Treated) 1 mM ACC Treated
There appears to be more lignin in the glume, lemma, and to an extent the rachis of the 1 mM ACC treated plants. Additionally, lignin seems
to preferentially accumulate at the rachis nodes in ‘Ning’ plants.
Special Thanks
• Scofield lab: – Steve Scofield – Mandy Brandt – Torrence Gill – Sara Reagan – Anshu Garg
• Herb Ohm • USDA-ARS • Purdue University • US Wheat and Barley Scab Initiative
An alternative explanation for the findings of Chen et al:
Inhibition of FHB on wheat treated with silver nitrate is due to the compound’s fungicidal activity rather than its inhibition of ET perception.
No AgNO3
0.25 mM AgNO3
0.5mM AgNO3
1mM AgNO3
1.5mM AgNO3
Chen et al. reported reduced FHB symptoms when detached heads of Bobwhite wheat were grown in 1.5mM AgNO3 compared to those in H2O. However, we have shown that F. graminearum cannot grow on plates with as little as 0.25mM AgNO3.
Figures from: Jun et al., (2004) Plant Cell Physiol. 45(3):281-289.
An alternative explanation for the findings of Chen et al:
Reduced EIN2 expression causes increased biosynthesis of ET and increased ET signaling.
Antisense EIN2 rice plants display ~3X increase in ET biosynthesis
1ppm 1-MCP Does Not Inhibit Fusarium Growth in vitro
Control 1 ppm 1-MCP
1mM ACC Does Not Inhibit Fusarium Growth in vitro
Control 1 mM ACC
Nicholson Group (Chen et al. 2009)Findings Regarding Fusarium and Ethylene
• Arabidopsis mutants compromised in ET signaling show less necrosis on the leaves, and ET overexpressing mutants have more.
• Detached wheat and barley leaves have less conidia when suspended in silver thiosulfate.
• Detached wheat heads immersed in silver nitrate are less susceptible to Fusarium.
• An EIN2 RNAi line of wheat is less susceptible to Fusarium.
Problems With These Findings
• Arabidopsis is not a natural host of Fusarium.
• Fusarium will infect the crown tissue of monocots (Crown Rot) or the floral tissue (Fusarium Head Blight).
• Detached heads senesce after a week making disease scoring difficult as it can take 2-3 weeks for infection to spread to the rest of the head from the inoculated floret.
• Silver nitrate fully inhibits Fusarium growth in vitro at concentrations well below those used.
• Silver thiosulfate causes Fusarium to lose its red pigmentation at low concentrations.
EIN2 may not be an ideal choice for silencing using RNAi.
• The Nicholson group used a 50% knockdown line from the Keller group (Travella et al. 2006).
• Keller’s data showed virtually no phenotypic difference between a 50% knockdown and the controls.
• Sung-Hoon et al. showed that antisense EIN2 plants in rice produced more ethylene leading to speculation that EIN2 is part of a feedback loop for ethylene biosynthesis.
• It is possible that the 50% knockdown line that the Nicholson group used is overproducing ethylene while still having enough EIN2 to signal, paradoxically leading to over signaling.
Additional, Circumstantial Evidence for a Positive Role for Ethylene in Fusarium
Resistance. • Use of strobilurin fungicides is not recommended
for Fusarium control as it can lead to a higher DON concentration in the grain. – Strobilurins inhibit ACC synthase, disrupting ethylene
biosynthesis, resulting in a “greening effect”
• The wheat cultivar ‘Apogee’ developed by NASA to be grown in space is extremely susceptible to Fusarium – Apogee is less sensitive to ethylene, as ethylene tends
to accumulate in confined spaces leading to pollen abort.
Effect of Silver Thiosulfate on Fusarium Growth in vitro
Concentration range from 0 – 1.5 mM STS
Control .25 mM STS .5 mM STS
1mM STS 1.5 mM STS
Effect of Ethephon on Fusarium growth in vitro
Concentration range from 0 – 10 mM Ethephon
Control 5mM Ethephon 10 mM Ethephon
Chen, X., Steed, A., Travella, S., Keller, B., Nicholson, P. Fusarium graminearum exploits ethylene signalling to colonize dicotyledonous and monocotyledonous plants. New Phytologist 182: 975–983. Sung-Hoon, J., Min-Jung, H., Shinyoung, L., Young, S., Woo, K., Gynheung, A. 2004. OsEIN2 is a Positive Component in Ethylene Signaling in Rice. Plant and Cell Physiology 45: 281-289. Travella, S., Klimm, TE., Keller, B. 2006. RNA interference-based gene silencing as an efficient tool for functional genomics in hexaploid bread wheat. Plant Physiology 142: 6–20.