Cotton is one of the world\'s most important economic crops. Verticillium wilt is a devastating cotton disease caused by Verticillium dahliae, significantly impacting cotton yield and quality. E3 ubiquitin ligases are essential components of the ubiquitin-mediated 26S proteasome system, responsible for recognizing ubiquitinated target proteins and promoting their degradation, which play a crucial regulatory role in plant immune responses. In this study, on the basis of the confirmation of differential expression of GhDIRP1, a RING-type E3 ubiquitin ligase encoding gene, in two cotton varieties resistant (Zhongzhimian 2) or susceptible (Jimian 11) to V. dahliae, we demonstrated that GhDIRP1 is a negative regulator of V. dahliae resistance because silencing GhDIRP1 in cotton and heterogeneously overexpressing the gene in Arabidopsis enhanced and compromised resistance to V. dahliae, respectively. The GhDIRP1-mediated immune response seemed to be realized through multiple physiological pathways, including hormone signaling, reactive oxygen species, and lignin biosynthesis. Based on the sequences of GhDIRP1 isolated from Zhongzhimian 2 and Jimian 11, we found that GhDIRP1 had identical coding but different promoter sequences in the two varieties, with the promoter of Zhongzhimian 2 being more active than that of Jimian 11 because the former drove a stronger expression of GUS and LUC reporter genes. The results link the ubiquitination pathway to multiple physiological pathways acting in the cotton immune response and provide a candidate gene for breeding cotton varieties resistant to V. dahliae.

Verticillium wilt, caused by the pathogenic fungus Verticillium dahliae, has emerged as a severe threat to cotton globally. However, little is known about the genetic diversity of this pathogen in an infected single cotton plant. In this study, we isolated three new V. dahliae strains from the disease stems of Gossypium hirsutum from the cotton field in Western China and assessed their pathogenicity to the cotton cultivar Xinnongmian-1 and its two transgenic lines, as well as two laboratory strains, VD592 and VD991. These three new V. dahliae strains were identified using DNA barcodes of tryptophan synthase (TS), actin (ACT), elongation factor 1-α (EF), and glyceraldehyde-3-phosphate dehydrogenase (GPD). Moreover, the haplotype analysis revealed that the three new races had distinct haplotypes at the TS locus. Furthermore, the results of culture features and genetic diversity of ISSR (inter-simple sequence repeat) revealed that there were separate V. dahliae strains, which were strong defoliating pathotypes belonging to race 2 type, as determined by particular DNA marker recognition. The identified strains demonstrated varied levels of pathogenicity by leaf disc and entire plant inoculation methods. Conservatively, these strains showed some pathogenicity on cotton lines, but were less pathogenic than the reference strains. The findings revealed that several strong defoliating V. dahliae pathotypes coexist on the same cotton plant. It indicats the importance of regular monitoring as an early warning system, as well as the detection and reporting of virulent pathogen strains and their effects on crop response.

Fusarium wilt (FW) is widespread in global cotton production, but the mechanism underlying FW resistance in superior-fiber-quality Sea Island cotton is unclear. This study reveals that FW resistance has been the target of genetic improvement of Sea Island cotton in China since the 2010s. The key nonsynonymous single nucleotide polymorphism (SNP, T/C) of gene Gbar_D03G001670 encoding protein phosphatase 2C 80 (PP2C80) results in an amino acid shift (L/S), which is significantly associated with FW resistance of Sea Island cotton. Silencing GbPP2C80 increases FW resistance in Sea Island cotton, whereas overexpressing GbPP2C80 reduces FW resistance in Arabidopsis. GbPP2C80 and GbWAKL14 exist synergistically in Sea Island cotton accessions with haplotype forms \"susceptible-susceptible\" (TA) and \"resistant-resistant\" (CC), and interact with each other. CRISPR/Cas9-mediated knockout of GbWAKL14 enhances FW and Verticillium wilt (VW) resistance in upland cotton and overexpression of GbWAKL14 and GbPP2C80 weakens FW and VW resistance in Arabidopsis. GbPP2C80 and GbWAKL14 respond to FW and VW by modulating reactive oxygen species (ROS) content via affecting MPK3 expression. In summary, two tandem genes on chromosome D03, GbPP2C80, and GbWAKL14, functions as cooperative negative regulators in cotton wilt disease defense, providing novel genetic resources and molecular markers for the development of resistant cotton cultivars.

