A DEK domain-containing protein GhDEK2D mediated Gossypium hirsutum enhanced resistance to Verticillium dahliae

ABSTRACT DEK is associated with DNA replication and break repair, mRNA splicing, and transcriptional regulation, which had been studied in humans and mammals. The function of DEK in plants was poorly understood. In this study, GhDEK2D was identified in Gossypium hirsutum by genome-wide and post-translational modifications. GhDEK2D had been phosphorylated, acetylated and ubiquitylated under Verticillium dahliae (Vd) challenge. The GhDEK2D-silenced cotton decreased resistance against Vd. In GhDEK2D-silenced cotton plants, the reactive oxygen species was activated, the callose, xylogen, hypersensitive reaction (HR) and expression levels of defense-related genes were reduced. Homozygous overexpressing-GhDEK2D transgenic Arabidopsis lines were more resistant to Verticillium wilt (Vw). We propose that GhDEK2D was a potential molecular target for improving resistance to Vw in cotton.


Introduction
Cotton is one of the most important economic, 1 textile fiber, and oilseed crop. 2 G. hirsutum is characterized by high yield and quality fiber, and becomes the most cultivated species in the world cotton production. 3 However, cotton production was affected by abiotic and biotic stresses in many regions of the world, among them, Vw is a destructive biotic stresses, which caused by the soil-borne fungus Vd. 4,5 For the past few years, in the wake of climatic change, long-term mono-cropping and frequent introduction of a fine variety led to break out Vw in cotton. 6 The fungus can form dormant structures called microsclerotia that can survive in the soil for years without a host and infect subsequent crops, so that Vw is difficult to control in cotton.
Post-translational modifications (PTMs) play a role in every aspect of cell biology by regulating many cellular processes. 7 Phosphorylation, acetylation, and ubiquitination are important PTMs. They are necessary for functional signal transduction and metabolism, and are involved in chromatin conformation regulation, transcription, and cell metabolism. 8 PTMs also affects cell localization, stability, interaction, and enzyme activity. 9 Activity of proteins Phosphorylation, one of the most extensive modifications of proteins, is a reversible process catalyzed by a kinase that transfers a phosphorylating group in ATP to the hydroxyl group of a specific serine, threonine or tyrosine residue in the target protein. 10,11 Phosphorylation is a key process that regulates plant growth, development and stress response. 12 Reversible phosphorylation of serine, threonine, and tyrosine residues performs many important functions, such as altering protein conformation and activation states, protein stability and degradation, subcellular localization, and interaction with protein substrates. 13 Mining resistance genes has become the major way to sustain cotton industry. Protein post-translational modifications is major defense mechanism for regulating plant immune. 14 A growing body of PTMs profiling has provide convenience to excavate new resources.
DEK was originally cloned from acute myeloid leukemia and is a bona fide oncoprotein associated with many different tumor types, as well as with stem cell and progenitor cell quality. [15][16][17] DEK was found in all multicellular organisms. 18 Chromatin structure plays an important role in various processes. 19 DEK proteins are involved in the control of various chromatin related processes. 20 The function of DEK has been widely studied in mammals, and it takes part in DNA replication and repair, mRNA splicing, transcription regulation, cell lifetime, division and differentiation. [21][22][23][24] In Arabidopsis, there are four DEK proteins, and named DEK1, DEK2, DEK3 and DEK4. DEK has been reported to be present in the nucleolus. DEK3 affects nucleosome occupancy and chromatin accessibility, and adjusts the expression levels of its target genes. 25 The correct expression of DEK3 can regulate the response of plants to stress conditions such as high salt and heat shock. Arabidopsis DEK3 and DEK4 prevent early flowering by activating transcription of key flowering inhibitors. 26 Silencing DEK2 or DEK4 in tomatoes accumulates more ROS during pathogen infection and alters the expression of PR1b, PR2, LaPA and PIN2. The results showed that DEK is involved in the regulation of defense response to pathogens. 27 In this study, there were six DEKs, which were highly conservative proteins in G. hirsutum. GhDEK2D had been phosphorylated, acetylated and ubiquitylated after Vd inoculation. The GhDEK2D-silenced cottons decreased resistance against Vd. In contrast, overexpressing-GhDEK2D enhanced Arabidopsis resistance to Vd. In GhDEK2D-silenced cotton plants, ROS, callose, xylogen, expression levels of defenserelated genes and HR genes were reduced. These all indicate that GhDEK2D involves in defense response' regulation to pathogen in G. hirsutum.

