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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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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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

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1-MCP ETR ETR

ETR

CTR1

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MAPK

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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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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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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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BobwhitePlantsSprayedwith1mMACCandPointInoculatedwithFusariumBecomeMoreResistant(TypeIIAssay)

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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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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.