BACKGROUND: Verticillium wilt, causes mainly by the soilborne pathogen Verticillium dahliae, is a devastated vascular disease resulting in huge financial losses in cotton, so research on improving V. dahliae stress tolerance in cotton is the utmost importance. Calcium as the second messenger acts as a crucial role in plant innate immunity. Cytosolic Ca2+during the pathogen infection is a significant increase in plant immune responses. Calcineurin B-like (CBL) proteins are widely known calcium sensors that regulate abiotic stress responses. However, the role of cotton CBLs in response to V. dahliae stress remains unclear.
OBJECTIVE: To discover and utilize the gene to Verticillium wilt resistance and defense response mechanism of cotton.
METHODS: Through screening the gene to Verticillium wilt resistance in cotton, four GhCBL3 copies were obtained from the current common cotton genome sequences. The protein domain and phylogenetic analyses of GhCBL3 were performed using NCBI Blast, DNAMAN, and MotifScan programs. Real-time RT-PCR was used to detect the expression of GhCBL3 gene in cotton seedlings under various stress treatments. The expression construct including GhCBL3 cDNA was transduced into Agrobacterium tumefaciens (GV3101) by heat shock method and transformed into cotton plants by Virus-Induced Gene Silencing (VIGS) method. The results of silencing of GhCBl3 on ROS accumulation and plant disease resistance in cotton plants were assessed.
RESULTS: A member of calcineurin B-like proteins (defined as GhCBL3) in cotton was obtained. The expression of GhCBL3 was significantly induced and raised by various stressors, including dahliae, jasmonic acid (JA) and H2O2 stresses. Knockdown GhCBL3 in cotton by Virus-Induced Gene Silencing analysis enhanced Verticillium wilt tolerance and changed the occurrence of reactive oxygen species. Some disease-resistant genes were increased in GhCBL3-silencing cotton lines.
CONCLUSIONS: GhCBL3 may function on regulating the Verticillium dahliae stress response of plants.

Verticillium wilt, caused by Verticillium dahliae, is a soil-borne disease affecting eggplant. Wild eggplant, recognized as an excellent disease-resistant resource against verticillium wilt, plays a pivotal role in grafting and breeding for disease resistance. However, the underlying resistance mechanisms of wild eggplant remain poorly understood. This study compared two wild eggplant varieties, LC-2 (high resistance) and LC-7 (sensitive) at the phenotypic, transcriptomic, and metabolomic levels to determine the molecular basis of their resistance to verticillium wilt. These two varieties exhibit substantial phenotypic differences in petal color, leaf spines, and fruit traits. Following inoculation with V. dahliae, LC-2 demonstrated significantly higher activities of polyphenol oxidase, superoxide dismutase, peroxidase, phenylalanine ammonia lyase, β-1,3 glucanase, and chitinase than did LC-7. RNA sequencing revealed 4,017 differentially expressed genes (DEGs), with a significant portion implicated in processes associated with disease resistance and growth. These processes encompassed defense responses, cell wall biogenesis, developmental processes, and biosynthesis of spermidine, cinnamic acid, and cutin. A gene co-expression analysis identified 13 transcription factors as hub genes in modules related to plant defense response. Some genes exhibited distinct expression patterns between LC-2 and LC-7, suggesting their crucial roles in responding to infection. Further, metabolome analysis identified 549 differentially accumulated metabolites (DAMs) between LC-2 and LC-7, primarily consisting of compounds such as flavonoids, phenolic acids, lipids, and other metabolites. Integrated transcriptome and metabolome analyses revealed the association of 35 gene-metabolite pairs in modules related to the plant defense response, highlighting the interconnected processes underlying the plant defense response. These findings characterize the molecular basis of LC-2 resistance to verticillium wilt and thus have potential value for future breeding of wilt-resistant eggplant varieties.

