GhLYK5-mediated GhNIT4B activates asparagine and ROS accumulation enhancing cotton Verticillium wilt resistance

Abstract

Verticillium wilt, caused by the soil-borne fungus Verticillium dahliae, is a destructive disease that significantly impairs cotton yield and fiber quality. Lysin motif (LysM) receptor-like kinases (LYKs), a subclass of plant pattern recognition receptors, are crucial for perceiving pathogen-derived molecules and activating immune responses. Here, we show that the transcription of GhLYK5, a gene encoding LYK protein in cotton, was rapidly upregulated following V. dahliae infection. Virus-induced gene silencing (VIGS) of GhLYK5 heightened cotton susceptibility to V. dahliae, whereas heterologous overexpression in Arabidopsis thaliana conferred enhanced resistance. RNA-seq analysis revealed that GhLYK5 silencing down-regulated defense-related genes and altered the expression of secondary metabolic pathways. Using yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC), luciferase complementation imaging (LCI), and co-inmunoprecipitation (Co-IP) assays, we demonstrated that GhLYK5 physically interacted with the nitrilase GhNIT4B, with both proteins localizing to the plasma membrane. Silencing of GhNIT4B reduced asparagine accumulation, decreased ROS (H₂O₂, NO) levels, suppressed defense gene expression, and increased plant susceptibility to V. dahliae, indicating that GhNIT4B acted as a positive immune regulator. In addition, exogenous asparagine significantly inhibited the infection of V. dahliae by activating the expression of defense genes (GhBAK1, GhNPR1, GhPR1, GhLac1). Our findings identified the GhLYK5-GhNIT4B module as a key signaling node in cotton immune responses and provide genetic targets for improving Verticillium wilt resistance in upland cotton.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13036-026-00628-x.

Introduction

Cotton is a cornerstone of the global agricultural and textile industries, providing a natural and sustainable source of fiber [1, 2]. Verticillium wilt (VW), a serious soil-borne vascular disease caused by the fungus Verticillium dahliae, represents one of the most destructive diseases affecting cotton production [3]. This pathogen can infect cotton plants throughout their entire growth cycle, severely constraining plant development and reducing yield [4]. In China, more than 40% of the cotton-growing area is affected by V. dahliae, leading to annual economic losses estimated at 1.5-2 billion RMB [5, 6]. In the United States alone, nearly 480 million bales were lost between 1990 and 2014 [7]. V. dahliae, widely distributed, which can infect more than 200 dicotyledonous plants such as Solanaceae, Leguminosae, Cruciferae, Malvaceae, Chenopodiaceae and cause Verticillium wilt [8–10], can also infect a few gramineous plants such as barley and wheat, and colonize in monocotyledonous plants in the form of endophytic fungi a broad host range [11]. Capable of surviving in soil for more than ten years in the absence of a host, the microsclerotia of V. dahliae render the pathogen extremely difficult to eradicate [12]. Furthermore, no effective chemical treatments are currently available to suppress the disease during cotton cultivation [13]. Therefore, elucidating the immune defense mechanisms of cotton and developing disease-resistant varieties through genetic engineering represent crucial strategies for sustainable management of cotton diseases.

As plants develop in environments rich with diverse microorganisms, they are frequently subjected to pathogenic invasions. In response, plants have evolved sophisticated immune systems to defend against pathogens. Pathogen-Associated Molecular Patterns (PAMPs) are conserved molecular markers of pathogens, which are recognized by plant pattern-recognition receptors Patterns pattern-triggered immunity (PRRs) and trigger PTI (Patterns pattern-triggered immunity) as the first line of defense. When pathogens inhibit PTI through effectors, plants recognize these effectors through nucleotide-binding leucine-rich repeat receptors (NLRs) and activate effector-triggered susceptibility (ETI), providing stronger and specific resistance. PTI and ETI together constitute the precise immune system of plants and evolve in the continuous “arms race ” [14, 15]. Most PRRs are either receptor-like kinases (RLKs) or receptor-like proteins (RLPs), typically comprising an extracellular ligand-binding domain, a transmembrane domain, and an intracellular kinase domain [16]. The extracellular domain specifically perceives PAMPs—such as bacterial lipopolysaccharides, flagellin, fungal chitin—transduces the signal via the transmembrane segment to activate intracellular kinase domains [17, 18]. Plant cell surface receptors include LRR (Leucine-Rich Repeat)-, G-lectin, wall-associated kinase (WAK), unknown functional domain 26 (Duf26) -, L-lectin, lysine motif (LysM) -, and RLKs and RLPs containing malik proteins [19].

As a PAMP receptor, LysM domain protein is a defense protein widely found in animals, plants and microorganisms and plays an important role in plant immunity [20]. It can recognize bacterial oligosaccharides, peptidoglycan (PGN) and fungal chitin, and respond by promoting plant defense [21]. The first identified LysM-domain PRRs in Arabidopsis thaliana, AtLYK1— also known as chitin elicitor receptor kinase 1 (CERK1)—participate in chitin-triggered immune response [22]. However, chitin perception in plants involves multiple receptors; mutations in AtLYK4 and AtLYK5 have also been shown to markedly impair chitin-induced immunity [23]. Notably, AtLYK5 displays a higher binding affinity for chitin than AtCERK1 and is capable of recognizing long-chain chitin oligosaccharides. It forms a complex with AtCERK1, leading to activation of the kinase domain and initiation of downstream immune signaling [24, 25].

Metabolic reprogramming is a cornerstone of plant immunity. A key aspect of this reprogramming involves the metabolism of cyanogenic compounds and associated nitrile hydrolysis. This pathway contributes to defense through cyanoglycoside turnover, hydrogen cyanide (HCN) homeostasis, and potential signaling functions [26]. Nitrile hydrolases (NITs) are widespread enzymes in plants that catalyze the hydrolysis of organic nitriles into carboxylic acids and ammonia [27]. In Arabidopsis, there are four nitrile hydrolase-encoding genes NIT1, NIT2, NIT3, and NIT4 [28]. Among these, NIT4 exhibits high substrate specificity for β-cyano-L-alanine and is mainly involved in cyanide detoxification [29]. In contrast, NIT1, NIT2, and NIT3 participate not only in detoxification processes but also in the biosynthesis of indole-3-acetic acid (IAA) from indole-3-acetonitrile (IAN), and positively regulate the disease resistance of Arabidopsis to Pst DC3000 [30]. The expression of NIT1-NIT3 is subject to developmental regulation and environmental influences [31]. Notably, NIT2 is specifically upregulated upon infection with Pseudomonas syringae, whereas NIT1, NIT3, and NIT4 show no induction in response to this pathogen [32].

