CRISPR/Cas9-based genome editing and functional analysis of SlHyPRP1 and SlDEA1 genes of Solanum lycopersicum L. in imparting genetic tolerance to multiple stress factors

Abstract

CRISPR/Cas is a breakthrough genome editing system because of its precision, target specificity, and efficiency. As a speed breeding system, it is more robust than the conventional breeding and biotechnological approaches for qualitative and quantitative trait improvement. Tomato (<i>Solanum lycopersicum</i> L.) is an economically important crop, but its yield and productivity have been severely impacted due to different abiotic and biotic stresses. The recently identified <i>SlHyPRP1</i> and <i>SlDEA1</i> are two potential negative regulatory genes in response to different abiotic (drought and salinity) and biotic stress (bacterial leaf spot and bacterial wilt) conditions in <i>S. lycopersicum</i> L. The present study aimed to evaluate the drought, salinity, bacterial leaf spot, and bacterial wilt tolerance response in <i>S. lycopersicum</i> L. crop through CRISPR/Cas9 genome editing of <i>SlHyPRP1</i> and <i>SlDEA1</i> and their functional analysis. The transient single- and dual-gene <i>SlHyPRP1</i> and <i>SlDEA1</i> CRISPR-edited plants were phenotypically better responsive to multiple stress factors taken under the study. The CRISPR-edited <i>SlHyPRP1</i> and <i>SlDEA1</i> plants showed a higher level of chlorophyll and proline content compared to wild-type (WT) plants under abiotic stress conditions. Reactive oxygen species accumulation and the cell death count per total area of leaves and roots under biotic stress were less in CRISPR-edited <i>SlHyPRP1</i> and <i>SlDEA1</i> plants compared to WT plants. The study reveals that the combined loss-of-function of <i>SlHyPRP1</i> along with <i>SlDEA1</i> is essential for imparting significant multi-stress tolerance (drought, salinity, bacterial leaf spot, and bacterial wilt) in <i>S. lycopersicum</i> L. The main feature of the study is the detailed genetic characterization of <i>SlDEA1</i>, a poorly studied 8CM family gene in multi-stress tolerance, through the CRISPR/Cas9 gene editing system. The study revealed the key negative regulatory role of <i>SlDEA1</i> that function together as an anchor gene with <i>SlHyPRP1</i> in imparting multi-stress tolerance in <i>S. lycopersicum</i> L. It was interesting that the present study also showed that transient CRISPR/Cas9 editing events of <i>SlHyPRP1</i> and <i>SlDEA1</i> genes were successfully replicated in stably generated parent-genome-edited line (GEd0) and genome-edited first-generation lines (GEd1) of <i>S. lycopersicum</i> L. With these upshots, the study's key findings demonstrate outstanding value in developing sustainable multi-stress tolerance in <i>S. lycopersicum</i> L. and other crops to cope with climate change.