SlWRKY16 and SlWRKY31 of tomato, negative regulators of plant defense, involved in susceptibility activation following root-knot nematode Meloidogyne javanica infection

The involvement of WRKY transcription factors in plant-nematode interactions, and in particular, how these WRKYs participate in regulating the complex morphological and physiological changes occurring after nematode infection, are the topic of active research. We characterized the functional role of the unstudied tomato WRKY genes SlWRKY16 and SlWRKY31 in regulating tomato roots’ response to infection by the root-knot nematode Meloidogyne javanica. Using promoter–GUS reporter gene fusions and qRT-PCR, we show that both SlWRKYs are predominantly expressed during the first half of the parasitic life stages, when feeding-site induction and construction occur. Expression of SlWRKY16 increased sharply 15 days after inoculation, whereas SlWRKY31 was already induced earlier, but reached its maximum expression at this time. Both genes were downregulated at the mature female stage. To determine biological function, we produced transgenic lines overexpressing SlWRKY16 and SlWRKY31 in tomato hairy roots. Overexpression of both genes resulted in enhanced M. javanica infection, reflected by increased galling occurrence and reproduction. Expression profiling of marker genes responsive to defense-associated phytohormones indicated reductions in salicylic acid defense-related PR-1 and jasmonic acid defense-related PI in inoculated roots overexpressing SlWRK16 and SlWRKY31, respectively. Our results suggest that SlWRKY16 and SlWRKY31 function as negative regulators of plant immunity induced upon nematode infection.

During the interaction between Meloidogyne spp.root-knot nematodes (RKNs) and their host, complex morphological and physiological changes occur in the infected plant tissue, ultimately resulting in the establishment of a nematode feeding site that supports nematode development 1 .Plants respond to root-knot nematode (RKN) infection by activating various immune responses that involve multiple recognition systems.One such system is the recognition of pathogen-associated molecular patterns (PAMPs) derived from plantparasitic nematodes by cell surface-localized pattern-recognition receptors, leading to pattern-triggered immunity (PTI).Additionally, plants can detect tissue and cell damage caused by RKN invasion or migration through the recognition of damage-associated molecular patterns by pattern-recognition receptors 2 .Furthermore, plants possess resistance proteins that can directly or indirectly recognize pathogen effectors, triggering effectortriggered immunity (ETI) [3][4][5] .In both PTI and ETI, a crucial step is the extensive reprogramming of gene expression, which is regulated by various transcription factors (TFs) that play important roles in plant innate immunity 6 .Research findings have revealed that within plant genomes, there exists a substantial number of transcription factors (TFs) or transcriptional regulators, estimated to be around 1000-3000.These TFs make up a significant proportion, ranging from 5 to 15%, of all the encoded proteins in plants 7,8 .Transcriptomic studies have provided a comprehensive understanding of TF gene expression during plant defense against pathogens, shedding light on their roles in global gene-expression networks involved in plant defense mechanisms 9 .This

Regulatory element analysis of WRKY promoters.
The upstream region of the SlWRKY genes, spanning 2.0 kb, was isolated from tomato genomic DNA obtained from the source (https:// solge nomics.net/).To conduct in-silico analysis of the promoter sequences, the PlantCARE 44 and PLACE 45 databases were utilized as tools.These databases provided access to cis-acting DNA regulatory elements that bind to the promoter region of the investigated WRKY genes.

