Unveiling Cotton's Defense: Harnessing GthZIM17-1 Inhibition for Verticillium Wilt Resistance

Unveiling Cotton's Defense: Harnessing GthZIM17-1 Inhibition for Verticillium Wilt Resistance | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Unveiling Cotton's Defense: Harnessing GthZIM17-1 Inhibition for Verticillium Wilt Resistance Mengying Yang, Richard Odongo Magwanga, Yuqing Hou, Muhammad Jawad Umer, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4517860/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Verticillium wilt (VW) is one of the major biotic stress factors in cotton production, causing a significant reduction in yields and quality reduction. Even though extensive molecular research has been carried out on V. wilt, the molecular basis of Verticillium dahliae host response has not been extensively explored. In this research work, the ZIM17 , a zinc finger motif protein, was investigated through genome-wide identification, and forward and reverse gene functional analysis to explore the role of ZIM17 in six cotton germplasms. Based on the transcription data, GthZIM17-1 was further explored through Virus-Induced gene silencing (VIGS), overexpression, and protein-protein interaction. Results A total of 23 ZIM17 genes were identified across the six cotton species, and were phylogenetically grouped into three clusters, designated A, B, and C. The entire gene family was characterized by Motif 1 and 3. The knockdown of the novel gene, GhZIM17-4 , revealed significantly enhanced resistance to V. wilt due to increased lignification with significantly low DAB staining, moreover, the overexpressed (OE) Arabidopsis thaliana , recorded the disease index (DI) percentage above 70% and above compared to the wild type. Moreover, disease-resistant genes GhPR1 , GhPR3 , and GhPDF1,2 were significantly upregulated in the VIGS-plants compared to the none VIGS-plants. Conclusion The findings therefore provide proof that the ZIM17 gene family plays an integral role of promoting Verticillium wilt, and suppression of its expression in the elite cotton cultivars will contribute significantly in reducing the V. Wilt infection, thereby improving the yield levels in cotton. Moreover, the ZIM17 has a homologous gene type in yeast, thus knockdown of the novel gene in cotton, has a similar effect to that of host-induced gene silencing (HIGS) mechanism. Verticillium wilt (VW) HIGS Disease index Overexpression (OE) ZIM17 Zinc finger motif protein Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1 Background Verticillium wilt is a soil-borne plant pathogenic fungus with a wide host range, so found to infect over 200 dicotyledonous plants, such as cotton [ 1 – 3 ], potato [ 4 , 5 ], and tomato [ 6 ]. The fungus is not restricted to only field crops, but also infects vegetables, fruits and ornamental plants species [ 7 ]. Verticillium wilt was first discovered in the United States of America (USA) in 1915, and first reported in China, twenty years later, in 1935, since then, it has had a global distribution, causing massive agricultural loss globally [ 8 ]. There six species of the pathogenic fungi however, two are known to be highly virulent, the Verticillium dahliae and Verticillium albo-atrum [ 9 ], the others species includes V. tricorpus , V. nigrescens , V. nubilum , and V. theobroma [ 10 ]. In China, the main pathogenic fungi is the Verticillium dahliae , V. dahliae is a semi-living nutrient pathogen, that can survive in the form of microsclerotia in the soil for many years, moreover, the microsclerotia are extremely adaptable to extremely harsh environments, and easily dispersed by wind or rain, which makes it difficult to control [ 11 ]. The longevity, effective disposal mechanism, and highly adaptive nature to harsh environmental conditions, have made the various control measures to be ineffective, furthermore, there is no effective fungicide that can be used in controlling V. dahliae infection in plants. Furthermore, the diseases caused by the V. dahliae is related to the human immunodeficiency virus (HIV), once the plants are infected, then no remedial measure. The two virulent forms of Verticillium spp . do cause plant diseases in conjunctions with other pathogenic organisms such as bacteria, nematodes and other forms of fungi [ 12 ]. The major symptoms of Verticillium wilt in plants includes necrosis, stunting, chlorosis, vascular discoloration, premature senescence, night recovery and wilting [ 13 ]. In cotton, V. wilt infection causes chlorosis, stem dryness, and root rot, which negatively affects the yield and quality of cotton [ 14 ]. In China, the economic loss due to V. wilt is estimated at 250 to 310 million US dollars annually [ 15 ]. Cotton production in China has transformed the economy and lives of millions of Chinese [ 16 ]. As an important economic crop, the yield and quality of cotton have been significantly affected by this terminal fungal infection, V. dahliae . Several attempts have been made to develop resistant crops to V. wilt, for instance, the use of biocontrol has been attempted, in which Verticillium isaacii Vt305 has been employed to play an antagonistic role to V. dahliae , however, the success level was very low due to different soil environments [ 17 ]. Moreover, conventional agronomic methods have been adopted, by intercropping cotton with either garlic and or onion, the level of V. wilt disease index was observed to be significantly reduced [ 18 ], however, this method is not sustainable on a large scale, where mechanization is required. Due to the nature of V. dahliae infection, the most suitable method is the utilization of resistant cultivars, so breeding for resistant crop varieties will contribute significantly to controlling Verticillium dahliae . Utilization of genes to confer tolerance to V. wilt has shown positive effects, the overexpression of GhIQD1 gene, which acts as a calmodulin-binding protein improved cotton resistance to V. wilt [ 19 ], moreover, a group of glutathione S-transferase (GST) genes, were found through transcriptome analysis to confer tolerance to V. dahliae infection [ 20 ]. However, research has shown that RNA interference (RNAi)-based host-induced gene silencing (HIGS) boost plants resistance to diseases by silencing essential genes for inducing pathogenicity, for instance, the silencing of V. dahliae gene encoding an exoglucanase (VdEXG, VDAG_02898) had positive effect on enhancing tolerance to V. dahliae [ 21 ]. The genetic diversity of cotton germplasm, has undergone drastic erosion, leading to massive narrowing of the genetic diversity [ 22 ]. The narrowing of the genetic base of the elite cotton has been worsened due to inbreeding and intensive selection for favorable agronomic traits [ 23 ]. In order to broaden the ever shrinking genetic diversity of the cultivated cotton, introgression of genes from the wild progenitor’s is key [ 24 ]. The wild cotton progenitors are the reservoirs for beneficial genes, more commonly referred to are the resistance (R) genes, for instance, Magwanga et al [ 25 ] evaluated Gossypium tomentosum in the identification of drought and salt stress genes. G. tomentosum is a wild progenitor of the elite cotton cultivars, furthermore, G. thurberi has been found to be highly resistant to V. dahliae infection and therefore survive under V. dahliae infested areas [ 26 , 27 ]. In this study, a novel gene, a ZIM17 (Zinc finger motif 17) was identified. The ZIM17 also known as TIM15 (translocase in the inner mitochondrial membrane) or Hep1 (MtHsp 70 escort protein 1) is a specific protein chaperone for mitochondrial 70 kDa heat shock protein (MtHsp 70) [ 28 ]. This gene has been extensively studied in yeast where it has been found to promote yeast cell growth, yeast being a fungus, its inhibition could have a positive effect in inhibiting the proliferation of V. dahliae , thereby reducing its virulence ability to cause V. wilt in cotton. Moreover, as a core component of the mitochondrial chaperone protein system, MtHsp70 not only provides ATP-driven force for polypeptide translocation reactions but also participates in the protein folding processes [ 28 , 29 ]. As a chaperone protein for MtHsp70, ZIM17 maintains the solubility of MtHsp70, prevents the self-aggregation of Hsp70 chaperone proteins SSC1 and SSQ1, and promotes the disassembly of aggregated proteins, thereby maintaining its function in mitochondrial protein import and Fe/S protein biosynthesis. Both in vivo and in vitro, ZIM17 assists MtHsp70 in importing proteins into the mitochondria [ 30 ]. However, there are currently no reports on the role of ZIM17 in cotton resistance to V. dahliae . In this study, a whole genome-wide identification and functional analysis of the ZIM17 genes were carried out. Moreover, the availability of the D genome sequence provides a perfect platform for the evaluation of the ZIM17 gene family. Moreover, forward and reverse genetics were employed in the model plant Arabidopsis thaliana and cotton, respectively to understand the putative role of the Gth ZIM17 -1 gene, further yeast two-hybrid and luciferase complementation imaging assays were conducted to screen and verify interacting proteins of Gth ZIM17 -1. The results provide fundamental steps to enhance research by exploiting the RNA interference (RNAi)-based host-induced gene silencing mechanisms in developing cotton germplasms which are highly versatile and resistant to various forms of biotic stress factors. 2 Material and methods Data acquisition, identification, and physicochemical analysis of ZIM17 family members in six cotton species. The whole genome data of Gossypium herbaceum (A 1 ) [ 31 ], G. arboreum (A 2 ) [ 32 ], G. thurberi (D 1 ) [ 33 ], G. raimondii (D 2 ) [ 34 ], G. hirsutum (AD) 1 [ 35 ], and G. barbadense (AD) 2 [ 36 ] from the Cottongen website ( https://www.cottongen.org/ ). A genome-wide blast was performed in the six cotton species using TBtools v1.106 [ 37 ] with Arabidopsis thaliana ZIM17 protein sequences downloaded from TAIR ( https://www.arabidopsis.org/ ) as reference sequences. The Hidden Markov Model (HMM) PF05180 of the ZIM17 family from Pfam ( https://pfam.xfam.org/ ) was downloaded. The candidate proteins were merged and deduplicated, and NCBI-CDD ( http://www.ncbi.nlm.nih.gov/Stru-cture/cdd/wrpsb.cgi ) was used to verify that all the sequences contained two conservative domains, zf-DNL , or zf-DNL superfamily. The physicochemical properties of the ZIM17 protein were further analyzed by predicting the amino acid number (aa), relative molecular weight (mw), isoelectric point ( pI ), and instability index of the protein using the ProtParam tool ( Expasy - ProtParam tool ) Phylogenetic tree construction of the ZIM17 genes family To construct the phylogenetic tree of the ZIM17 proteins in cotton, multiple sequence alignments of the ZIM17 protein sequence using the ClustalX tool in MEGA-X software were done [ 38 ]. The phylogenetic tree was constructed using the linkage method with a bootstrap set at 1000, using a p-distance model. The original file from MEGA was then exported to the EvolView website ( https://www.evolgenius.info/evolview/ ) for the visualization of the phylogenetic tree. Gene structure and conservative motif analysis of the ZIM17 genes in cotton The gene structure and conservative motif of the ZIM17 genes in cotton were analyzed by visualizing the exon-intron structure of each gene using the location information of family members on the TBtools software [ 37 ]. The conserved motif of the ZIM17 protein was identified through an online tool MEME ( http://www.meme-suite.org/ ) with a motifs number set at 12. The output file in XML format was downloaded and visualized through the TBtools [ 37 ]. Chromosome mapping of ZIM17 genes in different cotton species The location of ZIM17 genes on chromosomes was mapped by extracting the location of the genes on chromosomes using the TBtools [ 37 ] and mapped as per the genome and genome annotation information. Analysis of cis- acting elements of ZIM17 genes in different cotton species To analyze the cis- regulatory elements of the cotton ZIM17 ’s was analyzed from the upstream 2000 bp fragments. The cis- regulatory elements of the gene promoter region were predicted using an online tool, the PlantCARE ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ), and visualized using the TBtools [ 37 ]. Plant materials and growth The wild species G. thurberi and the cultivated species Zhongzhimian 2 were utilized. The two cotton species seeds were obtained from the wild cotton germplasm research group of the Institute of Cotton Research (ICR), Anyang, China. After de-fuzzing, cotton seeds were germinated using the sand culture method. Four Defuzzed seeds were germinated in boxes filled with sterilized wet sand, and a loose layer of sand approximately 1 cm thick was applied over the seeds. The boxes were then placed in a dark incubator at 29°C for 48 hours. Cotton seedlings with uniform growth were subsequently transplanted into pots filled with nutrient soil and grown in a greenhouse maintained at a constant temperature of 25°C with a 16-hr light/8-hr dark photoperiod. Pathogen cultivation and plant inoculation The fungal strain Vd991 was inoculated onto a potato dextrose agar (PDA) medium and cultured in darkness at 25°C for 7 days to achieve activation. Subsequently, 4–5 activated fungal blocks were aseptically excised using a sterile surgical blade and transferred to Czapek's medium containing streptomycin sulfate (0.05 g/ml). These blocks were further incubated in a shaking incubator at 25°C, 200 rpm/min, under dark conditions for 7 days. The fungal suspension was then filtered through four layers of sterile gauze, and the spore concentration was diluted to 1×10 7 cfu/ml using sterile double-distilled water (ddH 2 O) for subsequent use. RNA extraction and qRT-PCR analysis Plant tissues were collected in aluminum foil and rapidly frozen in liquid nitrogen. Using a mortar and pestle, the tissues were quickly ground into a fine powder, with timely replenishment of liquid nitrogen to prevent sample oxidation. Total RNA was extracted from cotton leaf tissues using the RNAprep Pure Plant Kit (TIANGEN, Beijing, China), with detailed extraction steps outlined in the kit's manual. The quality and quantity of the extracted RNA was determined through agarose gel electrophoresis. Reverse transcription was performed using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGene, Beijing, China). One microgram of RNA was used as the template, Real-time quantitative PCR experiments were conducted using the ABI 7500 Real-Time PCR System. Cotton GhUBQ7 and Arabidopsis AtACTIN2 genes were used as reference genes (Table S1 ), and the relative expression levels of different genes were calculated using the 2 −ΔΔCT method. Each sample had three biological and technical replicates. Subcellular localization of Gth ZIM17 -1 To validate the subcellular localization prediction, the Gth ZIM17 -1 618 bp fragment, excluding the stop codon, was cloned into the subcellular localization vector p2300-eGFP -Flag using the selected double restriction enzyme sites of Xba I and BamH I , resulting in the subcellular localization expression vector p2300-eGFP-Gth ZIM17 -1. The primer sequences are provided in (Table S1 ). The Agrobacterium tumefaciens strain harboring the subcellular localization vector p2300-eGFP-flag, preserved at -80°C, was activated by incubating in a 28°C shaking incubator for 2 hours. Subsequently, the strain was transferred into 50 mL of LB liquid medium containing Kana (50 mg/L) and Rif (50 mg/L) and cultured in a shaking incubator at 28°C and 200 rpm/min for 24–48 hours. Once the optical density measurements (OD) at a wavelength of 600 nm (OD600) of the Agrobacterium culture reached 1.0, the bacterial cells were collected by centrifugation and resuspended in a resuspension solution to an OD600 of approximately 1.0. The resuspended solution was incubated in the dark for 2 hours. Using a 1 mL sterile syringe, the bacterial suspension was injected into tobacco leaves. The tobacco plants were then cultured in the dark for 48 hours and observed under a laser confocal microscope. Virus-induced gene silencing (VIGS) of Gh ZIM17 -4 Using the cDNA of Zhongzhimian 2 as a template, specific primers for the VIGS 227 bp fragment were designed using Primer Premier 5 software (Table S1 ). The virus-induced gene silencing vector pTRV2 was double-digested using the enzyme EcoRI and BamHI digestion system. The vector pTRV2-GhZIM17-4 was obtained and then transformed into Agrobacterium tumefaciens LBA4404 and infiltrated in the cotton cotyledons. The infected plants were cultured in the dark for 24 hours before being transferred to a greenhouse for normal light conditions. Approximately two weeks later, the positive control plants exhibited albinism. Observations of disease development in cotton plants were made 20 days after inoculation. Photographs were taken, and disease severity indices were recorded at 25 days’ post-inoculation. The disease severity of seedlings was classified into five grades (0, 1, 2, 3, 4) according to the following criteria: Grade 0, healthy plants; Grade 1, 1%-33% diseased leaves; Grade 2, 34%-66% diseased leaves; Grade 3, 67%-99% diseased leaves; Grade 4, entire plant with diseased leaves or even dead and fallen leaves. The disease index (DI) was calculated using the formula: DI = [Σ (grade × number of plants at that grade) / (total number of plants × highest grade of 4)] × 100 [ 39 ]. Overexpression of Gth ZIM17 -1 gene in Arabidopsis thaliana and inoculation of the V. dahliae At the flowering stage of the wild-type A. thaliana , the pods and pollinated flowers that had already formed were trimmed off ahead of time. Wild-type Arabidopsis was then infected using the floral-dip method, with the buds fully immersed in the suspension containing the pCAMBIA2300-GthZIM17-1 recombinant vector for 1 minute. Following the infection, the plants were cultured in the dark for 24 hours. To enhance the infection efficiency, a second infection was conducted one week after the first infection. The obtained seeds were selected on 1/2 MS medium containing 50 mg/mL kanamycin, and homozygous lines were confirmed through RT-PCR and qRT-PCR until the T2 generation. Subsequent experiments were conducted on the plants with high expression levels. Before sowing, A. thaliana seeds underwent sterilization and disinfection. Initially, the seeds were treated with 75% ethanol for 3 minutes and rinsed three times with sterile ddH 2 O. Subsequently, they were sterilized with 15% NaClO solution for 5 minutes and rinsed five times with sterile ddH 2 O. Sterilized Arabidopsis seeds were then sown onto 1/2 MS medium, vernalized, and transferred to a constant temperature incubator maintained at 22°C with a 16-hour light/8-hour dark photoperiod. After approximately two weeks of growth on the medium, Arabidopsis plants were transplanted into pots filled with nutrient soil and grown in a greenhouse maintained at a constant temperature of 25°C with a 16-hour light/8-hour dark photoperiod for two weeks, before being inoculated by the spores of V. dahliae . Stem section detection, Histochemical staining of cotton stem lignin, DAB staining, and fungal recovery assay 2.1.1 Stem section detection On the 25th day after inoculation of cotton with V. dahliae , stem tissues from the same above-ground locations of both control and silenced plants were randomly selected and longitudinally sectioned using a disposable blade. These sections were then observed under a stereomicroscope to assess vascular bundle occlusion and browning. 