*3.2.4 Biotic stress resistance improvement*

Biotic stresses are one of the basic factor of crop losses in agriculture [47]. The main biotic stresses affecting crops include phytopathogens (viruses, bacteria, fungi), insect and pests (phytophagous insects, acari or nematodes). The strategy for dealing with biotic stresses is either in increasing of self defense mechanisms in plants or in introducing into the genome of constructs aimed against the pathogens [47, 48].

The producing of crops lines resistant to pathogens and pests using traditional breeding methods is based on increasing the own defense mechanisms in plants, but the introduction of constructs targeted pathogens into the genome is carried out by genetic engineering. The most of biotechnological crop lines resistant to biotic stresses created to date are obtained by transgenesis or RNA interference (RNAi) methods [47].

Nowadays, GE approaches are widely used to create new resistant lines [7–10, 44]. Herewith, it should be mentioned these methods makes possible to use both strategies to deal with biotic stresses [44, 47, 48]. Let us to consider some examples of the use several GE technologies to develop lines and varieties of crops resistant to biotic stresses.

*Targeting on plant susceptibility genes.* Plant susceptibility genes are essential to successful infection and development pathogens [49]. Thereat, affect these genes brings about development of plant resistance [7, 44, 49]. As targeting of TALENand CRISPR/Cas9-based *MLO* homologues providing resistance to powdery mildew allows obtaining resistant lines of barley, wheat, Arabidopsis, tomato and pea [7, 44, 49]. Additionally, the CRISPR/Cas9-mediated directed affecting of *DMR6* gene allows creating tomato lines resistant to *Pseudomonas syringae*, *Phytophthora capsici*, and *Xanthomonas* spp [49].

Targeting of disease susceptibility factors was also used for creation of virusresistant crops. As CRISPR/Cas9-based silencing of *eIF4e* factors associated with plant infection by positive sense RNA viruses allows creating virus-resistant plants. Herewith, CRISPR/Cas9-mediated disruption of *eIF4Es* gene function in *Arabidopsis* and cucumber promotes to develope potyvirus resistance [49]. Similarly, CRISPR/Cas9-based mutagenesis of *eIF4G* allows obtaining some rice lines resistant to rice tungro spherical virus (*RTSV*) [49].

To creation of crop lines resistant to biotic stress GE approaches are also used to impact on regulatory elements that can affect the process of pathogen proliferation [49]. So as, TALEN- and CRISPR/Cas9-mediated mutagenesis in effector binding site of promoter region of *OsSweet14* gene permits to develop *Xanthomonas oryzae* pv. *oryzae* resistant rice lines [7, 44, 49]. Also it was shown that Cas9/sgRNA-based targeting the effector binding element of *CsLOB1* gene in citrus provided the generation cancer-resistant citrus varieties [50].

*Targeting on genes of plant hormonal system.* It is known that hormonal signalling plays an important role in immune response of plants [51]. Herewith, salicylatemediated immune response develops in response to biotrophic and hemibiotrophic pathogens infection, and by jasmonate- and ethylene-mediated – against necrotrophic pathogens. Thus, directed effect on the genes of the hormonal response permit to create pathogen-resistant crop lines [49]. Recently this assumption has been confirmed experimentally. Herewith, CRISPR/Cas9-based targeting of the *SlJAZ2* gene in tomato resulted in genesis of resistance to *Pseudomonas syringae* pv. *tomato* (Pto) DC3000 [48].

*Targeting of pathogen genomes.* Another advanced strategy for pathogens defence is directed effect on pathogen genome. Currently, this strategy is mainly used against viral plant diseases [7, 44]. Thus, CRISPR/Cas9 system has been successfully applied to increase resistance to DNA-containing viruses, including tomato yellow leaf curl virus (*TYLCV*), beet root curl virus (*BCTV*), Merremia mosaic virus (*MeMV*), beans yellow dwarfism virus (*BeYDV*), cotton leaf curl virus and beet severe curly top virus (*BSCTV*) [44, 48]. It should be noted that one sgRNA designed to targeting a conserved region can mediate interference against a numerous DNA-containing viruses [8, 44]. Additionally, CRISPR/LshCas13a system capable to interfere of viral RNA turnip line is successfully applied to creation of resistant to RNA-containing turnip yellow mosaic virus (*TuMV*) [44].

#### *3.2.5 Abiotic stress resistance improvement*

Abiotic stresses are the main factors that negatively affect the yield of most crops [52]. In this regard, a creation of resistant to adverse environmental factors crop varieties is the urgent problem. However, the use of traditional breeding methods to develop such varieties is limited by the fact that the traits of resistance to abiotic stress are multigene controlled and have a complex inheritance type [52, 53]. The disadvantages of traditional breeding can be successfully overcome through the use of GE approaches. Herewith, a literature analysis has shown that the GE application

in various cultures allowed to increase their resistance to abiotic stresses [7, 44, 53, 54]. Let us consider detailed examples of the GE application to obtain varieties of crops resistant to abiotic stresses.

*Targeting of structural genes.* Structural genes are one of the most convenient targets to increase plant stress resistance. This class of genes can be divided into tolerance genes (T-genes) and sensitivity genes (S-genes). The T-genes encode enzymes of the antioxidant system, while S genes – negative regulators in plant defense mechanisms [54]. Therefore, "turn off" of S-genes allows to obtain drought-resistant crop varieties. This assumption was confirmed by CRISPR/Cas9 based targeting of *ARGOS8* (a negative regulator of ethylene response) in maize, that permit to obtain drought-resistant lines [53]. Similar results were obtained during directed mutagenesis of the *OST2* gene in *Arabidopsis*, *TaDREB2* gene in wheat (*Triticum aestivum*) and *OsDERFl*, *OsEPSPS* and *OsMSHl* genes in rice (*Oryza sativa* L.). In addition, CRISPR/Cas9-mediated targeting of *OsPDS*, *OsMPK2*, and *OsDEPl, OsAOXla*, *OsAOXlb*, *OsAOXlc* and *OsBEL* genes allows to develop rice lines resistant to a wide range of abiotic stresses [10, 44, 53, 54].

*Targeting of regulatory genes.* Regulatory genes, such as transcription factors, phosphatases and kinases are also involved in the regulation of intracellular signals during abiotic stresses and can be used as target for GE tools to create stress-resistant crop varieties [54]. Presently, CRISPR/Cas9-mediated silencing of *SlMAPK3* regulatory gene in tomato (*Solatium lycopersicum*), *TaERF3* gene in wheat, *OsSAPK2*, *OsPMS3*, *OsRAV2*, and *OsNAC.041* genes in rice permit to create lines of these crops that are resistant to abiotic stresses [44, 53].

Summarize the presented data, it should be noted that GE systems are successfully used to modify a wide range of economically valuable traits in main agricultural crops.