Culturing of Nematodes and preparation of inoculum for infection assay. Meloidogyne javanica
was cultured on tomato plants (Solanum lycopersicum cv.Avigail 870) in a greenhouse under controlled conditions.The plants were grown with a 16-h light regime at a temperature of 25 °C for a duration of 4 to 6 weeks.To extract the nematode eggs, a modified version of the method described by a previous study 46 was employed.Tomato roots were thoroughly washed, cut into segments, and macerated in a blender with a 0.05% (v/v) sodium hypochlorite (NaOCl) solution for 3 min.The resulting suspension was then passed through a set of three sieves with mesh sizes of 120, 60, and 30 µm.After discarding the debris collected on the sieves, the eggs deposited on the 30-µm sieve were transferred to a 50-mL test tube.Centrifugal flotation, following the method of Hussey and Barker 47 , was performed.The supernatant containing the eggs was poured onto a 30-µm sieve, washed with tap water, and the eggs were collected into MES buffer.The collected eggs were then sterilized using the procedure outlined by van Vuuren and Woodward 48 .Subsequently, the sterilized eggs were transferred back onto a 30-µm sieve and enclosed in a petri dish with 5 mL of MES buffer.The petri dish was placed in a growth chamber at a 28 °C in complete darkness, allowing the eggs to hatch over a period of 5 to 6 days.
Isolation of RNA and qRT-PCR analysis.Tomato roots were collected at various time points (0, 1, 2, 3, 10, 15, and 28 days post-infection) after M. javanica infection to isolate RNA.The RNA extraction was performed using Invitrogen TRIzol reagent (Thermo Fisher Scientific, Carlsbad, CA, US).To eliminate potential genomic DNA contamination, the RNA samples were subjected to TURBO DNA-free DNase treatment (Applied Biosystems, Foster City, CA) following the manufacturer's instructions.The quantification of gene transcripts for WRKY16 and WRKY31 was carried out using real-time reverse transcription PCR (qRT-PCR) on the total RNA obtained at the mentioned time points.Initially, the synthesis of the cDNA strand was performed using 1 µg of total RNA and the SuperScript III cDNA kit (Thermo Fisher Scientific).The qRT-PCR was carried out with SYBR-Green ROX Mix (ABgene, Epsom, UK), and primers for gene quantification were designed using primer3plus (https:// bioin fo.ut.ee/ prime r3-0.4.0/).The qRT-PCR reaction contained 2 µL of cDNA in a total volume of 10 µL was performed as per Chinnapandi et al. 38 , and the PCR cycles consisted of an initial step of 2 min at 50 °C, followed by 10 min at 95 °C, and then 40 two-step cycles of 10 s at 95 °C and 1 min at 60 °C.For the qRT-PCR, a mixture of all cDNAs was used as a template for calibration curves specific to each primer pair.Each reaction was conducted in triplicate, and the results represent the average of three independent biological experiments.The expression levels of two constitutively expressed genes, β-tubulin (GenBank accession no.NM_ 00124 7878.1), and β-actin (GenBank accession no.U60482.1)were used as endogenous controls for geneexpression analyses in tomato.The transcript levels of each sample were standardized by using the geometric mean of specific housekeeping genes 38 .To validate the qRT-PCR results, the expression of a subset of genes was assessed in two additional independent experiments, yielding consistent outcomes.The expression levels for each treatment were determined relative to the control noninoculated roots.