2.1.2 Histochemical staining of cotton stem lignin On the 25th day post-inoculation, stem tissues from the same locations of control and silenced cotton plants were cross-sectioned using the hand-slicing method. The sections were placed on slides and promptly stained with a drop of 3% phloroglucinol solution to ensure complete immersion. After 10 minutes, the sections were incubated in 6% hydrochloric acid for 5 minutes, rinsed twice with ddH 2 O, and then a drop of ddH 2 O was added to prevent dehydration and wrinkling. These stained sections were observed under a stereomicroscope. 2.1.3 3,3'-diaminobenzidine (DAB) staining On the 25th day of post-inoculation, cotton true leaves were randomly selected and immersed in an adequate amount of 3,3'-diaminobenzidine (DAB) staining solution. The leaves were incubated in the dark at room temperature for 8 hours. Subsequently, the DAB solution was removed, and the leaves were repeatedly decolorized with 95% ethanol until the chlorophyll was completely removed. After washing with sterile water, the leaves were photographed for observation. 2.1.4 Fungal recovery assay For the isolation of cotton stem segments, they were randomly selected from the same above-ground locations and sterilized in a laminar flow hood. This involved a 40-second immersion in 75% ethanol, followed by a 3-minute sterilization with 3% sodium hypochlorite. The segments were then rinsed three times with sterile ddH 2 O. Using a sterile surgical blade, the cotton stems were cut into approximately 0.5 cm segments and evenly placed on a potato dextrose agar (PDA) medium. Then were incubated in the dark at 25°C for 5–7 days. Yeast two-hybrid assays The full-length CoDing Sequence (CDS) of the target gene was cloned into the pGBKT7 vector, with EcoRⅠ and BamHI chosen as the double restriction enzyme sites, the yeast bait vector pGBKT7-Gth ZIM17 -1 was then obtained. The bait and prey plasmids were then co-transformed into yeast Y2H Gold competent cells using the co-transformation method for yeast two-hybrid point-to-point interaction verification. Firefly luciferase complementation imaging (LCI) assays The coding regions of Gth ZIM17 -1 and GthMOS4 sequences were cloned onto the pCAMBIA1300-35S-cLUC and pCAMBIA1300-35S-cLUC vector respectively (Table S1 ). Equal amounts of Agrobacterium cultures containing CLuc and NLuc constructs were mixed and then co-infiltrated into tobacco leaves. Before observation, tobacco leaves were picked and applied with 20 µL luciferase substrate (1 mmol/L D-luciferin sodium salt) in the injection area, and placed in a dark environment for 10 minutes. The leaves were placed under a fluorescence microscope to observe the fluorescence signal and take photos. All experiments were repeated at least three times for each plasmid combination. Data analysis All experimental data were analyzed; the mean values were obtained for at least three independent biological replicates. Data were analyzed using SPSS software, and statistical comparisons of differences were analyzed for significance of multiple data using ANOVA statistics, and plotted using GraphPad Prism 9.0. 3 Results Identification and analysis of the physiochemical properties of the ZIM17 gene family Three ZIM17 family members were identified in A. thaliana . Using the protein sequences for the Arabidopsis, a Blast comparison analysis was conducted across the 6 cotton species. Based on sequence similarity and conserved domains, a total of 11 ZIM17 genes were found for the 4 cotton species of the D genome, in which 2, 3, 3, and 3 ZIM17 family members were identified for G. herbaceum , G. arboreum , G. thurberi , and G. raimondii , respectively. Interestingly, among the tetraploid cotton G. hirsutum and G. barbadense , they harbored a similar number of the ZIM17 genes, with 6 ZIM17 family members in each. Based on the chromosomal location, the genes were annotated as follows; Gher ZIM17 -1-2 ( G. herbaceum ), Ga ZIM17 -1-3 ( G. arboreum ), Gth ZIM17 -1-3 ( G. thurberi ), Gr ZIM17 -1-3 ( G. raimondii ), Gh ZIM17 -1-6 ( G. hirsutum ), and Gb ZIM17 -1-6 ( G. barbadense ). Analysis of the physicochemical properties of the ZIM17 s revealed that the lengths of their open reading frames (ORFs) ranged from 447 to 621 bp, with amino acid (aa) counts varying from 148 to 206, molecular weights (MW) ranging from 16415.71 to 22861.54, isoelectric points ( pI ) from 6.06 to 8.63, and instability indices from 35.39 to 51.96 (Table 1). Over 70% of the ZIM17 proteins had instability indices above 40, an indication that ZIM17 proteins were significantly unstable, a common attribute of the heat shock proteins. Moreover, pI values showed that nearly all the proteins were nearly neutral, being the pI values ranging from 6.06 to 8.63 with only one with a pI value of 5.77, which further augments that their possible function within the membranes, mainly as heat shock proteins or chaperones. Table 1. Physiochemical properties of the ZIM17 proteins from the 6 cotton species. G. herbaceum , G. hirsutum , G. barbadense , G. arboreum , G. thurberi and G. raimondii . Gene Annotation Transcript ID ORF length (bp) Length (aa) Genomic location MW (Da) pI Instability index Subcellular localization GherZIM17-1 Ghe01G11880 621 206 Chr01:21275809–21276762+ 22755.42 6.06 37.75 Nucleus GherZIM17-2 Ghe05G32290 567 188 Chr05:39580088–39582567+ 20638.29 6.89 51.96 Chloroplast GaZIM17-1 Ga01G1171 621 206 Chr01:22307888–22308839+ 22767.47 6.06 40.3 Nucleus GaZIM17-2 Ga05G3179 450 149 Chr05:38291545–38294048+ 16548.97 8.63 43.44 Chloroplast GaZIM17-3 Ga12G1114 567 188 Chr12:12802258-12804577- 20638.29 6.89 51.96 Chloroplast GhZIM17-1 Gh_A01G1063 621 206 A01:20563176–20564123+ 22755.42 6.06 37.75 Nucleus GhZIM17-2 Gh_A05G2989 567 188 A05:36820554–36823031+ 20624.26 6.89 51.25 Chloroplast GhZIM17-3 Gh_A12G1915 450 149 A12:95617088–95619401+ 16532.97 8.63 43.25 Chloroplast GhZIM17-4 Gh_D01G1103 621 206 D01:16273743–16274691+ 22802.47 5.77 35.39 Nucleus GhZIM17-5 Gh_D05G3004 543 180 D05:30795668–30798118+ 19917.58 7.65 44.79 Chloroplast GhZIM17-6 Gh_D12G1914 447 148 D12:50817799–50820275+ 16444.75 6.5 47.76 Chloroplast GbZIM17-1 Gbar_A01G010000 621 206 A01:20316329–20317682+ 22755.42 6.06 37.75 Nucleus GbZIM17-2 Gbar_A05G029230 567 188 A05:35881431–35884182+ 20638.29 6.89 51.96 Chloroplast GbZIM17-3 Gbar_A12G018450 450 149 A12:90166096–90168769+ 16534.94 8.63 46.39 Chloroplast GbZIM17-4 Gbar_D01G010540 621 206 D01:16744844–16746191+ 22889.6 6.41 40.12 Nucleus GbZIM17-5 Gbar_D05G030090 561 186 D05:31265680–31268403+ 20457.17 7.65 45.74 Chloroplast GbZIM17-6 Gbar_D12G018590 447 148 D1248731799-48734648+ 16458.77 6.5 47.76 Chloroplast Phylogenetic analysis of the ZIM17 gene family To further investigate the evolutionary relationships among the ZIM17 family members, a phylogenetic tree was constructed using the 23 ZIM17 genes were identified from the 6 cotton species along with the three ZIM17 genes identified from the A. thaliana . Based on their evolutionary relationships, these family members were divided into three groups, designated as groups 1, 2, and 3 with 9, 8, and 9 ZIM17 proteins, respectively. In all the three groups, the protein distributions were nearly even, with a node value of 100, an indication that the grouping was a perfect fit. In all the three groups, gene pairing was observed, however, there was a unique observation was noted among the members of groups B and C, in which two homologous pairs were noted between Gr ZIM17 -3 and Gb ZIM17 -6 (group B) and Gb ZIM17 -2 and Ga ZIM17 -2 (Group C), these genes shared a common origin, however, ortholog gene pairs were also noted between G. hirsutum and G. barbadense in all the three groups, in group A (Gh ZIM17 -1 and Gb ZIM17 -1; Gr ZIM17 -1 and Gth ZIM17 -1), Group B (Gh ZIM17 -3 and Gb ZIM17 -3) and lastly Group C (Gh ZIM17 -5 and GbZIM71-5) (Fig. 1 ). The results showed that the genes were recently evolved and highly correlated. Plants do evolve genes to enhance their survival or mitigate the effects of the environmental stresses, thus the high threshold and infection intensity of V. dahliae might have led to this kind of evolution. Chromosomal mapping of the cotton ZIM17 genes To understand the distribution of ZIM17 gene family members on the chromosomes in different cotton species, a visualization tool was used by integrating the start and the end. The results showed that in the diploid A-genome cotton species, G. arboreum (A2), the ZIM17 genes were mapped on chromosomes 1, 5, and 12, however in G. herbaceum (A 1 ), with only 2 genes, the 2 genes were mapped on chromosome 1 and 5, the 2 diploid cotton species of the A sub genome showed consistency, though the reduction in gene number in G. herbaceum (A 1 ) is evident of gene loss. Among the cotton species of the D subgenomes, G. raimondii (D 5 ) and G. thurberi (D 1 ), had 3 genes, and were mapped in three different chromosomes, however, G. raimondii (D 5 ) chromosome mapping was in synchrony to the gene mapping in G. arboreum (A 2 ), in which the 3 genes were mapped on chromosome 1, 5 and 12 (Fig. 2 A i-ii), but on chromosome 2, 8 and 9 in G. thurberi (D 1 ) (Fig. 2 B i-ii), the deviation in chromosome mapping among the genes obtained for the D subgenomes could be attributed to chromosomal rearrangement in the course of their evolution. Moreover, consistency was observed among the AD genomes, G. hirsutum (AD) 1 and G. barbadense (AD) 2 , the 6 ZIM17 genes were mapped on chromosomes A01, A05, A12, D01, D05, and D12 (Fig. 2 C i-ii). This is in agreement with the already known hypothesis that AD emerged due to whole genome duplication between A and D, with specific reference to G. arboreum (A 2 ) and G. raimondii (D 5 ) These findings suggest that there may be conserved syntenic regions among different cotton species about the chromosomal distribution of ZIM17 genes. However, the unique distribution pattern observed in G. thurberi indicates that chromosomal rearrangements or gene duplications may have occurred during the evolution of this species, leading to variations in the chromosomal locations of ZIM17 genes. Understanding the chromosomal distribution of ZIM17 genes across cotton species can provide insights into their potential roles in cotton development and the evolution of gene families in plants. Analysis of gene structure and conserved motif of the ZIM17 gene family To further analyze the structure of the ZIM17 gene family, the gene structure and conserved motifs across the six species were examined. A total of 9 conserved motifs were identified and named Motif 1 to Motif 9 (Fig. 3 A). The results revealed that the number of conserved motifs within each group was generally consistent. However, there were some exceptions. For instance, Gth ZIM17 -1 in group A and Gth ZIM17 -2 in group B harbored an additional Motif 9 compared to other members, while Ga ZIM17 -2, Gb ZIM17 -2, Gher ZIM17 -2, and GH ZIM17 -2 in group C had an extra Motif 8 compared to the rest of group members. Notably, Motif 1 (TPISLLTKSPRRDLAVAFTCNVCGERTVRAINR EAYEKGVVFVQCGGCNNFHKJADNLGLF) and Motif 3 (PAISSFNVSRBSRYDVYRRLFDEEP D) were present across all the groups, an indication that the ZIM17 gene family could be identified and characterized by motif 1 and motif 3moreover, Motif 2 was exclusive to Group A, Motif 4 and Motif7 were unique to Group B, and Motif 5 and Motif 6 were specific to Group C. In relation to their gene structures, all the ZIM17 gene family contained introns and exons, with the number of exons ranging from 2 to 4 (Fig. 3 B). The number of exons within each group was consistent, with members of group A and group B having 4 exons, while members of group C had 2 exons. Furthermore, the distribution pattern of introns and exons within ZIM17 genes was highly uniform within each group. These findings suggest that ZIM17 gene family members share conserved structural features but also exhibit specific variations that may contribute to their functional diversity. The identification of conserved motifs and the analysis of gene structures provide insights into the evolution and potential functions of ZIM17 genes in cotton and potentially other plant species. Analysis of cis- acting elements of the ZIM17 gene To better understand the functional roles of the ZIM17 genes, 2000 bp upstream sequences of the coding regions were used for the prediction of the cis- acting elements. The results showed that the most abundant cis- acting elements in the upstream regions of ZIM17 genes were light-responsive elements. Additionally, several elements related to hormone and stress responses were also identified, including Abscisic acid (ABRE), jasmonic acid (CGTCA-motif and TGACG-motif), salicylic acid (TCA-element), drought (MBS), low temperature (LTR), metabolism (O2-site), and defense and stress (TC-rich repeats) response elements (Fig. 4 ). Further analysis revealed differences in the number and distribution of cis- acting elements among groups with different developmental branches. Compared to groups B and C, members of group A possessed a higher number of stress-responsive elements. Notably, defense and stress response elements were absent in both groups A and B, while low-temperature response elements were absent in groups B and C. Additionally, members of group B lacked Abscisic acid response elements, and members of group C did not contain MYB drought stress response elements. These findings suggest that ZIM17 genes may be involved in diverse biological processes, including light response, hormone signaling, and stress responses. The variations in the number and types of cis- acting elements among different groups indicate that ZIM17 genes may have specific functional roles that are tailored to the unique environmental conditions and developmental stages of the cotton species. Analysis of Gth ZIM17 -1 gene expression pattern To analyze whether the Gth ZIM17 -1 gene exhibits tissue-specific expression in G. thurberi , RNA samples were extracted from the roots, stems, and leaves of this cotton plant. Using the reverse-transcribed cDNA as a template, quantitative real-time PCR (qRT-PCR) was carried out to detect the expression levels of Gth ZIM17 -1 in different tissues. The results revealed that the Gth ZIM17 -1 gene was primarily expressed in the roots and leaves of G. thurberi , with relatively lower expression levels in the stems (Fig. 5 A). To investigate whether the Gth ZIM17 -1 gene responds to Verticillium wilt stress in G. thurberi , tissues were collected at time points (0h, 12h, 24h, and 48h) after inoculation with Verticillium dahliae . The results showed that within 48 hours of inoculation with V. dahliae , the expression of the Gth ZIM17 -1 gene exhibited a trend of initial increase followed by a decrease (Fig. 5 B). This significant change in expression suggests that Gth ZIM17 -1 responds to Verticillium wilt, being a negative regulator, the higher expression at initial stages could be to enhance the proliferation of the vegetative tissues of the V. dahliae , thus increasing their pathogenicity. Subcellular localization of the Gth ZIM17 -1 protein The ZIM17 protein serves as a mitochondrial protein import factor, and prior research indicates that the ZIM17 protein is primarily expressed and functions within mitochondria [ 28 ]. To corroborate its expression in mitochondria, we constructed a subcellular localization fusion vector, p2300-eGFP-GthZIM17-1 , tagged with a green fluorescent protein. This vector was co-transfected with a mitochondrial marker into tobacco cells, and the cellular expression of the gene was observed under a laser scanning confocal microscope. The results demonstrated that the Gth ZIM17 -1 protein is expressed in the nucleus, cytoplasm, and mitochondria (Fig. 6 ), indicating a broad expression profile within the cell and suggesting potential diverse functional roles within the plant. Gh ZIM17 -4 silencing enhanced the resistance of cotton to Verticillium dahliae To investigate the disease resistance function of the GthZIM17-1 gene in cotton, specific primers were designed based on the CDS sequence of Gh ZIM17 -4, the homolog of Gth ZIM17 -1 in Gossypium hirsutum. Two weeks after VIGS injection, positive control plants (TRV: PDS) exhibited an albino phenotype (Fig. 7 A), indicating the successful establishment of the virus induced gene silencing (VIGS) system. True leaves from three randomly selected silenced plants were collected, and the silencing efficiency was assessed using qRT-PCR. The expression levels of Gh ZIM17 -4 in the silenced plants were significantly reduced (Fig. 7 B), indicating successful interference of the gene. Under V. dahliae , the leaves of the empty vector plants exhibited more severe yellowing and wilting and had a higher disease index. Phloroglucinol staining of the stems showed more severe browning of the vascular bundles in the empty vector plants. Additionally, phloroglucinol staining was performed on the same region of the stem, approximately 1 cm above the ground, to observe changes in the vascular bundles. The results indicated less lignin accumulation in the silenced plants (Fig. 7 C-D). DAB staining of the leaves revealed more severe damage in the leaves of the empty vector plants (Fig. 7 E). Recovery experiments with V. dahliae showed less fungal colonization in the stems of the silenced plants (Fig. 7 F). moreover, the disease index (DI) was significantly higher in the none VIGS-plants compared to the VIGS-plants (Fig. 7 G) To further investigate the impact of GhZIM17 -4 gene silencing on the expression levels of genes associated with disease resistance pathways, qRT-PCR was employed to assess the expression profiles of GhPR1 and GhPR5 genes, the hallmark genes of the salicylic acid (SA) resistance pathway, as well as GhPR3 and GhPDF1.2 , genes related to the jasmonic acid (JA) resistance pathway. The results revealed that in silenced plants, the expression of the GhPR1 gene was significantly upregulated, while the expression of the GhPR5 gene was significantly downregulated (Fig. 7 H). Additionally, the expression levels of both GhPR3 and GhPDF1.2 genes were significantly upregulated (Fig. 7 I). The findings were in agreement with previous research in which, silencing of ABHYDROLASE-3 gene in Brassica napus , significantly improved plants tolerance to Sclerotinia sclerotiorum [ 40 ]. Overall, findings suggested that upon interference with GhZIM17-4 gene expression in cotton, the silenced plants exhibited enhanced disease resistance. Furthermore, the GhZIM17-4 gene may regulate the resistance of cotton to Verticillium wilt by modulating the expression of genes associated with both SA and JA resistance pathways. However, the specific disease resistance mechanisms underlying this regulation require further