Our previous study integrated transcriptomic and metabolic analyses of V. dahliae infected plants. Using BlastP, we identified the homolog of a differentially expressed gene (DEG) in Gossypium hirsutum, which is annotated in the NCBI database as lysin motif receptor-like kinase 5 (GhLYK5, Gene ID: LOC107913555), as a pathogenesis-induced candidate gene [33]. Here, we demonstrate that GhLYK5 is up-regulated in upland cotton upon V. dahliae infection. Subcellular localization confirmed that GhLYK5 was localized in the cell membrane, and further research showed that it positively regulated resistance against V. dahliae. To elucidate the specific signaling pathways governed by GhLYK5, we performed a new, targeted transcriptomic analysis in the current study. This analysis compared gene expression in GhLYK5-silenced plants versus control plants at 0.5 h post-inoculation (hpi)—the peak of GhLYK5 induction. This new transcriptome revealed that GhLYK5 contributes to disease resistance by modulating genes involved in secondary metabolite biosynthesis and plant immune defense pathways Additionally, we identified GhNIT4B as an interacting protein of GhLYK5, which also enhanced resistance to V. dahliae by modulating asparagine content and reactive oxygen species (ROS) levels. Moreover, exogenous application of asparagine can positively regulate plant resistance to VW by activating the expression of defense genes. This study provided valuable genetic targets for breeding improved resistance to VW in cotton.

Materials and methods

Fungal strain, plant materials and growth conditions

V. dahliae strain V991 was cultured as previously described, a highly virulent, defoliating pathogenic strain [34]. Subsequently, colonies of V991 were transferred into Czapek liquid medium for further cultivation [35].

Seeds of upland cotton (G. hirsutum) cv. Coker 312 were germinated in distilled water and sown in a 1:1 (v/v) mixture of nutrient soil and vermiculite. Plants were grown in a controlled-environment chamber maintained at 28 °C under a 16 h light/8 h dark photoperiod. Tobacco (Nicotiana benthamiana) seeds were cultivated under identical growth conditions.

Arabidopsis seeds were surface-sterilized and sown on half-strength Murashige and Skoog (1/2 MS) medium. After two weeks, seedlings were transplanted into nutrient soil and grown in a chamber set at 22/20°C (day/night), 60% relative humidity, and a 16/8 h (day/night) photoperiod.

Phylogenetic analysis and cis-element identification in the promoter region

Protein sequences with high homology to GhLYK5 from various species—including Arabidopsis thaliana, Morus notabilis, Triticum aestivum, Oryza sativa, Hordeum vulgare, Hordeum vulgare, Pisum sativum, and Vitis vinifera—were retrieved from the NCBI database (https://www.ncbi.nlm.nih.gov/). Multiple sequence alignment was performed using the ClustalW algorithm implemented in MAGA11. A phylogenetic tree was constructed using the neighbor-joining method with MAGA11 software. The three-dimensional structure of GhLYK5 was predicted using the SWISS-MODEL server (https://swissmodel.expasy.org/).

The promoter region of GhLYK5, defined as 2 kb genomic DNA fragment upstream of the transcription start site, was obtained from the CottonFGD database (https://cottonfgd.org/). Putative cis-acting regulatory elements within the promoter were identified using the PlantCARE database (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

RNA extraction, RT-qPCR and RNA-seq data analysis

Total RNA was extracted from cotton leaf samples 0.5 h post-inoculation (hpi) using the RNA Easy Fast Plant Tissue RNA Rapid Extraction Kit (Tiangen, Beijing, China) following the manufacturer’s instructions. Three biological replicates, each with three technical replicates, were included. RNA purity and integrity were assessed by electrophoresis on 1% (w/v) agarose gels.

Complementary DNA (cDNA) was synthesized from mRNA using HiScript III RT SuperMix for qPCR (+ gDNA wiper) (Vazyme, China). Quantitative real-time PCR (RT-qPCR) was performed with Taq Pro Universal SYBR qPCR Master Mix (Vazyme, China) on an ABI7500 Fast system. The cotton gene GhUBQ7 (LOC107925174) was used as an internal control. Gene expression levels were calculated using the 2^−ΔΔCt method [36].

RNA sequencing was conducted by Novogene Bioinformatics Institute (Beijing, China). Differentially expressed genes (DEGs) were identified using DESeq2 software V1.22.2 (http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html) with thresholds of |log2 fold change| ≥ 1 and adjusted p-value < 0.05. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on the DEGs. The DEGs are listed in Table S2. The original transcriptomic sequencing data in fastq format has been uploaded to NCBI SRA database (Accession number: PRJNA1390421 (https://www.ncbi.nlm.nih.gov/sra/PRJNA1390421).

Virus-induced gene silencing (VIGS)

An approximately 300 bp fragment from the coding sequence (CDS) of GhLYK5 and GhNIT4B was amplified and cloned into the pTRV2 vector using EcoRI and BamHI restriction sites (Figure S1A, B). The constructed vectors were introduced into Agrobacterium tumefaciens and infiltrated into cotyledons of cotton seedlings as previously described [37]. Approximately 10 days post-infiltration, once the positive control plants (TRV2:CLA1) exhibited an albino phenotype in the second true leaf, RNA was extracted from both silenced and control plants. cDNA was synthesized, and silencing efficiency was evaluated via RT-qPCR.

Arabidopsis transformation

The coding sequence of GhLYK5 was amplified and inserted into the recombinant vector pCAMBIA2300 under the control of the CaMV 35 S promoter (Figure S1C). The constructed overexpression vector was introduced into Arabidopsis ecotype Columbia (Col-0) via the floral dip method [38]. Transgenic T1 seedlings were selected on 1/2 MS medium supplemented with kanamycin. Genomic DNA was extracted from surviving T1 plants and subjected to PCR analysis to confirm positive transformants. Genetic segregation analysis was performed on the T2 generation to identify lines with single-copy insertions. Total RNA was extracted from T2 transgenic plants, and GhLYK5 expression levels were quantified using RT-qPCR. Transgenic lines harboring single-copy insertions were advanced to the T3 generation to obtain homozygous seeds.

