ARGOS8 variants generated by CRISPR-Cas9 improve maize grain yield under field drought stress conditions

Abstract

Maize ARGOS8 is a negative regulator of ethylene responses. A previous study has shown that transgenic plants constitutively overexpressing ARGOS8 have reduced ethylene sensitivity and improved grain yield under drought stress conditions. To explore the targeted use of ARGOS8 native expression variation in drought-tolerant breeding, a diverse set of over 400 maize inbreds was examined for ARGOS8 mRNA expression, but the expression levels in all lines were less than that created in the original ARGOS8 transgenic events. We then employed a CRISPR-Cas-enabled advanced breeding technology to generate novel variants of ARGOS8. The native maize GOS2 promoter, which confers a moderate level of constitutive expression, was inserted into the 5'-untranslated region of the native ARGOS8 gene or was used to replace the native promoter of ARGOS8. Precise genomic DNA modification at the ARGOS8 locus was verified by PCR and sequencing. The ARGOS8 variants had elevated levels of ARGOS8 transcripts relative to the native allele and these transcripts were detectable in all the tissues tested, which was the expected results using the GOS2 promoter. A field study showed that compared to the WT, the ARGOS8 variants increased grain yield by five bushels per acre under flowering stress conditions and had no yield loss under well-watered conditions. These results demonstrate the utility of the CRISPR-Cas9 system in generating novel allelic variation for breeding drought-tolerant crops.

Keywords: ARGOS; CRISPR-Cas9; drought tolerance; genome editing; grain yield; maize.

Figure 1

Maize ARGOS8 reduces plant responses to ethylene when overexpressed in transgenic Arabidopsis plants. (a) Ethylene triple response of Arabidopsis ARGOS8 transgenic plants ( ARGOS8) and wild‐type (WT) controls to 0.5 μm of the ethylene precursor aminocyclopropane‐1‐carboxylic acid (ACC). A short version of ARGOS8 was overexpressed under control of the cauliflower mosaic virus 35S promoter (35S). Composite image of representative 3‐day‐old etiolated seedlings. Bar = 2 mm. (b) Hypocotyl and root lengths of etiolated Arabidopsis seedlings overexpressing the short version of ARGOS8. Four transgenic lines (E1, E2, E4 and E12) and wild‐type (WT) controls were grown in the dark in the presence of indicated ACC concentrations for 3 days. Data are means ± SD, n = 15. Significant differences of the transgenic plants from the WT are denoted by asterisks (*P < 0.05, **P < 0.01, ANOVA, Tukey's HSD).

Figure 2

Editing the ARGOS8 genomic sequence using the CRISPR/Cas9 system to generate variants with constitutive expression. (a) Schematic drawing illustrating the insertion of GOS2 PRO into the 5′‐UTR of ARGOS8 and the promoter swap. CTS, CRISPR‐RNA target site; HA, homology arm; HDR, homology‐directed repair; GOS2 PRO, maize GOS2 promoter and the 5′‐UTR with an intron. (b) Genomic sequence of the ARGOS8 5′‐UTR and the upstream region. The CRISPR‐RNA target sites (CTS) are highlighted in red, and the protospacer adjacent motifs (PAM) are shown in blue font. The ARGOS8 coding region is shown in bold font. (c) Diagram showing primers used in junction PCR for genotyping regenerated shoots and long PCR for amplifying and sequencing the entire modification region in homozygous plants. The relative position and direction of PCR primers (P) are indicated by arrows. P1 and P2 for the HR1 junction; P5 and P4 for the HR2 junction; P1 and P4 for the long PCR. (d) Junction PCR analysis of regenerated shoots. Agarose gel images are shown for representative regenerated shoots positive for one junction or two junctions and shoots negative in the junction PCR assay. JP1, HR1 junction PCR with the primer P1 and P2; JP2, HR2 junction PCR with P5 and P4. (e) PCR screening regenerated shoots for deletion in the ARGOS8 locus. An agarose gel image is shown for PCR products amplified with the primer P1 and P4 in representative shoots (Lanes 1‐14) generated from the CRISPR RNA‐3 and RNA‐1 transformation. M, DNA molecular weight markers.

Figure 3

Maize genome‐edited ARGOS8 variants. (a) Genomic sequence upstream of the ARGOS8 coding region in three genome‐edited variants. The entire modification region in homozygous F2 plants was amplified using long PCR, and the PCR products were sequenced. Part of the GOS2 5′‐UTR sequence (blue font) and the remaining 5′‐UTR of ARGOS8 as well as the 5′‐terminus of ARGOS8 coding sequence are shown. In the promoter deletion variant ARGOS8‐v3, the remnant CTS3 and CTS1 sequences are highlighted. (b) Relative expression levels of ARGOS8 in leaves as measured by qRT‐PCR. Means ± SD are shown for F2 plants of 14‐day‐old ARGOS8‐v1 and 18‐day‐old ARGOS8‐v2; n = 10–24. WT, wild‐type; Hete, Heterozygote; Homo, homozygote.

Figure 4

Comparison of the ARGOS8 expression in genome‐edited variants and wild‐type maize plants. (a) Relative expression of ARGOS8 in a selection of maize tissues and stages. mRNA was quantified with qRT‐PCR. Six individual plants were analysed for the genome‐edited variants and two plants for WT controls. DAP, days after pollination. (b) ARGOS8 protein expression in developing kernels. Immature kernels (21 DAP) were analysed by immunoblotting using a monoclonal anti‐ARGOS8 antibody.