exploration, however, the upregulation of disease-resistant genes in VIGS-plants showed that the downregulation of Gh ZIM17 -4 improved the ability of the VIGS-plants to induct other disease regulatory genes to be mobilized, thereby enhancing their ability to significantly reduce the disease severity in VIGS-plants compared to the non VIGS-plants. These results further validate the notion that host-induced gene silencing improves the plant performance under biotic stress conditions. Overexpression of the Gth ZIM17 -1 gene in Arabidopsis reduced the resistance of Arabidopsis to Verticillium dahliae infection In the transformation of the GthZIM17-1 gene into the model plant, the gene was effectively transformed in 10 plants, designated as OE1 to OE10 (Fig. 8 A). validation of the expression was further confirmed through RT-qPCR in which OE1 and OE10 were found to have the highest level of upregulation of the overexpressed gene (Fig. 8 B). The overexpressed lines, OE1 and OE10 lines were further used to evaluate the OE lines response towards V. dahliae infection. The overexpressing Arabidopsis plants (OE1 and OE10) were transferred to nutrient soil for continued cultivation for one week and then inoculated with 5 µL of V. dahliae spore suspension at the roots. Two weeks later, the disease incidence of the Arabidopsis was observed and recorded. Consistent with the results obtained in the medium, the overexpressing Arabidopsis lines exhibited more severe disease symptoms following infection with V. dahliae (Fig. 8 C-D), with a higher disease severity index (Fig. 8 E-F). The high threshold of the overexpressed gene, accelerated the proliferation of the disease-causing organism, V. dahliae , resulting in enhanced disease index among the OE plants compared to the wild type. The results obtained were in agreement with previous findings in which overexpression of necrosis and ethylene-inducing peptide 1-like Protein (NLP) gene significantly increased the virulence of Verticillium dahliae in eggplant, tomato, and cotton [ 41 ], which, which showed that the increased copy of VdNEP acerbated the virulence factor in plants, thus enhancing their susceptibility to pathogens. Protein-protein interaction, Gth ZIM17 -1 interacts with GthMOS4 Through screening of a yeast library, GthMOS4 was identified to have an interaction with Gth ZIM17 -1. Validation of this interaction using yeast two-hybrid point-to-point assays revealed that the positive control ( pGBKT7-53 + pGADT7-T ) and negative control ( pGBKT7-Lam + pGADT7-T ) grew normally on SD/-Leu-Trp solid medium, indicating successful plasmid co-transformation in yeast. The positive control grew normally and turned the yeast colonies blue on SD/-His-Leu-Trp and SD/-His-Leu-Trp-Ade defective media containing X-α-gal , while the negative control did not grow, confirming the successful establishment of the yeast two-hybrid system. Co-transformed yeast cells expressing MOS4 and DEG15 were able to grow on SD/-His-Leu-Trp and SD/-His-Leu-Trp-Ade solid media and turned the yeast colonies blue, while SDR1 did not grow. This suggested a potential interaction between MOS4 and the ZIM17 -1 protein. Similarly, LUC fusion vectors were constructed containing the target proteins and transiently transformed into tobacco using Agrobacterium. The results showed that only one experimental combination exhibited a distinct fluorescent signal (Fig. 9 A-B). No fluorescent signal was observed in the three control regions of the tobacco leaves. However, a prominent fluorescent signal was detected in the injection area where the combination of pCAMBIA1300-GthZIM17-1-nLUC and pCAMBIA1300-GthMOS4-cLUC was used, indicating an interaction between the ZIM17 and MOS4 proteins. Basically, the MOS4 proteins function as suppressers of the autoimmunity-related phenotypes in plants [ 42 ], thus the positive interaction between ZIM17 and MOS4 is a clear indication that the ZIM17 protein has inhibitory role in enhancing plants tolerance to pathogens. 4 Discussion Cotton being an important economic crop, its production has dwindled over time due to the effects of biotic and abiotic stress factors. Significant efforts have been made to develop ecologically and area-specific cotton germplasms to alleviate the localized effects of environmental factors. However, due to the complex nature of biotic factor, a diverse strategy is required to reduce the effects of various forms of biotic factors. One of the major biotic factor with deleterious effects on cotton is the causative agent for Verticillium Wilt, known as Verticillium dahliae , a soil-borne fungi with the ability to remain dormant over a long period. The utilization of genes to improve cotton performance towards V. dahliae has helped in mitigating its effects. In this research work, the host-induced gene silencing approach was explored to understand the putative role of the ZIM17 gene a member of the plant zinc finger proteins. A ZIM17 is a novel zinc finger protein that has been extensively studied in yeast. Is critical in promoting cell division, cell growth, and proliferation of the yeast tissues, thus essential for the viability of the yeast cells [ 28 ]. Furthermore, ZIM17/TIM5 also known as the HEP1have has been found to regulate recombination, and its loss results into hyper recombinant phenotypes which occur due to severe genomic instability as a result of inhibition in mitochondrial iron-sulfur clusters biosynthesis [ 43 ]. In plants, the ZIM17 acts as protein chaperones for heat shock proteins (Hsp70) and functions in the mitochondrial protein importation process. However, in plants, it does not function as a transcription factor but instead serves as a dedicated chaperone protein for Hsp70, facilitating the mitochondrial protein importation process. Studies have shown that members of the cotton heat shock protein family, Hsp70s, can be induced by both biotic and abiotic stresses such as salt, drought, and Verticillium wilt [ 44 , 45 ]. The evolution of polyploid species often involves the loss of a large number of genes or the duplication of gene copies, leading to increased genetic diversity and plasticity in polyploid species [ 46 ]. A genome-wide identification of the ZIM17 gene family in the four diploid cotton species with A and D genomes, as well as in two tetraploid cotton species of the AD genome revealed a total of 23 ZIM17 genes. In either of the A and D subgenomes, the maximum number of genes was three while in the AD genome, only 6 genes were identified. The distribution of the ZIM17 genes in the 6 selected cotton species, revealed low volumes of the ZIM17 genes in cotton, this is contrary to other genome wide studies conducted in cotton, for instance the whole genome identification of the plant U-box gene (PUB), 93, 96, 185 and 208 PUBs genes were identified for G. raimondii (D5), G. arboreum (A2), G. hirsutum (AD1) and G. barbadense (AD2), respectively, [ 47 ]. Moreover, in the identification of genes involved in the flowering 636, 673 and 350 genes were obtained for G. hirsutum , G. barbadense and G. arboreum , respectively [ 48 ]. The low ZIM17 gene number across all the various cotton genomes, (A, D and AD) points towards gene loss over time, the finding is in agreement, to a study conducted on soybean, in which 3,765 genes were found to be absent from the reference genome assembly, and further found a significant reduction in the average number of the protein-coding genes, the reduction or loss was attributed to frequency associated with selection for agronomic traits [ 49 ]. The deleterious effect of the ZIM17 genes could either have led to their significant loss or reduction across the various genomes including the wild types, such as the G. thurberi , despite their higher ability to resist V. dahliae infection. Due to low gene density, their mapping was skewed to specific chromosomes, for instance in A sub genome, the highest number was observed in G. arboreum with three genes, which were located on chromosomes 01,05 and 12 similar gene mapping was also observed for the D sub genome, however the most intriguing thing is that despite the higher number of genes in AD genome, the chromosome mapping was similar, in either of the (AD) 1 and or (AD) 2 , the genes were mapped on chromosome A01, A05, A12, D01, D05 and D12. It is worth to note that in series of quantitative trait loci (QTL) analysis for V. wilt in cotton, only chromosome none of the identified QTLs were found to be mapped on the chromosomes in which the ZIM17 genes were found to be mapped except for chromosome 5, for instance, previous studies on mapping QTLS for V. wilt resistance in cotton, 23 QTL clusters were mapped on 15 chromosomes (Chr03, Chr04, Chr05, Chr06, Chr07, Chr11, Chr14, Chr15, Chr16, Chr19, Chr20, Chr23, Chr24, Chr25, and Chr26). Moreover, 28 QTL hotshots were found to be associated with different disease resistance traits including one hotspot on chromosome 4 for Verticillium wilt resistance, 3 hotspots on chromosome 19 for the resistance to Verticillium wilt and Fusarium wilt, and micronaire under drought stress conditions [ 50 ]. Additionally, determination of the QTLs for Verticillium wilt resistance in backcross inbred lines, four QTLs were found to be associated with enhancing resistance to V. wilt, in which they were equally distributed on chromosomes 2 and 4 [ 51 ]. The inability to detect any of the ZIM17 genes in some of the vital chromosomes, further points towards chromosomal rearrangement being in the resistant variety, G. thurberi the three ZIM17 genes were found to be mapped onto totally unparalled chromosomes compared to the other five cotton genotypes ( G. herbaceum , G. arboreum , G. hirsutum , G. barbadense and G. raimondii ), the three genes were mapped on chromosomes, Chr02, Chr08 and Chr09. A clear indication of chromosomal rearrangement. The study of cis- acting elements and their roles in plant growth, development, and stress response has been a focal point in plant biology. The absence of these cis- acting elements can potentially affect gene expression and, consequently, their functions [ 52 ]. In this study, the results revealed that, apart from abundant light-responsive elements, numerous stress and hormone-related response elements, such as Abscisic acid (ABRE), methyl jasmonate (CGTCA-motif and TGACG-motif), salicylic acid (TCA-element), drought (MBS), and low temperature (LTR) response elements were observed. These findings suggest that ZIM17 s may be involved in plant growth, development, and stress responses. The presence of these diverse cis- acting elements in the upstream promoter region of ZIM17 s implies that they may regulate the expression of these genes in response to various environmental cues and hormonal signals. Future functional studies on these ZIM17 s will be crucial to understanding their exact roles in cotton and potentially other plants, and how they contribute to plant adaptation and survival under diverse environmental conditions. Moreover, plant hormones are signal transducers in the disease resistance processes [ 53 ], for instance, salicylic acid (SA) and jasmonic acid (JA) are signaling molecules involved in plant disease resistance [ 54 ]. Through gene silencing mechanisms, the Gh ZIM17 -4 expression level was downregulated, and the expression level of GhPR1 , a gene was significantly upregulated despite of playing a critical role in the SA pathway, while the expression of GHPR5 decreased. Salicylic acid and jasmonic acid do have an antagonistic role, thus the variation in expression is in agreement with previous findings in which SA induces resistance against biotrophic and hemitrophic while jasmonic acid induces resitance towards necrotrophic pathogens [ 55 ]. even though there was a variation in the expression levels of the biotic stress responsive genes, there was a higher lignification of the VIGS-plants, lignin is a protective layer, being the V. dahliae infects the plants through the roots, the higher lignification at the stem region could be to enhance physical barrier to block the growth of the fungi. The results were in agreement to previous research which found that lignification of cell wall appositions is a conserved basal defense mechanism in the plant innate immune response, and overexpression of GhLAC15 gene improved cell wall lignin deposition which in turn improved transgenic A. thaliana resistance to Verticillium wilt [ 56 ]. The plant resistance proteins enhance protection by recognizing pathogenic effector molecules and initiating downstream defense. Through yeast hybridization, there was a positive association between the knocked gene and MOS4, MOS4 is a small protein with a coiled-coil domain. Previous studies on Arabidopsis snc1 mutant as an autoimmune model showed that mos4-1 snc1 reduced the enhanced resistance of the snc1 mutant to pathogens [ 57 ]. Moreover, the constructed mos4-1 npr1-1 double mutants and mos4-1 npr1-1 snc1 triple mutants, revealed that the defect in MOS4 blocked the NPR1-independent immune pathway downstream of SNC1 . These findings demonstrated that MOS4 suppresses plant innate immunity (Palma et al., 2007). Furthermore, MOS4 is a core component of the spliceosome-associated complex (MAC), MOS4 in association with MOS12, affects the alternative splicing of plant immune-related R genes, thereby influencing plant immune defense responses [ 58 ]. The interaction between Gth ZIM17 -1 and GthMOS4 provides further evidence of the putative role of Gth ZIM17 -1 in negatively regulating the plant immune defense processes through similar mechanisms. However, a deeper understanding of the interaction mechanism requires further investigation. Future studies could focus on elucidating the specific roles of Gth ZIM17 -1 and GthMOS4 in plant immunity, as well as exploring their potential involvement in the regulation of alternative splicing and immune-related gene expression. 5 Conclusions The low gene density observed among the cotton genomes revealed an element of gene loss, moreover gene loss is a pervasive source of genetic variation among the organisms. The ZIM17 genes were significantly low with 2, 3, 3, 3, 6, and 6 members of the ZIM17 gene family identified among the four diploid cotton species, G. herbaceum (A 1 ), G. arboreum (A 2 ), G. thurberi (D 1 ), and G. raimondii (D 5 ), as well as in two tetraploid cotton species, G. hirsutum (AD) 1 and G. barbadense (AD) 2 , respectively. The expression profiling of the ZIM17 genes, showed that Gth ZIM17 -1 gene was primarily expressed in the root and leaf tissues of Gossypium thurberi . The root is the first organ to be into contact with the pathogenic fungi, while the leaves are the organ with the highest effect of V. dahliae infection. The proteins encoded by the ZIM17 genes are localized in various cellular compartments including cytoplasm, nucleus, and mitochondria. Using virus-induced gene silencing (VIGS) to interfere with the expression of its homologous gene GhZIM17-4 , there was increased lignin accumulation, reduced vascular bundle browning, fewer V. dahliae colonization in the stem, and a lower disease index in the silenced plants, indicating a significant enhancement in disease resistance. Furthermore, the expression of SA signaling pathway-related genes GhPR1 was significantly upregulated, while GhPR5 was downregulated in the silenced plants. Additionally, the expression of JA signaling pathway-related genes GhPR3 and GhPDF1.2 was also significantly upregulated, suggesting that the GhZ IM17 -4 gene regulates cotton disease resistance through SA and JA signaling pathways. Disease resistance assays conducted on Gth ZIM17 -1 overexpressed Arabidopsis plants revealed that overexpressing plants displayed a higher disease index and increased sensitivity to Verticillium wilt following inoculation with Verticillium dahliae . These results suggest that the GthZIM17-1 gene may play a negative regulatory role in plant disease resistance. Moreover, using yeast two-hybrid (Y2H) and luciferase complementation imaging (LCI) experiments revealed an interaction between GthMOS4 and GthZIM17-1 proteins. Declarations Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Consent for publication Not applicable Ethics approval and consent to participate The wild species G. thurberi and the cultivated species Zhongzhimian 2 utilized in this experiment were obtained from the wild cotton germplasm research group of the Institute of Cotton Research (ICR), Anyang, China. The fungal isolates used in this experiment were obtained from soil infested by the fungi, under the management of the Institute of the Cotton Research (ICR), Anyang, China. All methods used in this experiment were performed in accordance with the relevant guidelines, regulations and legislation. Funding This research was funded by National Natural Science Foundation of China (2171994, 332072023, 32272090) and Nanfan Special Project of National Nanfan Research Institute of Chinese Academy of Agriculture Sciences (YBXM2439, YBXM2324, YBXM2309), Author Contribution MYY, ROM. JNK, ZZ, and LF; methodology, MYY, ROM, YX, ZZ.; software, MYY and ROM.; validation, MYY, ROM, ZZ, YX and ZZ.; formal analysis, MYY and ROM.; investigation, MYY.; resources, ZZ, XC, YYZ, QKL and JZ.; data curation, MYY, ROM, JNK, MJU, YL, JH, HW, QKL and LF; writing—original draft preparation, MYY, and ROM.; writing—review and editing, ROM.; All authors have read and agreed to the published version of the manuscript. Acknowledgement We acknowledge the enormous support provided by the entire research group of the wild cotton germplasm resources, of the institute of cotton research (ICR), Anyang, China, the lab technicians and laboratory managers for the support accorded to us during this research work. Data Availability Statement All data that support this publication are fully provided within the text and its supplementary files References Zhang T, Xie Z, Zhou J, Feng H, Zhang T. Temperature impacts on cotton yield superposed by effects on plant growth and verticillium wilt infection in China. Int J Biometeorol. 2024;68:199–209. Zhang G, Zhao Z, Ma P, Qu Y, Sun G, Chen Q. Integrative transcriptomic and gene co-expression network analysis of host responses upon Verticillium dahliae infection in Gossypium hirsutum. Sci Rep. 2021;11:1–13. Zhang Y, Chen B, Sun Z, Liu Z, Cui Y, Ke H, et al. 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Gao W, Long L, Zhu L-F, Xu L, Gao W-H, Sun L-Q, et al. Proteomic and Virus-induced Gene Silencing (VIGS) Analyses Reveal That Gossypol, Brassinosteroids, and Jasmonic acid Contribute to the Resistance of Cotton to Verticillium dahliae . Mol Cell Proteom. 2013;12:3690–703. Wytinck N, Ziegler DJ, Walker PL, Sullivan DS, Biggar KT, Khan D, et al. Host induced gene silencing of the Sclerotinia sclerotiorum ABHYDROLASE-3 gene reduces disease severity in Brassica napus. PLoS ONE. 2022;17(8 August):1–24. Triantafyllopoulou A, Tzima AK, Chronopoulou EG, Labrou NE, Kang S, Paplomatas EJ. Overexpression of an NLP protein family member increases virulence of Verticillium dahliae. Plant Pathol 2024; August 2023:1264–75. Palma K, Zhao Q, Yu TC, Bi D, Monaghan J, Cheng W, et al. Regulation of plant innate immunity by three proteins in a complex conserved across the plant and animal kingdoms. Genes Dev. 2007;21:1484–93. 