Molecular Plant Breeding 2024, Vol.15, No.3, 144-154 http://genbreedpublisher.com/index.php/mpb 148 4.3 Case studies of identifiedRgenes in pines Several R genes have been identified and characterized in pine species, providing valuable insights into their mechanisms of action. For instance, the meta-analysis of 314 cloned R genes has revealed that the majority encode cell surface or intracellular receptors, which can detect pathogen-derived molecules either directly or indirectly (Kourelis and Hoorn, 2018). These receptors often belong to the NBS-LRR family, which is the largest group of plant resistance genes and is crucial for initiating immune responses. Specific case studies in pines have demonstrated the effectiveness of pyramiding multiple Rgenes to enhance resistance durability, thereby reducing the need for chemical pesticides and promoting sustainable forestry practices (Fernandez-Gutierrez and Gutierrez-Gonzalez, 2021). The identification of these genes and their functional validation through various genomic and bioinformatic approaches underscores the potential for rational engineering of disease-resistant pine varieties. Fernandez-Gutierrez and Gutierrez-Gonzalez (2021) presents an overview of four protocols--RenSeq, MutRenSeq, MutChromSeq, and AgRenSeq--for discovering nucleotide-binding leucine-rich repeat (NLR) genes. Each pipeline starts with different inputs: wild-type plants, mutagenesis, and a diversity panel. All protocols involve DNA fragmentation, hybridization with biotinylated oligonucleotide baits, and enrichment of target sequences, followed by next-generation sequencing (NGS). RenSeq directly identifies NLRs from wild-type samples. MutRenSeq and MutChromSeq include mutagenesis steps and focus on mapping to wild-type assemblies for single nucleotide variant (SNV) identification. AgRenSeq uses a diversity panel and k-mer filtering to associate genetic variations with phenotypes. These protocols, with their similarities and differences, offer robust methods for NLRgene discovery, each tailored for specific research needs in plant genetics and disease resistance studies. 5 Strategies for Precise Gene Editing 5.1 Targeted mutagenesis Targeted mutagenesis is a powerful strategy for precise gene editing, primarily utilizing the CRISPR/Cas9 system. This method involves creating specific mutations at targeted genomic loci to disrupt gene function, which can confer disease resistance in plants. For instance, the CRISPR/Cas9 system was employed to knockout the Os8N3 gene in rice, resulting in enhanced resistance to Xanthomonas oryzae pv. oryzae (Xoo). The mutations were stably transmitted across generations, and the edited plants displayed no significant differences in agronomic traits compared to non-transgenic controls (Kim et al., 2019). Similarly, CRISPR/Cas9-mediated mutagenesis of the VvMLO3 gene in grapevine led to enhanced resistance to powdery mildew, demonstrating the effectiveness of this approach in improving disease resistance in economically important crops (Wan et al., 2020). 5.2 Homology-directed repair (HDR) Homology-Directed Repair (HDR) is another precise gene editing strategy that leverages the cell's natural repair mechanisms. When a double-strand break (DSB) is introduced at a specific genomic location, a donor template with homologous sequences can be provided to guide the repair process, allowing for precise insertion, deletion, or replacement of DNA sequences. This method is particularly useful for introducing specific genetic changes or correcting mutations. Although HDR is less efficient than non-homologous end joining (NHEJ), advancements in CRISPR/Cas9 technology and the development of novel donor templates are improving its efficiency and applicability in plant genome editing. 5.3 Base editing and prime editing Base editing and prime editing are innovative gene editing techniques that enable precise nucleotide changes without introducing DSBs. Base editing uses engineered deaminases to convert specific DNA bases, such as cytosine to thymine or adenine to guanine, directly at the target site. This method has been successfully applied to create point mutations that confer disease resistance in plants. Prime editing, on the other hand, uses a reverse transcriptase enzyme fused to a modified Cas9 protein to introduce small insertions, deletions, or base substitutions guided by a prime editing guide RNA (pegRNA). These techniques offer higher precision and reduced off-target effects compared to traditional CRISPR/Cas9 methods, making them promising tools for functional genomics and crop improvement.
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