Plant disease resistance assay

Plant inoculation was performed using the root-dip method. Roots of 4-week-old transgenic Arabidopsis, cotton, and empty vector control plants were immersed in a V. dahliae spore suspension at a concentration of 10⁷ spores/mL for 5 min. Control plants were dipped in ddH₂O. Disease symptoms were assessed 15 day post-inoculation (dpi). The disease index (DI) was evaluated based on the following grading scale, Grade 0: healthy plants; Grade 1: 0–25% of leaves showing vein yellowing; Grade 2: 25–50% of leaves exhibiting chlorosis or wilting; Grade 3: 50–75% of leaves showing chlorosis or wilting; Grade 4: 75–100% of leaves affected. Disease index (DI) = ∑ (disease level × number of diseased plants)/(number of investigated plants × 4) × 100% [39].

Fungal biomass was quantified by extracting genomic DNA from inoculated Arabidopsis and cotton plants. The ITS1 and ITS2 regions of the V. dahliae ribosomal RNA gene (accession Z29511) were amplified using specific primers Vd-ITS-F/R, with AtUBQ10 serving as the internal reference gene (Supplementary Table S1).

Analysis of conserved protein motifs and gene structure

The physicochemical properties of the GhLYK5 protein were predicted using the ExPASy ProtParam online tool (https://web.expasy.org/protparam/). A structural model of the GhLYK5 protein was built employing AlphaFold2 [40]. Functionally conserved domains were identified via the Conserved Domain Database (CDD) on the NCBI website. Multiple protein sequence alignment was carried out using the ClustalW algorithm. The secondary structure of GhLYK5 was predicted using the AlphaFold2 server and the SOPMA online tool (https://npsa-prabi.ibcp.fr/NPSA/npsa_sopma.html). Results from these analyses were integrated and visualized using Espript 3.0 to generate a comparative sequence diagram. Transmembrane domains were predicted with the TMHMM Server v.2.0 (https://services.healthtech.dtu.dk/services/TMHMM-2.0/).

Subcellular localization

The CDS sequences of GhLYK5 and GhNIT4B were amplified using specific primers (pYBA1132-GhLYK5/GhNIT4B -F/R; see Supplementary Table S1) and cloned into the BamHI and EcoRI sites of the pYBA1132 expression vector, which contained a green fluorescent protein (GFP) tag (Figure S1D, E). The recombinant plasmids were introduced into A. tumefaciens strain GV3101 (pSoup-19) and transiently expressed in 4 ~ 5 true-leaf stage tobacco (N. benthamiana) plants. The lower epidermal layers of tobacco leaves were co-infiltrated with combinations of pYBA1132-GhLYK5/GhNIT4B and the plasma membrane marker pYBA1132-PIP2A, or the empty vector control pYBA1132-EV with pYBA1132-PIP2A. After 48 hpi, GFP fluorescence was observed using an LSM980 confocal laser scanning microscope (Zeiss, Jena, Germany).

Yeast two-hybrid (Y2H) assay

The CDS sequence of GhLYK5 was amplified using specific primers GhLYK5-BD-F/R (Supplementary Table S1) and cloned into the pGBKT7 vector via the EcoRI and BamHI restriction sites to generate the pGBKT7-GhLYK5 construct (Figure S1F). Self-activation of the bait protein was tested on TDO medium (SD/-Trp/-Leu/-His). A V. dahliae-induced cotton cDNA library was screened in the Y2H Gold yeast strain harboring pGBKT7-GhLYK5 to identify interacting proteins. Yeast cells were co-transformed with the following plasmid combinations: pGADT7-GhNIT4B + pGBKT7-GhLYK5 (test group), pGADT7-ADT + pGBKT7-P53 (positive control), pGADT7-ADT + pGBKT7-Lam (negative control). Transformants were selected on SD/-Trp/-Leu plates and incubated at 30 ℃ for 4–5 days. Monoclonal cultures were serially diluted (10X, 100X, 1000X dilutions) and spotted onto SD/-Trp/-Leu/-His/-Ade medium supplemented with X-α-gal to assess protein-protein interactions based on colony growth of color development.

Bimolecular fluorescence complementation (BiFC)

The CDS regions of GhLYK5 and GhNIT4B were amplified using primer pairs BifC-GhLYK5-F/R and BifC-GhNIT4B-F/R (Supplementary Table S1), respectively. These fragments were inserted into the BiFC vectors 35 S-SPYNE and 35 S-SPYCE using BamHI and SalI sites, yielding the following constructs: GhLYK5-cYFP (C-terminal YFP fusion), GhLYK5-nYFP (N-terminal YFP fusion), GhNIT4B-cYFP, and GhNIT4B-nYFP (Figure S2A-D). Constructs were introduced into A. tumefaciens GV3101 (pSoup-19) and transiently co-expressed in 4 ~ 5 true-leaf stage tobacco plants. After 48 hpi, YFP fluorescence was visualized using an LSM980 confocal laser scanning microscope (Zeiss, Germany) with excitation at 489 nm and emission detection at 510 nm.

Luciferase complementation assay (LCI)

The CDS sequences of GhLYK5 and GhNIT4B were amplified using primers LUC-GhLYK5-F/R and LUC-GhNIT4B-F/R (Supplementary Table S1) and inserted into pCAMBIA1300-nLUC and pCAMBIA1300-cLUC vectors via KpnI and SalI restriction sites. The resulting constructs included: GhLYK5-nLUC (Figure S2E), cLUC-GhLYK5 (Figure S2F), GhNIT4B-nLUC (Figure S2G), cLUC-GhNIT4B (Figure S2H). A. tumefaciens strains carrying these constructs were mixed in a 1:1 ratio and co-infiltrated into the abaxial side of 3-week-old tobacco leaves. The following combinations were tested: cLUC-GhLYK5 + GhNIT4B-nLUC, GhLYK5-nLUC + cLUC, nLUC + cLUC-GhNIT4B, nLUC + cLUC (negative control). Each combination was infiltrated at distinct sites on the same leaf. After infiltration, plants were kept in the dark for 24 h and then moved to normal growth conditions. A solution of 1 mM potassium salt beetle fluorescein was applied uniformly to the infiltrated areas, and luminescence signals were captured using a Night SHADE LB 985 multimodal imaging system (Berthold, Germany).

