homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew Yanpeng Wang Xi Cheng Qiwei Shan Yi Zhang Jinxing Liu Caixia Gao amp Jin Long Qiu Evan Perkins ID: 573143
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Slide1
Simultaneous editing of three
homoeoalleles inhexaploid bread wheat confers heritable resistanceto powdery mildew
Yanpeng Wang, Xi Cheng Qiwei Shan, Yi Zhang, Jinxing Liu, Caixia Gao & Jin-Long Qiu
Evan Perkins
April 15, 2017Slide2
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.Slide3
Blumeria
graminis f. sp. tritici “Powdery Mildew”Obligate biotrophPowdery conidia on upper surfaceChlorosis and eventually death of tissueDark chasmothecia
with ascospores as disease progressesSlide4
B. graminis
lifecyclePolycyclic disease capable of rapidly spreading through fields.Chasmothesia are durable, allow for overwintering in soilCan also overwinter as mycelium on debris.Slide5
Current controls
Use partially resistant cultivarsDo not sow wheat into wheatChemical fungicidesRemove volunteers between plantingsMaintain a loose canopy and space rows.Slide6
Why is this important?
Bread wheat contributes to ~20% of worldwide caloriesAllohexaploidy, 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.Slide7
TALEN
Transcription activator-like effector nucleasesRestriction enzymes engineered to cut specific sequences of DNAIn situ gene editingSlide8
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.Slide9
Procedure
Pair of TALENs used to target a conserved region in exon 2PCR-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)Slide10Slide11
PCR primer for maize
Ubiquitin 1 promoter used in TALENAssay failed to detect Ubiquitin 1 promoter in 45/652 T1 and 22/105 T2.GFP insertion more reliable with TALENsTwo TALEN free tamlo-aabbdd mutants obtained by 2 generations of
selfing T0-3 plant.Slide12Slide13
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 hexaploidyMost mutations within the TALEN target were deletions of 1-10bpFrom 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 MendelianSlide14
Results
Self T0 with mutations and genotype T1T0 homozygote transmission was 100%T0 heterozygotes mostly separated 1:2:1T1 -> T2 shows the same patternSome 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.Slide15
aabbdd
showed significantly less disease than any combination of 1 or 2 allele knockouts.Slide16
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 genesSlide17
Questions
What’s up with T0-6 and T0-9 (mutation in somatic cells?)Mutation percentages seem low, is this normal?