Cotton Genomics and Genetics 2025, Vol.16, No.1, 29-38 http://cropscipublisher.com/index.php/cgg 32 the CC-NBS-LRR type. By activating the salicylic acid pathway, it can also drive a group of resistance genes to "go online" together, and its defense against Verticillium wilt is very obvious (Li et al., 2021). However, not all genes are so "cooperative". For example, TIR-NB-LRRgenes like GhRVD1 have a double TIR structure. Although it is relatively rare, it is closely related to the type of immune response that is particularly strong. It often appears in the mechanism that triggers cell death after pathogen infection and is critical for long-term resistance (Zhang et al., 2023c). 4.2 Bt and related transgenic loci for insect resistance To protect cotton from insects, traditional methods alone are not enough. The introduction of genetically modified Bt technology is a turning point. Bt toxins can directly make chewing pests "lose their fighting power", so Bt cotton can express one or more Bt genes, and the effect is indeed good (Zafar et al., 2020). But this technology has not been without trouble. For example, in some places, cotton bollworms have gene mutations (such as HaTSPAN1), and as a result, Bt toxins are not so effective against them (Guan et al., 2020). In order to prevent resistance from being "cracked", scientists have come up with many ways. Some stack multiple Bt genes together, and some simply use Bt and RNAi technology together, with the aim of controlling different pests while making the effect more stable and less likely to fail. 4.3 Metabolic and signaling genes associated with resistance Not all genes that help with resistance look like NB-LRR. Some genes are usually "silent", but they are critical in regulating metabolism and signal transduction. For example, GhnsLTPsA10, this gene encodes a lipid transport protein, which doesn't sound very "disease-resistant", but it does make cotton more "resistant" to Verticillium wilt and Fusarium wilt. It can also regulate phenylpropanoid metabolism, thereby affecting the synthesis of flavonoids and lignin, and has some effect on fighting cotton aphids and cotton bollworms (Chen et al., 2021). Similar ones include GhCPK33 and GhCPK74, which belong to the calcium-dependent protein kinase (CDPK) family. Mutants made by CRISPR-Cas9 have also confirmed that they are indeed involved in cotton's defense response (Wang et al., 2024). Therefore, in addition to the traditional "main" resistance genes, these "auxiliary" genes related to signals and metabolism cannot be ignored. Combining them may be a long-term solution for disease and insect resistance. 5 Functional Validation and Gene Editing Technologies 5.1 Gene silencing and overexpression studies In order to verify whether the candidate resistance genes in cotton are effective, the commonly used methods are gene silencing and overexpression. Now there are some rapid methods, such as the transient expression system based on protoplasts, which can quickly detect whether the gene is effective without the need for stable transformation (Zhang et al., 2023a). These systems can also help us study how genes are expressed, where proteins are located, and whether there are interactions between proteins. These works have saved a lot of time for the study of cotton functional genomics. 5.2 CRISPR/Cas-based genome editing in cotton Not all editing tools are suitable for cotton, especially this complex allotetraploid crop. However, the emergence of CRISPR/Cas has indeed made things much simpler. Systems such as CRISPR/Cas9 and Cpf1 (also called Cas12a) can now precisely edit a single gene or even different copies of multiple genes in cotton. It is quite smooth to use and has high efficiency, sometimes reaching about 87% (Gao et al., 2017; Li et al., 2022a). More importantly, the rate of accidental injury is not high, and the chance of error is relatively small. Of course, not every editing is so "ideal". In order to make the efficiency more stable, some studies have begun to use viruses to deliver sgRNA, and some have used geminiviruses as vectors (Li et al., 2019; Lei et al., 2022). Although these methods sound complicated, they do improve the accuracy in practice. Now, mutants made with these tools have been widely used in functional studies. Which genes are really useful in disease and insect resistance? Which ones have improved agronomic traits? Through these editing experiments, we can basically figure it out (Zhu et al., 2018).
RkJQdWJsaXNoZXIy MjQ4ODYzNA==