Co-immunoprecipitation (Co-IP) assays

The CDS of GhLYK5 was cloned into the pCAMBIA1300-FLAG vector, and GhNIT4B was inserted into the pCAMBIA1302-GFP plant expression vector (Figure S2I-J). These recombinant plasmids were introduced into A. tumefaciens GV3101 and co-infiltrated into the leaves of 4-week-old tobacco plants. Total protein was extracted from infiltrated leaf tissues 3 dpi. Co-IP was performed using anti-Flag (TT0053, Abmart) and anti-GFP (M20004, Abmart) antibodies to detect protein interactions [41].

Asparagine extraction and analysis by high-performance liquid chromatography (HPLC)

Asparagine (purity ≥ 97%) was obtained from Macklin (Shanghai, China). Leaves from TRV2:00 (control) and TRV2:GhNIT4B cotton plants were harvested 12 hpi, freeze-dried using LGJ-12 A lyophilizer (Beijing Sihuan Qihang Technology Co., Ltd, Beijing, China), and ground to a fine powder. For extraction, 50 mg of powdered tissue was mixed with 1mL of 0.1 mol/L HCl in a 5 mL centrifuge tube. The mixture was sonicated at 25 °C for 30 min and centrifuged at 12,000 ×g for 5 min. The supernatant was collected and stored at − 80 °C until analysis. High-performance liquid chromatography (HPLC) was performed according to previously established methods [42–44].

Measurement of ROS metabolism-related parameters

To visualize ROS accumulation, cotton leaves were collected 12 hpi with V991 and stained with 2’,7’-dichlorodihydrofluorescein diacetate (H₂DCFDA) or 3,3’-diaminobenzidine (DAB; 1 mg/mL, pH 7.5) for 8 h, following the previously described protocol [45].

Hydrogen peroxide (H₂O₂) and nitric oxide (NO) levels were quantified using commercial assay kits (Solarbio Science & Technology Co., Ltd., Beijing, China) according to the manufacturer’s instructions.

Plant treatment with asparagine

Two-week-old cotton leaves were sprayed with 1 mM asparagine, and the control plants were sprayed with sterile water. After three days of spraying, the cotton was inoculated with the root soaking method. The leaves at 0 hpi and 12 hpi were immediately frozen in liquid nitrogen and stored at − 80 ° C for RNA isolation.

Statistical analysis

Data are presented as mean values. Statistical significance was evaluated using GraphPad Prism 8 (GraphPad, MA, USA). For all assays, means and standard deviation (SD) values were calculated, and significances were determined by Student’s t test or ANOVA followed by Tukey’s or Dunnett’s multiple comparison test.

Results

Response to V. dahliae and identification of GhLYK5

Chitin, a key component of fungal cell wall, is recognized to enhance immunity against fungal pathogens [46]. In Arabidopsis, AtLYK5 served as the primary chitin receptor and triggered PTI defense response [24]. Phylogenetic analysis revealed that GhLYK5 from G. hirsutum was the closest ortholog of AtLYK5 (Fig. 1A). Temporal expression pattern showed that GhLYK5 expression was rapidly induced upon V. dahliae infection, peaking at 0.5 hpi (Fig. 1B), indicating its role in early pathogen perception. The sequence of 2000 bp upstream of the gene transcription start site was analyzed by PlantCARE online website for cis-acting element analysis. The promoter region of GhLYK5 contained seven key cis-acting elements related to hormone signaling, defense, and stress response, including two methyl jasmonate (MeJA)-responsive elements (Fig. 1C), suggesting its involvement in immune-related hormonal pathways. Protein domain analysis predicted a conserved LysM domain (200–243 aa) and a C-terminal kinase domain (Figure S3). The three-dimensional structure of GhLYK5 further supported its identity as a transmembrane protein (Fig. 1D). Subcellular localization confirmed that GhLYK5 was localized in the plasma membrane (Fig. 1E). Collectively, these findings implied that GhLYK5 plays a critical role in cotton defense against V. dahliae.

Fig. 1.

Response to V. dahliae and identification of GhLYK5. (A) Phylogenetic tree of LYK proteins from Gossypium hirsutum (Gh), Solanum lycopersicum (Sl), Arabidopsis thaliana (At), Glycine max (Gm), Oryza sativa (Os), and Malus domestica (Md). (B) Temporal expression profiles of GhLYK5 in roots of G. hirsutum cv. Coker 312 following infection with V. dahliae at different time points (0, 0.5, 1, 2, 4, 8, and 12 hpi). Values represent mean ± SE of three biological replicates. GhUBQ7 was used as the internal reference gene, and relative expression was calculated using the 2^−∆∆Ct method. Significant differences compared to the 0 hpi control were indicated with asterisks (Student’s t-test: ^*, P < 0.05; ^**, P < 0.01). (C) Cis-acting regulatory elements in the promoter region of GhLYK5. (D) Predicted tertiary structure of the GhLYK5 protein. (E) Subcellular localization of GhLYK5. A. tumefaciens carrying pYBA1132-GFP (control) or pYBA1132-GhLYK5-GFP were transiently expressed in leaves of 4-week-old N. benthamiana plants co-expressing the plasma membrane marker PIP2A-mCherry. BF, the bright field. Scale bars = 20 μm

GhLYK5 positively regulated cotton resistance to V. dahliae

To further investigate the role of GhLYK5 in the defense response, virus-induced gene silencing (VIGS) was employed to knock down GhLYK5 expression in cotton. Plants infiltrated with TRV2:CLA1 exhibited albino phenotypes (Fig. 2A), and it was proven that the VIGS system had successfully inhibited the expression of the target gene. The gene silencing efficiency was analyzed by RT-qPCR. The results showed that the expression of GhLYK5 in the silenced plants was significantly reduced, and the silencing efficiency was more than 60%. (Fig. 2C). Upon inoculation with V. dahliae, GhLYK5-silenced plants displayed more severe wilting, yellowing, and necrosis symptoms compared to wild-type (WT) and TRV2:00 control plants at 15 dpi. Vascular browning was also more pronounced in silenced plants (Fig. 2B). The disease index (DI) of GhLYK5-silenced plants was significantly higher than WT (46.67) and TRV2:00 (48.33) plants (Fig. 2D). Consistent with this, fungal biomass was markedly increased in silenced plants (Fig. 2E). These results indicate that silencing of GhLYK5 enhanced susceptibility to V. dahliae.

