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The potential of genome editing to improve the agronomic performance of crops is often limited by low plant regeneration efficiencies and few transformable genotypes. Here, we show that expression of a fusion protein combining wheat GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1) substantially increases the efficiency and speed of regeneration in wheat, triticale and rice and increases the number of transformable wheat genotypes. GRF4-GIF1 transgenic plants were fertile and without obvious developmental defects. Moreover, GRF4-GIF1 induced efficient wheat regeneration in the absence of exogenous cytokinins, which facilitates selection of transgenic plants without selectable markers. We also combined GRF4-GIF1 with CRISPR-Cas9 genome editing and generated 30 edited wheat plants with disruptions in the gene Q (AP2L-A5). Finally, we show that a dicot GRF-GIF chimera improves regeneration efficiency in citrus, suggesting that this strategy can be applied to dicot crops. Recent studies have reported improvements in the efficiency of plant regeneration from tissue culture by overexpression of plant developmental regulators, including LEAFY COTYLEDON1 (refs. 1,2), LEAFY COTYLEDON2 (ref. 3), WUSCHEL (WUS) 4 and BABY BOOM (BBM) 5. These genes promote the generation of somatic embryos or the regeneration of shoots. For example, overexpression of the maize developmental regulators BBM and WUS2 produces high transformation frequencies in previously non-transformable maize inbred lines and other monocot species 6-8. Another strategy uses different combinations of developmental regulators to induce de novo meristems in dicotyledonous species without tissue culture 9. However, there remains a need for new methods that provide efficient transformation, increased ease of use and suitability for a broader range of recalcitrant species and genotypes. GRF transcription factor genes are highly conserved in angiosperms, gymnosperms and moss 10. They encode proteins with conserved QLQ and WRC domains that mediate protein-protein and protein-DNA interactions, respectively 11-13. Many angiosperm and gymnosperm GRF genes carry a target site for microRNA miR396, which reduces the function of GRFs in mature tissues 14. The GRF proteins form complexes with GIF cofactors that also interact with chromatin remodeling complexes in vivo 15,16. Multiple levels of regulation control the efficiency of functional GRF-GIF complex assembly in vivo 17. Loss-of-function mutations in GIF genes mimic the reduced organ size observed in GRF loss-of-function mutants

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Summary CRISPR /Cas9 has been widely used for genome editing in many organisms, including important crops like wheat. Despite the tractability in designing CRISPR /Cas9, efficacy in the application of this powerful genome editing tool also depends on DNA delivery methods. In wheat, the biolistics based transformation is the most used method for delivery of the CRISPR /Cas9 complex. Due to the high frequency of gene silencing associated with co‐transferred plasmid backbone and low edit rate in wheat, a large T 0 transgenic plant population are required for recovery of desired mutations, which poses a bottleneck for many genome editing projects. Here, we report an Agrobacterium ‐delivered CRISPR /Cas9 system in wheat, which includes a wheat codon optimized Cas9 driven by a maize ubiquitin gene promoter and a guide RNA cassette driven by wheat U6 promoters in a single binary vector. Using this CRISPR /Cas9 system, we have developed 68 edit mutants for four grain‐regulatory genes, Ta CKX 2‐1 , Ta GLW 7 , Ta GW 2, and Ta GW 8 , in T 0 , T 1 , and T 2 generation plants at an average edit rate of 10% without detecting off‐target mutations in the most Cas9‐active plants. Homozygous mutations can be recovered from a large population in a single generation. Different from most plant species, deletions over 10 bp are the dominant mutation types in wheat. Plants homozygous of 1160‐bp deletion in Ta CKX 2‐D1 significantly increased grain number per spikelet. In conclusion, our Agrobacterium ‐delivered CRISPR /Cas9 system provides an alternative option for wheat genome editing, which requires a small number of transformation events because CRISPR /Cas9 remains active for novel mutations through generations.

To form nitrogen-fixing symbioses, legume plants recognize a bacterial signal, Nod Factor (NF). The legume Medicago truncatula has two predicted NF receptors that direct separate downstream responses to its symbiont Sinorhizobium meliloti. NOD FACTOR PERCEPTION encodes a putative low-stringency receptor that is responsible for calcium spiking and transcriptional responses. LYSIN MOTIF RECEPTOR-LIKE KINASE3 (LYK3) encodes a putative high-stringency receptor that mediates bacterial infection. We localized green fluorescent protein (GFP)-tagged LYK3 in M. truncatula and found that it has a punctate distribution at the cell periphery consistent with a plasma membrane or membrane-tethered vesicle localization. In buffer-treated control roots, LYK3:GFP puncta are dynamic. After inoculation with compatible S. meliloti, LYK3:GFP puncta are relatively stable. We show that increased LYK3:GFP stability depends on bacterial NF and NF structure but that NF is not sufficient for the change in LYK3:GFP dynamics. In uninoculated root hairs, LYK3:GFP has little codistribution with mCherry-tagged FLOTILLIN4 (FLOT4), another punctate plasma membrane-associated protein required for infection. In inoculated root hairs, we observed an increase in FLOT4:mCherry and LYK3:GFP colocalization; both proteins localize to positionally stable puncta. We also demonstrate that the localization of tagged FLOT4 is altered in plants carrying a mutation that inactivates the kinase domain of LYK3. Our work indicates that LYK3 protein localization and dynamics are altered in response to symbiotic bacteria. INTRODUCTIONPlants in the family Fabaceae (legumes) form symbiotic relationships with nitrogen-fixing rhizobial bacteria. The bacteria live in association with plant roots inside morphologically unique structures called nodules. In this mutualistic interaction, the bacteria reduce (or fix) molecular dinitrogen to ammonia, a form of nitrogen that plants can use; in exchange, the plants supply the bacteria with carbon sources. Through this intimate metabolic partnership, both plant and bacteria benefit.An exchange of signals initiates symbiosis between legumes and bacteria. Plant roots secrete flavonoids, which cause induction of bacterial genes required for synthesis of a lipochitooligosaccharide called Nod Factor (NF) (Fisher and Long, 1992). Within minutes of application of bacteria or purified NF, root hair membrane depolarization occurs (Ehrhardt et al., 1992). Within the first hour after NF treatment, cellular calcium levels begin to oscillate in root hairs and root epidermal cells (called calcium spiking) (Ehrhardt et al., 1996). A number of phenotypic changes occur in root hairs following calcium spiking. First, actively elongating root hairs stop growing and swell. The root hairs then reinitiate tip growth and grow to form a curl around a bacterial colony. Bacteria enter root hairs through plant-derived intracellular structures called infection threads. These events coincide with transcriptional changes that are largely NF dependent (Mitra e...