CRISPR/Cas9-mediated targeted mutagenesis of GmLHY genes alters plant height and internode length in soybean

Abstract

BackgroundSoybean (Glycine max) is an economically important oil and protein crop. Plant height is a key trait that significantly impacts the yield of soybean; however, research on the molecular mechanisms associated with soybean plant height is lacking. The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated system 9) system is a recently developed technology for gene editing that has been utilized to edit the genomes of crop plants.ResultsHere, we designed four gRNAs to mutate four LATE ELONGATED HYPOCOTYL (LHY) genes in soybean. In order to test whether the gRNAs could perform properly in transgenic soybean plants, we first tested the CRISPR construct in transgenic soybean hairy roots using Agrobacterium rhizogenes strain K599. Once confirmed, we performed stable soybean transformation and obtained 19 independent transgenic soybean plants. Subsequently, we obtained one T1 transgene-free homozygous quadruple mutant of GmLHY by self-crossing. The phenotypes of the T2-generation transgene-free quadruple mutant plants were observed, and the results showed that the quadruple mutant of GmLHY displayed reduced plant height and shortened internodes. The levels of endogenous gibberellic acid (GA3) in Gmlhy1a1b2a2b was lower than in the wild type (WT), and the shortened internode phenotype could be rescued by treatment with exogenous GA3. In addition, the relative expression levels of GA metabolic pathway genes in the quadruple mutant of GmLHY were significantly decreased in comparison to the WT. These results suggest that GmLHY encodes an MYB transcription factor that affects plant height through mediating the GA pathway in soybean. We also developed genetic markers for identifying mutants for application in breeding studies.ConclusionsOur results indicate that CRISPR/Cas9-mediated targeted mutagenesis of four GmLHY genes reduces soybean plant height and shortens internodes from 20 to 35 days after emergence (DAE). These findings provide insight into the mechanisms underlying plant height regulatory networks in soybean.

Progresses, Challenges, and Prospects of Genome Editing in Soybean (Glycine max)

“…each of them in a separate plasmid as shown in previous reports (Jiang et al, 2013;Li et al, 2013;Brooks et al, 2014). In most cases in CRISPR system for soybean, the Streptococcus pyogenes Cas9 gene (Cai et al, 2015;Cheng et al, 2019), human codonoptimized Cas9 gene (Campbell et al, 2019;Do et al, 2019) or a soybean codon optimized Cas9 gene (Michno et al, 2015;Sun et al, 2015) was driven by commonly used promoters in soybean transformation, such as ubiquitin (soybean ubiquitin-3, Campbell et al, 2019), Arabidopsis egg-cell specific promoter (Zheng et al, 2020) and cauliflower mosaic virus 35S (CaMV 35S) promoters (Cai et al, 2015;Jacobs et al, 2015;Michno et al, 2015;Bao et al, 2019;Chen F. et al, 2019;Cheng et al, 2019;Do et al, 2019;Han et al, 2019). Some endogenous promoters, such as proGmSCREAM M4 (pM4), proGmSCREAM M8 (pM8) (Bai et al, 2020) and soybean ELONGATION FACTOR1 ALPHA2 (EF1A2) gene constitutive promoter (Li et al, 2015), were used as well.…”

“…Plant architecture can be modified for improving yield. Cheng et al (2019) mutated 4 Late Elongated Hypocotyl (LHY) genes again in soybean and obtained a homozygous quadruple mutant of GmLHY which is similar to GmLCLa1a2b1b2 described above and showed reduced plant height and shortened internodes. As a class A gene in the ABCE model in plant, APETALA1 (AP1) involves in floral organ development.…”

“…Transient expression systems such as the hairy root induction system via Agrobacterium rhizogenes K599, callus tissue expression via biolistic delivery, agro-infiltration using leaves and protoplast transfection system, have been employed for the purpose (Figure 2). The assay for editing activity in hairy roots is a popular transient system for soybean (Curtin et al, 2011;Haun et al, 2014;Jacobs et al, 2015;Du et al, 2016;Cheng et al, 2019;Do et al, 2019;Bai et al, 2020), A. rhizogenes K599 containing editing reagents were delivered into seedlings of target genotypes and hairy roots can be recovered within 15 days. Editing activity of the reagents can be evaluated in those hairy roots.…”

“…PCR/RE assay detection method was used frequently in soybean (Michno et al, 2015;Sun et al, 2015;Kanazashi et al, 2018;Di et al, 2019). T7EI assay was occasionally used (Cai et al, 2015;Du et al, 2016) and PCR/Sequencing assay is now the most popular method used to detect mutation in soybean (Haun et al, 2014;Campbell et al, 2019;Cheng et al, 2019;Do et al, 2019;Han et al, 2019;Li R. et al, 2019;Wang J. et al, 2020). PCR/RE and T7EI are cost efficient method when large number putative mutations need to be screened, but limitation of restriction enzyme cut sites can restrict design of sgRNA.…”

“…For SDN1 mutations in soybean, Agrobacterium-mediated transformation system is currently an efficient and popular method (Table 2) due to convenient mature seed used as explants. GE with multiplex have been achieved in soybean to recover simultaneously target multiple genes or genomic sites in a single transformation event through either making one construct with the expression of multiple sgRNAs using a single Cas9 (Bao et al, 2019;Do et al, 2019) or infecting target tissue with pooled Agrobacterium strains each containing one sgRNA (Kanazashi et al, 2018;Cheng et al, 2019;Do et al, 2019;Wang J. et al, 2020;Bai et al, 2020). Biolistic delivery plus embryogenesis-based regeneration system is an alternative method to recover GE evens with various outcomes.…”