Fig. 2.

The GhLYK5 gene positively regulated cotton resistance to VW. (A) The positive control of VIGS in which the cotton CLA1 gene was silenced to produce the albino phenotype. (B) The disease phenotypes and stem vascular browning of GhLYK5-silenced plants (TRV2:GhLYK5), non-silenced plants (TRV2:00), and WT plants at 14 dpi. The sterile water was used as a mock treatment. (C) GhLYK5 expression in TRV2:GhLYK5 and control plants, GhUBQ7 was used as the internal reference gene. The GhLYK5 abundance values in the TRV2:00 plants were arbitrarily designated as ‘1’. (D) Disease index (DI) of GhLYK5-silenced plants and control plants at 14 dpi. 1, 2, 3, and 4 represent the disease degree. Statistical analysis of the DI and the rate of diseased plants, with each count including at least 25 plants at 14 dpi. (E) Fungal biomass of GhLYK5-silenced plants at 14 dpi. The fungal biomass was expressed as the ratio of the DNA level of V. dahliae ITS1 to the DNA level of the GhUBQ7 gene. (F) Disease symptoms induced with V. dahliae strain on the rosette leaves of WT, OE-2/4(GhLYK5 overexpression plants). 3-week-old seedlings were inoculated with V. dahliae. Photos were taken at 15 dpi, bar = 1 cm. (G) DI of WT, OE-2/4 plants. Sterile water was used as a mock treatment. (H) Fungal biomass of Arabidopsis plants at 15 dpi. AtUBQ10 was used as the standard internal gene. Data represent mean ± SE of three biological replicates. Different letters indicate significant differences at P < 0.05, as determined using ANOVA

To further validate the function of GhLYK5, a transgenic Arabidopsis line overexpressing GhLYK5 was generated. Two homozygous overexpression lines (OE-2, OE-4) were selected based on high transgene expression levels as determined by semi-quantitative RT-PCR (Figure S4). At 14 dpi, OE-2, OE-4, and OE-7 plants exhibited significantly enhanced resistance to V. dahliae compared to WT controls (Fig. 2F). The DI of OE-2, and OE-4 were 38.75 and 30.5 respectively, significantly lower than that of WT plants, 63.75 (Fig. 2G). Additionally, fungal biomass in the roots of overexpression lines was substantially reduced to about one-third of that in the control (Fig. 2H). These results demonstrated that heterologous expression of GhLYK5 enhanced resistance to V. dahliae in Arabidopsis.

GhLYK5-enabled resistance to V. dahliae involves secondary metabolism and MAPK pathway

GhLYK5 responds rapidly to V. dahliae invasion, and its expression peaked at 0.5 hpi. Therefore, to further elucidate the role of GhLYK5 in cotton immunity, RNA-Seq was conducted to compare transcriptome profiles between GhLYK5-silenced (VIGS_GhLYK5) and WT plants for 0.5 hpi. A total of 741 differentially expressed genes (DEGs) were identified, including 436 up-regulated and 305 down-regulated genes (Figure S5, Table S2). Gene Ontology (GO) enrichment analysis indicated that GhLYK5 was involved in regulating metabolic processes, particularly those related to this biosynthesis of secondary metabolites, such as the aromatic amino acid metabolism and organic acid biosynthesis (Fig. 3A). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed significant enrichment of DEGs in flavonoid biosynthesis; tropane, piperidine, and pyridine alkaloid biosynthesis; phenylalanine, tyrosine and tryptophan biosynthesis; phenylpropanoid biosynthesis; phenylalanine metabolism; glycolysis/gluconeogenesis; and (mitogen-activated protein kinase) MAPK signaling pathway (Fig. 3B). A heatmap of resistance-related DEGs showed altered expression patterns upon GhLYK5-silencing (Fig. 3C). Several genes involved in secondary metabolites were down-regulated, including fumarylacetoacetase (GhFAH), caffeoyl-CoAO-methyltransferase (GhCCOAOMT7) and chalcone synthase 1 (GhCAS). Similarly, key defense-related genes—such as a putative disease resistance protein (GhSUT1) and calmodulin-like protein (GhCML46)—were significantly down-regulated. These expression changes suggest that silencing of GhLYK5 may impair disease resistance by disrupting secondary metabolite synthesis and suppressing the expression of critical defense genes.

Fig. 3.

Transcriptome analysis of GhLYK5-silenced (VIGS_GhLYK5) and control (Coker 312) under V. dahliae infection by RNA-sequencing. (A) Gene ontology (GO) enrichment analysis of the 741 DEGs between VIGS_GhLYK5 and WT. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the DEGs between VIGS_GhLYK5 and WT. (C) The heat map showed the expression pattern of typical genes of GhLYK5 involved in the regulation of the pathogen defense pathway. (D) The expression of plant secondary metabolism and plant immune defense-related genes in TRV2: GhLYK5 and TRV2: 00 plants. GhUBQ7 is the internal reference. Data were expressed as mean ± SD, n = 3 (* P < 0.05, ** P < 0.01, *** P < 0.001. Student ‘s t-test)

GhLYK5 interacts with GhNIT4B in vitro and in vivo

To identify proteins interacting with GhLYK5, a yeast two-hybrid (Y2H) screening was performed using a V. dahliae-induced cotton cDNA library. This screening revealed that GhLYK5 nitrilase interacted with GhNIT4B (Fig. 4A). The interaction was confirmed by luciferase complementation imaging (LCI) assay. Strong luciferase activity was detected when GhLYK5-cLUC and GhNIT4B-nLUC were co-expressed, while no signal was observed in negative controls (GhLYK5-cLUC+nLUC, GhNIT4B-nLUC+cLUC, and nLUC+cLUC) (Fig. 4B). BiFC assays in N. benthamiana leaves showed that co-expression of GhLYK5-cYFP and GhNIT4B-nYFP resulted in reconstitution of YFP fluorescence at the plasma membrane. No fluorescence was detected in control combinations (GhLYK5-cYFP+nYFP, GhNIT4B-nYFP+cYFP) (Fig. 4C). Additionally, co-immunoprecipitation (Co-IP) experiments in plants co-expressing 35 S: FLAG-GhLYK5 and 35 S: GFP-GhNIT4B demonstrated that GhNIT4B was specifically immunoprecipitated by GhLYK5 (Fig. 4D). Together, these results indicated that GhLYK5 interacted with GhNIT4B in planta and that their interaction occurred at the plasma membrane.