Plasmid construction and generation of transgenic hairy roots.
For the promoter-GUS reporter assays, the promoter regions (about 1.5 kb upstream of the ATG) of SlWRKY16 (1436 bp) and SlWRKY31 (1496 bp) were amplified from S. lycopersicum genomic DNA using the gene-specific primers (Table S1).After amplification, the genomic fragments obtained were cloned into the pGEMT vector (Promega) and validated through sequencing.Subsequently, the promoter fragment was amplified from the pGEMT vector in both orientations using primers containing appropriate attB sites.The amplified fragment was then cloned into the pDONR221 vector through recombination using a Gateway BP kit (Invitrogen).Finally, the promoter fragment was transferred into the pKGWFS7 49 destination vector through recombination using a Gateway LR kit, resulting in the generation of transcriptional fusions that drive GFP-GUS expression.
To facilitate the overexpression of SlWRKY16 and SlWRKY31, the coding sequences were amplified from tomato plant cDNA derived from isolated total RNA.Gene-specific primers containing attB sites (Supplementary Table S1) were utilized to flank the coding sequences.The resulting PCR products, encompassing the full-length SlWRKY16 (968 bp) and SlWRKY31 (888 bp) sequences, were then inserted into the pDONR221 vector using BP clonase (Invitrogen, Carlsbad, CA).Subsequently, the pDONR221 vector harboring both WRKY genes underwent recombination with the pK7WG2D1 49 , vector using LR clonase (Invitrogen).The resulting constructs were transformed into Escherichia coli (DH5α) cells, and individual colonies were subjected to PCR screening to confirm the presence of inserts.Selected clones were further verified by sequencing analysis.These constructed vectors, including an empty vector control, were subsequently employed for Rhizobium rhizogenes-mediated root transformation, following the provided protocol.All primers utilized in this study were synthesized by integrated DNA technologies.
The transformation of roots using R. rhizogenes and the generation of hairy root cultures.The binary vectors for promoter analysis, pKGWFS7::WRKY16:GUS and pKGWFS7::WRKY31:GUS, together with pK7WG2D,1::OEWKRY16, pK7WG2D,1::OEWKRY31 and empty vector controls (pKGWFS7 and pK7WG2D,1) were introduced into R. rhizogenes ATCC 15834 through electrotransformation 50 .Cotyledons were individually extracted from tomato seedlings aged 15 to 20 days and submerged in a suspension of R. rhizogenes that had been incubated for 2 days at 28 °C, with agitation at 100 rpm.The excised cotyledons were then placed on standard-strength Gamborg's B5 salts medium for a co-cultivation period of 3 days.Subsequently, they were transferred to B5 agar media supplemented with kanamycin at 50 mg/mL (Duchefa, Haarlem, the Netherlands) and timentin (15:1) at 300 mg/mL (Duchefa).Following an incubation period of 7-10 days in the dark at 25 °C, roots began to emerge from the wounded surface of the cotyledons, indicating successful transformation.The hairy roots obtained were subsequently transferred to Gamborg's B5 medium (GB) supplemented with 0.8% (w/v) Gelrite and kanamycin at 50 mg/mL.To confirm the presence of transgenic lines, DNA and RNA from the respective constructs were analyzed using vector-specific GUS and GFP primers (Supplementary Table S1).In the nematode-infection experiments, the transformed roots were subcultured in media without antibiotics for a duration of 2 weeks.Following this, 200 freshly hatched sterile M. javanica juveniles were introduced to inoculate the transgenic root lines.At specified time intervals, root samples were collected for GUS assessment and disease evaluation.The presence and expression of the transgenes in the tomato hairy roots overexpressing WRKY16 and WRKY31 were verified using qRT-PCR analysis, employing the primers provided in Table S2.
Localization of GUS activity through histochemical staining and microscopic examination.The 15-days-old transformed promoter-GUS root lines were subjected to M. javanica infection using the provided protocol.GUS activity was evaluated at 2, 5, 10, 15, and 28 days post-infection (dpi), with non-infected plants serving as controls in the promoter-GUS experiment.At the designated time points, infected and non-infected transgenic roots were collected from Gamborg's media and incubated with GUS buffer (composed of 50 mM sodium phosphate pH 7.0, 10 mM EDTA, 5 mM K 4 [Fe 2 (CN) 6 ], 5 mM K 3 [Fe 2 (CN) 6 ], 0.2% v/v Triton X-100, and 2 mM 5-bromo-4-chloro-3-indolyl β-d-glucuronide) for 12 h at 37 ºC 51,52 .Afterward, the roots were washed twice with distilled water.GUS expression was assessed in a minimum of 15-20 infected transgenic roots at each time point.To examine the cellular localization of GUS expression in giant cells, thin sections were prepared from infected roots at 15 and 28 dpi and examined under a microscope following the established procedure described in reference 53 .
To prepare the GUS-stained roots for histological sectioning, they were first dehydrated in a solution containing 0.25% (w/v) glutaraldehyde and 4% (v/v) paraformaldehyde in 50 mM PBS at pH 7.2.Subsequently, the dehydrated roots were embedded in Technovit 7100 according to the manufacturer's instructions, following the protocol described by Chinnapandi et al. 38 .For capturing GUS-stained root samples at 15 and 28 dpi, a stereomicroscope (Leica MZFLIII, Leica Microsystems GmbH) equipped with a Nikon DS-Fi1 camera was utilized.To conduct the wounding treatment, transgenic roots were initially subcultured in GB media for one week.Using sterile forceps, mechanical wounds were created at various locations along the length of the roots.The wounded roots were then returned to the GB plates and kept in a dark environment.At specific time intervals of 0, 9, and 24 h after GUS staining, the wounded roots were collected and photographed for further analysis.

