RAPID IDENTIFICATION OF GENES CONTRIBUTING TOFHB RESISTANCE IN WHEAT

Steve ScofieldUSWBSI National Forum

December 3, 2008Crop Production and

Pest Control UnitWest Lafayette, IN

The Gene Discovery and Engineering Resistance (GDER) RA is urgently working to develop strategies that can be used tosignificantly improve FHB resistance in wheat and barley.

Although the FHB1 locus is the best tool currently availablefor scab resistance in wheat, it has several limitations.

1. FHB1-mediated resistance can be breakdown under heavy pathogenpressure.

2. Recently, it has been shown that FHB1 has low genetic penetrancein some genotypes.

For these reasons we firmly believe that novel strategies must be found for engineering scab resistance.

My lab’s approach is to identify the wheat genes that are essential for FHB resistance in wheat. These genes will then be used to engineerimproved FHB resistance.

The isolation of wheat genes with particular functions has been very difficult

Wheat’s hexaploid genome often prevents conventional mutational analyses

The wheat genome is enormous - 16,000 Mbp genome (128X Arabidopsis)

Wheat is very difficult to transform. This prevents the use of many modern approaches to gene isolation, T-DNA libraries and transposon gene tagging

Genetic analysis of recessive mutations in diploids and hexaploids

Y y

XY 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

Capabilities that would be very useful for wheat molecular genetics:

The ability to specifically turn off genes (knockout) so thattheir functions can be inferred from the knockout phenotype.

The ability to efficiently generate gene knockouts despite the hexaploidy of wheat.

The ability to create knockouts without needing to transform wheat.

An efficient virus-induced gene silencing (VIGS) system for wheat should satisfy these requirements.

OUTLINE FOR THE REMAINDER OF THIS PRESENTATION:

1. What is VIGS?

2. Development of the BSMV-VIGS system for wheat

3. Using BSMV-VIGS to functionally identify genes in FHBand other resistance pathways of wheat.

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 tocarry a fragment of plant

gene X

RNA silencing mechanismtargets viral RNA for degradation

Transcripts of plant gene Xare also targeted for destruction

Plant gene X

General mechanism of RNA-induced gene silencing

DicerAAAARDRP

siRNAs miRNAs

AGO

AAAA AAAA

RNA Interference Silencing Complex (RISC)

DNA/Histone Methylation mRNA Degradation Translation BlockTGS VIGS, PTGS, RNAi Developmental Processes

dsRNA dsRNA

Barley Stripe Mosaic VirusBSMV is a member of Hordeivirus

family

Positive-sense single strand RNA virus

Tripartite genome: α, β, γ RNAs

Hosts: barley, wheat, oat, rice and tobacco

First DNA clones of BSMV genomeconstructed by Petty and Jackson(1989) Virology 171: 342-349.

Use of BSMV for VIGS in barleyfirst demonstrated by Holzberget. al., (2002) Plant J. 30: 315-327.

Barley Stripe Mosaic VirusBSMV Genomic Organization & Protein Function:

: Sub-genomic promoter

β

γ

α

1 3239

βcβdβbβa

1 2790γ a γ b

1 3647α a

replicase

replicase

movement proteinscoat protein

suppressor of silencing

VIGS targeting sequence

Clone 200 - 500bp cDNA fragment from target gene into BSMV γ plasmid in antisense orientation.

Linearize α, β and γ DNA plasmids.

In vitro transcribe 5’ capped α, β and γ RNAs.

Mix RNAs 1:1:1 in 25μl inoculation buffer (~0.5 μg of each RNA/inoculation).

Rub inoculate RNAs onto wheat leaves.

Observe VIGS phenotype; confirm silencing of intended target gene.

BSMV-VIGS Procedure

Silencing fragment

α β γ

T7 promoter

T7 promoter

T7 promoter

BSMV:00(carries no plant cDNA)

BSMV:PDS4

VIGS in Hexaploid Wheat: Silencing the PDS gene results inthe appearance of photobleached tissue

The first test using BSMV-VIGS in the dissection of a resistance pathway

Lr21 provides hypersensitive resistance to leaf rust caused by Puccinia triticina.

Lr21 was cloned by Li Huang in Bikram Gill’s group at Kansas State University.

(Huang et. al., 2003 Genetics 164: 655-664).

A cloned R-gene would provide a very useful positive control.

Silencing the R-gene should cause a resistant plant to become susceptible.

Design for the Leaf Rust VIGS Experiment

Two wheat genotypes tested: WI = Susceptible; WGRC7 = Resistant

10 plants inoculated for each treatment; 2 experimental blocks.

Treatments: WI and WGRC7 infected with: BSMV:00BSMV:PDS4BSMV:Lr21BSMV:RAR1BSMV:SGT1BSMV:HSP90

Plants inoculated with BSMV transcripts 7 days after germination.