Fig. 4.

Interaction between GhLYK5 and GhNIT4B. (A) Yeast two-hybrid assay showing the interaction between GhLYK5 and GhNIT4B. Serial dilutions (10X, 100X, and 1000X) of yeast cultures are shown. (B) Bimolecular fluorescence complementation (BiFC) assay in N. benthamiana leaves. YFP fluorescence indicates interaction at the plasma membrane. Images were captured 48 hpi. Scale bars = 20 μm. (C) Luciferase complementation imaging (LCI) of GhLYK5 and GhNIT4B interaction. The color scale represents luminescence intensity from low (blue) to high (red). (D) Co-immunoprecipitation (Co-IP) of GhLYK5 and GhNIT4B in N. benthamiana leaves. Total protein extracts (Input) and anti-FLAG immunoprecipitates (IP: α-FLAG) from leaves co-expressing 35 S: FLAG-GhLYK5 and 35 S: GFP-GhNIT4B were immunoblotted with anti-FLAG and anti-GFP antibodies. Data were representative of three biological replicates

GhNIT4B positively regulated cotton resistance to V. dahliae

The role of GhNIT4B in cotton resistance to VW has not been previously reported. Phylogenetic analysis classified the NIT family proteins in upland cotton into three subgroups (I-III), which contained TCP superfamily domains and at least one intron (Figure S6). Promoter analysis of NIT family genes revealed the presence of defense- and stress-responsive cis-elements (Figure S7). Following V. dahliae infection, GhNIT4B expression in root tissues was transiently up-regulated at 0.5 hpi, gradually increased, and peaked at 12 hpi (Fig. 5A), indicating its responsiveness to pathogen challenge and potential involvement in cotton defense. Subcellular localization experiments indicated that GhNIT4B was localized to the cell membrane (Fig. 5B), indicate that it may function primarily at this site.

Fig. 5.

GhNIT4B acted as a positive regulator in cotton defense against V. dahliae. (A) The temporal expression of GhNIT4B inoculated with V. dahliae at different inoculation time points (0, 0.5, 1, 2, 4, 8, 12, and 24 hpi) in Coker 312 cotton. (B) Subcellular localization of GhNIT4B. Agrobacteria carrying pYBA1132-GFP (control) or pYBA1132-GhNIT4B-GFP were transiently expressed in leaves of 4-week-old N. benthamiana plants co-expressing the plasma membrane marker PIP2A-mCherry. BF, the bright field. Scale bars = 20 μm. (C) Albino phenotypes in TRV2:CLA1-positive control plants at 14 dpi. (D) Disease symptoms of WT, empty vector control (TRV2:00), and GhNIT4B-silenced (TRV2:GhNIT4B) plants after inoculation with V. dahliae. (E) Silencing efficiency of GhNIT4B in VIGS plant, determined by RT-qPCR. (F) DI of WT, TRV2:00, and TRV2:GhNIT4B plants at 14 dpi. Rated on a 0–4 disease severity scale. (G) Fungal biomass quantified by RT-qPCR in stem tissues at 14 dpi. GhUBQ7 was used as the endogenous reference gene. Data represent mean ± SE of three biological replicates. Different letters indicate significant differences at P < 0.05, as determined using ANOVA

Furthermore, VIGS system was employed to investigate the role of GhNIT4B. Successful silencing was confirmed by the albino phenotypes in TRV2:CLA1 plants (Fig. 5C) and significant down-regulation of GhNIT4B expression (Fig. 5E). Compared to WT, GhNIT4B-silenced plants exhibited more severe disease symptoms, including wilting, yellowing, necrosis, and leaf abscission, along with enhanced vascular browning (Fig. 5D). DI and fungal biomass were significantly higher in GhNIT4B-silenced plants than in WT controls (Fig. 5F, G). These results demonstrated that GhNIT4B positively regulates plant immune defense in cotton against V. dahliae.

Silencing GhNIT4B reduced disease resistance by suppressing asparagine biosynthesis and ROS accumulation

NIT4 exhibits strong substrate specificity for β-cyano-L-alanine, and catalyzes to formation of asparagine [47]. GhNIT4B was significantly induced at 12 hpi. Therefore, to clarify whether GhNIT4B is involved in the regulation of cyanide metabolism. Using HPLC-MS/MS, we measured asparagine (Asn) content and found that, before inoculation, Asn levels in TRV2:00 plants was twice those in TRV2:GhNIT4B plants. Following 12 hpi, asparagine content increased significantly in TRV2:00 plants, but there was no significant difference in TRV2:GhNIT4B plants (Fig. 6A). These results suggested that silencing of GhNIT4B impaired Asn accumulation, likely due to disruption of the β-cyanoalanine synthase (β-CAS) metabolic pathway.

Fig. 6.

