Bioscience Methods 2024, Vol.15, No.6, 315-326 http://bioscipublisher.com/index.php/bm 317 2020). Leaf rust, on the other hand, is known for its widespread occurrence and ability to cause yield losses of up to 50% under favorable conditions for the pathogen (Kumar et al., 2020; Wang and Li, 2024). The economic impact of these diseases is profound, necessitating the development of resistant wheat varieties to ensure food security. The evolution of virulent pathotypes of these rusts poses a continuous threat to wheat production. For instance, the emergence of new virulent strains of stripe rust has led to frequent epidemics, challenging the durability of existing resistant varieties (Figure 1) (Kumar et al., 2020). Similarly, leaf rust has shown a high degree of adaptability, with new races overcoming previously effective resistance genes. This dynamic nature of pathogen evolution underscores the need for innovative approaches, such as CRISPR/Cas9, to develop durable disease resistance in wheat. Figure 1 Pie chart representation of seedling response against (A) five pathotypes of stripe rust (YR), (B) six pathotypes of leaf rust (LR), and (C) seven pathotypes of stem rust (SR) of rust association mapping panel (RAMP) (Adopted from Kumar et al., 2020) Image caption: The color legend on the right side of each pie chart represents the infection type (IT) score. The magnitude of arc length is directly proportional to the frequency of genotypes showing corresponding IT scores (Adopted from Kumar et al., 2020) 3.2 Case Studies on using CRISPR/Cas9 to knock out susceptibility genes for enhanced disease resistance CRISPR/Cas9 technology has been successfully employed to enhance disease resistance in wheat by targeting and knocking out susceptibility genes. One notable example is the targeting of the TaHRC and Tsn1 genes, which confer susceptibility to Fusariumhead blight (FHB) and tan spot, respectively. By using CRISPR/Cas9-mediated genome editing, researchers were able to generate wheat plants with mutations in these genes, resulting in enhanced resistance to these diseases (Karmacharya et al., 2023). This approach not only improves disease resistance but also provides insights into the functional roles of these genes in wheat-pathogen interactions. Another significant case study involves the identification and disruption of 33 susceptibility genes (S genes) related to various wheat diseases, including stripe rust, leaf rust, and powdery mildew. The down-regulation or deletion of these S genes using CRISPR/Cas9 has been shown to improve disease tolerance in wheat. This strategy highlights the potential of CRISPR/Cas9 to target multiple genes simultaneously, thereby providing a robust and comprehensive approach to enhancing disease resistance in wheat (Taj et al., 2022). 3.3 Identification and targeted editing strategies of disease resistance genes The identification of disease resistance genes is a critical step in developing resistant wheat varieties. Genome-wide association studies (GWAS) have been instrumental in mapping resistance genes for major wheat diseases. For instance, a study identified several quantitative trait loci (QTLs) associated with resistance to stripe rust, leaf rust, and stem rust in a diverse panel of spring wheat genotypes. These QTLs, located on various chromosomes, provide valuable targets for CRISPR/Cas9-mediated editing to enhance disease resistance (Kumar et al., 2020).
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