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The advancement of precision engineering for crop trait improvement is important in the face of rapid population growth, climate change, and disease. To this end, targeted double-stranded break technology using RNA-guided Cas9 has been adopted widely for genome editing in plants. Agrobacterium or particle bombardment-based delivery of plasmids encoding Cas9 and guide RNA (gRNA) is common, but requires optimization of expression and often results in random integration of plasmid DNA into the plant genome. Recent advances have described gene editing by the delivery of Cas9 and gRNA as pre-assembled ribonucleoproteins (RNPs) into various plant tissues, but with moderate efficiency in resulting regenerated plants. In this report we describe significant improvements to Cas9-RNP mediated gene editing in wheat. We demonstrate that Cas9-RNP assays in protoplasts are a fast and effective tool for rational selection of optimal gRNAs for gene editing in regenerable immature embryos (IEs), and that high temperature treatment enhances gene editing rates in both tissue types. We also show that Cas9-mediated editing persists for at least 14 days in gold particle bombarded wheat IEs. The regenerated edited wheat plants in this work are recovered at high rates in the absence of exogenous DNA and selection. With this method, we produce knockouts of a set of three homoeologous genes and two pathogenic effector susceptibility genes, engineering insensitivity to corresponding necrotrophic effectors produced by Parastagonospora nodorum . The establishment of highly efficient, exogenous DNA-free gene editing technology holds promise for accelerated trait diversity production in an expansive array of crops., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Poddar, Tanaka, Running, Kariyawasam, Faris, Friesen, Cho, Cate and Staskawicz.)

The necrotrophic fungal pathogen Pyrenophora tritici-repentis ( Ptr ) causes the foliar disease tan spot in both bread wheat and durum wheat. Wheat lines carrying the tan spot susceptibility gene Tsc1 are sensitive to the Ptr -produced necrotrophic effector (NE) Ptr ToxC. A compatible interaction results in leaf chlorosis, reducing yield by decreasing the photosynthetic area of leaves. Developing genetically resistant cultivars will effectively reduce disease incidence. Toward that goal, the production of chlorosis in response to inoculation with Ptr ToxC-producing isolates was mapped in two low-resolution biparental populations derived from LMPG-6 × PI 626573 (LP) and Louise × Penawawa (LouPen). In total, 58 genetic markers were developed and mapped, delineating the Tsc1 candidate gene region to a 1.4 centiMorgan (cM) genetic interval spanning 184 kb on the short arm of chromosome 1A. A total of nine candidate genes were identified in the Chinese Spring reference genome, seven with protein domains characteristic of resistance genes. Mapping of the chlorotic phenotype, development of genetic markers, both for genetic mapping and marker-assisted selection (MAS), and the identification of Tsc1 candidate genes provide a foundation for map-based cloning of Tsc1 ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Running, Momotaz, Kariyawasam, Zurn, Acevedo, Carter, Liu and Faris.)