GhNIT4B regulated amino acid metabolism and ROS accumulation in response to V. dahliae infection. (A) The contents of asparagine in TRV2:00 and TRV2: GhNIT4B before and 12 hpi were determined by high-performance liquid chromatography (HPLC). (B) DAB staining was used to detect the accumulation of ROS in TRV2:00 and TRV2: GhNIT4B cotton leaves before and 12 hpi. (C) H₂O₂ content in leaf tissues. (D) Nitric oxide (NO) levels. (E-G) The expression levels of ROS-related genes (GhRobhD, GhNOA1, GhRboh5) in TRV2:00 and TRV2:GhNIT4B cotton leaves. (H-I) The expression levels of plant immune defense-related genes (GhPR3, GhHIN1, GhPR1) in cotton leaves of TRV2:00 and TRV2:GhNIT4B. Data are presented as mean ± SE. Asterisks indicate significant difference compared with the control group as determined by Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001)

ROS burst is an early defense response against pathogen infection [48]. To assess whether GhNIT4B participated in ROS regulation, we monitored ROS accumulation in TRV2:00 and TRV2:GhNIT4B cotton plants after inoculation with V991. DAB staining revealed weaker and more restricted staining in GhNIT4B-silenced plants compared to TRV2:00 control (Fig. 6B). Consistent with this, both H₂O₂ and NO levels were significantly lower in GhNIT4B-silenced plants (Fig. 6C-D), indicating an attenuated ROS burst. Furthermore, the expression of ROS-related genes— GbRbohD, GbRboh5, and GhNOA1—was markedly down-regulated in TRV2:GhNIT4B plants (Fig. 6E-G). The transcript levels of hypersensitive response (HR) marker genes GhPR1, GhPR3, and GhHIN1 were also significantly suppressed in silenced plants (Fig. 6H-J). In conclusion, GhNIT4B positively regulated cotton resistance to VW by promoting asparagine biosynthesis, facilitating ROS accumulation, and activating the expression of key defense-related genes.

Exogenous application of Asn activated defense responses and enhanced cotton resistance to VW

GhNIT4B is a positive regulator of plant resistance to VW. Silencing GhNIT4B can significantly inhibit the synthesis of asparagine at 12 hpi. To further study the regulatory role of NIT4B in cotton resistance to VW, cotton was treated with exogenous Asn in this study. Two-week-old cotton leaves were sprayed with 1 mM asparagine (or H₂O as a control). After three days of treatment, the plants were inoculated with Verticillium dahliae using the root-dip method, and leaf samples were collected at 0 and 12 hpi for subsequent analysis. Compared to water-treated controls, Asn-treated plants exhibited reduced symptoms of VW, including less leaf wilting and yellowing, along with attenuated vascular browning (Fig. 7A). Disease grade and fungal biomass also confirmed that Asn application significantly enhanced resistance to V.dahliae. We further quantified the expression of key defense-related genes— GhBAK1 [49], GhNPR1 [50], GhPR1 [51], and GhLac1 [52]— in both H₂O and Asn-treated plants before and 12 hpi using RT-qPCR. Asn treatment markedly activated the expression of these defense genes, with significant induction observed at 12 hpi (Fig. 7D-G). In contrast, the Asn application did not strongly induce the expression of ROS-related genes (GhRbohD, GhRboh5, GhNOA1) or the MAPK gene (GhMAPK3, GhMAPK6) in H₂O treated plants (Figure S8). In conclusion, exogenous Asn enhanced cotton disease resistance by activating the expression of defense genes rather than ROS or MAPK pathway genes.

Fig. 7.

Asparagine treatment enhanced cotton resistance to V. dahliae. (A) Disease phenotypes and vascular browning in cotton plants sprayed with Asn or H₂O, followed by V. dahliae inoculation. Symptoms were assessed 14 dpi. Results are representative of three independent experiments. (B) Disease index and disease grade of Asn- and H₂O-treated plants after inoculation with V. dahliae. (C) Fungal biomass in cotton, determined by qPCR analysis of the V. dahliae ITS1 region, normalized to the GhUBQ7 gene. (D-G) Expression levels of VW resistance-related genes (GhPR1, GhLac1, GhBAK1, GhNPR1) in leaves of Asn- and H₂O-treated plants at 0 hpi and 12 hpi. Data represent mean ± SE, asterisks indicate significant differences based on Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001)

Discussion

VW, caused by the soil-borne fungus V. dahliae, is a destructive vascular disease that severely limits cotton yield and impairs fiber quality [53]. Developing effective control strategies requires a deeper understanding of the molecular mechanisms underlying cotton resistance. In this study, we investigated the role of LysM receptor-like kinase (LYK) genes, which are implicated in plant defense but remain poorly characterized in cotton. Our results demonstrate that GhLYK5 was rapidly induced following V. dahliae infection. Silencing of GhLYK5 in cotton led to enhanced susceptibility, whereas heterologous overexpression in Arabidopsis conferred increased resistance, confirming that GhLYK5 acts as a positive regulator of immunity against V. dahliae. These findings are consistent with previous reports indicating that other LysM family members in cotton, such as GhLYK1, GhLYK2, GhLYP1, GhLYK7, and GhLysMe3, also contribute to VW resistance through activation of salicylic acid (SA), jasmonic acid (JA), and reactive oxygen species (ROS) signaling pathways [54, 55]. However, the role of LysM proteins in fungal resistance is not universally positive; for example, the Arabidopsis cerk1 mutant exhibits increased susceptibility to some fungi but remains resistant to others [56, 57], highlighting functional specificity across species and pathosystems.

The MAPK cascade serves as a central signaling node in plant immunity, amplifying defense signals through phosphorylation of downstream substrates [58]. In cotton, several MAPK members—GhMKK4, GhMKK6, and GhMKK9—have been shown to positively regulate VW resistance [59]. Our transcriptomic data revealed that silencing of GhLYK5 led to significant down-regulation of MAPK pathway genes, suggesting that GhLYK5 may modulate cotton immunity through MAPK signaling. In Arabidopsis, MPK4 interacts with and phosphorylates WRKY33 upon pathogen infection [60], illustrating cross-talk between MAPK cascades and transcription factors. Several WRKY family genes were identified among the DEGs in our study. WRKY transcription factors in cotton can enhance flavonoid accumulation and resistance to fungal pathogens by binding to W-box elements in promoters of defense-related genes—such as GhMKK2—thereby activating MAPK cascades involving GhNTF6 and GhMYC2 [61]. Flavonoid biosynthetic genes are known to respond to V. dahliae infection [62], and our results show that this pathway is enriched among up-regulated DEGs. Thus, we propose that WRKY transcription factors may serve as core regulators downstream of GhLYK5, integrating MAPK signaling and flavonoid metabolism to confer resistance to VW.