Fungal strain, plant lines, and culture conditions
In this study, a virulent, defoliating Vd Vd080 was used to infect plants, 28 and its cultural method referenced Zhou et al. 5 A G. hirsutum cultivar Zhongzhimian NO. 2 is resistance to Vd. They were grown in a conditioned greenhouse with a 16 h/ 8 h photoperiod at 22°C to 28°C. A. thaliana (Columbia) was grown in a conditioned greenhouse with a 16 h/8 h photoperiod at 22°C. The culture method of Nicotiana benthamiana is the same as that of Zhongzhimian NO. 2.

Phylogenetic, gene structure, conserved domain analysis and chromosomal mapping
A total of 4 Arabidopsis DEK protein sequences were downloaded from TAIR (https://www.arabidopsis.org/). Multiple sequence alignments of all identified DEK from cotton, P. trichocarpa, Z. mays, S. lycopersicum, S. tuberosum, L. usitatissimum and Arabidopsis were performed in ClustalX 2.0. After conducting a model test, a maximum likelihood (ML) phylogenic tree was constructed with the best substitution model using MEGA X software. 42 TBtools was extracted to draw the gene structure of DEK based on the whole-genome sequence and annotation datas. 43 Conserved motifs of DEK proteins were identified using the Multiple Em for Motif Elicitation Version 5.4.1 (https://memesuite.org/meme/tools/meme) with the default parameters. 44 The conserved domains of DEK proteins were identified by Batch Web CD-Search Tool (https://www.ncbi.nlm.nih.gov/ Structure/bwrpsb/bwrpsb.cgi). 45,46 The figures of gene structure, conserved motifs and domains were drew by TBtools software. 47 Chromosomal position information about cotton DEKs was obtained from the CottonFGD webpage. GhDEK, GrDEK, GaDEK and GbDEK genes synteny and collinearity were determined and analyzed by the MCScanX software. 48 And the distribution of DEK genes were draw used TBtools.

Transcriptome analysis of GhDEK in tissues of cotton and responded to Vd
The expression patterns of GhDEKs were exhibited in tissues, TM-1 transcriptome data from (Accession codes, SRA: PRJNA490626, https://www.ncbi.nlm.nih.gov/bioproject/?term= PRJNA490626). 49 The Zhongzhimian NO. 2 plants treated with Vd at 12 and 24 hpi, quantitative reverse transcription PCR (RT-qPCR) was used to determine GhDEKs expression levels.

Virus-induced gene silencing (VIGS) of GhDEK2D in cotton
VIGS were performed to inhibit expression of GhDEK2D, the experimentation as Feng et al. 50

Arabidopsis thaliana transformation
In order to express GhDEK2D in A. thaliana, the primers OE-GhDEK2D-F and OE-GhDEK2D-R (Table S2) were used to clone the GhDEK2D. The GhDEK2D was inserted into the pCAMBIA3300-eGFP vector containing a glufosinate (Basta) resistance gene for overexpression studies. T 0 -T 3 transgenic seeds were sprayed with 0.01% Basta and PCR to select positive transformants. The high GhDEK2D expression T 4 lines were used to study.

