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Verticillium wilt (VW), a fungal disease caused by Verticillium dahliae , currently devastates cotton fiber yield and quality seriously, yet few resistance germplasm resources have been discovered in Gossypium hirsutum . The cotton variety Nongda601 with suitable VW resistance and high yield was developed in our lab, which supplied elite resources for discovering resistant genes. Early nodulin-like protein (ENODL) is mainly related to nodule formation, and its role in regulating defense response has been seldom studied. Here, 41 conserved ENODLs in G. hirsutum were identified and characterized, which could divide into four subgroups. We found that GhENODL6 was upregulated under V. dahliae stress and hormonal signal and displayed higher transcript levels in resistant cottons than the susceptible. The GhENODL6 was proved to positively regulate VW resistance via overexpression and gene silencing experiments. Overexpression of GhENODL6 significantly enhanced the expressions of salicylic acid (SA) hormone-related transcription factors and pathogenicity-related (PR) protein genes, as well as hydrogen peroxide (H 2 O 2 ) and SA contents, resulting in improved VW resistance in transgenic Arabidopsis . Correspondingly, in the GhENODL6 silenced cotton, the expression levels of both phenylalanine ammonia lyase (PAL) and 4-coumarate-CoA ligase (4CL) genes significantly decreased, leading to the reduced SA content mediating by the phenylalanine ammonia lyase pathway. Taken together, GhENODL6 played a crucial role in VW resistance by inducing SA signaling pathway and regulating the production of reactive oxygen species (ROS). These findings broaden our understanding of the biological roles of GhENODL and the molecular mechanisms underlying cotton disease resistance.

Verticillium wilt (VW) is a destructive disease that results in great losses in cotton yield and quality. Identifying genetic variation that enhances crop disease resistance is a primary objective in plant breeding. Here we reported a GWAS of cotton VW resistance in a natural-variation population, challenged by different pathogenicity stains and different environments, and found 382 SNPs significantly associated with VW resistance. The associated signal repeatedly peaked in chromosome Dt11 (68 798 494-69 212 808) containing 13 core elite alleles undescribed previously. The core SNPs can make the disease reaction type from susceptible to tolerant or resistant in accessions with alternate genotype compared to reference genotype. Of the genes associated with the Dt11 signal, 25 genes differentially expressed upon Verticillium dahliae stress, with 21 genes verified in VW resistance via gene knockdown and/or overexpression experiments. We firstly discovered that a gene cluster of L-type lectin-domain containing receptor kinase (GhLecRKs-V.9) played an important role in VW resistance. These results proved that the associated Dt11 region was a major genetic locus responsible for VW resistance. The frequency of the core elite alleles (FEA) in modern varieties was significantly higher than the early/middle varieties (12.55% vs 4.29%), indicating that the FEA increased during artificial selection breeding. The current developmental resistant cultivars, JND23 and JND24, had fixed these core elite alleles during breeding without yield penalty. These findings unprecedentedly provided genomic variations and promising alleles for promoting cotton VW resistance improvement., (© 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Lipids are major and essential constituents of plant cells and provide energy for various metabolic processes. However, the function of the lipid signal in defence against Verticillium dahliae, a hemibiotrophic pathogen, remains unknown. Here, we characterized 19 conserved stearoyl-ACP desaturase family proteins from upland cotton (Gossypium hirsutum). We further confirmed that GhSSI2 isoforms, including GhSSI2-A, GhSSI2-B, and GhSSI2-C located on chromosomes A10, D10, and A12, respectively, played a dominant role to the cotton 18:1 (oleic acid) pool. Suppressing the expression of GhSSI2s reduced the 18:1 level, which autoactivated the hypersensitive response (HR) and enhanced cotton Verticillium wilt and Fusarium wilt resistance. We found that low 18:1 levels induced phenylalanine ammonia-lyase-mediated salicylic acid (SA) accumulation and activated a SA-independent defence response in GhSSI2s-silenced cotton, whereas suppressing expression of GhSSI2s affected PDF1.2-dependent jasmonic acid (JA) perception but not the biosynthesis and signalling cascade of JA. Further investigation showed that structurally divergent resistance-related genes and nitric oxide (NO) signal were activated in GhSSI2s-silenced cotton. Taken together, these results indicate that SA-independent defence response, multiple resistance-related proteins, and elevated NO level play an important role in GhSSI2s-regulated Verticillium wilt resistance. These findings broaden our knowledge regarding the lipid signal in disease resistance and provide novel insights into the molecular mechanism of cotton fungal disease resistance., (© 2021 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Background: Verticillium wilt is a widespread and destructive disease, which causes serious loss of cotton yield and quality. Long non-coding RNA (lncRNA) is involved in many biological processes, such as plant disease resistance response, through a variety of regulatory mechanisms, but their possible roles in cotton against Verticillium dahliae infection remain largely unclear., Results: Here, we measured the transcriptome of resistant G. hirsutum following infection by V. dahliae and 4277 differentially expressed lncRNAs (delncRNAs) were identified. Localization and abundance analysis revealed that delncRNAs were biased distribution on chromosomes. We explored the dynamic characteristics of disease resistance related lncRNAs in chromosome distribution, induced expression profiles, biological function, and these lncRNAs were divided into three categories according to their induced expression profiles. For the delncRNAs, 687 cis-acting pairs and 14,600 trans-acting pairs of lncRNA-mRNA were identified, which indicated that trans-acting was the main way of Verticillium wilt resistance-associated lncRNAs regulating target mRNAs in cotton. Analyzing the regulation pattern of delncRNAs revealed that cis-acting and trans-acting lncRNAs had different ways to influence target genes. Gene Ontology (GO) enrichment analysis revealed that the regulatory function of delncRNAs participated significantly in stimulus response process, kinase activity and plasma membrane components. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that delncRNAs participated in some important disease resistance pathways, such as plant-pathogen interaction, alpha-linolenic acid metabolism and plant hormone signal transduction. Additionally, 21 delncRNAs and 10 target genes were identified as being involved in alpha-linolenic acid metabolism associated with the biosynthesis of jasmonic acid (JA). Subsequently, we found that GhlncLOX3 might regulate resistance to V. dahliae through modulating the expression of GhLOX3 implicated in JA biosynthesis. Further functional analysis showed that GhlncLOX3-silenced seedlings displayed a reduced resistance to V. dahliae, with down-regulated expression of GhLOX3 and decreased content of JA., Conclusion: This study shows the dynamic characteristics of delncRNAs in multiaspect, and suggests that GhlncLOX3-GhLOX3-JA network participates in response to V. dahliae invasion. Our results provide novel insights for genetic improvement of Verticillium wilt resistance in cotton using lncRNAs.

