Knockout Mutants of OsPUB7 Generated Using CRISPR/Cas9 Revealed Abiotic Stress Tolerance in Rice

Abstract

Plants produce and accumulate stress-resistant substances when exposed to abiotic stress, which involves a protein conversion mechanism that breaks down stress-damaged proteins and supplies usable amino acids. Eukaryotic protein turnover is mostly driven by the ubiquitination pathway. Among the three enzymes required for protein degradation, E3 ubiquitin ligase plays a pivotal role in most cells, as it determines the specificity of ubiquitination and selects target proteins for degradation. In this study, to investigate the function of OsPUB7 (Plant U-box gene in Oryza sativa), we constructed a CRISPR/Cas9 vector, generated OsPUB7 gene-edited individuals, and evaluated resistance to abiotic stress using gene-edited lines. A stress-tolerant phenotype was observed as a result of drought and salinity stress treatment in the T2 OsPUB7 gene-edited null lines (PUB7-GE) lacking the T-DNA. In addition, although PUB7-GE did not show any significant change in mRNA expression analysis, it showed lower ion leakage and higher proline content than the wild type (WT). Protein–protein interaction analysis revealed that the expression of the genes (OsPUB23, OsPUB24, OsPUB66, and OsPUB67) known to be involved in stress increased in PUB7-GE and this, by forming a 1-node network with OsPUB66 and OsPUB7, acted as a negative regulator of drought and salinity stress. This result provides evidence that OsPUB7 will be a useful target for both breeding and future research on drought tolerance/abiotic stress in rice.

Arabidopsis ubiquitin ligase PUB41 is a positive regulator of the ABA response

“…Interestingly, pub mutants reported to be affected in drought resilience were both hyper- or hypo-sensitive: single gene mutants of the Arabidopsis paralogs AtPUB46 and AtPUP48 (Adler et al 2017), and the Atpub8 mutant (Adler 2011) are drought hypersensitive whereas Arabidopsis Atpub18, Atpub19, Atpub23 , and Atpub24 mutants exhibit increased drought tolerance compared with wild-type plants (Seo et al 2012). Also, the rice OsPUB7 knockout mutant and the Ospub41 suppression mutant, and Ubi:RNAi-OsPUB41 knock-down lines showed enhanced survival to drought (Kim et al 2023; Seo et al 2021). (Despite sharing a common gene name OsPUB41 is not orthologous to AtPUB41 , but shows the highest similarity to AtPUB20 and AtPUB21 ).…”

Creating Climate-Resilient Crops by Increasing Drought, Heat, and Salt Tolerance

“…The concerns about introducing foreign genes into plants are eliminated in this technique as it precisely edits the target gene at nucleotide level without any transgene intervention [142]. Knockout mutants of diverse genes involved in abiotic stress response created by the CRISPR technique resulted in enhanced tolerance in a range of species such as rice [143], tomato [144], tobacco [145], soybean [146], and wheat [147]. To ensure the gene stability in the modified plants, a lipofection-mediated delivery into protoplasts [148] was developed along with other approaches such as Agrobacterium-mediated transformation [149] and particle bombardment [150].…”

CRISPR/Cas9 genome editing tool for rice crop improvement

“…CRISPR/Cas9 employed targeted mutagenesis in the OsRR22 was improved the salinity tolerance in the japonica rice cultivar WPB106 . Kim et al (2023) paved the way of generating targeted mutagenesis in the gene OsPUB7 through CRISPR/Cas9 to develop drought tolerance and abiotic stress rice in the future. Alam et al (2022) reported that knocking out the OsbHLH024 transcription factor conferred salinity tolerance.…”