Insights into the molecular mechanism of the hard-to-cook defect towards genetic improvement of common bean (phaseolus vulgaris L.) through crispr-cas9 gene editing optimization

The hard-to-cook (HTC) defect is characterized by prolonged cooking of common bean (Phaseolus vulgarisL.) due to difficulty in achieving cell separation in the cotyledons. This study aimed to unveil insights into the molecular mechanism of the HTC defect and optimize a CRISPR-Cas9 knockout system for HTC candidate genes in P. vulgaris. Using RNA sequencing, numerous differentially expressed genes (DEGs) were established between a slow-and a fast-cooking bean, providing a wide gene pool for selection of candidate genes of the HTC defect. Molecular processes relevant to the HTC defect were uncovered, including up-regulated DEGs in the slow-cooking bean within the cell periphery. The cell wall has predominantly featured as a critical contributor to the HTC defect due to the dynamics of pectin adhesion. In this study, some cell-wall modifying enzymes including a pectin methyl-esterase (PME) were differentially expressed. Further investigations of the contribution of PMEs to the HTC defect revealed that the PMEs and their inhibitor proteins belonged to a large multi-gene family in P. vulgaris, with low expression in seeds. Notably, two genes encoding a PME and a PME inhibitor (PMEI) were highly expressed in the fast-and slow-cooking beans, respectively. Subsequent analyses revealed that PMEs show increased gene expression and enzymatic activity in fast-cooking beans and may be necessary for promoting the fast-cooking phenotype in P. vulgaris, since the action of PME may further encourage the activity of cell wall modifying enzymes which soften the cell wall. We hypothesized that the high expression of the PMEI in the slow-cooking bean could result in the inhibition of the activity of PME, causing the HTC phenotype. Therefore, both the PME and PMEI genes were targeted for knockout using CRISPR-Cas9. However, common bean is highly recalcitrant to transformation and in vitro regeneration, with no successful CRISPR studies reported yet. Using aRhizobium rhizogenes-mediated hairy root assay, the efficiency of the designed sgRNAs to induce mutations in the target genes was rapidly determined. Further, successful induction of callus and subsequent transformation using Agrobacterium tumefaciens was accomplished. This protocol was successfully used to induce potentially useful mutations in the PME and PMEI sequences in the calli of the fast-and slow-cooking beans respectively. Calli from the slow-cookingbeans were able to regenerate into whole plantlets with shoots containing the CRISPR constructs. In conclusion, results from this study unveiled molecular processes effectuating the HTC defect in common beans, including the contribution of pectin methyl-esterase enzyme and its inhibitor protein. An efficient callus induction and transformation protocol for the common bean is provided here, putting the crop in a position to benefit from improved technologies such as CRISPR-Cas9.