Verticillium dahliae seriously affects the yield of cotton. Here, V. dahliae infection induced the significant reduction of protein concentration in cotton xylem sap (CXS), suggesting that the protein composition have changed. Thus, the proteomics in CXS from resistant Gossypium hirsutum cv. ND601 and susceptible CCRI8 infected by V. dahliae were analyzed using the label-free method. A total of 3047 proteins were identified across all four CXS sample groups. 1717 and 1476 proteins were differentially accumulated in ND601 and CCRI8 after infection with V. dahliae, respectively. The majority of up-accumulated and induced proteins belongs to pathogenesis-related proteins and associates with cell wall (CWRPs). Down-accumulated and disappeared proteins were principally related to plant growth and development. Differentially accumulated CWRPs from ND601 and CCRI8 in type and quantity were not entirely consistent with each other, leading to different cell wall dynamics and strength, which were partly proved by the measurement of stem mechanical strength. Most of proteins related to growth and development were down-accumulated in ND601 compared to CCRI8, suggesting that the resistant variety may transfer more energy for defense responses or reduce nutrient acquisition of V. dahliae for colonization more effectively than the susceptible. SIGNIFICANCE: Verticillium wilt, mainly caused by V. dahliae, is one of the most destructive diseases in cotton. V. dahliae usually penetrates the root epidermis, reaches vascular tissues, and eventually extends to the above-ground tissues along the xylem vessels. Obviously, xylem is an important battlefront for plant defense to V. dahliae. Therefore, we analyzed the proteome profiles of xylem saps from resistant and susceptible cotton cultivars. Our findings provide valuable insights into the molecular mechanism underlying the interaction between V. dahliae and cotton., (Copyright © 2019. Published by Elsevier B.V.)