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Li X, Wu Y, Chi H, Wei H, Wang H, Yu S. Genomewide Identification and Characterization of the Genes Involved in the Flowering of Cotton. Int J Mol Sci. 2022;23. Bayer PE, Valliyodan B, Hu H, Marsh JI, Yuan Y, Vuong TD, et al. Sequencing the USDA core soybean collection reveals gene loss during domestication and breeding. Plant Genome. 2022;15:1–12. Abdelraheem A, Liu F, Song M, Zhang JF. A meta-analysis of quantitative trait loci for abiotic and biotic stress resistance in tetraploid cotton. Mol Genet Genomics. 2017;292. Zhang J, Yu J, Pei W, Li X, Said J, Song M et al. Genetic analysis of Verticillium wilt resistance in a backcross inbred line population and a meta-analysis of quantitative trait loci for disease resistance in cotton. BMC Genomics. 2015;16. Schmitz RJ, Grotewold E, Stam M. Cis-regulatory sequences in plants: Their importance, discovery, and future challenges. Plant Cell. 2022;34:718–41. Zhu Y, Zhao M, Li T, Wang L, Liao C, Liu D, et al. Interactions between Verticillium dahliae and cotton: pathogenic mechanism and cotton resistance mechanism to Verticillium wilt. Front Plant Sci. 2023;14:1–16. Sultana R, Imam Z, Kumar RR, Banu VS, Nahakpam S, Bharti R, et al. Signaling and Defence Mechanism of Jasmonic and Salicylic Acid Response in Pulse Crops: Role of WRKY Transcription Factors in Stress Response. J Plant Growth Regul. 2024. https://doi.org/10.1007/s00344-023-11203-9 . Tamaoki D, Seo S, Yamada S, Kano A, Miyamoto A, Shishido H et al. Jasmonic acid and salicylic acid activate a common defense system in rice. Plant Signal Behav. 2013;8. Zhang Y, Wu L, Wang X, Chen B, Zhao J, Cui J, et al. The cotton laccase gene GhLAC15 enhances Verticillium wilt resistance via an increase in defence-induced lignification and lignin components in the cell walls of plants. Mol Plant Pathol. 2019;20:309–22. Cai Q, Liang C, Wang S, Hou Y, Gao L, Liu L, et al. The disease resistance protein SNC1 represses the biogenesis of microRNAs and phased siRNAs. Nat Commun. 2018;9:1–14. Xu F, Xu S, Wiermer M, Zhang Y, Li X. The cyclin L homolog MOS12 and the MOS4-associated complex are required for the proper splicing of plant resistance genes. Plant J. 2012;70:916–28. Additional Declarations No competing interests reported. Supplementary Files TableS1.Primersusedinthisstudy.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4517860","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":312603576,"identity":"c428781a-03b2-458e-80b0-865cb5fccf10","order_by":0,"name":"Mengying Yang","email":"","orcid":"","institution":"Zhengzhou Research Base, National Key Laboratory of Cotton Bio-breeding and Integrated Utilization, Zhengzhou University/Institute of Cotton Research, Chinese Academy of Agricultural Science","correspondingAuthor":false,"prefix":"","firstName":"Mengying","middleName":"","lastName":"Yang","suffix":""},{"id":312603577,"identity":"fa8920e2-891b-4554-8fbf-d044b73ace24","order_by":1,"name":"Richard Odongo Magwanga","email":"","orcid":"","institution":"Zhengzhou Research Base, National Key Laboratory of Cotton Bio-breeding and Integrated Utilization, Zhengzhou University/Institute of Cotton Research, Chinese Academy of Agricultural Science","correspondingAuthor":false,"prefix":"","firstName":"Richard","middleName":"Odongo","lastName":"Magwanga","suffix":""},{"id":312603578,"identity":"8dacb2b0-0805-4500-baa4-17841caeb844","order_by":2,"name":"Yuqing Hou","email":"","orcid":"","institution":"Zhengzhou Research Base, National Key Laboratory of Cotton Bio-breeding and Integrated Utilization, Zhengzhou University/Institute of Cotton Research, Chinese Academy of Agricultural Science","correspondingAuthor":false,"prefix":"","firstName":"Yuqing","middleName":"","lastName":"Hou","suffix":""},{"id":312603579,"identity":"2810d7e0-77e1-4093-9b12-4704de83a4ff","order_by":3,"name":"Muhammad Jawad Umer","email":"","orcid":"","institution":"Zhengzhou Research Base, National Key Laboratory of Cotton Bio-breeding and Integrated Utilization, Zhengzhou University/Institute of Cotton Research, Chinese Academy of Agricultural Science","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"Jawad","lastName":"Umer","suffix":""},{"id":312603580,"identity":"72f81e91-5e63-43ca-bbac-9b28e814efb9","order_by":4,"name":"Heng Wang","email":"","orcid":"","institution":"Zhengzhou Research Base, National Key Laboratory of Cotton Bio-breeding and Integrated Utilization, Zhengzhou University/Institute of Cotton Research, Chinese Academy of Agricultural 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Cai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqklEQVRIiWNgGAWjYNACAxsefv4G0rSkyUjOOECaNYdtDBoSiFTLPyPH8HNFwXkeA4YDjB8+5hChReLMGWPJMwa3ecyZG5glZ24jQosBe48ZYwNQi2XDATZmXqK0MPOAtJzjMTiQQKwWiC0HSNAiceZYsWSDQTKP5IyDzcT5hX9G8saPDX/s7Pn5mw9++EiMFgYGDgMog7GBKPVAwP6AWJWjYBSMglEwUgEAJwEw+UMK714AAAAASUVORK5CYII=","orcid":"","institution":"Zhengzhou Research Base, National Key Laboratory of Cotton Bio-breeding and Integrated Utilization, Zhengzhou University/Institute of Cotton Research, Chinese Academy of Agricultural Science","correspondingAuthor":true,"prefix":"","firstName":"Xiaoyan","middleName":"","lastName":"Cai","suffix":""}],"badges":[],"createdAt":"2024-06-02 17:08:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4517860/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4517860/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58953101,"identity":"a5cf5b23-0200-4c15-88ad-d3a79d0e24e1","added_by":"auto","created_at":"2024-06-24 14:24:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":368725,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePhylogenetic tree of the cotton and Arabidopsis ZIM17 proteins. At- Arabidopsis thaliana, Gth-G. thurberi, Gh- G. hirsutum, Ga- G. arboreum, Gr. G. raimondii, and Gb- G. barbadense,\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4517860/v1/42c83e6ddd5c57358252a667.png"},{"id":58953104,"identity":"6784fde9-f4bb-4a8f-be6b-6cded3489930","added_by":"auto","created_at":"2024-06-24 14:24:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":269812,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eChromosomal mapping of the ZIM17 genes in the six cotton species. (A) G. herbaceum, (B) G. arboreum, (C) G. thurberi, (D). G. raimondii; (E) G. hirsutum and G. barbadense. The vertical bar located on the left side indicates the chromosome sizes in megabases (Mb), the chromosome number is located on the left side of each chromosome, and the cotton genome corresponding is located above each part.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4517860/v1/3e80b9f91ac01415122e5663.png"},{"id":58953864,"identity":"1190d250-e043-4c8b-af67-1a55ced5a74c","added_by":"auto","created_at":"2024-06-24 14:32:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":168412,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eThe conserved motif and exon-intron structure of ZIM17 genes in cotton. (A) Phylogenetic analysis of ZIM17 proteins. (B) Analysis of conserved motifs of ZIM17 gene sequences. Different motifs are shown in a specific color. (C) Intron and exon analysis of ZIM17 genes. Exons and introns are represented by green boxes and thin lines, respectively. The UTR is shown in a yellow box.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4517860/v1/a72f33c22b69cf38748dfc2a.png"},{"id":58953105,"identity":"79b024a5-2b50-4aad-9fc2-79e77fbf9fa5","added_by":"auto","created_at":"2024-06-24 14:24:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":328484,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePromoter analyses of ZIM17 genes in different cotton species. The colour coding denotes different Cis-acting elements.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4517860/v1/81ac3ba7d6106a8adc002eb8.png"},{"id":58953102,"identity":"57a2b081-d8c7-4696-a42e-9c5bba9ccfa4","added_by":"auto","created_at":"2024-06-24 14:24:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":159784,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eExpression analysis of the GtZIM17-1 gene. (A) Expression levels on different plant tissues, roots, stem, and leaf; (B). Expression of the gene under V. dahliae infection at time intervals 0h, 12h,24h and 48h. Error bars represent the standard deviation of three biological replicates. Student’s t-test was used for significance analysis, *: p \u0026lt; 0.05; **: p \u0026lt; 0.01.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4517860/v1/47a5d1b28e9fa40c9f1679f8.png"},{"id":58953103,"identity":"6d6cd24c-4241-4390-a131-9124d6417da4","added_by":"auto","created_at":"2024-06-24 14:24:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1887910,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSubcellular localization of GthZIM17-1 in tobacco epidermal cells. Red colour denotes subcellular localization within the mitochondria, purple denotes chloroplast. the presence of both red and purple denotes the detection of the ZIM17-1 proteins both in the mitochondria and chloroplast.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-4517860/v1/f81c68671bf39ae2fceacdfe.png"},{"id":58953107,"identity":"03f5391f-8241-490a-90fb-4b8843590aa2","added_by":"auto","created_at":"2024-06-24 14:24:03","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1933557,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSilencing of the GhZIM17-4 gene leads to increased resistance against Verticillium wilt in cotton. (A) Plant albino phenotype and the phenotypes of the positive control and silenced plants; B: VIGS interference detection of GhZIM17-4 gene expression level; C: Stem longitudinal sections text; D: Phloroglucinol staining; E: DAB staining of plant leaves; F: Fungal recovery assay; G: The disease index. H: Expression of SA pathogenesis-related genes; I: Expression of JA pathogenesis-related genes; At least three biological replicates were conducted for each experiment. Student’s t test was used for significance analysis, *: p \u0026lt; 0.05; **: p \u0026lt; 0.01; ***: p \u0026lt; 0.001; ****: p \u0026lt; 0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-4517860/v1/ac66d5f15cd4849e0c469c26.png"},{"id":58953863,"identity":"607dddfc-685d-4262-bc11-01b3533aa069","added_by":"auto","created_at":"2024-06-24 14:32:03","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePhenotypes of wild-type (WT) and GthZIM17-transgenic Arabidopsis plants in response to V. dahliae infection. (A). Polymerase chain reaction (PCR) analysis was performed to check 621 bp coding sequence (CDS) integration in transformed T2 generation, number 1–10 transgenic lines, and WT is the negative control (wild-type). (B). The transcript levels of the GthZIM17 of T3 transgenic lines were analyzed through qRT-PCR. (C). Phenotypes of wild-type and Overexpression A. thaliana infected with V. dahliae grown on 0.5 MS medium; (D). Phenotypes of wild-type and Overexpression Arabidopsis plants infected with V. dahliae in soil; (E). Disease index statistics of wild-type and Overexpression A. thaliana grown on 0.5 MS medium; (F). Disease index statistics of wild type and mutant A. thaliana growing in soil. At least three biological replicates were conducted for each experiment. At least three biological replicates were conducted for each experiment\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"placeholderimage.png","url":"https://assets-eu.researchsquare.com/files/rs-4517860/v1/f3712c7c117c68f3ee05c17f.png"},{"id":58953109,"identity":"286264eb-4dca-474d-a479-b116202d9464","added_by":"auto","created_at":"2024-06-24 14:24:03","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1404408,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eInteraction between GthZIM17-1 and GthMOS4 protein\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.9.png","url":"https://assets-eu.researchsquare.com/files/rs-4517860/v1/3b886b70ef552ca77145d8ca.png"},{"id":91000179,"identity":"137ba95b-2b2c-4f94-9815-8a786d950954","added_by":"auto","created_at":"2025-09-10 13:32:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9354603,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4517860/v1/8db49f8a-12cf-491b-8ccb-9c7bb1143648.pdf"},{"id":58953111,"identity":"e6bee73e-b932-4770-abb1-8227510508b8","added_by":"auto","created_at":"2024-06-24 14:24:03","extension":"docx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":19173,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.Primersusedinthisstudy.docx","url":"https://assets-eu.researchsquare.com/files/rs-4517860/v1/c3deaa3fc6e3cf9e3e1fbbf7.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Unveiling Cotton's Defense: Harnessing GthZIM17-1 Inhibition for Verticillium Wilt Resistance","fulltext":[{"header":"1 Background","content":"\u003cp\u003eVerticillium wilt is a soil-borne plant pathogenic fungus with a wide host range, so found to infect over 200 dicotyledonous plants, such as cotton [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], potato [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], and tomato [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The fungus is not restricted to only field crops, but also infects vegetables, fruits and ornamental plants species [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Verticillium wilt was first discovered in the United States of America (USA) in 1915, and first reported in China, twenty years later, in 1935, since then, it has had a global distribution, causing massive agricultural loss globally [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. There six species of the pathogenic fungi however, two are known to be highly virulent, the \u003cem\u003eVerticillium dahliae\u003c/em\u003e and \u003cem\u003eVerticillium albo-atrum\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], the others species includes \u003cem\u003eV. tricorpus\u003c/em\u003e, \u003cem\u003eV. nigrescens\u003c/em\u003e, \u003cem\u003eV. nubilum\u003c/em\u003e, and \u003cem\u003eV. theobroma\u003c/em\u003e [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In China, the main pathogenic fungi is the \u003cem\u003eVerticillium dahliae\u003c/em\u003e, \u003cem\u003eV. dahliae\u003c/em\u003e is a semi-living nutrient pathogen, that can survive in the form of microsclerotia in the soil for many years, moreover, the microsclerotia are extremely adaptable to extremely harsh environments, and easily dispersed by wind or rain, which makes it difficult to control [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The longevity, effective disposal mechanism, and highly adaptive nature to harsh environmental conditions, have made the various control measures to be ineffective, furthermore, there is no effective fungicide that can be used in controlling \u003cem\u003eV. dahliae\u003c/em\u003e infection in plants. Furthermore, the diseases caused by the \u003cem\u003eV. dahliae\u003c/em\u003e is related to the human immunodeficiency virus (HIV), once the plants are infected, then no remedial measure.\u003c/p\u003e \u003cp\u003eThe two virulent forms of Verticillium \u003cem\u003espp\u003c/em\u003e. do cause plant diseases in conjunctions with other pathogenic organisms such as bacteria, nematodes and other forms of fungi [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The major symptoms of Verticillium wilt in plants includes necrosis, stunting, chlorosis, vascular discoloration, premature senescence, night recovery and wilting [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In cotton, V. wilt infection causes chlorosis, stem dryness, and root rot, which negatively affects the yield and quality of cotton [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In China, the economic loss due to V. wilt is estimated at 250 to 310\u0026nbsp;million US dollars annually [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Cotton production in China has transformed the economy and lives of millions of Chinese [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. As an important economic crop, the yield and quality of cotton have been significantly affected by this terminal fungal infection, \u003cem\u003eV. dahliae\u003c/em\u003e. Several attempts have been made to develop resistant crops to V. wilt, for instance, the use of biocontrol has been attempted, in which \u003cem\u003eVerticillium isaacii\u003c/em\u003e Vt305 has been employed to play an antagonistic role to \u003cem\u003eV. dahliae\u003c/em\u003e, however, the success level was very low due to different soil environments [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Moreover, conventional agronomic methods have been adopted, by intercropping cotton with either garlic and or onion, the level of V. wilt disease index was observed to be significantly reduced [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], however, this method is not sustainable on a large scale, where mechanization is required. Due to the nature of \u003cem\u003eV. dahliae\u003c/em\u003e infection, the most suitable method is the utilization of resistant cultivars, so breeding for resistant crop varieties will contribute significantly to controlling \u003cem\u003eVerticillium dahliae\u003c/em\u003e. Utilization of genes to confer tolerance to V. wilt has shown positive effects, the overexpression of \u003cem\u003eGhIQD1\u003c/em\u003e gene, which acts as a calmodulin-binding protein improved cotton resistance to V. wilt [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], moreover, a group of glutathione S-transferase (GST) genes, were found through transcriptome analysis to confer tolerance to \u003cem\u003eV. dahliae\u003c/em\u003e infection [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, research has shown that RNA interference (RNAi)-based host-induced gene silencing (HIGS) boost plants resistance to diseases by silencing essential genes for inducing pathogenicity, for instance, the silencing of \u003cem\u003eV. dahliae\u003c/em\u003e gene encoding an exoglucanase (VdEXG, VDAG_02898) had positive effect on enhancing tolerance to \u003cem\u003eV. dahliae\u003c/em\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe genetic diversity of cotton germplasm, has undergone drastic erosion, leading to massive narrowing of the genetic diversity [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The narrowing of the genetic base of the elite cotton has been worsened due to inbreeding and intensive selection for favorable agronomic traits [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In order to broaden the ever shrinking genetic diversity of the cultivated cotton, introgression of genes from the wild progenitor\u0026rsquo;s is key [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The wild cotton progenitors are the reservoirs for beneficial genes, more commonly referred to are the resistance (R) genes, for instance, Magwanga et al [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] evaluated \u003cem\u003eGossypium tomentosum\u003c/em\u003e in the identification of drought and salt stress genes. \u003cem\u003eG. tomentosum\u003c/em\u003e is a wild progenitor of the elite cotton cultivars, furthermore, \u003cem\u003eG. thurberi\u003c/em\u003e has been found to be highly resistant to \u003cem\u003eV. dahliae\u003c/em\u003e infection and therefore survive under \u003cem\u003eV. dahliae\u003c/em\u003e infested areas [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In this study, a novel gene, a ZIM17 (Zinc finger motif 17) was identified. The ZIM17 also known as TIM15 (translocase in the inner mitochondrial membrane) or Hep1 (MtHsp 70 escort protein 1) is a specific protein chaperone for mitochondrial 70 kDa heat shock protein (MtHsp 70) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This gene has been extensively studied in yeast where it has been found to promote yeast cell growth, yeast being a fungus, its inhibition could have a positive effect in inhibiting the proliferation of \u003cem\u003eV. dahliae\u003c/em\u003e, thereby reducing its virulence ability to cause V. wilt in cotton. Moreover, as a core component of the mitochondrial chaperone protein system, MtHsp70 not only provides ATP-driven force for polypeptide translocation reactions but also participates in the protein folding processes [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. As a chaperone protein for MtHsp70, ZIM17 maintains the solubility of MtHsp70, prevents the self-aggregation of Hsp70 chaperone proteins SSC1 and SSQ1, and promotes the disassembly of aggregated proteins, thereby maintaining its function in mitochondrial protein import and Fe/S protein biosynthesis. Both in vivo and in vitro, ZIM17 assists MtHsp70 in importing proteins into the mitochondria [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. However, there are currently no reports on the role of ZIM17 in cotton resistance to \u003cem\u003eV. dahliae\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn this study, a whole genome-wide identification and functional analysis of the \u003cem\u003eZIM17\u003c/em\u003e genes were carried out. Moreover, the availability of the D genome sequence provides a perfect platform for the evaluation of the \u003cem\u003eZIM17\u003c/em\u003e gene family. Moreover, forward and reverse genetics were employed in the model plant \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and cotton, respectively to understand the putative role of the Gth\u003cem\u003eZIM17\u003c/em\u003e-1 gene, further yeast two-hybrid and luciferase complementation imaging assays were conducted to screen and verify interacting proteins of Gth\u003cem\u003eZIM17\u003c/em\u003e-1. The results provide fundamental steps to enhance research by exploiting the RNA interference (RNAi)-based host-induced gene silencing mechanisms in developing cotton germplasms which are highly versatile and resistant to various forms of biotic stress factors.