Plants sprayed with avirulent P. triticina (PRTUS6) 8 days later.

Plants scored 10 days later.

Control experiment: Infection with BSMV:00 does not alter resistance or

susceptibility to Puccinia triticina

Wi SusceptibleBSMV:00

WGRC7ResistantBSMV:00

WGRC7BSMV:RAR1

WGRC7BSMV:Lr21

WGRC7BSMV:SGT1

WGRC7BSMV:00

Silencing Lr21, RAR1, SGT1 or HSP90 converts incompatible interactions to compatible, indicating that each is essential for

Lr21-mediated resistance

WGRC7BSMV:HSP90

Resistance to FHB

The most useful known sources of FHB resistance are QTL-based.

In wheat, the strongest QTL is FHB1 from chromosome 3B of Sumai-3.

FHB1 conditions type II resistance that limits the spread of the fungus.

Type II resistance is ssayed by single floret inoculation.

FHB1 resistance is inadequate during FHB epidemics.

We are working to identify the genes involved in resistance so that we can improve resistance.

Our current approach is to use VIGS to test genes that are differentially expressed during FHB interactions, or have annotations suggesting function in resistance.

Demonstration of BSMV:VIGS in adult wheat plants

BSMV:00 BSMV:PDS

BSMV:VIGS of PDS in the awns of wheat

BS

MV:00

BS

MV:P

DS

qRTPCR analysis of PDS expression in wheat awns

Relative P

DS

expressionqRTPCR normalized with GAPD

Infection with BSMV:00 does not interfere with type II resistance of Ning 7840

Single floret Fusarium graminearum inoculation of BSMV:00 infected Ning 7840 wheat

Inoculated floret

Y-axis: Barley - Fusarium Exp SS 1, Default InterpretationColored by: Time 24 WaterGene List: Strongest Up Chitinases (3)

24.0 48.0 72.0 96.0 144.0 24.0 48.0 72.0 96.0 144.0Water Fusarium

Time

1

0.6

0.7

0.8

0.9

2

3

4

5 Normalized Intensity(log scale)

24.0 48.0 72.0 96.0 144.0 24.0 48.0 72.0 96.0 144.0Water Fusarium

Time

1

0.6

0.7

0.8

0.9

2

3

4

5 Normalized Intensity(log scale)

Expression of chitinase genes after infection with Fusarium

Water Fusarium graminearum

Barley microarray data from Boddu et. al., 2006 MPMI 19: 407-417 and www.plexdb.org

Ning 7840+ BSMV:080+ Fusarium

Ning 7840+ BSMV:098+ Fusarium

Ning 7840+ BSMV:097+ Fusarium

Chitinase

080097098

ATG TAG

Ning 7840+ BSMV:00 (Control)

+ Fusarium

VIGS assays using three different fragments of chitinase to targetgene silencing result in Ning 7840 becoming susceptible to FHB.

097-1 *

097-10

097-12 *

097-13

097-14

097-15 *

097-9

097-7

Poo

led

Con

trols

Expression of wheat chitinase after silencing by BSMV:097

Chitinase097 VIGS fragment

* Plants scored as susceptible to FHB

qRTPCR productR

elat

ive

Chi

tinas

e E

xpre

ssio

n

Recently, the Muehlbauer lab demonstrated that over-expression of chitinase increases FHB resistance in

transgenic wheat.

Shin et al., (2008) J. Exp. Bot. 59: 2371-2378

Tryptophan decarboxylase

Anthranilate synthase α subunit

Tryptophan synthase α chainIndole-3-glycerolphosphate synthasePhophoribosyl anthranilte transferase

Phosphoribosyl anthranilate isomerase

Water Fusarium

Anthranilate synthase β subunit

My group is testing whether a wide range of genes, which are differentially expressed during challenge by F. graminearum, make significant

contributions to FHB resistance.

VIGS analysis has shown that genes that act to promote ethylene biosynthesis and ethylene-dependent signaling

play essential roles in FHB resistance.

EthyleneBiosynthesis

EthyleneSignal

Transduction

Conclusions

We have obtained strong evidence that BSMV-VIGS is an effective tool for the rapid identification of genes functioning in wide range of wheat disease resistance pathways.

We have proven this system’s utility for probing the FHB resistance pathways, and are actively testing a range of new candidate genes.

We are very interested in testing whether genes from the Ethylene signaling pathway can be used to engineer FHB resistance.

BSMV-VIGS can be used to test candidate genes identified on the basisof differential expression, candidates identified in genome-walking experiments, or candidates identified from other functional screens performed in systems (ie., yeast or Physcomitrella).

Acknowledgements

VIGS DevelopmentMandy BrandtCahid Cakir

Lr21 PathwayLi Huang Bikram Gill

Fusarium Head BlightMegan GillespieLingrang KongJoe AndersonHerb OhmGary Muehlbauer

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US Wheat and Barley Scab Initiative