Verticillium dahliae is a phytopathogenic fungal pathogen that causes vascular wilt diseases responsible for considerable decreases in cotton yields. The lignification of cell wall appositions is a conserved basal defence mechanism in the plant innate immune response. However, the function of laccase in defence-induced lignification has not been described. Screening of an SSH library of a resistant cotton cultivar, Jimian20, inoculated with V. dahliae revealed a laccase gene that was strongly induced by the pathogen. This gene was phylogenetically related to AtLAC15 and contained domains conserved by laccases; therefore, we named it GhLAC15. Quantitative reverse transcription-polymerase chain reaction indicated that GhLAC15 maintained higher expression levels in tolerant than in susceptible cultivars. Overexpression of GhLAC15 enhanced cell wall lignification, resulting in increased total lignin, G monolignol and G/S ratio, which significantly improved the Verticillium wilt resistance of transgenic Arabidopsis. In addition, the levels of arabinose and xylose were higher in transgenic plants than in wild-type plants, which resulted in transgenic Arabidopsis plants being less easily hydrolysed. Furthermore, suppression of the transcriptional level of GhLAC15 resulted in an increase in susceptibility in cotton. The content of monolignol and the G/S ratio were lower in silenced cotton plants, which led to resistant cotton cv. Jimian20 becoming susceptible. These results demonstrate that GhLAC15 enhances Verticillium wilt resistance via an increase in defence-induced lignification and arabinose and xylose accumulation in the cell wall of Gossypium hirsutum. This study broadens our knowledge of defence-induced lignification and cell wall modifications as defence mechanisms against V. dahliae., (© 2018 The Authors. Molecular Plant Pathology published by BSPP and John Wiley & Sons Ltd.)

Verticillium wilt, caused by Verticillium dahliae Kleb., is a serious threat to cotton (Gossypium spp.) crop production. To enhance our understanding of the plant's complex defensive mechanism, we examined colonization patterns and interactions between V. dahliae and two cotton species, the resistant G. barbadense and the susceptible G. hirsutum. Microscopic examinations and grafting experiments showed that the progression of infection was restricted within G. barbadense. At all pre- and postinoculation sampling times, levels of salicylic acid (SA) were also higher in that species than in G. hirsutum. Comparative RNA-Seq analyses indicated that infection induced dramatic changes in the expression of thousands of genes in G. hirsutum, whereas those changes were fewer and weaker in G. barbadense. Investigations of the morphological and biochemical nature of cell-wall barriers demonstrated that depositions of lignin, phenolic compounds, and callose were significantly higher in G. barbadense. To determine the contribution of a known resistance gene to these processes, we silenced GbEDS1 and found that the transformed plants had decreased SA production, which led to the upregulation of PLASMODESMATA-LOCATED PROTEIN (PDLP) 1 and PDLP6. This was followed by a decline in callose deposition in the plasmodesmata, which then led to increased pathogen susceptibility. This comparison between resistant and susceptible species indicated that both physical and chemical mechanisms play important roles in the defenses of cotton against V. dahliae.