Plant disease resistance assay
Plants (A. tumefaciens and cotton) disease resistance assay as Zhou et al. 5

Visualization and quantification of H 2 O 2 , lignification and callose staining
To visualize the accumulation of H 2 O 2 , cotton leaves were collected 48 h after Vd080 inoculation and incubated in DAB solution (1 mg/mL, pH 7.5) for 8 h. leaves were decolorized in 95% ethanol until the green color was completely removed in boiling water bath. ROS accumulation of leaves in 70% glycerol was observed under a microscope.
Cotton lignification was examined with phloroglucinol staining. The experimental procedure referenced Zhou et al. 5 Thickness measurement were manipulated using ImageJ software.
Cotton callose deposition was visualized by aniline blue staining. The experimental procedure referenced Zhou et al. 5 Thickness measurement were manipulated using ImageJ software.

Expression of defense-related genes by RT-qPCR
Cotton leaves were excised 0, 3, 6, 9, 12, 24, and 48 h post inoculation for RT-qPCR. The RNA was extracted by RNAprep Pure Plant Kit (TIANGEN, Beijing, China). The cDNA was synthesized by TransScript® All-in-One first-strand cDNA Synthesis Super Mix for qPCR reverse transcription kit (TransGen, Beijing, China). qPCR was execute by TransStart® Tip Green qPCR Super Mix kit (TransGen, Beijing, China). The specific primers of cotton defense-related genes were listed in Table S4. Technical replicates of three independent biological samples were performed.

Subcellular localization
The A. tumefaciens strain GV3101 containing pCAMBIA3300-GhDEK2D-eGFP vector was used for subcellular localization. The experimentation as Feng et al. 50 DAPI was used to mark cell nucleus.

Identification and phylogenetic analysis of DEK genes family in cotton
To identify putative DEK family genes in cotton, 4 Arabidopsis DEK protein sequences were used as queries, and then conducted a homologous blast search against G. hirsutum (CRI), G. raimondii (JGI), G. arboretum (CRI) and G. barbadense (CRI) protein databases (https://cottonfgd.org/sequenceser ver/). The potential DEK sequences were further inspected whether containing the conserved DEK motif by the InterProScan databases (https://www.ebi.ac.uk/interpro/ search/sequence/). We obtained 6, 2, 3, and 7 DEKs in G. hirsutum, G. raimondii, G. arboretum and G. barbadense, respectively. The lengths of DEK proteins ranged from 360 to 642 amino acids, the molecular weight (MW) ranged from 41.333 kDa to 73.261 kDa, and the isoelectric point (IP) ranged from 4.571 to 8.033 (Table S1).
To further explore the evolutionary relationship of DEKs, 6 GhDEKs, 2 GrDEKs, 3 GaDEKs, 7 GbDEK, 6 GdDEK, 6 GtDEK, 5 GmDEK from cotton, 3 PtDEK from P. trichocarpa, 4 ZmDEK from Z. mays, 4 MeDEK from M. esculenta, 2 SlDEK from S. lycopersicum, 4 StDEK from S. tuberosum, 4 LuDEK from L. usitatissimum and 4 AtDEKs from Arabidopsis were used for constructing a phylogenetic tree by MEGA X software using the maximum likelihood (ML) method ( Figure 1). The DEK family can be divided into four groups (I to IV), Group I only contained AtDEKs, Group II only contained 2 ZmDEKs, Group III contained 20 DEKs of 11 species, Group IV contained 30 DEKs of 12 species. The results suggested DEKs were highly conservative in respective species. The DEKs ID and names were listed in Table S2.

