CRISPR-Cas9 mediated genome editing of drought and salt tolerance (OsDST) gene in indica mega rice cultivar MTU1010

Abstract

Development of abiotic stress tolerant rice cultivars is necessary for sustainable rice production under the scenario of global climate change, dwindling fresh water resources and increase in salt affected areas. Several genes from rice have been functionally validated by using EMS mutants and transgenics. Often, many of these desirable alleles are not available indica rice which is mainly cultivated, and where available, introgression of these alleles into elite cultivars is a time and labour intensive process, in addition to the potential introgression of non-desirable genes due to linkage. CRISPR-Cas technology helps development of elite cultivars with desirable alleles by precision gene editing. Hence, this study was carried out to create mutant alleles of drought and salt tolerance (DST) gene by using CRISPR-Cas9 gene editing in indica rice cv. MTU1010. We used two different gRNAs to target regions of DST protein that might be involved in protein-protein interaction and successfully generated different mutant alleles of DST gene. We selected homozygous dst mutant with 366 bp deletion between the two gRNAs for phenotypic analysis. This 366 bp deletion led to the deletion of amino acid residues from 184 to 305 in frame, and hence the mutant was named as dst D184-305 . The dst D184-305 mutation induced by CRISPR-Cas9 method in DST gene in indica rice cv. MTU1010 phenocopied EMS-induced dst (N69D) mutation reported earlier in japonica cultivar. The dst D184-305 mutant produced leaves with broader width and reduced stomatal density, and thus enhanced leaf water retention under dehydration stress. Our study showed that the reduction in stomatal density in loss of function mutants of dst is, at least, in part due to downregulation of stomatal developmental genes SPCH1, MUTE and ICE1. The Cas9-free dst D184-305 mutant exhibited moderate level tolerance to osmotic stress and high level of salt stress in seedling stage. Thus, dst mutant alleles generated in this study will be useful for improving drought and salt tolerance and grain yield in indica rice cultivars.

Genome editing in cereal crops: an overview

“…In a recent study, CRISPR/Cas9 targeted mutagenesis of the zinc finger transcription factor OsDST (DROUGHT AND SALT TOLERANCE) was used to develop mutant lines of indica mega rice cv. MTU1010 with tolerance to both drought and salinity stress (Santosh Kumar et al 2020). Under drought conditions, OsDST mutant plants produced broader leaves with a lower stomatal density, thus showing improved water retention under dehydration stress (Santosh Kumar et al 2020).…”

“…MTU1010 with tolerance to both drought and salinity stress (Santosh Kumar et al 2020). Under drought conditions, OsDST mutant plants produced broader leaves with a lower stomatal density, thus showing improved water retention under dehydration stress (Santosh Kumar et al 2020). The reduction of stomatal density in rice plants increases its ability to conserve water, as observed in the IR64 mutant lines overexpressing the rice epidermal patterning factor OsEPF1 (EPIDERMAL PATTERNING FACTOR) .…”

Mechanistic insights of CRISPR/Cas‐mediated genome editing towards enhancing abiotic stress tolerance in plants

“…The CRISPR/Cas9 system was used to develop drought‐tolerant tomato plants mutant for the slnpr1 gene (Li et al, 2019). The indica mega rice cultivar MTU1010 dst mutant generated by CRISPR‐Cas9 editing of drought and salt tolerance ( OsDST ) gene was found having broader leaves with reduced stomatal density towards enhancing leaf water retention under drought stress (Ganie et al, 2021; Santosh Kumar et al, 2020).…”

Rice breeding in the new era: comparison of useful agronomic traits

“…In contrast, salt tolerance in varieties like IR29, Fatmawati, Pokkali, Cheriviroppu, FL478, IR651, CSR27, FL30, Fontan, SR86, IR9884-54-3 results from ion exclusion, osmotic and tissue tolerance with multiple genes involved in the process, which confers agronomic stability of this trait (Qin et al, 2020;Reddy et al, 2017;Takagi et al, 2015;. The orchestrated stress system can be targeted for achieving salt tolerance by mutating genes encoding key transcription factors, specifically OsRR22 (Os06g0183100), STL1 (Os04g0110600), and the zinc finger transcription factor encoded by DST (Os03g0786400) (Cui et al, 2015;Santosh Kumar et al, 2020;Yuan et al, 2020;. Other transcription factors are critical in stress adaptation, which results in stress sensitivity when inactivated.…”