Molecular Pathogens, 2025, Vol.16, No.6, 285-293 http://microbescipublisher.com/index.php/mp 286 editing, this study aims to provide a sustainable and effective approach for cotton disease management, restore and enhance the disease resistance of cotton plants, and promote crop protection and global cotton production. 2 Pathogenic Mechanisms and Key Targets in Drug-Resistant Pathogens 2.1 Overview of resistance-related genes in major cotton pathogens Not all cotton pathogenic bacteria can be easily suppressed by pesticides or plant defenses. Problem pathogens like Trichoderma lucidum and Fusarium acuminatum have long evolved multiple sets of "weapons" to counter various external disturbances. Genes such as VDAG_04757, VDAG_06462, VDAG_03218 and VDAG_08487 have been shown to be associated with enhanced pathogenicity and adaptability to host stress in the Pseudomonas aeruginosa (Zhang et al., 2025). In addition, signal regulatory factors like VdRGS1 also play a crucial role in maintaining the reproductive capacity and virulence of pathogens. Cotton itself is not powerless to fight back. For example, GhAMT2, GhFAR3 and some CC-NBS-LRR type genes will initiate defense responses when pathogens invade, strengthen cell walls and transmit disease resistance signals (Wang et al., 2025; Wubben et al., 2025). However, the problem lies in that the expression of these resistance genes is not stable among different cotton varieties. 2.2 Key virulence factors and signaling regulators in pathogenic pathways How do pathogenic bacteria manage to make plants "unable to cope"? The signal system behind it is extremely complex. Transcription factor networks such as the MAPK pathway, G protein-related signaling systems (VdRGS1 being one of them), and WRKY and MYB are all involved in regulation. For instance, genes such as GhWRKY70D13, GhWRKY53, and GhODO1 are not only related to signaling pathways such as jasmonic acid, salicylic acid, and ethylene in plants, but also associated with lignin synthesis and REDOX reactions (Xiong et al., 2020; Zhu et al., 2022; Li et al., 2023). These links themselves may be independent of each other, but their interaction is the core that makes pathogenic bacteria more infectious. Once such regulatory mechanisms form a "series effect", it is often very difficult to interrupt them through a single means. 2.3 Role of functional gene targets in maintaining and spreading resistance Some functional genes have attracted much attention because they not only determine the adaptability of pathogenic bacteria but also may influence the spread of resistance genes in the population. In pathogenic bacteria, those genes that control virulence, signal transduction and even "bypass" the host immune mechanism are all key to supporting their long-term survival and spread. Conversely, genes like GhUKL4 and GhGDH2 in cotton regulate the hormone balance and oxidation status within the plant, thereby affecting its ability to resist diseases continuously. These also happen to be the target points that many CRISPR editing strategies aim to "precisely strike" (Xiong et al., 2021; Cheng et al., 2025). However, theory is theory, but in practical operation, to accurately hit these targets, a large number of experiments are still needed for verification. 3 Current Applications of the CRISPR/Cas System in Microbial Pathogens 3.1 Editing mechanisms of CRISPR/Cas9 in fungi and bacteria Nowadays, the practice of "cutting and modifying" genes in pathogenic microorganisms increasingly relies on systems like CRISPR/Cas9. However, this technology itself is not complicated. The core lies in guiding the Cas9 nuclease to the target gene's location through sgRNA and then creating a break there. For filamentous fungi, in addition to the common Cas9, Cas12a is sometimes also used, and its efficiency is even higher (Figure 1). Nowadays, many laboratories can rapidly knockout a single gene or even an entire chromosomal region through this method (Yuan et al., 2024). In the case of bacteria, the situation is similar. Often, several genes are edited together. Especially when it comes to removing fragments related to drug resistance, CRISPR tools are particularly useful (Vivekanandan et al., 2025). Of course, the prerequisite for these operations is that the sgRNA design is accurate and the import methods should also keep up. 3.2 CRISPR-based strategies for target gene screening and validation To figure out which gene in the pathogenic bacteria is causing trouble, it is really too slow to screen it all by traditional methods. CRISPR technology has greatly accelerated in this regard. It can not only batch knockout
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