Chromosomal location and gene synteny analysis of DEK genes in Gossypium
The chromosomal locations of genes were investigated by the complete Gossypium genome sequence. A total of six GhDEK genes were distributed throughout the Gh_A04, Gh_A07, Gh_A12, Gh_D05, Gh_D07, and Gh_D12 subgenome. Three GaDEKs were distributed throughout the Ga_Chr05, Ga_Chr07, and Ga_Chr12 subgenome. Two GrDEKs were distributed throughout the Gr_Chr08 and Gr_Chr09 subgenome. Seven GbDEKs were distributed throughout the Gb_A04, Gb_A07, Gb_A12, Gb_D01, Gb_D05, Gb_D07 and Gb_D12 subgenome ( Figure 2). Syntenic analysis were performed to studies the relationships of DEK genes among the G. hirsutum, G. raimondii, G. arboretum, and G. barbadense (Figure 2). Among the DEK genes, six GhDEKs were the orthologous genes of the two GrDEKs, five GbDEKs and three GaDEKs, respectively. Five GbDEKs were the orthologous genes of the two GrDEKs and three GaDEKs, respectively. Two GrDEKs were the orthologous genes of the three GaDEKs. Some DEKs had not only one orthologous gene, such as GrDEK1 had four genes (GhDEK1A, GhDEK1D, GhDEK2A and GhDEK2D) in G. hirsutum, three genes (GbDEK1A, GbDEK2A and GbDEK2D) in G. barbadense, and two genes (GaDEK2 and GaDEK1) in G. arboretum. GrDEK4 had two genes (GhDEK4A and GhDEK4D) in G. hirsutum, two genes (GbDEK3D, and GbDEK4D) in G. barbadense. More orthologous genes were displayed in Table S5. In addition, there were two paralogous gene pairs (GhDEK1A/2A/1D/2D and GhDEK4A/4D) in G. hirsutum, one paralogous gene pair (GaDEK1/2) in G. arboretum, two paralogous gene pairs (GhDEK1A/2A/1D/ 2D and GhDEK3D/4A/4D) in G. barbadense, and zero paralogous gene pair in G. raimondii genome (Table S3). Furthermore, all cotton DEKs were classified into WGD or segmental duplications (Table S3). In the expansion of the DEK gene family, WGD or segmental duplication might play a crucial role.

Gene structure, conserved motifs and domain of DEKs
To better reveal the evolutionary relationships of the DEK gene family, a separate unrooted phylogenetic tree was constructed with DEK DNA sequences (Figure 3a). The comparative analysis of intron-exon structure showed that GhDEK gene length varied significantly. DEK genes had a highly rich distribution of introns in the regions, except GbDEK3D possessed 12 exons, other DEK genes possessed 11 exons (Figure 3b). In addition, closely related genes had more similar arrangements of gene structure. There were 15 conserved motifs (motif 1 to 15) in DEKs were identified by  , the group II DEK (GbDEK3D) protein had nine motifs (motif 1, 2, 3, 4, 5, 10, 11, 12 and 13), and the group III DEK proteins contained all motifs (Figure 3c), indicating that they are highly conserved among DEKs. We also identified domains in the DEKs using Batch Web CD-Search Tool, so as to gene structure, closely related proteins had more similar arrangements of motifs and domains (Figure 3d).

Prediction of cis-acting elements in the promoters of GhDEKs
The 2.0 kb upstream promoter regions of GhDEKs were obtained and analyzed to predict the possible biological functions. Response to various stresses were our main focus, for instance salicylic acid, abscisic acid, defense, auxin, methyl jasmonate (MeJA), drought, etc., ( Table 1). The abundance of cis-acting showed that GhDEK genes might perform different biological functions.

Expression patterns of GhDEKs in tissues of cotton
To explore the functions of the GhDEKs, the expression patterns of the six GhDEKs were analyzed in leaf, bract, filament, pental, anther, root, torus, stem, and sepal. GhDEK4A and GhDEK4D were higher than other genes in all tissues. All the genes expression were the highest in root (Figure 4a). Vd is a soil-borne fungus, which infests cotton roots, the abundance of DEKs in roots portend that DEKs might play an important role in against soil-borne fungus.

Expression patterns and post-translational modifications of GhDEKs under V. dahliae challenge
In the Vw-resistant cotton (Zhongzhimian NO. 2) plants treated with Vd080, at 12 hpi and 24 hpi, GhDEKs expression had no significant change compared with 0 hpi (Figure 4b). Under Vd challenge, only GhDEK2D had been phosphorylated, acetylated and ubiquitylated. A day after inoculation, GhDEK2D was phosphorylated, deubiquitylated in cotton resistant cultivar. GhDEK2D acetylation was activated in susceptible cultivar one and two days after inoculation. The mass spectrometry results showed that phosphorylation sites were Thr327 and Ser329, acetylation and ubiquitylation sites were Lys273 (Fig.  S1). The results suggested that the GhDEK2D was important in response to Vd infection.