LysM proteins function as essential pattern recognition receptors in plant immunity, as evidenced in model systems such as rice and Arabidopsis [63, 64]. They activate various receptor-like cytoplasmic kinases, initiate downstream MAPK cascades, and transmit immune signals through diverse oligomerization states [20]. In Arabidopsis, chitin perception involves the formation of a complex between AtCERK1, AtLYK4, and AtLYK5, resulting in phosphorylation and membrane retention of AtCERK1 [65]. Additionally, chitin-induced phosphorylation by AtCERK1 promotes internalization of AtLYK5 [66]. In upland cotton, GhLYK5 recognizes chitin, interacts with GhCERK1, and activates defense responses including ROS burst, MAPK signaling, and the expression of defense-related genes [39]. In this study, we demonstrated that GhLYK5 interacted with GhNIT4B and that both proteins acted as positive regulators of VW resistance. Interestingly, in Arabidopsis, AtLYK5 participates in the AtCERK1-mediated signaling pathway and regulates the expression of defense genes such as AtNit4 and AtWRKY75 via interaction with the receptor-like cytoplasmic kinase AtPBL1, which phosphorylates AtMAPKKK4 [67]. These findings highlight both conserved and species-specific roles of LysM proteins in immune regulation across plants, further enriching the network of plant immune signaling. However, whether GhLYK5 competes for binding with GhCERK1 and GhNIT4B remains an open question. It also remains unclear how the GhLYK5-GhNIT4B interaction influences ROS production and MAPK activation in the cotton immune pathway.

NIT enzymes are widely distributed in plants and play pivotal roles in nitrile detoxification, nitrogen assimilation, phytohormone biosynthesis, and plant-microbe interactions [68–70]. In Arabidopsis, NIT1, NIT2, and NIT3 enhance resistance to PstDC3000 and oomycete pathogens through the metabolism of nitrile compounds and modulation of growth regulators [71]. In our study, silencing of GhNIT4B reduced endogenous asparagine levels and increased sensitivity to VW. Accumulation of asparagine has been shown to activate salicylic acid (SA) signaling and enhance disease resistance in tobacco [72]. Furthermore, exogenous asparagine application can attenuate pathogen pathogenicity, underscoring its emerging role as a central component of plant immune network [73, 74]. Our results clearly demonstrated that GhNIT4B is directly involved in asparagine biosynthesis and contributes to cotton immunity against to VW by modulating this metabolic pathway. In summary, this study provided novel insights into the direct involvement of NIT genes in plant immune responses and expands the regulatory landscape of NIT proteins in plant defense.

ROS in plants can disrupt the cytoplasmic membranes of pathogens, leading to the disintegration and inactivation of cell structures, while also functioning as signaling molecules to activate downstream immune responses [75, 76]. This study revealed that silencing GhNIT4B suppresses the expression of key ROS biosynthetic genes and attenuates the ROS burst in cotton upon fungal infection. Additionally, asparagine, which serves as a nitrogen transport and storage molecule, may restrict pathogen access to nitrogen sources during infection, while also modulating the tricarboxylic acid (TCA) cycle and mitochondrial electron transport chain activity to minimize ROS leakage and maintain cellular redox homeostasis [77, 78]. Silencing of GhNIT4B resulted in reduced accumulation of asparagine, which is known to participate in stress responses, potentially through ROS-mediated mechanisms. Therefore, whether GhNIT4B contributes to the positive regulation of cotton resistance to VW by directly modulating ROS accumulation warrants further investigation.

Amino acid metabolism level has a significant effect on plant disease defense ability [74]. Exogenous glutamate (Glu) treatment can enhance the resistance to Magnaporthe oryzae in rice leaves [79]. Tryptophan (Try) is synthesized as a new antiviral substance as a substrate, which can induce SA and antioxidant system [80, 81]. It has been proved that the application of asparagine can increase the resistance of rice to Magnaporthe oryzae, and the external application of asparagine can activate the salicylic acid signaling pathway to significantly inhibit tobacco mosaic virus [72, 79]. Consistently, we also found that exogenous application of asparagine can enhance the disease resistance of cotton to V. dahliae and activate plant immune defense genes. And we found that asparagine treatment did not lead to significant growth and development changes in plants, so these findings have great potential for the future application of asparagine in the control of VW.

Conclusions

Based on our findings, we propose a model for GhLYK5-GhNIT4B-mediated resistance against V. dahliae in cotton (Fig. 8). This study demonstrates that GhLYK5 acts as a positive regulator of VW resistance. Further analysis indicates that GhLYK5 enhances immunity primarily by modulating the expression of defense-related and secondary metabolism-related genes. We also confirmed that GhLYK5 physically interacts with GhNIT4B at the plasma membrane. GhNIT4B contributes to resistance by regulating asparagine accumulation, maintaining ROS homeostasis, and activating defense-related gene expression. Moreover, asparagine can improve the disease resistance of plants by activating the expression of defense genes. Together, GhLYK5 and GhNIT4B form a novel signaling module that orchestrates early defense responses, offering valuable molecular targets for improving VW resistance in G. hirsutum.

Fig. 8.

Proposed model for the roles of GhLYK5 and GhNIT4B in cotton defence against V. dahliae. Infection by V. dahliae rapidly induced the expression of GhLYK5, which affected the expression of secondary metabolic genes and activated MAPK cascade signaling and defense-related gene expression. GhLYK5 interacted with GhNIT4B at the plasma membrane. GhNIT4B enhanced resistance to VW by modulating asparagine biosynthesis, promoting ROS accumulation, and regulating the expression of defense-related genes

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

All authors contributed to the study’s conception. Na Li: Formal analysis, Investigation, Writing – original draft. Wenfang Guo: Data Curation, Investigation, Writing – review & editing, Validation, Supervision. Guoqiang Pan: Methodology, Conceptualization, Investigation. Xingfen Wang: Methodology, Investigation. Zhengwen Sun: Validation, Data curation. Huiming Guo: Project administration, Supervision. Hongmei Gheng: Writing – review & editing, Validation, Funding acquisition, Data curation, Conceptualization. Xiaofeng Su: Writing – review & editing, Validation, Funding acquisition, Data curation, Conceptualization.

Funding

This study was supported by the National Key Research and Development Program of China (2022YFD1200300), the National Natural Science Foundation of China (32372515), Winall Hi-tech Seed Co., Ltd., China (GMLM2023), Hebei Key Technology R&D program (21326314D), and the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences (CAAS-ZDRW202308).

Data availability

Data will be made available on request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors agree with publishing this article within The Journal of Biological Engineering.

Competing interests

The authors declare no competing interests.