Plant virus-mediated sgRNA delivery and expression have great advantages; sgRNA expression can rapidly expand and accumulate along with virus replication and movement, resulting in efficient gene editing efficiency. In this study, a VIGE system based on cotton leaf crumple virus (CLCrV) was established using cotton overexpressing Cas9 (Cas9-OE) as the VIGE receptor. CLCrV-mediated VIGE could not only target and knock out the GhMAPKKK2, GhCLA1 and GhPDS genes subgroup A and D genome sequences but also achieve double mutation of GhCLA1 and GhPDS genes at the same time. These results verified the effectiveness and efficiency of this system. In addition, the off-target effect assay demonstrated that the CLCrV-mediated VIGE system not only has high gene editing efficiency but also high gene editing specificity in cotton. We further explored whether the FT-sgRNA strategy could transport sgRNA to cotton apical meristem (SAM) over long distances to avoid using tissue culture to obtain stable genetic mutants. The results showed that the sgRNA fused with FT mRNA at the 5’ end could also efficiently achieve targeted editing of endogenous genes in cotton, but it was difficult to detect heritable mutant progeny. The above results showed that the CLCrV-mediated VIGE system provided an accurate and rapid validation tool for screening effective sgRNAs in cotton.

As a plant-specific Rho-like small G protein, the ROP (Rho-related GTPase of plants) protein regulates the growth and development of plants and various stress responses in the form of molecular switches. Drought is a major abiotic stress that limits cotton yield and fiber quality. In this study, virus-induced gene silencing (VIGS) technology was used to analyze the biological function of GhROP3 in cotton drought stress tolerance. Meanwhile, we used yeast two-hybrid and bimolecular fluorescence complementation assays to examine the interaction between GhROP3 and GhGGB. GhROP3 has a high expression level in cotton true leaves and roots, and responds to drought, high salt, cold, heat stress, and exogenous abscisic acid (ABA) and auxin (IAA) treatments. Silencing GhROP3 improved the drought tolerance of cotton. The water loss rates (WLR) of detached leaves significantly reduced in silenced plants. Also, the relative water content (RWC) and total contents of chlorophyll (Chl) and proline (Pro) of leaves after drought stress and the activities of three antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) significantly increased, whereas the contents of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) significantly reduced. In the leaves of silenced plants, the expression of genes related to ABA synthesis and its related pathway was significantly upregulated, and the expression of decomposition-related GhCYP707A gene and genes related to IAA synthesis and its related pathways was significantly downregulated. It indicated that GhROP3 was a negative regulator of cotton response to drought by participating in the negative regulation of the ABA signaling pathway and the positive regulation of the IAA signaling pathway. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that the GhROP3 protein interacted with the GhGGB protein in vivo and in vitro. This study provided a theoretical basis for the in-depth investigation of the drought resistance–related molecular mechanism of the GhROP3 gene and the biological function of the GhGGB gene.

Cotton is one of the most economically important crops in the world, and drought is a key abiotic factor that can significantly reduce cotton yield. MADS-box transcription factors play essential roles in various aspects of plant growth and development as well as responses to biotic and abiotic stress. However, the use of MADS-box transcription factors to regulate water stress responses has not been fully explored in cotton. Here, we showed that GhAGL16 acts as a negative regulator of water deficit in cotton, at least in part by regulating ABA signaling. GhAGL16-overexpressing (GhAGL16-OE) transgenic Arabidopsis had lower survival rates and relative water contents (RWCs) under water stress. Isolated leaves of GhAGL16-OE Arabidopsis had increased water loss rates, likely attributable to their increased stomatal density. GhAGL16-OE Arabidopsis also showed reduced primary root lengths in response to mannitol treatment and decreased sensitivity of seed germination to ABA treatment. By contrast, silencing GhAGL16 in cotton enhanced tolerance to water deficit by increasing proline (Pro) content, increasing superoxide dismutase (SOD) and peroxidase (POD) activities, and reducing malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) contents under water stress. Subcellular localization and transcriptional activation assays confirmed that GhAGL16 is a nuclear protein that lacks transcriptional self-activation activity. The expression of ABA biosynthesis-related genes (GhNCED3/7/14), a catabolism-related gene (GhCYP707A), and a gene related to the ABA signaling pathway (GhABF4) was altered in GhAGL16-silenced plants. Taken together, our data demonstrate that GhAGL16 plays an important role in cotton resistance to water stress.

Fire blight of pear is caused by Erwinia amylovora, which can cause devastating damage to pear trees and other pome fruit trees worldwide. Fire blight has resulted in a reduction in the production of Korla fragrant pears in Xinjiang, China, as well as a decrease in their quality and taste, causing severe economic losses. To this end, a comparative transcriptome analysis of common Korla fragrant pear (KFP, susceptible) and a bud mutation line (1910, resistant) at three inoculation periods was conducted. Clustering and principal component analysis (PCA) of the RNA-seq data revealed that the differences between lines were greater than those within lines. A total of 7271 DEGs were identified in the bud mutation line, while 11,937 DEGs were identified in KFP. Between KFP and the resistant material bud mutation line, 11,937 DEGs were identified, which were significantly enriched in the pathways of photosynthesis, jasmonic acid metabolic process, flavonoid biosynthesis, and starch and sucrose metabolism. A total of 8 clusters were identified for all (17,354) DEGs via k-means, and KEGG pathway annotations were performed for each individual cluster. In addition, the 1027 differentially expressed transcription factors (TFs) were clustered into five clusters, and the TFs with the largest fold change in each cluster were identified. A gene coexpression network was further constructed through weighted correlation network analysis (WGCNA), and 15 key genes that determine the fire blight resistance of Korla fragrant pear were identified. These research results provide a theoretical basis for a deeper understanding of the molecular mechanism of Korla fragrant pear resistance to fire blight and provide new genetic resources for the study of Korla fragrant pear resistance to fire blight.

Verticillium wilt (VW), caused by Verticillium dahliae, poses a significant threat to global cotton production. Through analysis of public transcriptome databases, this study identified GhSTZ, a C2H2 zinc finger protein transcription factor gene, which was significantly induced by V. dahliae. Suppressing GhSTZ expression via virus-induced gene silencing significantly enhanced cotton resistance to VW. This resistance manifested as a 1.2-fold increase in lignin deposition, optimized ROS (reactive oxygen species) homeostasis, and a 1.3-fold elevation in glucose levels. Transcriptome analysis revealed 338 differentially expressed genes in GhSTZ-silenced plants, with 97 upregulated and 241 downregulated. Key downregulated genes included PME (pectin methylesterase) and PG1-pec (polygalacturonase) in the pentose phosphate pathway, while the key upregulated genes comprised C4H (cinnamate 4-hydroxylase) and C3H (p-coumarate 3-hydroxylase) in the phenylpropanoid biosynthesis pathway. Notably, in the plant–pathogen interaction signaling pathway, approximately half of the genes exhibited upregulated expression while the other half showed downregulation. Protein–protein interaction network analysis further revealed cooperative interaction between PME and the secoisolariciresinol dehydrogenase SIRD. This study is the first to elucidate GhSTZ as a negative regulator that compromises cotton disease resistance through a tripartite mechanism. These findings offer a novel approach to enhancing crop disease resistance by targeting the negative regulatory genes.