A GRF-GIF chimeric protein improves the regeneration efficiency of transgenic plants

Abstract

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.

Figure 1.. GRF4-GIF1 chimera

A) Schematic representation of the GRF4 (blue)-GIF1 (pink) chimera. The black region represents a four amino acid spacer. B) The GRF4-GIF1 transgenic wheat plants were normal and fertile. C) Representative transformation showing higher frequency of regenerated shoots during Kronos transformation in the presence of the GRF4-GIF1 chimera than in the control. D-H) Box-plots showing regeneration frequencies of transgenic Kronos plants and their respective controls. The box shows the range from first to third quartiles, and is divided by the median. The whiskers span down to the minimum, and up to the maximum observation. Results from individual experiments are indicated by empty black circles. All experiments include the empty pLC41 vector as control and the wheat GRF4-GIF1 chimera. Numbers below the genotypes are total number of inoculated embryos and different letters above bars indicate significant differences (P < 0.05, Tukey test). D) Control vs. GRF4-GIF1, n= 15 experiments (*** P < 0.001, square root transformation). E) Control, GRF4-GIF1 and vector including GRF4 and GIF1 driven by separate maize UBIQUITIN promoters (GRF4+GIF1), n = 5 experiments (contrast GRF4-GIF1 vs. GRF4+GIF1, ** P = 0.0064, the empty-vector control was included only in two experiments). F) Control, GRF4-GIF1 and vectors including only GIF1 or only GRF4, n = 5 experiments (contrast GRF4-GIF1 vs. combined GRF4 & GIF1 P = 0.0007). G) Control and GRF4 chimeras fused to either GIF1, GIF2 or GIF3, n = 3 experiments (contrast chimeras with GIF1 vs. combined GIF2 and GIF3 ** P = 0.0046). H) Control and chimeras combining different wheat GRF genes fused with GIF1 (n= 4 experiments, except for GRF5 n=3). ** P = 0.0064 in contrast comparing combined GRF4-GIF1 and GRF5-GIF1 chimeras (evolutionary related) with combined GRF1-GIF1 and GRF9-GIF1 chimeras (more distantly related). In all tests, normality of residuals was confirmed by Shapiro-Wilk’s test and homogeneity of variances by Levene’s test (raw-data is available in Supplementary Table 3).

Figure 2.. The GRF4-GIF1 chimera induces embryogenesis in the absence of cytokinins.

A) Schematic representation of the different steps of wheat transformation. B). Representative calli in auxin media with no hygromycin. Note growing green shoots in callus transformed with the wheat GRF4-GIF1 chimera in the absence of cytokinins (red arrows). Control: pLC41. C) Transgenic specific PCR product (yellow arrow) amplified with primers pLC41–1064 and pLC41–1061 (Supplementary Table S1). In this first experiment (out of three), we identified five transgenic plants among nine regenerated from the GRF4-GIF1 marker-free vector and no transgenic plants among four regenerated from the control.

Figure 3.. High frequency of genome edited plants using combined GRF4-GIF1 – CRISPR-Cas9 technology.

A) Technologies combined in a single vector. B) Region of the gene Q (AP2L-A5) targeted with the guide RNA (gRNA) and schematic representation of the vector combining both technologies (LB = left border, Hyg. = hygromycin resistance, RB = right border). C) Kronos shoot regeneration of embryos transformed with an empty vector and with the combined GRF4-GIF1 - CRISPR-Cas9-gRNA-AP2L-A5 construct (93.7 % regeneration efficiency). D) All 10 sequenced transgenic T₀ plants showed AP2L-A5 editing. Seven of the 10 plants (T#1 to T#10) carried two different mutations (a1 and a2), documenting high editing efficiency. E) Edited T₀ plants showed increased number of florets per spikelet (characteristic of q-null plants).