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With increasing frequency and severity of droughts in various parts of the world, agricultural productivity may suffer major setbacks. Among all the abiotic factors, drought is likely to have one of the most detrimental effects on soil organisms and plants. Drought is a major problem for crops because it limits the availability of water, and consequently nutrients which are crucial for plant growth and survival. This results in reduced crop yields, stunted growth, and even plant death, according to the severity and duration of the drought, the plant’s developmental stage, and the plant’s genetic background. The ability to withstand drought is a highly complex characteristic that is controlled by multiple genes, making it one of the most challenging attributes to study, classify, and improve. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) technology has opened a new frontier in crop enhancement, revolutionizing plant molecular breeding. The current review provides a general understanding of principles as well as optimization of CRISPR system, and presents applications on genetic enhancement of crops, specifically in terms of drought resistance and yield. Moreover, we discuss how innovative genome editing techniques can aid in the identification and modification of genes conferring drought tolerance.

Plant protein inhibitors counteract the activity of cell wall–degrading enzymes (CWDEs) secreted by pathogens to breach the plant cell-wall barrier. Transgenic plants expressing a single protein inhibitor restrict pathogen infections. However, since pathogens secrete a number of CWDEs at the onset of infection, we combined more inhibitors in a single wheat genotype to reinforce further the cell-wall barrier. We combined polygalacturonase (PG) inhibiting protein (PGIP) and pectin methyl esterase inhibitor (PMEI), both controlling the activity of PG, one of the first CWDEs secreted during infection. We also pyramided PGIP and TAXI-III, a xylanase inhibitor that controls the activity of xylanases, key factors for the degradation of xylan, a main component of cereal cell wall. We demonstrated that the pyramiding of PGIP and PMEI did not contribute to any further improvement of disease resistance. However, the presence of both pectinase inhibitors ensured a broader spectrum of disease resistance. Conversely, the PGIP and TAXI-III combination contributed to further improvement of Fusarium head blight (FHB) resistance, probably because these inhibitors target the activity of different types of CWDEs, i.e., PGs and xylanases. Worth mentioning, the reduction of FHB symptoms is accompanied by a reduction of deoxynivalenol accumulation with a foreseen great benefit to human and animal health.

Xylanase (E.C. 3.2.1.8) degrades β-1, 4 xylan by cleaving β-1, 4 glycosidic linkages randomly, resulting in the generation of xylose and xylo-oligosaccharides. Xylanases are produced by organisms including fungi, bacteria, yeast, marine algae, protozoans, snails, crustaceans and insects. Xylanases present considerable industrial interest for their use in paper manufacturing, improvement of animal feed digestibility, and clarification of fruit juices. In addition, this enzyme is the component of cell wall-degrading enzymes (CWDEs) during plant–pathogen interaction. Thus, considering their various applications in plant defence and also in industry, the characterization of xylanase activity becomes an important aspect. Conventionally, xylanase activity is determined by radial gel diffusion assay using Congo red staining (Emami and Hack, 2001) and by DNSA assay which is a colorimetric method for xylanase activity (McLauchlan et al., 1999; Kutasi et al., 2001). Comparatively, radial gel diffusion assay using Congo red staining is a qualitative assay whereas DNSA method is a quantitative assay. Moreover, Congo red is a chemical considered as hazardous category 1B (Carcinogenicity) and category 12 (Reproductive toxicity) by the 2012 OSHA Hazard Communication Standard (29 CFR 1910.1200). In the present study, the proposed method enables qualitative detection of xylanase activity using ethanol precipitation in the radial gel diffusion assay which is safer and simpler. The ethanol precipitation in agar plate has been adapted from the method for detecting xylanase activity in polyacrylamide gels (Royer and Nakas, 1990).

Although wheat is a staple food for most of the human population, some of its components trigger adverse reactions. Among wheat components, the alpha-amylase/trypsin inhibitors (ATI) are important triggers of several allergies and activators of innate immunity. ATI are a group of exogenous protease inhibitors and include several polypeptides. The three ATI polypeptides named CM3, CM16 and 0.28 are considered major allergens, and might also play a role in other common wheat-related pathologies, such as Non Celiac Wheat Sensitivity and even Celiac Disease. On this basis, we pointed to obtain high amounts of them in purity and to evaluate their allergenicity potential. We thus isolated the mRNA corresponding to the three ATI genes CM3, CM16 and 0.28 from 28 days post-anthesis wheat kernels and the corresponding cDNAs were used for heterologous expression in Pichia pastoris. The three purified proteins were tested in degranulation assay against human sera of patients with food allergy to wheat. A large range of degranulation values was observed for each protein according to the sera tested. All of the three purified proteins CM3, CM16 and 0.28 were active as allergens because they were able to induce basophils degranulation on wheat allergic patients’ sera, with the highest values of β-hexosaminidase release observed for CM3 protein.