Download Slides - Barley World

Simultaneous editing of three homoeoalleles in
hexaploid bread wheat confers heritable resistance
to powdery mildew
Yanpeng Wang, Xi Cheng Qiwei Shan, Yi Zhang, Jinxing Liu, Caixia Gao &
Jin-Long Qiu
Evan Perkins
April 15, 2017
Objective
• Loss of function in MLO alleles in barley, Arabidopsis, and tomato
confers broad-spectrum resistance to powdery mildew. Does this
apply to wheat?
• Use of TALEN and CRISPR-Cas9 to simultaneously edit 3 homologous
alleles to create durable and heritable resistance to Powdery Mildew.
Blumeria graminis f. sp. tritici “Powdery
Mildew”
• Obligate biotroph
• Powdery conidia on upper surface
• Chlorosis and eventually death of tissue
• Dark chasmothecia with ascospores as disease progresses
B. graminis lifecycle
• Polycyclic disease
capable of rapidly
spreading through
fields.
• Chasmothesia are
durable, allow for
overwintering in soil
• Can also overwinter as
mycelium on debris.
Current controls
• Use partially resistant cultivars
• Do not sow wheat into wheat
• Chemical fungicides
• Remove volunteers between plantings
• Maintain a loose canopy and space rows.
Why is this important?
• Bread wheat contributes to ~20% of worldwide calories
• Allohexaploidy, large genome (17,000 mb), and high content of
repetitive DNA (80-90%) make it resistant to forward and reverse
genetic analysis.
• Difficult to use genetic tools to improve desired traits.
• No spontaneous or induced mlo mutants to date in bread wheat.
TALEN
• Transcription activator-like
effector nucleases
• Restriction enzymes engineered
to cut specific sequences of DNA
• In situ gene editing
CRISPR-Cas9
• Cas9 nuclease complexed with
synthetic guide RNA (gRNA)
delivered into a cell.
• Genome is cut at the location
determined by the Cas9 nuclease,
allowing for removal or addition of
genes.
Procedure
• Pair of TALENs used to target a conserved region in exon 2
• PCR-RE demonstrated the occurrence of indel mutations at the target
site with efficiencies of 23%-38% (not affected by SNPs between
alleles)
• T-MLO plasmid transformed into wheat embryo by particle
bombardment (biolistics)
• Seedling generated from callus and MLO sites amplified (PCR-RE)
• PCR primer for maize Ubiquitin
1 promoter used in TALEN
• Assay failed to detect Ubiquitin
1 promoter in 45/652 T1 and
22/105 T2.
• GFP insertion more reliable with
TALENs
• Two TALEN free tamlo-aabbdd
mutants obtained by 2
generations of selfing T0-3
plant.
Results
• 27 mutations in 450 T0 transgenic lines in winter wheat (5
transformation experiments)
• 8 mutations out of 237 T0 transgenic lines in spring wheat (one
transformation experiment)
• TaMLO-A1/B1/D1 amplified (PCR-RE): mutations were in all 3
genomes of the hexaploidy
• Most mutations within the TALEN target were deletions of 1-10bp
• From 27 T0 plants: 12 het for A1, 8 het for D1, 1 het for B1 but homo
for A1, 3 het for A1 & D1, 1 het for all 3. T0-6 and T0-9 not Mendelian
Results
• Self T0 with mutations and genotype T1
• T0 homozygote transmission was 100%
• T0 heterozygotes mostly separated 1:2:1
• T1 -> T2 shows the same pattern
• Some mutations not detected in the T0 were present in T1, indicating
that the TALENs may remain active in progeny.
• NHEJ is feasible for target gene insertions.
aabbdd showed significantly
less disease than any
combination of 1 or 2 allele
knockouts.
Discussion
• TALEN can efficiently created targeted mutations in the wheat genome.
(slide 7)
• TALEN-mediated gene modifications observed in T0 can be stably
transmitted to subsequent generations (slide 10)
• TALEN-free plant with only desired DNA can be obtained through genetic
segregation (Slide 14)
• Promising results using CRISPR-Cas9 but have only tested mutation in one
allele so far.
• Only tamlo-aabbdd showed significant reduction in mildew infection
• Also showed strong resistance to 2 additional bgt races (not shown)
• Loss of function alleles may provide more durable resistance than R genes
Questions
• What’s up with T0-6 and T0-9 (mutation in somatic cells?)
• Mutation percentages seem low, is this normal?