GhDEK2D silencing reduced the resistance of cotton to Vd
To verify the function of GhDEK2D resistance to Vd in cotton, the transcript activity of GhDEK2D was restrained by VIGS. When Zhongzhimian NO. 2 seedlings had grown for 10 days, we infiltrated cotyledons with TRV::00, TRV:: GhDEK2D or TRV::PDS (a visible gene marker). When the albino phenotype appeared in TRV::GhPDS infected newly true leaves (Figure 5a). After 1 week of treatment with Vd080 by root dipping method. Compared with the TRV::00 plants, the morbidity of silencing plants was higher, such as wilted leaves and dark vascular bundles (Figure 5b,c). The disease index ( Figure 5e) and disease levels ( Figure 5f) were consistent with the plant phenotype. The results showed that the GhDEK2D silencing could attenuate plant resistance to Vd infection.

GhDEK2D promotes plant defensive reaction
In order to explore the mechanism of GhDEK2D resistance to Vd infection, the cotton xylem was observed. A noticeable increase in the thickness of xylem in the control plants was observed (Figure 6a). The thickness of xylem were 52.99 μm and 115.09 μm in TRV::GhDEK2D and TRV::00, respectively (Figure 6b). A decreased density of callose depositions (number per mm 2 ) was visualized in the leaves of silencing plants by staining with aniline blue (Figure 6c,d). The ROS changed in the control group was stronger (Figure 6e). Thesa results suggested that GhDEK2D silencing could reduce physical defense. The expressions of cotton defense-related genes were tested by RT-qPCR in root. NOA is a gene related to nitricoxide synthesis pathway, NOA1 expression of TRV::00 plants were significantly higher than TRV::GhDEK2D at 3, 9, 12, 24 and 48 hpi (Figure 7a) HIN1 and HSR203J are HR marker gene. HIN1 expression of TRV::GhDEK2D were inhibited all the time (Figure 7b). HSR203J expression of TRV::GhDEK2D were inhibited at 3, 6, 12, 24 and 48 hpi (Figure 7c). PAL, PPO and POD play important roles in plant immunology. In our study, PAL expression of TRV::00 plants were significantly higher than TRV::GhDEK2D all the time (Figure 7d); PPO expression of TRV::00 plants were significantly higher than TRV:: GhDEK2D at 3, 24 and 48 hpi (Figure 7e). At 6, 12, 24 and 48 hpi, POD expression was significant increase in TRV::00 plants, but it also had a quick low afterward (Figure 7f). The results suggested that the expressions of defense-related genes were broke in silenced cotton plants.

Overexpression of GhDEK2D enhances Arabidopsis resistance to Vd
In order to analyze the subcellular localization of GhDEK2D, by heterologous expression in tobacco leaves, we observed GhDEK2D was located on the cell nucleus of tobacco cells (Figure 8a). The resistance of overexpression of GhDEK2D transgenic plants to Vd was assessed with an in vitro technique. GhDEK2D-overexpressing Arabidopsis were more resistant to Vw than the WT plants (Figure 8b,c). These results indicated that GhDEK2D overexpression enhances Arabidopsis resistance to Vd.