\u003c/p\u003e"},{"header":"2 Material and methods","content":"\u003cp\u003eData acquisition, identification, and physicochemical analysis of ZIM17 family members in six cotton species.\u003c/p\u003e \u003cp\u003eThe whole genome data of \u003cem\u003eGossypium herbaceum\u003c/em\u003e (A\u003csub\u003e1\u003c/sub\u003e) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], \u003cem\u003eG. arboreum\u003c/em\u003e (A\u003csub\u003e2\u003c/sub\u003e) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], \u003cem\u003eG. thurberi\u003c/em\u003e (D\u003csub\u003e1\u003c/sub\u003e) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], \u003cem\u003eG. raimondii\u003c/em\u003e (D\u003csub\u003e2\u003c/sub\u003e) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], \u003cem\u003eG. hirsutum\u003c/em\u003e (AD)\u003csub\u003e1\u003c/sub\u003e [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], and \u003cem\u003eG. barbadense\u003c/em\u003e (AD)\u003csub\u003e2\u003c/sub\u003e [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] from the Cottongen website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cottongen.org/\u003c/span\u003e\u003cspan address=\"https://www.cottongen.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). A genome-wide blast was performed in the six cotton species using TBtools v1.106 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] with \u003cem\u003eArabidopsis thaliana ZIM17\u003c/em\u003e protein sequences downloaded from TAIR (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.arabidopsis.org/\u003c/span\u003e\u003cspan address=\"https://www.arabidopsis.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) as reference sequences. The Hidden Markov Model (HMM) PF05180 of the ZIM17 family from Pfam (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pfam.xfam.org/\u003c/span\u003e\u003cspan address=\"https://pfam.xfam.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was downloaded. The candidate proteins were merged and deduplicated, and NCBI-CDD (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/Stru-cture/cdd/wrpsb.cgi\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/Stru-cture/cdd/wrpsb.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to verify that all the sequences contained two conservative domains, \u003cem\u003ezf-DNL\u003c/em\u003e, or \u003cem\u003ezf-DNL\u003c/em\u003e superfamily. The physicochemical properties of the \u003cem\u003eZIM17\u003c/em\u003e protein were further analyzed by predicting the amino acid number (aa), relative molecular weight (mw), isoelectric point (\u003cem\u003epI\u003c/em\u003e), and instability index of the protein using the ProtParam tool (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eExpasy - ProtParam tool\u003c/span\u003e)\u003c/p\u003e \u003cp\u003ePhylogenetic tree construction of the \u003cem\u003eZIM17\u003c/em\u003e genes family\u003c/p\u003e \u003cp\u003eTo construct the phylogenetic tree of the ZIM17 proteins in cotton, multiple sequence alignments of the \u003cem\u003eZIM17\u003c/em\u003e protein sequence using the ClustalX tool in MEGA-X software were done [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The phylogenetic tree was constructed using the linkage method with a bootstrap set at 1000, using a p-distance model. The original file from MEGA was then exported to the EvolView website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.evolgenius.info/evolview/\u003c/span\u003e\u003cspan address=\"https://www.evolgenius.info/evolview/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for the visualization of the phylogenetic tree.\u003c/p\u003e \u003cp\u003eGene structure and conservative motif analysis of the \u003cem\u003eZIM17\u003c/em\u003e genes in cotton\u003c/p\u003e \u003cp\u003eThe gene structure and conservative motif of the \u003cem\u003eZIM17\u003c/em\u003e genes in cotton were analyzed by visualizing the exon-intron structure of each gene using the location information of family members on the TBtools software [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The conserved motif of the ZIM17 protein was identified through an online tool MEME (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.meme-suite.org/\u003c/span\u003e\u003cspan address=\"http://www.meme-suite.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with a motifs number set at 12. The output file in XML format was downloaded and visualized through the TBtools [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eChromosome mapping of \u003cem\u003eZIM17\u003c/em\u003e genes in different cotton species\u003c/p\u003e \u003cp\u003eThe location of \u003cem\u003eZIM17\u003c/em\u003e genes on chromosomes was mapped by extracting the location of the genes on chromosomes using the TBtools [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] and mapped as per the genome and genome annotation information.\u003c/p\u003e \u003cp\u003eAnalysis of \u003cem\u003ecis-\u003c/em\u003eacting elements of \u003cem\u003eZIM17\u003c/em\u003e genes in different cotton species\u003c/p\u003e \u003cp\u003eTo analyze the \u003cem\u003ecis-\u003c/em\u003eregulatory elements of the cotton \u003cem\u003eZIM17\u003c/em\u003e\u0026rsquo;s was analyzed from the upstream 2000 bp fragments. The \u003cem\u003ecis-\u003c/em\u003eregulatory elements of the gene promoter region were predicted using an online tool, the PlantCARE (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and visualized using the TBtools [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePlant materials and growth\u003c/p\u003e \u003cp\u003eThe wild species \u003cem\u003eG. thurberi\u003c/em\u003e and the cultivated species Zhongzhimian 2 were utilized. The two cotton species seeds were obtained from the wild cotton germplasm research group of the Institute of Cotton Research (ICR), Anyang, China. After de-fuzzing, cotton seeds were germinated using the sand culture method. Four Defuzzed seeds were germinated in boxes filled with sterilized wet sand, and a loose layer of sand approximately 1 cm thick was applied over the seeds. The boxes were then placed in a dark incubator at 29\u0026deg;C for 48 hours. Cotton seedlings with uniform growth were subsequently transplanted into pots filled with nutrient soil and grown in a greenhouse maintained at a constant temperature of 25\u0026deg;C with a 16-hr light/8-hr dark photoperiod.\u003c/p\u003e \u003cp\u003ePathogen cultivation and plant inoculation\u003c/p\u003e \u003cp\u003eThe fungal strain Vd991 was inoculated onto a potato dextrose agar (PDA) medium and cultured in darkness at 25\u0026deg;C for 7 days to achieve activation. Subsequently, 4\u0026ndash;5 activated fungal blocks were aseptically excised using a sterile surgical blade and transferred to Czapek's medium containing streptomycin sulfate (0.05 g/ml). These blocks were further incubated in a shaking incubator at 25\u0026deg;C, 200 rpm/min, under dark conditions for 7 days. The fungal suspension was then filtered through four layers of sterile gauze, and the spore concentration was diluted to 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e cfu/ml using sterile double-distilled water (ddH\u003csub\u003e2\u003c/sub\u003eO) for subsequent use.\u003c/p\u003e \u003cp\u003eRNA extraction and qRT-PCR analysis\u003c/p\u003e \u003cp\u003ePlant tissues were collected in aluminum foil and rapidly frozen in liquid nitrogen. Using a mortar and pestle, the tissues were quickly ground into a fine powder, with timely replenishment of liquid nitrogen to prevent sample oxidation. Total RNA was extracted from cotton leaf tissues using the RNAprep Pure Plant Kit (TIANGEN, Beijing, China), with detailed extraction steps outlined in the kit's manual. The quality and quantity of the extracted RNA was determined through agarose gel electrophoresis. Reverse transcription was performed using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGene, Beijing, China). One microgram of RNA was used as the template,\u003c/p\u003e \u003cp\u003eReal-time quantitative PCR experiments were conducted using the ABI 7500 Real-Time PCR System. Cotton \u003cem\u003eGhUBQ7\u003c/em\u003e and Arabidopsis \u003cem\u003eAtACTIN2\u003c/em\u003e genes were used as reference genes (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), and the relative expression levels of different genes were calculated using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method. Each sample had three biological and technical replicates.\u003c/p\u003e \u003cp\u003eSubcellular localization of Gth\u003cem\u003eZIM17\u003c/em\u003e-1\u003c/p\u003e \u003cp\u003eTo validate the subcellular localization prediction, the Gth\u003cem\u003eZIM17\u003c/em\u003e-1 618 bp fragment, excluding the stop codon, was cloned into the subcellular localization vector \u003cem\u003ep2300-eGFP\u003c/em\u003e-Flag using the selected double restriction enzyme sites of \u003cem\u003eXba I\u003c/em\u003e and \u003cem\u003eBamH I\u003c/em\u003e, resulting in the subcellular localization expression vector p2300-eGFP-Gth\u003cem\u003eZIM17\u003c/em\u003e-1. The primer sequences are provided in (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain harboring the subcellular localization vector p2300-eGFP-flag, preserved at -80\u0026deg;C, was activated by incubating in a 28\u0026deg;C shaking incubator for 2 hours. Subsequently, the strain was transferred into 50 mL of LB liquid medium containing Kana (50 mg/L) and Rif (50 mg/L) and cultured in a shaking incubator at 28\u0026deg;C and 200 rpm/min for 24\u0026ndash;48 hours. Once the optical density measurements (OD) at a wavelength of 600 nm (OD600) of the Agrobacterium culture reached 1.0, the bacterial cells were collected by centrifugation and resuspended in a resuspension solution to an OD600 of approximately 1.0. The resuspended solution was incubated in the dark for 2 hours. Using a 1 mL sterile syringe, the bacterial suspension was injected into tobacco leaves. The tobacco plants were then cultured in the dark for 48 hours and observed under a laser confocal microscope.\u003c/p\u003e \u003cp\u003eVirus-induced gene silencing (VIGS) of Gh\u003cem\u003eZIM17\u003c/em\u003e-4\u003c/p\u003e \u003cp\u003eUsing the cDNA of Zhongzhimian 2 as a template, specific primers for the VIGS 227 bp fragment were designed using Primer Premier 5 software (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The virus-induced gene silencing vector pTRV2 was double-digested using the enzyme \u003cem\u003eEcoRI\u003c/em\u003e and \u003cem\u003eBamHI\u003c/em\u003e digestion system. The vector \u003cem\u003epTRV2-GhZIM17-4\u003c/em\u003e was obtained and then transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e LBA4404 and infiltrated in the cotton cotyledons. The infected plants were cultured in the dark for 24 hours before being transferred to a greenhouse for normal light conditions. Approximately two weeks later, the positive control plants exhibited albinism.\u003c/p\u003e \u003cp\u003eObservations of disease development in cotton plants were made 20 days after inoculation. Photographs were taken, and disease severity indices were recorded at 25 days\u0026rsquo; post-inoculation. The disease severity of seedlings was classified into five grades (0, 1, 2, 3, 4) according to the following criteria: Grade 0, healthy plants; Grade 1, 1%-33% diseased leaves; Grade 2, 34%-66% diseased leaves; Grade 3, 67%-99% diseased leaves; Grade 4, entire plant with diseased leaves or even dead and fallen leaves. The disease index (DI) was calculated using the formula: DI = [Σ (grade \u0026times; number of plants at that grade) / (total number of plants \u0026times; highest grade of 4)] \u0026times; 100 [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOverexpression of Gth\u003cem\u003eZIM17\u003c/em\u003e-1 gene in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and inoculation of the \u003cem\u003eV. dahliae\u003c/em\u003e\u003c/p\u003e \u003cp\u003eAt the flowering stage of the wild-type \u003cem\u003eA. thaliana\u003c/em\u003e, the pods and pollinated flowers that had already formed were trimmed off ahead of time. Wild-type Arabidopsis was then infected using the floral-dip method, with the buds fully immersed in the suspension containing the \u003cem\u003epCAMBIA2300-GthZIM17-1\u003c/em\u003e recombinant vector for 1 minute. Following the infection, the plants were cultured in the dark for 24 hours. To enhance the infection efficiency, a second infection was conducted one week after the first infection. The obtained seeds were selected on 1/2 MS medium containing 50 mg/mL kanamycin, and homozygous lines were confirmed through RT-PCR and qRT-PCR until the T2 generation. Subsequent experiments were conducted on the plants with high expression levels.\u003c/p\u003e \u003cp\u003eBefore sowing, \u003cem\u003eA. thaliana\u003c/em\u003e seeds underwent sterilization and disinfection. Initially, the seeds were treated with 75% ethanol for 3 minutes and rinsed three times with sterile ddH\u003csub\u003e2\u003c/sub\u003eO. Subsequently, they were sterilized with 15% NaClO solution for 5 minutes and rinsed five times with sterile ddH\u003csub\u003e2\u003c/sub\u003eO. Sterilized Arabidopsis seeds were then sown onto 1/2 MS medium, vernalized, and transferred to a constant temperature incubator maintained at 22\u0026deg;C with a 16-hour light/8-hour dark photoperiod. After approximately two weeks of growth on the medium, Arabidopsis plants were transplanted into pots filled with nutrient soil and grown in a greenhouse maintained at a constant temperature of 25\u0026deg;C with a 16-hour light/8-hour dark photoperiod for two weeks, before being inoculated by the spores of \u003cem\u003eV. dahliae\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eStem section detection, Histochemical staining of cotton stem lignin, DAB staining, and fungal recovery assay\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1.1 Stem section detection\u003c/h2\u003e \u003cp\u003eOn the 25th day after inoculation of cotton with \u003cem\u003eV. dahliae\u003c/em\u003e, stem tissues from the same above-ground locations of both control and silenced plants were randomly selected and longitudinally sectioned using a disposable blade. These sections were then observed under a stereomicroscope to assess vascular bundle occlusion and browning.\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.2 Histochemical staining of cotton stem lignin\u003c/h2\u003e \u003cp\u003eOn the 25th day post-inoculation, stem tissues from the same locations of control and silenced cotton plants were cross-sectioned using the hand-slicing method. The sections were placed on slides and promptly stained with a drop of 3% phloroglucinol solution to ensure complete immersion. After 10 minutes, the sections were incubated in 6% hydrochloric acid for 5 minutes, rinsed twice with ddH\u003csub\u003e2\u003c/sub\u003eO, and then a drop of ddH\u003csub\u003e2\u003c/sub\u003eO was added to prevent dehydration and wrinkling. These stained sections were observed under a stereomicroscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.1.3 3,3'-diaminobenzidine (DAB) staining\u003c/h2\u003e \u003cp\u003eOn the 25th day of post-inoculation, cotton true leaves were randomly selected and immersed in an adequate amount of 3,3'-diaminobenzidine (DAB) staining solution. The leaves were incubated in the dark at room temperature for 8 hours. Subsequently, the DAB solution was removed, and the leaves were repeatedly decolorized with 95% ethanol until the chlorophyll was completely removed. After washing with sterile water, the leaves were photographed for observation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.1.4 Fungal recovery assay\u003c/h2\u003e \u003cp\u003eFor the isolation of cotton stem segments, they were randomly selected from the same above-ground locations and sterilized in a laminar flow hood. This involved a 40-second immersion in 75% ethanol, followed by a 3-minute sterilization with 3% sodium hypochlorite. The segments were then rinsed three times with sterile ddH\u003csub\u003e2\u003c/sub\u003eO. Using a sterile surgical blade, the cotton stems were cut into approximately 0.5 cm segments and evenly placed on a potato dextrose agar (PDA) medium. Then were incubated in the dark at 25\u0026deg;C for 5\u0026ndash;7 days.