Evaluation of tomato overexpression roots in response to nematode infection.
To assess the nematode infection in both control and WRKY-overexpressing transgenic lines, root samples were collected from the monoxenic culture after 28 days post-infection (dpi).The degree of nematode infection was determined by counting the number of galls and eggs per gram of root weight, following the methodology described in previous studies [54][55][56][57] .The average values of galls and eggs were obtained by analyzing 10-15 replicates per line.The root-infection experiments were replicated twice, and the results of one experiment are presented here.The data were statistically analyzed for significance, and differences among the means were determined using the Tukey-Kramer test with an alpha level of 0.05, using JMP software (SAS Institute, Cary, NC).To investigate the expression of defense genes in the overexpressing lines after infection, RNA was extracted from the roots 24 h post-infection with M. javanica, following the previously described protocol.www.nature.com/scientificreports/Genomic DNA and cDNA isolation.For the amplification of the promoter regions of WRKY16 and WRKY31, genomic DNA was isolated from 1-month-old soil-grown tomato cv.Avigail 870 seedlings using the cetyltrimethylammonium bromide method, following the procedure described by Goetz et al. 58 .To generate full-length cDNA of SlWRKY16 and SlWRKY31, tomato roots were finely ground in liquid nitrogen, and total RNA was extracted using Invitrogen TRIzol reagent (Thermo Fisher Scientific, Carlsbad, CA).To eliminate potential genomic DNA contamination, the RNA samples were subjected to TURBO DNA-free DNase treatment (Applied Biosystems, Foster City, CA) following the manufacturer's instructions.Subsequently, the DNA-free RNA was reverse transcribed into first-strand cDNA using the Verso cDNA kit.

Statement of compliance.
All experimental research on plants and nematodes described here complies with relevant institutional, national, and international guidelines and legislation.Formal ethical approval is not required.