Disscussion
Chromatin plays a major role in the regulation of DNAdependent processes. DEK as an architectural chromatin protein, which is associated with DNA, chromatin, and histone binding as well as DNA-folding activities. 51 in Arabidopsis thaliana, there are four DEK genes, they are named DEK1, DEK2, DEK3, and DEK4, respectively. 52 In  G. hirsutum, six DEK proteins were named GhDEK1A, GhDEK1D, GhDEK2A, GhDEK2D, GhDEK4A, and GhDEK4D. The 60 DEK proteins were used for constructing a phylogenetic tree from 14 species. The same species proteins were clustered together, such as: SlDEKs, AtDEKs, LuDEKs, and so on (Figure 1). In cottons, gene structure, conserved motifs and domain of DEKs were semblable ( Figure 3). The results suggested DEKs were conserved at the evolution.
Post-translationally can regulate a lot of plant receptors and critical signaling nodes, so that plant can rapidly and appropriately respond to biotic stresses. Protein is phosphorylated on serine, threonine, and tyrosine residues, which is a reversible reaction. 53 Phosphorylation is major defense mechanism for controlling plant immune. 14 The phosphorylation of WRKY33 upregulates camalexin biosynthesis in Arabidopsis upon pathogen infection. 54 MPK3 and MPK6 phosphorylated ERF72 and improved its transactivation activity, resulting in increased camalexin concentration and increased resistance to Botrytis cinerea. 55 The DEK (human chromatin protein) is phosphorylated by the protein kinase CK2. 56 Acetylation of DEK affects the sub-nuclear localization and the ability to bind DNA. 57 Fbxw7 acts as a tumor suppressor targeting multiple transcriptional activators and may target DEK for degradation to influence murine intestinal homeostasis and cancer by DEK ubiquitination. 58 The intricate network of phosphorylation, acetylation, and ubiquitination mediated plant immunity had been reported. However, phosphorylation, acetylation, and ubiquitination of DEK hasn't been studied. In this study, GhDEK2D had been phosphorylated, acetylated, and ubiquitylated under Vd challenge. As a positive regulator disease-resistant protein, GhDEK2D activated immunity by phosphorylated in cotton-resistant cultivar, and it was influenced by ubiquitylated in cotton-susceptible cultivar. GhDEK2D acetylation was activated in susceptible cultivar one and two days after inoculation, however, the function of DEK acetylation was unclear. The network of GhDEK2D phosphorylation, acetylation, and ubiquitination still needs further research.
In GhDEK2D-silenced cotton plants, the disease index was increscent ( Figure 5), and the accumulation of ROS and callose in leaves, and xylogen in stems were reduced ( Figure 6). ROS is an early events responses and a signal that activates downstream biochemical reaction. 59 Deposition of callose at the cell wall is used in response to stress. 60 Xylogen is a physical mechanism of plant stress resistance. 61 Nitric oxide associated factor (NOA) is related to nitricoxide synthesis pathway, and indirectly regulates nitric oxide (NO) accumulation and response to stress. 62,63 HIN1 and HSR203J are another specific marker gene for HR cell death. 64,65 In the current study, the expression of GhNOA1, GhHIN1, and GhHSR203J were inhibited in silenced cotton plants (Figure 7a,b,c). PAL, POD and PPO as defense enzymes to involve in the formation of barriers against pathogens, 66-68 the expression of disease-resistant genes in GhDEK2D silenced cottons were significantly decreased after Vd inoculation (Figure 7d,e,f). These changes lead to Vd was more likely to infect silenced cotton.
In Arabidopsis, DEK plays a central role in balancing the response between growth and arrest. 69 Arabidopsis DEK3 and DEK4 facilitate the transcription of key flowering repressors by interacting with chromatin, and prevent precocious flowering. Tomato DEK involves in defense response regulation to Botrytis cinerea and Pseudomonas syringae. 27 Overexpression  The GhDEK2D was transiently expressed in tobacco leaves, GFP and DAPI staining were observed using confocal microscopy. GhDEK2D was located on cell nucleus of tobacco cells. DAPI is a cell nucleus-specific fluorescent probe. Scale bar, 50 μm. Symptoms of wild-type (WT), OE-GhDEK2D plants in nutrient soil at three weeks (b) after inoculation with Vd. The disease index (c) and disease levels (d) of WT and transgenic plants. Error bars indicate the SD of three biological replicates. The data represent the mean ± SD, n = 3 (biological replicates) (*p < .05; **p < .01, t test).

e2024738-10
of GhDEK2D enhances Arabidopsis resistance to Vd. However, the molecular mechanism of GhDEK2D against pathogen, which we need do in the future.

Disclosure statement
No potential conflict of interest was reported by the author(s).