\u003c/p\u003e \u003cp\u003eYeast two-hybrid assays\u003c/p\u003e \u003cp\u003eThe full-length CoDing Sequence (CDS) of the target gene was cloned into the \u003cem\u003epGBKT7\u003c/em\u003e vector, with \u003cem\u003eEcoRⅠ\u003c/em\u003e and \u003cem\u003eBamHI\u003c/em\u003e chosen as the double restriction enzyme sites, the yeast bait vector pGBKT7-Gth\u003cem\u003eZIM17\u003c/em\u003e-1 was then obtained. The bait and prey plasmids were then co-transformed into yeast \u003cem\u003eY2H\u003c/em\u003e Gold competent cells using the co-transformation method for yeast two-hybrid point-to-point interaction verification.\u003c/p\u003e \u003cp\u003eFirefly luciferase complementation imaging (LCI) assays\u003c/p\u003e \u003cp\u003eThe coding regions of Gth\u003cem\u003eZIM17\u003c/em\u003e-1 and GthMOS4 sequences were cloned onto the \u003cem\u003epCAMBIA1300-35S-cLUC\u003c/em\u003e and \u003cem\u003epCAMBIA1300-35S-cLUC\u003c/em\u003e vector respectively (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Equal amounts of Agrobacterium cultures containing \u003cem\u003eCLuc\u003c/em\u003e and \u003cem\u003eNLuc\u003c/em\u003e constructs were mixed and then co-infiltrated into tobacco leaves. Before observation, tobacco leaves were picked and applied with 20 \u0026micro;L luciferase substrate (1 mmol/L D-luciferin sodium salt) in the injection area, and placed in a dark environment for 10 minutes. The leaves were placed under a fluorescence microscope to observe the fluorescence signal and take photos. All experiments were repeated at least three times for each plasmid combination.\u003c/p\u003e \u003cp\u003eData analysis\u003c/p\u003e \u003cp\u003eAll experimental data were analyzed; the mean values were obtained for at least three independent biological replicates. Data were analyzed using SPSS software, and statistical comparisons of differences were analyzed for significance of multiple data using ANOVA statistics, and plotted using GraphPad Prism 9.0.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cp\u003eIdentification and analysis of the physiochemical properties of the \u003cem\u003eZIM17\u003c/em\u003e gene family\u003c/p\u003e\n\u003cp\u003eThree ZIM17 family members were identified in \u003cem\u003eA. thaliana\u003c/em\u003e. Using the protein sequences for the Arabidopsis, a Blast comparison analysis was conducted across the 6 cotton species. Based on sequence similarity and conserved domains, a total of 11 \u003cem\u003eZIM17\u003c/em\u003e genes were found for the 4 cotton species of the D genome, in which 2, 3, 3, and 3 ZIM17 family members were identified for \u003cem\u003eG. herbaceum\u003c/em\u003e, \u003cem\u003eG. arboreum\u003c/em\u003e, \u003cem\u003eG. thurberi\u003c/em\u003e, and \u003cem\u003eG. raimondii\u003c/em\u003e, respectively. Interestingly, among the tetraploid cotton \u003cem\u003eG. hirsutum\u003c/em\u003e and \u003cem\u003eG. barbadense\u003c/em\u003e, they harbored a similar number of the \u003cem\u003eZIM17\u003c/em\u003e genes, with 6 ZIM17 family members in each. Based on the chromosomal location, the genes were annotated as follows; Gher\u003cem\u003eZIM17\u003c/em\u003e-1-2 (\u003cem\u003eG. herbaceum\u003c/em\u003e), Ga\u003cem\u003eZIM17\u003c/em\u003e-1-3 (\u003cem\u003eG. arboreum\u003c/em\u003e), Gth\u003cem\u003eZIM17\u003c/em\u003e-1-3 (\u003cem\u003eG. thurberi\u003c/em\u003e), Gr\u003cem\u003eZIM17\u003c/em\u003e-1-3 (\u003cem\u003eG. raimondii\u003c/em\u003e), Gh\u003cem\u003eZIM17\u003c/em\u003e-1-6 (\u003cem\u003eG. hirsutum\u003c/em\u003e), and Gb\u003cem\u003eZIM17\u003c/em\u003e-1-6 (\u003cem\u003eG. barbadense\u003c/em\u003e). Analysis of the physicochemical properties of the \u003cem\u003eZIM17\u003c/em\u003es revealed that the lengths of their open reading frames (ORFs) ranged from 447 to 621 bp, with amino acid (aa) counts varying from 148 to 206, molecular weights (MW) ranging from 16415.71 to 22861.54, isoelectric points (\u003cem\u003epI\u003c/em\u003e) from 6.06 to 8.63, and instability indices from 35.39 to 51.96 (Table\u0026nbsp;1). Over 70% of the ZIM17 proteins had instability indices above 40, an indication that ZIM17 proteins were significantly unstable, a common attribute of the heat shock proteins. Moreover, pI values showed that nearly all the proteins were nearly neutral, being the pI values ranging from 6.06 to 8.63 with only one with a pI value of 5.77, which further augments that their possible function within the membranes, mainly as heat shock proteins or chaperones.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\n\u003cp\u003eTable 1. Physiochemical properties of the ZIM17 proteins from the 6 cotton species. \u003cem\u003eG. herbaceum\u003c/em\u003e, \u003cem\u003eG. hirsutum\u003c/em\u003e, \u003cem\u003eG. barbadense\u003c/em\u003e, \u003cem\u003eG. arboreum\u003c/em\u003e, \u003cem\u003eG. thurberi\u003c/em\u003e and \u003cem\u003eG. raimondii\u003c/em\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Taba\" border=\"1\"\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGene Annotation\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTranscript ID\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eORF length (bp)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eLength (aa)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGenomic location\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMW (Da)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003epI\u003c/em\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eInstability index\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSubcellular localization\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGherZIM17-1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGhe01G11880\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e621\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e206\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr01:21275809\u0026ndash;21276762+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e22755.42\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e6.06\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e37.75\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNucleus\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGherZIM17-2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGhe05G32290\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e567\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e188\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr05:39580088\u0026ndash;39582567+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e20638.29\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e6.89\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e51.96\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChloroplast\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGaZIM17-1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGa01G1171\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e621\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e206\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr01:22307888\u0026ndash;22308839+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e22767.47\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e6.06\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e40.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNucleus\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGaZIM17-2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGa05G3179\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e450\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e149\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr05:38291545\u0026ndash;38294048+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e16548.97\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e8.63\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e43.44\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChloroplast\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGaZIM17-3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGa12G1114\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e567\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e188\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr12:12802258-12804577-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e20638.29\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e6.89\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e51.96\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChloroplast\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGhZIM17-1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGh_A01G1063\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e621\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e206\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eA01:20563176\u0026ndash;20564123+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e22755.42\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e6.06\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e37.75\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNucleus\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGhZIM17-2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGh_A05G2989\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e567\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e188\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eA05:36820554\u0026ndash;36823031+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e20624.26\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e6.89\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e51.25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChloroplast\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGhZIM17-3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGh_A12G1915\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e450\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e149\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eA12:95617088\u0026ndash;95619401+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e16532.97\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e8.63\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e43.25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChloroplast\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGhZIM17-4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGh_D01G1103\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e621\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e206\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eD01:16273743\u0026ndash;16274691+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e22802.47\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e5.77\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e35.39\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNucleus\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGhZIM17-5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGh_D05G3004\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e543\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e180\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eD05:30795668\u0026ndash;30798118+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e19917.58\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e7.65\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e44.79\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChloroplast\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGhZIM17-6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGh_D12G1914\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e447\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e148\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eD12:50817799\u0026ndash;50820275+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e16444.75\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e6.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e47.76\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChloroplast\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGbZIM17-1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGbar_A01G010000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e621\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e206\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eA01:20316329\u0026ndash;20317682+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e22755.42\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e6.06\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e37.75\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNucleus\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGbZIM17-2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGbar_A05G029230\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e567\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e188\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eA05:35881431\u0026ndash;35884182+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e20638.29\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e6.89\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e51.96\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChloroplast\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGbZIM17-3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGbar_A12G018450\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e450\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e149\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eA12:90166096\u0026ndash;90168769+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e16534.94\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e8.63\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e46.39\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChloroplast\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGbZIM17-4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGbar_D01G010540\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e621\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e206\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eD01:16744844\u0026ndash;16746191+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e22889.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e6.41\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e40.12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNucleus\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGbZIM17-5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGbar_D05G030090\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e561\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e186\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eD05:31265680\u0026ndash;31268403+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e20457.17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e7.65\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e45.74\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChloroplast\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGbZIM17-6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGbar_D12G018590\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e447\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e148\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eD1248731799-48734648+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e16458.77\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e6.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e47.76\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChloroplast\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003ePhylogenetic analysis of the \u003cem\u003eZIM17\u003c/em\u003e gene family\u003c/p\u003e\n\u003cp\u003eTo further investigate the evolutionary relationships among the ZIM17 family members, a phylogenetic tree was constructed using the 23 \u003cem\u003eZIM17\u003c/em\u003e genes were identified from the 6 cotton species along with the three \u003cem\u003eZIM17\u003c/em\u003e genes identified from the \u003cem\u003eA. thaliana\u003c/em\u003e. Based on their evolutionary relationships, these family members were divided into three groups, designated as groups 1, 2, and 3 with 9, 8, and 9 ZIM17 proteins, respectively. In all the three groups, the protein distributions were nearly even, with a node value of 100, an indication that the grouping was a perfect fit. In all the three groups, gene pairing was observed, however, there was a unique observation was noted among the members of groups B and C, in which two homologous pairs were noted between Gr\u003cem\u003eZIM17\u003c/em\u003e-3 and Gb\u003cem\u003eZIM17\u003c/em\u003e-6 (group B) and Gb\u003cem\u003eZIM17\u003c/em\u003e-2 and Ga\u003cem\u003eZIM17\u003c/em\u003e-2 (Group C), these genes shared a common origin, however, ortholog gene pairs were also noted between \u003cem\u003eG. hirsutum\u003c/em\u003e and \u003cem\u003eG. barbadense\u003c/em\u003e in all the three groups, in group A (Gh\u003cem\u003eZIM17\u003c/em\u003e-1 and Gb\u003cem\u003eZIM17\u003c/em\u003e-1; Gr\u003cem\u003eZIM17\u003c/em\u003e-1 and Gth\u003cem\u003eZIM17\u003c/em\u003e-1), Group B (Gh\u003cem\u003eZIM17\u003c/em\u003e-3 and Gb\u003cem\u003eZIM17\u003c/em\u003e-3) and lastly Group C (Gh\u003cem\u003eZIM17\u003c/em\u003e-5 and GbZIM71-5) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The results showed that the genes were recently evolved and highly correlated. Plants do evolve genes to enhance their survival or mitigate the effects of the environmental stresses, thus the high threshold and infection intensity of \u003cem\u003eV. dahliae\u003c/em\u003e might have led to this kind of evolution.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eChromosomal mapping of the cotton \u003cem\u003eZIM17\u003c/em\u003e genes\u003c/p\u003e\n\u003cp\u003eTo understand the distribution of \u003cem\u003eZIM17\u003c/em\u003e gene family members on the chromosomes in different cotton species, a visualization tool was used by integrating the start and the end. The results showed that in the diploid A-genome cotton species, \u003cem\u003eG. arboreum\u003c/em\u003e (A2), the \u003cem\u003eZIM17\u003c/em\u003e genes were mapped on chromosomes 1, 5, and 12, however in \u003cem\u003eG. herbaceum\u003c/em\u003e (A\u003csub\u003e1\u003c/sub\u003e), with only 2 genes, the 2 genes were mapped on chromosome 1 and 5, the 2 diploid cotton species of the A sub genome showed consistency, though the reduction in gene number in \u003cem\u003eG. herbaceum\u003c/em\u003e (A\u003csub\u003e1\u003c/sub\u003e) is evident of gene loss. Among the cotton species of the D subgenomes, \u003cem\u003eG. raimondii\u003c/em\u003e (D\u003csub\u003e5\u003c/sub\u003e) and \u003cem\u003eG. thurberi\u003c/em\u003e (D\u003csub\u003e1\u003c/sub\u003e), had 3 genes, and were mapped in three different chromosomes, however, \u003cem\u003eG. raimondii\u003c/em\u003e (D\u003csub\u003e5\u003c/sub\u003e) chromosome mapping was in synchrony to the gene mapping in \u003cem\u003eG. arboreum\u003c/em\u003e (A\u003csub\u003e2\u003c/sub\u003e), in which the 3 genes were mapped on chromosome 1, 5 and 12 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA i-ii), but on chromosome 2, 8 and 9 in \u003cem\u003eG. thurberi\u003c/em\u003e (D\u003csub\u003e1\u003c/sub\u003e) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB i-ii), the deviation in chromosome mapping among the genes obtained for the D subgenomes could be attributed to chromosomal rearrangement in the course of their evolution. Moreover, consistency was observed among the AD genomes, \u003cem\u003eG. hirsutum\u003c/em\u003e (AD)\u003csub\u003e1\u003c/sub\u003e and \u003cem\u003eG. barbadense\u003c/em\u003e (AD)\u003csub\u003e2\u003c/sub\u003e, the 6 \u003cem\u003eZIM17\u003c/em\u003e genes were mapped on chromosomes A01, A05, A12, D01, D05, and D12 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC i-ii). This is in agreement with the already known hypothesis that AD emerged due to whole genome duplication between A and D, with specific reference to \u003cem\u003eG. arboreum\u003c/em\u003e (A\u003csub\u003e2\u003c/sub\u003e) and \u003cem\u003eG. raimondii\u003c/em\u003e (D\u003csub\u003e5\u003c/sub\u003e)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese findings suggest that there may be conserved syntenic regions among different cotton species about the chromosomal distribution of \u003cem\u003eZIM17\u003c/em\u003e genes. However, the unique distribution pattern observed in \u003cem\u003eG. thurberi\u003c/em\u003e indicates that chromosomal rearrangements or gene duplications may have occurred during the evolution of this species, leading to variations in the chromosomal locations of \u003cem\u003eZIM17\u003c/em\u003e genes. Understanding the chromosomal distribution of \u003cem\u003eZIM17\u003c/em\u003e genes across cotton species can provide insights into their potential roles in cotton development and the evolution of gene families in plants.