Reduced gene expression of potato apoplastic invertase inhibitor gene on CRISPR/Cas9 targeting and analyzing its transformation efficiency parameters

Abstract

Abstract Background Pathogen infections that affect potato yield cause severe economic losses every year. Several studies point the role of apoplastic (cell wall) invertase (CWIN) enzyme in plant defense mechanisms, and that apoplastic invertase inhibitor (INVINH1) post-translationally regulates CWIN. Nevertheless, the role of INVINH1 needs to be elucidated for several effects in plant transformation parameters and its gene expression which we sought to explore using CRISPR/Cas9 technology. Methods and Results In this study, we sequenced the first exon of INVINH1 gene in cv. Desiree and Solanum chacoense M6. We identified in the first exon two alleles for StINVINH1 gene in cv. Desiree and one allele for ScINVINH1 gene in S. chacoense M6. We designed two single-guided RNAs (sgRNAs) to target INVINH1 gene from diploid S. chacoense M6 and tetraploid S. tuberosum cv. Desiree using CRISPR/Cas9 based technology. In our earlier study, we have already optimized transformation protocol for M6 and cv. Desiree using Agrobacterium strains, based on which Agrobacterium strain AGL1 was chosen for CRISPR/Cas9 experiment. Our experimentation showed that heat stress at 37°C could increase the mutagenesis capability, and CRISPR/Cas9 targeting affected plant transformation parameters. It was found from the knockout experiment that the indels were present in the calli, and the candidate regenerated plants showed reduced gene expression level conducted via RT-qPCR. Conclusion Our study demonstrated that INVINH1 targeting affected the calli induction and regeneration rates, was effective under heat stress, and reduced its gene expression level. More studies are required to comprehend the function of INVINH1 enzyme in potato stress response and defense mechanism.