Most of the disease resistance genes already characterized in plants encode nucleotide-binding site-leucine rich repeat (NBS-LRR) proteins that have key roles in resistance to Verticillium dahliae. Using a cDNA library and RACE protocols, we cloned a coiled-coil (CC)-NBS-LRR-type gene, GbRVd, from a resistant tetraploid cotton species, Gossypium barbadense (RVd=Resistance to V. dahliae). We also applied RT-qPCR and VIGS technologies to analyze how expression of GbRVd was induced upon attack by V. dahliae. Its 2862-bp ORF encodes a predicted protein containing 953 amino acid residues, with a predicted molecular weight of 110.17kDa and an isoelectric point of 5.87. GbRVd has three domains - CC, NBS, and LRR - and is most closely related to Gossypium raimondii RVd (88% amino acid identity). Profiling demonstrated that GbRVd is constitutively expressed in all tested tissues, and transcript levels are especially high in the leaves. In plants inoculated with V. dahliae, GbRVd was significantly up-regulated when compared with the control, with expression peaking at 48h post-inoculation. Silencing of GbRVd in cotton through VIGS dramatically down-regulated SA, NO, and H2O2 production, resulting in greater susceptibility to V. dahliae. Taken together, these results suggest that GbRVd has an important role in protecting G. barbadense against infection by V. dahliae., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Verticillium dahliae is a destructive, soil-borne fungal pathogen that causes vascular wilt disease in many economically important crops worldwide. A polyamine oxidase (PAO) gene was identified and cloned by screening suppression subtractive hybridisation and cDNA libraries of cotton genotypes tolerant to Verticillium wilt and was induced early and strongly by inoculation with V. dahliae and application of plant hormone. Recombinant cotton polyamine oxidase (GhPAO) was found to catalyse the conversion of spermine (Spm) to spermidine (Spd) in vitro. Constitutive expression of GhPAO in Arabidopsis thaliana produced improved resistance to V. dahliae and maintained putrescine, Spd and Spm at high levels. Hydrogen peroxide (H2 O2 ), salicylic acid and camalexin (a phytoalexin) levels were distinctly increased in GhPAO-overexpressing Arabidopsis plants during V. dahliae infection when compared with wild-type plants, and Spm and camalexin efficiently inhibited growth of V. dahliae in vitro. Spermine promoted the accumulation of camalexin by inducing the expression of mitogen-activated protein kinases and cytochrome P450 proteins in Arabidopsis and cotton plants. The three polyamines all showed higher accumulation in tolerant cotton cultivars than in susceptible cotton cultivars after inoculation with V. dahliae. GhPAO silencing in cotton significantly reduced the Spd level and increased the Spm level, leading to enhanced susceptibility to infection by V. dahliae, and the levels of H2 O2 and camalexin were distinctly lower in GhPAO-silenced cotton plants after V. dahliae infection. Together, these results suggest that GhPAO contributes to resistance of the plant against V. dahliae through the mediation of Spm and camalexin signalling., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)

Key Message: Overexpression of a cotton defense-related gene GbSTK in Arabidopsis resulted in enhancing pathogen infection and oxidative stress by activating multiple defense-signaling pathways., Abstract: Serine/threonine protein kinase (STK) plays an important role in the plant stress-signaling transduction pathway via phosphorylation. Most studies about STK genes have been conducted with model species. However, their molecular and biochemical characterizations have not been thoroughly investigated in cotton. Here, we focused on one such member, GbSTK. RT-PCR indicated that it is induced not only by Verticillium dahliae Kleb., but also by signaling molecules. Subcellular localization showed that GbSTK is present in the cell membrane, cytoplasm, and nucleus. Overexpression of GbSTK in Arabidopsis resulted into the enhanced resistance to V. dahliae. Moreover, Overexpression of GbSTK elevated the expression of PR4, PR5, and EREBP, conferring on transgenic plants enhanced reactive oxygen species scavenging capacity and oxidative stress tolerance. Our results suggest that GbSTK is active in multiple defense-signaling pathways, including those involved in responses to pathogen infection and oxidative stress.

Verticillium wilt causes enormous loss to yield or quality in many crops. In an effort to help controlling this disease through genetic engineering, we first cloned and characterized a Verticillium wilt resistance gene (GbVe) from cotton (Gossypium barbadense) and analyzed its function in Arabidopsis thaliana. Its nucleotide sequence is 3,819 bp long, with an open reading frame of 3,387 bp, and encoding an 1,128-aa protein precursor. Sequence analysis shows that GbVe produces a leucine-rich repeat receptor-like protein. It shares identities of 55.9% and 57.4% with tomato Ve1 and Ve2, respectively. Quantitative real-time PCR indicated that the Ve gene expression pattern was different between the resistant and susceptible cultivars. In the resistant Pima90-53, GbVe was quickly induced and reached to a peak at 2 h after inoculation, two-fold higher than that of control. We localized the GbVe-GFP fusion protein to the cytomembrane in onion epidermal cells. By inserting GbVe into Arabidopsis via Agrobacterium-mediated transformation, T(3) transgenic lines were obtained. Compared with the wild-type control, GbVe-overexpressing plants had greater levels of resistance to V. dahliae. This suggests that GbVe is a useful gene for improving the plant resistance against fungal diseases.