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Major crop production does not yet match the population growth rate because multiple abiotic stresses hamper the growth and yield of these crops. Most of the plants can tolerate adverse climatic conditions by performing some adaptive machineries but to certain extent. Till date various biotechnological and molecular breeding research approaches have been directed towards developing resistance to single stress factor. However, development of crop plants resistant to a single stress is not an ideal solution in the current agriculture scenario. Occurrence of multiple stresses at a single point of time makes it difficult to formulate research strategies. Considering the huge loss of crop productivity due to these environmental factors, there is an urgent need to direct our research focus towards developing sustainable multi-stress resistance in plants to counter the adverse effect of climate change on the productivity of crops. RNA helicases are ubiquitous proteins that are found in both prokaryotes and eukaryotes. The largest RNA helicase family comprises the DEAD-box RNA helicases which are involved in many aspects of RNA metabolism and in diverse biological processes in plants including regulation of multiple abiotic stress responses. The DEAD-box RNA helicases can be considered as means to identify pathways involved in multiple abiotic stress tolerance. In this review, we summarize the recent advances in elucidating the functions of the DEAD-box RNA helicases in multiple abiotic stress responses and future challenges. We also briefly discussed about our recent research efforts (published and on-going) in this direction. This review would help to formulate new research endeavours utilizing the DEAD-box helicase genes in development of multiple abiotic stress tolerant plants through genetic engineering and biotechnology. Target specific multiplex and multigene CRISPR/Ca9 genome editing would be ideal approach to edit different abiotic stress responsive DEAD-box RNA helicase genes to develop sustainable multiple abiotic stress tolerance in crop plants.

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Owing to rapid global climate change, the occurrence of multiple abiotic stresses is known to influence the outburst of biotic stress factors which affects crop productivity. Therefore, it is essential to understand the molecular and cell biology of key genes associated with multiple stress responses in crop plants. SlHyPRP1 and DEA1, the members of eight-cysteine motif (8CM) family genes have been recently identified as putative regulators of multiple stress responses in tomato (Solanum lycopersicum L.). In order to gain deeper insight into cell and molecular biology of SlHyPRP1 and DEA1, we performed their expression analysis in three tomato cultivars and in vivo cell biological analysis. The semi-quantitative PCR and qRT-PCR results showed the higher expression of SlHyPRP1 and DEA1 in leaf, stem, flower and root tissues as compared to fruit and seed tissues in all three cultivars. The expression levels of SlHyPRP1 and DEA1 were found to be relatively higher in a wilt susceptible tomato cultivar (Arka Vikas) than a multiple disease resistant cultivar (Arka Abhed). In vivo cell biological analysis through Gateway cloning and Bi-FC assay revealed the Electronic supplementary material The online version of this article (

Fusarium wilt is a major devastating fungal disease of tomato (Solanum lycopersicum L.) caused by Fusarium oxysporum f. sp. lycopersici (Fol) which reduces the yield and production. Xylem sap protein 10 (XSP10) and Salicylic acid methyl transferase (SlSAMT) are two putative negative regulatory genes associated with Fusarium wilt of tomato. Fusarium wilt tolerance in tomato can be developed by targeting these susceptible (S) genes. Due to its efficiency, high target specificity, and versatility, CRISPR/Cas9 has emerged as one of the most promising techniques for knocking out disease susceptibility genes in a variety of model and agricultural plants to increase tolerance/resistance to various plant diseases in recent years. Though alternative methods, like RNAi, have been attempted to knock down these two S genes in order to confer resistance in tomato against Fusarium wilt, there has been no report of employing the CRISPR/Cas9 system for this specific intent. In this study, we provide a comprehensive downstream analysis of the two S genes via CRISPR/Cas9-mediated editing of single (XSP10 and SlSAMT individually) and dual-gene (XSP10 and SlSAMT simultaneously). Prior to directly advancing on to the generation of stable lines, the editing efficacy of the sgRNA-Cas9 complex was first validated using single cell (protoplast) transformation. In the transient leaf disc assay, the dual-gene editing showed strong phenotypic tolerance to Fusarium wilt disease with INDEL mutations than single-gene editing. In stable genetic transformation of tomato at the GE1 generation, dual-gene CRISPR transformants of XSP10 and SlSAMT primarily exhibited INDEL mutations than single-gene-edited lines. The dual-gene CRISPR-edited lines (CRELs) of XSP10 and SlSAMT at GE1 generation conferred a strong phenotypic tolerance to Fusarium wilt disease compared to single-gene-edited lines. Taken together, the reverse genetic studies in transient and stable lines of tomato revealed that, XSP10 and SlSAMT function together as negative regulators in conferring genetic tolerance to Fusarium wilt disease.