\u003c/p\u003e\n\u003cp\u003eAnalysis of gene structure and conserved motif of the \u003cem\u003eZIM17\u003c/em\u003e gene family\u003c/p\u003e\n\u003cp\u003eTo further analyze the structure of the \u003cem\u003eZIM17\u003c/em\u003e gene family, the gene structure and conserved motifs across the six species were examined. A total of 9 conserved motifs were identified and named Motif 1 to Motif 9 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). The results revealed that the number of conserved motifs within each group was generally consistent. However, there were some exceptions. For instance, Gth\u003cem\u003eZIM17\u003c/em\u003e-1 in group A and Gth\u003cem\u003eZIM17\u003c/em\u003e-2 in group B harbored an additional Motif 9 compared to other members, while Ga\u003cem\u003eZIM17\u003c/em\u003e-2, Gb\u003cem\u003eZIM17\u003c/em\u003e-2, Gher\u003cem\u003eZIM17\u003c/em\u003e-2, and GH\u003cem\u003eZIM17\u003c/em\u003e-2 in group C had an extra Motif 8 compared to the rest of group members. Notably, Motif 1 (TPISLLTKSPRRDLAVAFTCNVCGERTVRAINR EAYEKGVVFVQCGGCNNFHKJADNLGLF) and Motif 3 (PAISSFNVSRBSRYDVYRRLFDEEP D) were present across all the groups, an indication that the \u003cem\u003eZIM17\u003c/em\u003e gene family could be identified and characterized by motif 1 and motif 3moreover, Motif 2 was exclusive to Group A, Motif 4 and Motif7 were unique to Group B, and Motif 5 and Motif 6 were specific to Group C. In relation to their gene structures, all the \u003cem\u003eZIM17\u003c/em\u003e gene family contained introns and exons, with the number of exons ranging from 2 to 4 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). The number of exons within each group was consistent, with members of group A and group B having 4 exons, while members of group C had 2 exons. Furthermore, the distribution pattern of introns and exons within \u003cem\u003eZIM17\u003c/em\u003e genes was highly uniform within each group. These findings suggest that \u003cem\u003eZIM17\u003c/em\u003e gene family members share conserved structural features but also exhibit specific variations that may contribute to their functional diversity. The identification of conserved motifs and the analysis of gene structures provide insights into the evolution and potential functions of \u003cem\u003eZIM17\u003c/em\u003e genes in cotton and potentially other plant species.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnalysis of \u003cem\u003ecis-\u003c/em\u003eacting elements of the \u003cem\u003eZIM17\u003c/em\u003e gene\u003c/p\u003e\n\u003cp\u003eTo better understand the functional roles of the \u003cem\u003eZIM17\u003c/em\u003e genes, 2000 bp upstream sequences of the coding regions were used for the prediction of the \u003cem\u003ecis-\u003c/em\u003eacting elements. The results showed that the most abundant \u003cem\u003ecis-\u003c/em\u003eacting elements in the upstream regions of \u003cem\u003eZIM17\u003c/em\u003e genes were light-responsive elements. Additionally, several elements related to hormone and stress responses were also identified, including Abscisic acid (ABRE), jasmonic acid (CGTCA-motif and TGACG-motif), salicylic acid (TCA-element), drought (MBS), low temperature (LTR), metabolism (O2-site), and defense and stress (TC-rich repeats) response elements (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurther analysis revealed differences in the number and distribution of \u003cem\u003ecis-\u003c/em\u003eacting elements among groups with different developmental branches. Compared to groups B and C, members of group A possessed a higher number of stress-responsive elements. Notably, defense and stress response elements were absent in both groups A and B, while low-temperature response elements were absent in groups B and C. Additionally, members of group B lacked Abscisic acid response elements, and members of group C did not contain MYB drought stress response elements.\u003c/p\u003e\n\u003cp\u003eThese findings suggest that \u003cem\u003eZIM17\u003c/em\u003e genes may be involved in diverse biological processes, including light response, hormone signaling, and stress responses. The variations in the number and types of \u003cem\u003ecis-\u003c/em\u003eacting elements among different groups indicate that \u003cem\u003eZIM17\u003c/em\u003e genes may have specific functional roles that are tailored to the unique environmental conditions and developmental stages of the cotton species.\u003c/p\u003e\n\u003cp\u003eAnalysis of Gth\u003cem\u003eZIM17\u003c/em\u003e-1 gene expression pattern\u003c/p\u003e\n\u003cp\u003eTo analyze whether the Gth\u003cem\u003eZIM17\u003c/em\u003e-1 gene exhibits tissue-specific expression in \u003cem\u003eG. thurberi\u003c/em\u003e, RNA samples were extracted from the roots, stems, and leaves of this cotton plant. Using the reverse-transcribed cDNA as a template, quantitative real-time PCR (qRT-PCR) was carried out to detect the expression levels of Gth\u003cem\u003eZIM17\u003c/em\u003e-1 in different tissues. The results revealed that the Gth\u003cem\u003eZIM17\u003c/em\u003e-1 gene was primarily expressed in the roots and leaves of \u003cem\u003eG. thurberi\u003c/em\u003e, with relatively lower expression levels in the stems (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo investigate whether the Gth\u003cem\u003eZIM17\u003c/em\u003e-1 gene responds to Verticillium wilt stress in \u003cem\u003eG. thurberi\u003c/em\u003e, tissues were collected at time points (0h, 12h, 24h, and 48h) after inoculation with \u003cem\u003eVerticillium dahliae\u003c/em\u003e. The results showed that within 48 hours of inoculation with \u003cem\u003eV. dahliae\u003c/em\u003e, the expression of the Gth\u003cem\u003eZIM17\u003c/em\u003e-1 gene exhibited a trend of initial increase followed by a decrease (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). This significant change in expression suggests that Gth\u003cem\u003eZIM17\u003c/em\u003e-1 responds to Verticillium wilt, being a negative regulator, the higher expression at initial stages could be to enhance the proliferation of the vegetative tissues of the \u003cem\u003eV. dahliae\u003c/em\u003e, thus increasing their pathogenicity.\u003c/p\u003e\n\u003cp\u003eSubcellular localization of the Gth\u003cem\u003eZIM17\u003c/em\u003e-1 protein\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eZIM17\u003c/em\u003e protein serves as a mitochondrial protein import factor, and prior research indicates that the \u003cem\u003eZIM17\u003c/em\u003e protein is primarily expressed and functions within mitochondria [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. To corroborate its expression in mitochondria, we constructed a subcellular localization fusion vector, \u003cem\u003ep2300-eGFP-GthZIM17-1\u003c/em\u003e, tagged with a green fluorescent protein. This vector was co-transfected with a mitochondrial marker into tobacco cells, and the cellular expression of the gene was observed under a laser scanning confocal microscope. The results demonstrated that the Gth\u003cem\u003eZIM17\u003c/em\u003e-1 protein is expressed in the nucleus, cytoplasm, and mitochondria (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e), indicating a broad expression profile within the cell and suggesting potential diverse functional roles within the plant.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGh\u003cem\u003eZIM17\u003c/em\u003e-4 silencing enhanced the resistance of cotton to \u003cem\u003eVerticillium dahliae\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the disease resistance function of the \u003cem\u003eGthZIM17-1\u003c/em\u003e gene in cotton, specific primers were designed based on the CDS sequence of Gh\u003cem\u003eZIM17\u003c/em\u003e-4, the homolog of Gth\u003cem\u003eZIM17\u003c/em\u003e-1 in Gossypium hirsutum. Two weeks after VIGS injection, positive control plants (TRV: PDS) exhibited an albino phenotype (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA), indicating the successful establishment of the virus induced gene silencing (VIGS) system. True leaves from three randomly selected silenced plants were collected, and the silencing efficiency was assessed using qRT-PCR. The expression levels of Gh\u003cem\u003eZIM17\u003c/em\u003e-4 in the silenced plants were significantly reduced (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB), indicating successful interference of the gene.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUnder \u003cem\u003eV. dahliae\u003c/em\u003e, the leaves of the empty vector plants exhibited more severe yellowing and wilting and had a higher disease index. Phloroglucinol staining of the stems showed more severe browning of the vascular bundles in the empty vector plants. Additionally, phloroglucinol staining was performed on the same region of the stem, approximately 1 cm above the ground, to observe changes in the vascular bundles. The results indicated less lignin accumulation in the silenced plants (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC-D). DAB staining of the leaves revealed more severe damage in the leaves of the empty vector plants (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eE). Recovery experiments with \u003cem\u003eV. dahliae\u003c/em\u003e showed less fungal colonization in the stems of the silenced plants (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eF). moreover, the disease index (DI) was significantly higher in the none VIGS-plants compared to the VIGS-plants (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eG)\u003c/p\u003e\n\u003cp\u003eTo further investigate the impact of \u003cem\u003eGhZIM17\u003c/em\u003e-4 gene silencing on the expression levels of genes associated with disease resistance pathways, qRT-PCR was employed to assess the expression profiles of \u003cem\u003eGhPR1\u003c/em\u003e and \u003cem\u003eGhPR5\u003c/em\u003e genes, the hallmark genes of the salicylic acid (SA) resistance pathway, as well as \u003cem\u003eGhPR3\u003c/em\u003e and \u003cem\u003eGhPDF1.2\u003c/em\u003e, genes related to the jasmonic acid (JA) resistance pathway. The results revealed that in silenced plants, the expression of the \u003cem\u003eGhPR1\u003c/em\u003e gene was significantly upregulated, while the expression of the \u003cem\u003eGhPR5\u003c/em\u003e gene was significantly downregulated (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eH). Additionally, the expression levels of both \u003cem\u003eGhPR3\u003c/em\u003e and \u003cem\u003eGhPDF1.2\u003c/em\u003e genes were significantly upregulated (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eI). The findings were in agreement with previous research in which, silencing of \u003cem\u003eABHYDROLASE-3\u003c/em\u003e gene in \u003cem\u003eBrassica napus\u003c/em\u003e, significantly improved plants tolerance to \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eOverall, findings suggested that upon interference with \u003cem\u003eGhZIM17-4\u003c/em\u003e gene expression in cotton, the silenced plants exhibited enhanced disease resistance. Furthermore, the \u003cem\u003eGhZIM17-4\u003c/em\u003e gene may regulate the resistance of cotton to Verticillium wilt by modulating the expression of genes associated with both SA and JA resistance pathways. However, the specific disease resistance mechanisms underlying this regulation require further exploration, however, the upregulation of disease-resistant genes in VIGS-plants showed that the downregulation of Gh\u003cem\u003eZIM17\u003c/em\u003e-4 improved the ability of the VIGS-plants to induct other disease regulatory genes to be mobilized, thereby enhancing their ability to significantly reduce the disease severity in VIGS-plants compared to the non VIGS-plants. These results further validate the notion that host-induced gene silencing improves the plant performance under biotic stress conditions.\u003c/p\u003e\n\u003cp\u003eOverexpression of the Gth\u003cem\u003eZIM17\u003c/em\u003e-1 gene in Arabidopsis reduced the resistance of Arabidopsis to \u003cem\u003eVerticillium dahliae\u003c/em\u003e infection\u003c/p\u003e\n\u003cp\u003eIn the transformation of the \u003cem\u003eGthZIM17-1\u003c/em\u003e gene into the model plant, the gene was effectively transformed in 10 plants, designated as OE1 to OE10 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA). validation of the expression was further confirmed through RT-qPCR in which OE1 and OE10 were found to have the highest level of upregulation of the overexpressed gene (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eB). The overexpressed lines, OE1 and OE10 lines were further used to evaluate the OE lines response towards \u003cem\u003eV. dahliae\u003c/em\u003e infection. The overexpressing Arabidopsis plants (OE1 and OE10) were transferred to nutrient soil for continued cultivation for one week and then inoculated with 5 \u0026micro;L of \u003cem\u003eV. dahliae\u003c/em\u003e spore suspension at the roots. Two weeks later, the disease incidence of the Arabidopsis was observed and recorded. Consistent with the results obtained in the medium, the overexpressing Arabidopsis lines exhibited more severe disease symptoms following infection with \u003cem\u003eV. dahliae\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eC-D), with a higher disease severity index (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eE-F). The high threshold of the overexpressed gene, accelerated the proliferation of the disease-causing organism, \u003cem\u003eV. dahliae\u003c/em\u003e, resulting in enhanced disease index among the OE plants compared to the wild type. The results obtained were in agreement with previous findings in which overexpression of necrosis and ethylene-inducing peptide 1-like Protein (NLP) gene significantly increased the virulence of \u003cem\u003eVerticillium dahliae\u003c/em\u003e in eggplant, tomato, and cotton [\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e], which, which showed that the increased copy of VdNEP acerbated the virulence factor in plants, thus enhancing their susceptibility to pathogens.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eProtein-protein interaction, Gth\u003cem\u003eZIM17\u003c/em\u003e-1 interacts with GthMOS4\u003c/p\u003e\n\u003cp\u003eThrough screening of a yeast library, GthMOS4 was identified to have an interaction with Gth\u003cem\u003eZIM17\u003c/em\u003e-1. Validation of this interaction using yeast two-hybrid point-to-point assays revealed that the positive control (\u003cem\u003epGBKT7-53\u0026thinsp;+\u0026thinsp;pGADT7-T\u003c/em\u003e) and negative control (\u003cem\u003epGBKT7-Lam\u0026thinsp;+\u0026thinsp;pGADT7-T\u003c/em\u003e) grew normally on \u003cem\u003eSD/-Leu-Trp\u003c/em\u003e solid medium, indicating successful plasmid co-transformation in yeast. The positive control grew normally and turned the yeast colonies blue on \u003cem\u003eSD/-His-Leu-Trp\u003c/em\u003e and \u003cem\u003eSD/-His-Leu-Trp-Ade\u003c/em\u003e defective media containing \u003cem\u003eX-\u0026alpha;-gal\u003c/em\u003e, while the negative control did not grow, confirming the successful establishment of the yeast two-hybrid system. Co-transformed yeast cells expressing MOS4 and DEG15 were able to grow on \u003cem\u003eSD/-His-Leu-Trp\u003c/em\u003e and \u003cem\u003eSD/-His-Leu-Trp-Ade\u003c/em\u003e solid media and turned the yeast colonies blue, while SDR1 did not grow. This suggested a potential interaction between MOS4 and the \u003cem\u003eZIM17\u003c/em\u003e-1 protein.\u003c/p\u003e\n\u003cp\u003eSimilarly, LUC fusion vectors were constructed containing the target proteins and transiently transformed into tobacco using Agrobacterium. The results showed that only one experimental combination exhibited a distinct fluorescent signal (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eA-B). No fluorescent signal was observed in the three control regions of the tobacco leaves. However, a prominent fluorescent signal was detected in the injection area where the combination of \u003cem\u003epCAMBIA1300-GthZIM17-1-nLUC\u003c/em\u003e and \u003cem\u003epCAMBIA1300-GthMOS4-cLUC\u003c/em\u003e was used, indicating an interaction between the \u003cem\u003eZIM17\u003c/em\u003e and MOS4 proteins. Basically, the MOS4 proteins function as suppressers of the autoimmunity-related phenotypes in plants [\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e], thus the positive interaction between \u003cem\u003eZIM17\u003c/em\u003e and MOS4 is a clear indication that the \u003cem\u003eZIM17\u003c/em\u003e protein has inhibitory role in enhancing plants tolerance to pathogens.