Understanding the extent of heritability of a plant-associated microbiome (phytobiome) is critically important for exploitation of phytobiomes in agriculture. Two crosses were made between pairs of cotton cultivars (Z2 and J11, L1 and Z49) with differential resistance to Verticillium wilt. F2 plants were grown in a field, together with the four parents to study the heritability of cotton rhizosphere microbiome. Amplicon sequencing was used to profile bacterial and fungal communities in the rhizosphere. F2 offspring plants of both crosses had higher average alpha diversity indices than the two parents; parents differed significantly from F2 offspring in Bray-Curtis beta diversity indices as well. Two types of data were used to study the heritability of rhizosphere microbiome: principal components (PCs) and individual top microbial operational taxonomic units (OTUs). For the L1 × Z49 cross, the variance among the F2 progeny genotypes (namely, genetic variance, VT) was significantly greater than the random variability (VE) for 12 and 34 out of top 100 fungal and bacterial PCs, respectively. For the Z2 × J11 cross, the corresponding values were 10 and 20 PCs. For 29 fungal OTUs and 10 bacterial OTUs out of the most abundant 100 OTUs, genetic variance (VT) was significantly greater than VE for the L1 × Z49 cross; the corresponding values for the Z2 × J11 cross were 24 and one. The estimated heritability was mostly in the range of 40% to 60%. These results suggested the existence of genetic control of polygenic nature for specific components of rhizosphere microbiome in cotton. KEY POINTS: • F2 offspring cotton plants differed significantly from parents in rhizosphere microbial diversity. • Specific rhizosphere components are likely to be genetically controlled by plants. • Common PCs and specific microbial groups are significant genetic components between the two crosses.

Terpene synthases (TPSs) are enzymes responsible for catalyzing the production of diverse terpenes, the largest class of secondary metabolites in plants. Here, we identified 107 TPS gene loci encompassing 92 full-length TPS genes in upland cotton (Gossypium hirsutum L.). Phylogenetic analysis showed they were divided into six subfamilies. Segmental duplication and tandem duplication events contributed greatly to the expansion of TPS gene family, particularly the TPS-a and TPS-b subfamilies. Expression profile analysis screened out that GhTPSs may mediate the interaction between cotton and Verticillium dahliae. Three-dimensional structures and subcellular localizations of the two selected GhTPSs, GhTPS6 and GhTPS47, which belong to the TPS-a subfamily, demonstrated similarity in protein structures and nucleus and cytoplasm localization. Virus-induced gene silencing (VIGS) of the two GhTPSs yielded plants characterized by increased wilting and chlorosis, more severe vascular browning, and higher disease index than control plants. Additionally, knockdown of GhTPS6 and GhTPS47 led to the down-regulation of cotton terpene synthesis following V. dahliae infection, indicating that these two genes may positively regulate resistance to V. dahliae through the modulation of disease-resistant terpene biosynthesis. Overall, our study represents a comprehensive analysis of the G. hirsutum TPS gene family, revealing their potential roles in defense responses against Verticillium wilt.