Results
In-silico identification and characterization of SlWRKY16 and SlWRKY31.To gain a deeper understanding of the involvement of WRKY TFs in the process of RKN parasitism in tomato, we conducted a study to explore the functional characteristics of two WRKY genes that showed differential expression in tomato roots following M. javanica infection, as revealed by a previous RNA-Seq analysis 37,46 .These two WRKY genes are SlWRKY16 and SlWRKY31.In-silico analysis revealed that SlWRKY16 is composed of 323 amino acids spanning three exons, featuring a single WRKY domain (WRKYGQK) and a zinc finger-like motif ligand, C-X4-C-X23-H-X1-H.Similarly, SlWRKY31 consists of 295 amino acids distributed across three exons, with a single WRKY domain (WRKYGQK) and a zinc finger-like motif ligand C-X4-C-X23-H-X1-H.Both proteins carry a nuclear localization signal, RKYGQK, at positions 190 and 160 of SlWRKY16 and SlWRKY31, respectively.According to Huang et al. 21, both SlWRKY16 and SlWRKY31 were classified to WRKY group IIc.Phylogenetic analysis of both studied SlWRKYs was conducted by neighbor-joining method using a bootstrap value of 1000.A genome-wide NCBI-BLAST search of WRKY16 and WRKY31 revealed their homologs in tomato, rice and Arabidopsis.WRKY16 shared the highest sequence homology with three other WRKY proteins of tomato-SlWRKY71, SlWRKY31 and SlWRKY28, with 49.14, 42.15 and 58.75% similarity, respectively.Similarly, WRKY31 had the highest sequence homology with three other tomato WRKY proteins-SlWRKY23, SlWRKY47 and SlWRKY16, with 36.64,51.13 and 42.15% similarity, respectively.Orthologs of SlWRKY16 were reported in rice (OsWRKY3, WRKY8 and WRKY16) and Arabidopsis (AtWRKY57, WRKY23, WRKY48, WRKY28, WRKY8 and WRKY71; Fig. 1); similarly, SlWRKY31 had several orthologs in rice (OsWRKY49, WRKY16, WRKY8 and WRKY3) and Arabidopsis (AtWRKY28, WRKY71, WRKY48 and WRKY23) (Fig. 1).Interestingly, SlWRKY16 and SlWRKY31 exhibited significant homology (40.62% and 43.06%, respectively) with the well-characterized AtWRKY48, a negative regulator of plant immunity against the bacterial pathogen Pseudomonas syringae.
In-silico promoter analysis of SlWRKY16 and SlWRKY31.A 1436-bp and 1496-bp 5′-flanking sequence upstream of the SlWRKY16 and SlWRKY31 translation site (ATG), respectively, were analyzed using the PlantCARE and PLACE databases to investigate the regulatory elements and potential core sequences of the studied promoters (Table 1).The analysis revealed the presence of two primary types of cis-acting elements, specifically stress-responsive and hormone-responsive regulatory elements, within both promoters (as indicated www.nature.com/scientificreports/ in Table 1).Notably, several putative cis-acting elements associated with stress responses were identified, including the TATC-box, TCA element, TGA element, and the pathogenesis-related element BIHD1OS.Additionally, various motifs related to hormone responsiveness were discovered, such as the gibberellin-responsive elements TATC-box, WRKY71OS, and PYRIMIDINEBOXOSRAMY1A; the SA-responsive elements TCA element and WBOXATNPR1; the auxin-responsive TGA element; the ethylene-responsive element ERE; and the abscisic acid-responsive element ABRE.All cis-elements, along with their respective numbers and genomic locations, are listed in Table 1.

Spatiotemporal expression pattern of SlWRKY16 and SlWRKY31 during M. javanica root infection.
The full-length promoter fragments were obtained from tomato DNA and utilized in subsequent cloning procedures, resulting in the generation of binary vectors containing a promoter-GUS fusion construct.These vectors were then employed for the production of WRKY16::GUS and WRKY31::GUS tomato hairy root reporter gene lines through Rhizobium rhizogenes-mediated transformation, as described in detail in the Materials and Methods section.In order to examine the expression patterns of SlWRKY genes following inoculation, GUS signal in the root tissues was assessed at 2, 5, 10, 15, and 28 days post-inoculation (dpi) with second-stage juveniles (J2).The observed GUS expression in the inoculated root lines was compared with that in the respective noninoculated control root lines (Fig. 2a).WRKY16::GUS lines induced clear GUS expression (as observed by the blue signal at 2-15 dpi), associated with penetration and migration in the root-elongation zones in which tissue swelling occurred.At a later time point during nematode maturation and feeding-site establishment, GUS signal associated with the generated gall decreased significantly (28 dpi).GUS signal was primarily detected in the root-elongation zone and along the vasculature of noninoculated control roots (Fig. 2a).
To investigate the role of SlWRKY16 in feeding-site establishment, thin sections of galls expressing SlWRKY16 promoter-GUS constructs were examined at 15 and 28 dpi.At 15 dpi, SlWRKY16 exhibited expression in the endodermis, pericycle cells, and phloem, with a noticeable signal in the developing feeding sites, as indicated by the red precipitate observed under dark-field optics (Fig. 2b).At 28 dpi, the expression level became significantly weaker in the pericycle cells and phloem, as evidenced by the GUS signal observed under light-and dark-field microscopy (Fig. 2b).These findings were consistent with the observations made on the entire root samples.GUS analysis of WRKY31::GUS lines at 2 dpi showed increased expression in the root-elongation zone at the site of penetration and nematode migration (Fig. 3a).At 5 dpi, a pronounced GUS signal was detected in both the vascular system and the swollen root tissue, indicating the presence of nematode invasion (Fig. 3a); this signal remained very strong until 15 dpi.At 28 dpi, GUS signal in all infected root parts gradually diminished, with only residual GUS signal remaining in the developed galls (Fig. 3a).The control roots showed GUS expression in the apical meristem and the elongation zone at all-time points (Fig. 3a).
Thin sections of the SlWRKY31::GUS gall clearly showed high expression of WRKY31 at 15 dpi.The expression was particularly prominent in the cortex and pericycle cells adjacent to the protoxylem poles, encompassing the developing giant cells.This expression pattern was clearly visualized through light-and darkfield microscopy, as evident from the intense GUS signal (Fig. 3b).Notably, the giant cells within the feeding sites associated with the developing nematodes at 15 dpi also displayed a strong GUS signal (Fig. 3b).At 28 dpi, the expression levels decreased such that only a weak signal remained.
In summary, it appears that SlWRKY16 and SlWRKY31 GUS expression peaked at 15 dpi and then declined, indicating that these genes' expression underlies the changes following RKN infection.