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eCotton being an important economic crop, its production has dwindled over time due to the effects of biotic and abiotic stress factors. Significant efforts have been made to develop ecologically and area-specific cotton germplasms to alleviate the localized effects of environmental factors. However, due to the complex nature of biotic factor, a diverse strategy is required to reduce the effects of various forms of biotic factors. One of the major biotic factor with deleterious effects on cotton is the causative agent for Verticillium Wilt, known as \u003cem\u003eVerticillium dahliae\u003c/em\u003e, a soil-borne fungi with the ability to remain dormant over a long period. The utilization of genes to improve cotton performance towards \u003cem\u003eV. dahliae\u003c/em\u003e has helped in mitigating its effects. In this research work, the host-induced gene silencing approach was explored to understand the putative role of the \u003cem\u003eZIM17\u003c/em\u003e gene a member of the plant zinc finger proteins. A ZIM17 is a novel zinc finger protein that has been extensively studied in yeast. Is critical in promoting cell division, cell growth, and proliferation of the yeast tissues, thus essential for the viability of the yeast cells [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Furthermore, ZIM17/TIM5 also known as the HEP1have has been found to regulate recombination, and its loss results into hyper recombinant phenotypes which occur due to severe genomic instability as a result of inhibition in mitochondrial iron-sulfur clusters biosynthesis [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In plants, the ZIM17 acts as protein chaperones for heat shock proteins (Hsp70) and functions in the mitochondrial protein importation process. However, in plants, it does not function as a transcription factor but instead serves as a dedicated chaperone protein for Hsp70, facilitating the mitochondrial protein importation process. Studies have shown that members of the cotton heat shock protein family, Hsp70s, can be induced by both biotic and abiotic stresses such as salt, drought, and Verticillium wilt [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The evolution of polyploid species often involves the loss of a large number of genes or the duplication of gene copies, leading to increased genetic diversity and plasticity in polyploid species [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. A genome-wide identification of the \u003cem\u003eZIM17\u003c/em\u003e gene family in the four diploid cotton species with A and D genomes, as well as in two tetraploid cotton species of the AD genome revealed a total of 23 \u003cem\u003eZIM17\u003c/em\u003e genes. In either of the A and D subgenomes, the maximum number of genes was three while in the AD genome, only 6 genes were identified. The distribution of the \u003cem\u003eZIM17\u003c/em\u003e genes in the 6 selected cotton species, revealed low volumes of the \u003cem\u003eZIM17\u003c/em\u003e genes in cotton, this is contrary to other genome wide studies conducted in cotton, for instance the whole genome identification of the plant U-box gene (PUB), 93, 96, 185 and 208 PUBs genes were identified for \u003cem\u003eG. raimondii\u003c/em\u003e (D5), \u003cem\u003eG. arboreum\u003c/em\u003e (A2), \u003cem\u003eG. hirsutum\u003c/em\u003e (AD1) and \u003cem\u003eG. barbadense\u003c/em\u003e (AD2), respectively, [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Moreover, in the identification of genes involved in the flowering 636, 673 and 350 genes were obtained for \u003cem\u003eG. hirsutum\u003c/em\u003e, \u003cem\u003eG. barbadense\u003c/em\u003e and \u003cem\u003eG. arboreum\u003c/em\u003e, respectively [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The low \u003cem\u003eZIM17\u003c/em\u003e gene number across all the various cotton genomes, (A, D and AD) points towards gene loss over time, the finding is in agreement, to a study conducted on soybean, in which 3,765 genes were found to be absent from the reference genome assembly, and further found a significant reduction in the average number of the protein-coding genes, the reduction or loss was attributed to frequency associated with selection for agronomic traits [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The deleterious effect of the \u003cem\u003eZIM17\u003c/em\u003e genes could either have led to their significant loss or reduction across the various genomes including the wild types, such as the \u003cem\u003eG. thurberi\u003c/em\u003e, despite their higher ability to resist \u003cem\u003eV. dahliae\u003c/em\u003e infection.\u003c/p\u003e \u003cp\u003eDue to low gene density, their mapping was skewed to specific chromosomes, for instance in A sub genome, the highest number was observed in \u003cem\u003eG. arboreum\u003c/em\u003e with three genes, which were located on chromosomes 01,05 and 12 similar gene mapping was also observed for the D sub genome, however the most intriguing thing is that despite the higher number of genes in AD genome, the chromosome mapping was similar, in either of the (AD)\u003csub\u003e1\u003c/sub\u003e and or (AD)\u003csub\u003e2\u003c/sub\u003e, the genes were mapped on chromosome A01, A05, A12, D01, D05 and D12. It is worth to note that in series of quantitative trait loci (QTL) analysis for V. wilt in cotton, only chromosome none of the identified QTLs were found to be mapped on the chromosomes in which the \u003cem\u003eZIM17\u003c/em\u003e genes were found to be mapped except for chromosome 5, for instance, previous studies on mapping QTLS for V. wilt resistance in cotton, 23 QTL clusters were mapped on 15 chromosomes (Chr03, Chr04, Chr05, Chr06, Chr07, Chr11, Chr14, Chr15, Chr16, Chr19, Chr20, Chr23, Chr24, Chr25, and Chr26). Moreover, 28 QTL hotshots were found to be associated with different disease resistance traits including one hotspot on chromosome 4 for Verticillium wilt resistance, 3 hotspots on chromosome 19 for the resistance to Verticillium wilt and Fusarium wilt, and micronaire under drought stress conditions [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Additionally, determination of the QTLs for Verticillium wilt resistance in backcross inbred lines, four QTLs were found to be associated with enhancing resistance to V. wilt, in which they were equally distributed on chromosomes 2 and 4 [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The inability to detect any of the \u003cem\u003eZIM17\u003c/em\u003e genes in some of the vital chromosomes, further points towards chromosomal rearrangement being in the resistant variety, \u003cem\u003eG. thurberi\u003c/em\u003e the three \u003cem\u003eZIM17\u003c/em\u003e genes were found to be mapped onto totally unparalled chromosomes compared to the other five cotton genotypes (\u003cem\u003eG. herbaceum\u003c/em\u003e, \u003cem\u003eG. arboreum\u003c/em\u003e, \u003cem\u003eG. hirsutum\u003c/em\u003e, \u003cem\u003eG. barbadense\u003c/em\u003e and \u003cem\u003eG. raimondii\u003c/em\u003e), the three genes were mapped on chromosomes, Chr02, Chr08 and Chr09. A clear indication of chromosomal rearrangement.\u003c/p\u003e \u003cp\u003eThe study of \u003cem\u003ecis-\u003c/em\u003eacting elements and their roles in plant growth, development, and stress response has been a focal point in plant biology. The absence of these \u003cem\u003ecis-\u003c/em\u003eacting elements can potentially affect gene expression and, consequently, their functions [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. In this study, the results revealed that, apart from abundant light-responsive elements, numerous stress and hormone-related response elements, such as Abscisic acid (ABRE), methyl jasmonate (CGTCA-motif and TGACG-motif), salicylic acid (TCA-element), drought (MBS), and low temperature (LTR) response elements were observed. These findings suggest that \u003cem\u003eZIM17\u003c/em\u003es may be involved in plant growth, development, and stress responses. The presence of these diverse \u003cem\u003ecis-\u003c/em\u003eacting elements in the upstream promoter region of \u003cem\u003eZIM17\u003c/em\u003es implies that they may regulate the expression of these genes in response to various environmental cues and hormonal signals. Future functional studies on these \u003cem\u003eZIM17\u003c/em\u003es will be crucial to understanding their exact roles in cotton and potentially other plants, and how they contribute to plant adaptation and survival under diverse environmental conditions. Moreover, plant hormones are signal transducers in the disease resistance processes [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], for instance, salicylic acid (SA) and jasmonic acid (JA) are signaling molecules involved in plant disease resistance [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Through gene silencing mechanisms, the Gh\u003cem\u003eZIM17\u003c/em\u003e-4 expression level was downregulated, and the expression level of \u003cem\u003eGhPR1\u003c/em\u003e, a gene was significantly upregulated despite of playing a critical role in the SA pathway, while the expression of GHPR5 decreased. Salicylic acid and jasmonic acid do have an antagonistic role, thus the variation in expression is in agreement with previous findings in which SA induces resistance against biotrophic and hemitrophic while jasmonic acid induces resitance towards necrotrophic pathogens [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. even though there was a variation in the expression levels of the biotic stress responsive genes, there was a higher lignification of the VIGS-plants, lignin is a protective layer, being the \u003cem\u003eV. dahliae\u003c/em\u003e infects the plants through the roots, the higher lignification at the stem region could be to enhance physical barrier to block the growth of the fungi. The results were in agreement to previous research which found that lignification of cell wall appositions is a conserved basal defense mechanism in the plant innate immune response, and overexpression of \u003cem\u003eGhLAC15\u003c/em\u003e gene improved cell wall lignin deposition which in turn improved transgenic \u003cem\u003eA. thaliana\u003c/em\u003e resistance to Verticillium wilt [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe plant resistance proteins enhance protection by recognizing pathogenic effector molecules and initiating downstream defense. Through yeast hybridization, there was a positive association between the knocked gene and MOS4, MOS4 is a small protein with a coiled-coil domain. Previous studies on Arabidopsis snc1 mutant as an autoimmune model showed that \u003cem\u003emos4-1 snc1\u003c/em\u003e reduced the enhanced resistance of the snc1 mutant to pathogens [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Moreover, the constructed \u003cem\u003emos4-1 npr1-1\u003c/em\u003e double mutants and \u003cem\u003emos4-1 npr1-1 snc1\u003c/em\u003e triple mutants, revealed that the defect in MOS4 blocked the NPR1-independent immune pathway downstream of \u003cem\u003eSNC1\u003c/em\u003e. These findings demonstrated that MOS4 suppresses plant innate immunity (Palma et al., 2007). Furthermore, MOS4 is a core component of the spliceosome-associated complex (MAC), MOS4 in association with MOS12, affects the alternative splicing of plant immune-related R genes, thereby influencing plant immune defense responses [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. The interaction between Gth\u003cem\u003eZIM17\u003c/em\u003e-1 and GthMOS4 provides further evidence of the putative role of Gth\u003cem\u003eZIM17\u003c/em\u003e-1 in negatively regulating the plant immune defense processes through similar mechanisms. However, a deeper understanding of the interaction mechanism requires further investigation. Future studies could focus on elucidating the specific roles of Gth\u003cem\u003eZIM17\u003c/em\u003e-1 and GthMOS4 in plant immunity, as well as exploring their potential involvement in the regulation of alternative splicing and immune-related gene expression.\u003c/p\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eThe low gene density observed among the cotton genomes revealed an element of gene loss, moreover gene loss is a pervasive source of genetic variation among the organisms. The \u003cem\u003eZIM17\u003c/em\u003e genes were significantly low with 2, 3, 3, 3, 6, and 6 members of the \u003cem\u003eZIM17\u003c/em\u003e gene family identified among the four diploid cotton species, \u003cem\u003eG. herbaceum\u003c/em\u003e (A\u003csub\u003e1\u003c/sub\u003e), \u003cem\u003eG. arboreum\u003c/em\u003e (A\u003csub\u003e2\u003c/sub\u003e), \u003cem\u003eG. thurberi\u003c/em\u003e (D\u003csub\u003e1\u003c/sub\u003e), and \u003cem\u003eG. raimondii\u003c/em\u003e (D\u003csub\u003e5\u003c/sub\u003e), as well as in two tetraploid cotton species, \u003cem\u003eG. hirsutum\u003c/em\u003e (AD)\u003csub\u003e1\u003c/sub\u003e and \u003cem\u003eG. barbadense\u003c/em\u003e (AD)\u003csub\u003e2\u003c/sub\u003e, respectively. The expression profiling of the \u003cem\u003eZIM17\u003c/em\u003e genes, showed that Gth\u003cem\u003eZIM17\u003c/em\u003e-1 gene was primarily expressed in the root and leaf tissues of \u003cem\u003eGossypium thurberi\u003c/em\u003e. The root is the first organ to be into contact with the pathogenic fungi, while the leaves are the organ with the highest effect of \u003cem\u003eV. dahliae\u003c/em\u003e infection. The proteins encoded by the \u003cem\u003eZIM17\u003c/em\u003e genes are localized in various cellular compartments including cytoplasm, nucleus, and mitochondria. Using virus-induced gene silencing (VIGS) to interfere with the expression of its homologous gene \u003cem\u003eGhZIM17-4\u003c/em\u003e, there was increased lignin accumulation, reduced vascular bundle browning, fewer \u003cem\u003eV. dahliae\u003c/em\u003e colonization in the stem, and a lower disease index in the silenced plants, indicating a significant enhancement in disease resistance. Furthermore, the expression of SA signaling pathway-related genes \u003cem\u003eGhPR1\u003c/em\u003e was significantly upregulated, while \u003cem\u003eGhPR5\u003c/em\u003e was downregulated in the silenced plants. Additionally, the expression of JA signaling pathway-related genes \u003cem\u003eGhPR3\u003c/em\u003e and \u003cem\u003eGhPDF1.2\u003c/em\u003e was also significantly upregulated, suggesting that the GhZ\u003cem\u003eIM17\u003c/em\u003e-4 gene regulates cotton disease resistance through SA and JA signaling pathways. Disease resistance assays conducted on Gth\u003cem\u003eZIM17\u003c/em\u003e-1 overexpressed Arabidopsis plants revealed that overexpressing plants displayed a higher disease index and increased sensitivity to Verticillium wilt following inoculation with \u003cem\u003eVerticillium dahliae\u003c/em\u003e. These results suggest that the \u003cem\u003eGthZIM17-1\u003c/em\u003e gene may play a negative regulatory role in plant disease resistance. Moreover, using yeast two-hybrid (Y2H) and luciferase complementation imaging (LCI) experiments revealed an interaction between GthMOS4 and GthZIM17-1 proteins.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConsent for publication\u003c/h2\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eThe wild species \u003cem\u003eG. thurberi\u003c/em\u003e and the cultivated species Zhongzhimian 2 utilized in this experiment were obtained from the wild cotton germplasm research group of the Institute of Cotton Research (ICR), Anyang, China. The fungal isolates used in this experiment were obtained from soil infested by the fungi, under the management of the Institute of the Cotton Research (ICR), Anyang, China. All methods used in this experiment were performed in accordance with the relevant guidelines, regulations and legislation.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was funded by National Natural Science Foundation of China (2171994, 332072023, 32272090) and Nanfan Special Project of National Nanfan Research Institute of Chinese Academy of Agriculture Sciences (YBXM2439, YBXM2324, YBXM2309),\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMYY, ROM. JNK, ZZ, and LF; methodology, MYY, ROM, YX, ZZ.; software, MYY and ROM.; validation, MYY, ROM, ZZ, YX and ZZ.; formal analysis, MYY and ROM.; investigation, MYY.; resources, ZZ, XC, YYZ, QKL and JZ.; data curation, MYY, ROM, JNK, MJU, YL, JH, HW, QKL and LF; writing\u0026mdash;original draft preparation, MYY, and ROM.; writing\u0026mdash;review and editing, ROM.; All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe acknowledge the enormous support provided by the entire research group of the wild cotton germplasm resources, of the institute of cotton research (ICR), Anyang, China, the lab technicians and laboratory managers for the support accorded to us during this research work.\u003c/p\u003e\u003ch2\u003eData Availability Statement\u003c/h2\u003e \u003cp\u003eAll data that support this publication are fully provided within the text and its supplementary files\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhang T, Xie Z, Zhou J, Feng H, Zhang T. 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Plant J. 2012;70:916\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Verticillium wilt (VW), HIGS, Disease index, Overexpression (OE), ZIM17 Zinc finger motif protein","lastPublishedDoi":"10.21203/rs.3.rs-4517860/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4517860/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eVerticillium wilt (VW) is one of the major biotic stress factors in cotton production, causing a significant reduction in yields and quality reduction. Even though extensive molecular research has been carried out on V. wilt, the molecular basis of \u003cem\u003eVerticillium dahliae\u003c/em\u003e host response has not been extensively explored. In this research work, the \u003cem\u003eZIM17\u003c/em\u003e, a zinc finger motif protein, was investigated through genome-wide identification, and forward and reverse gene functional analysis to explore the role of \u003cem\u003eZIM17\u003c/em\u003e in six cotton germplasms. Based on the transcription data, \u003cem\u003eGthZIM17-1\u003c/em\u003e was further explored through Virus-Induced gene silencing (VIGS), overexpression, and protein-protein interaction.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eA total of 23 \u003cem\u003eZIM17\u003c/em\u003e genes were identified across the six cotton species, and were phylogenetically grouped into three clusters, designated A, B, and C. The entire gene family was characterized by Motif 1 and 3. The knockdown of the novel gene, \u003cem\u003eGhZIM17-4\u003c/em\u003e, revealed significantly enhanced resistance to V. wilt due to increased lignification with significantly low DAB staining, moreover, the overexpressed (OE) \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, recorded the disease index (DI) percentage above 70% and above compared to the wild type. Moreover, disease-resistant genes \u003cem\u003eGhPR1\u003c/em\u003e, \u003cem\u003eGhPR3\u003c/em\u003e, and \u003cem\u003eGhPDF1,2\u003c/em\u003e were significantly upregulated in the VIGS-plants compared to the none VIGS-plants.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe findings therefore provide proof that the \u003cem\u003eZIM17\u003c/em\u003e gene family plays an integral role of promoting Verticillium wilt, and suppression of its expression in the elite cotton cultivars will contribute significantly in reducing the V. Wilt infection, thereby improving the yield levels in cotton. Moreover, the ZIM17 has a homologous gene type in yeast, thus knockdown of the novel gene in cotton, has a similar effect to that of host-induced gene silencing (HIGS) mechanism.\u003c/p\u003e","manuscriptTitle":"Unveiling Cotton's Defense: Harnessing GthZIM17-1 Inhibition for Verticillium Wilt Resistance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-24 14:23:58","doi":"10.21203/rs.3.rs-4517860/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d1508b31-b826-4632-8bc0-d0988388539c","owner":[],"postedDate":"June 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-10T13:24:01+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-24 14:23:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4517860","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4517860","identity":"rs-4517860","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}