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?