Mitogen-activated protein kinase (MAPK) cascade is the center of plant signal transduction system that amplify immune signals into cellular responses by phosphorylating diverse substrates. The MAPK cascade consisting of MAPK kinase kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs is well characterized in plants, in which Raf-like kinases are generally regarded as MAPKKKs. However, it is rarely reported that Raf-like MAPKKKs function as middle regulators to link MAPK and its downstream transcription factors in plant immunity. Verticillium wilt, caused by the soil-borne vascular fungus Verticillium dahliae, is a serious disease in many plants, including cotton. The previous studies showed that GhMPK9 (a MAPK) is involved in the response to Verticillium wilt. Here, the Raf-like kinase GhRAF39_1 is reported as helper regulates the phosphorylation of WRKY transcription factor GhWRKY40a by GhMPK9. The phosphorylated GhWRKY40a can further activate the transcription of GhERF1b to up-regulate defense-related genes while inhibit the transcription of GhABF2 to regulate the stomatal opening, thus improving the resistance to Verticillium wilt in cotton. This study reveals a new signaling module of GhMPK9-GhRAF39_1-GhWRKY40a to regulate GhERF1b- and GhABF2-mediated defense responses, which triggers plant defense against Verticillium wilt.

BACKGROUND: Cotton is globally important crop. Verticillium wilt (VW), caused by Verticillium dahliae, is the most destructive disease in cotton, reducing yield and fiber quality by over 50% of cotton acreage. Breeding resistant cotton cultivars has proven to be an efficient strategy for improving the resistance of cotton to V. dahliae. However, the lack of understanding of the genetic basis of VW resistance may hinder the progress in deploying elite cultivars with proven resistance.
RESULTS: We planted the VW-resistant Gossypium hirsutum cultivar Zhongzhimian No.2 (ZZM2) in an artificial greenhouse and disease nursery. ZZM2 cotton was subsequently subjected to transcriptome sequencing after Vd991 inoculation (6, 12, 24, 48, and 72 h post-inoculation). Several differentially expressed genes (DEGs) were identified in response to V. dahliae infection, mainly involved in resistance processes, such as flavonoid and terpenoid quinone biosynthesis, plant hormone signaling, MAPK signaling, phenylpropanoid biosynthesis, and pyruvate metabolism. Compared to the susceptible cultivar Junmian No.1 (J1), oxidoreductase activity and reactive oxygen species (ROS) production were significantly increased in ZZM2. Furthermore, gene silencing of cytochrome c oxidase subunit 1 (COX1), which is involved in the oxidation-reduction process in ZZM2, compromised its resistance to V. dahliae, suggesting that COX1 contributes to VW resistance in ZZM2.
CONCLUSIONS: Our data demonstrate that the G. hirsutum cultivar ZZM2 responds to V. dahliae inoculation through resistance-related processes, especially the oxidation-reduction process. This enhances our understanding of the mechanisms regulating the ZZM2 defense against VW.

Cotton Verticillium wilt is mainly caused by the fungus Verticillium dahliae, which threatens the production of cotton. Its pathogen can survive in the soil for several years in the form of microsclerotia, making it a destructive soil-borne disease. The accurate, sensitive, and rapid detection of V. dahliae from complex soil samples is of great significance for the early warning and management of cotton Verticillium wilt. In this study, we combined the loop-mediated isothermal amplification (LAMP) with CRISPR/Cas12a technology to develop an accurate, sensitive, and rapid detection method for V. dahliae. Initially, LAMP primers and CRISPR RNA (crRNA) were designed based on a specific DNA sequence of V. dahliae, which was validated using several closely related Verticillium spp. The lower detection limit of the LAMP-CRISPR/Cas12a combined with the fluorescent visualization detection system is approximately ~10 fg/μL genomic DNA per reaction. When combined with crude DNA-extraction methods, it is possible to detect as few as two microsclerotia per gram of soil, with the total detection process taking less than 90 min. Furthermore, to improve the method\'s user and field friendliness, the field detection results were visualized using lateral flow strips (LFS). The LAMP-CRISPR/Cas12a-LFS system has a lower detection limit of ~1 fg/μL genomic DNA of the V. dahliae, and when combined with the field crude DNA-extraction method, it can detect as few as six microsclerotia per gram of soil, with the total detection process taking less than 2 h. In summary, this study expands the application of LAMP-CRISPR/Cas12a nucleic acid detection in V. dahliae and will contribute to the development of field-deployable diagnostic productions.