Expression of SlWRKY16 and SlWRKY31 is associated with the first half of the parasitic stage.
To determine when, after inoculation, WRKY16 and WRKY31 exhibit their main role, the expression pattern of both genes was evaluated using qRT-PCR in noninoculated control tomato roots and those 1, 2, 3, 10, and 15 dpi.SlWRKY16 transcript was dramatically induced at 15 dpi (in the presence of third-and fourthstage juveniles), increasing 18.44-fold compared to noninoculated roots and earlier time points post-infection www.nature.com/scientificreports/(Fig. 4a).Expression of SlWRKY31 increased gradually after inoculation, peaking at 15 dpi (27.1-fold change) (Fig. 4b).Expression of both genes declined at 28 dpi (Supplementary Fig. S1).These results further support the promoter-GUS assay, indicating that WRKY16 and WRKY31 are involved in plant processes that occur mainly at the infection stage when mature feeding sites have been constructed.

Wound-response cues differentially regulate SlWRKY16 and SlWRKY31 expression.
To explore the possible regulation of SlWRKY16 and SlWRKY31 promoter by wounding, we performed expression analyses using reporter gene GUS constructs fused to the promoters of SlWRKY16 and SlWRKY31 genes.Analysis of WRKY16 in nonwounded control roots showed uniform and constant GUS expression throughout the branching roots (Fig. 5a), whereas no activity of the WRKY16 promoter was detected at the site of wounding, both at 9 h (Fig. 5b) and 24 h (Fig. 5c) post-wounding.These findings imply that the expression of WRKY16 is specifically suppressed at the site of mechanical damage, indicating a localized response to the wound.In contrast, the promoter of WRKY31 did not show a significant increase in GUS activity at 9 h or 24 h after wounding compared to the unwounded control root (Fig. 5d-f).
Overexpression of SlWRKY16 and SlWRKY31 promotes susceptibility to the RKN M. javanica in tomato.To investigate the potential involvement of WRKY16 and WRKY31 in regulating plant responses to nematode infection, tomato plants were transformed using R. rhizogenes-mediated transformation with a plant binary vector containing a 969-bp and 888-bp fragment of SlWRKY16 and SlWRKY31, respectively, under the control of the CaMV35S promoter.A total of 15 putative transformants resistant to kanamycin were obtained and maintained on selective media.Among the kanamycin-resistant transgenic lines carrying the 35S:SlWRKY16 construct, two lines (WRKY16-OE-E2 and WRKY16-OE-E5) with high transcript levels, as confirmed by qRT-PCR analysis (Fig. S2a), were selected for further investigation.Similarly, two lines (WRKY31-OE-E1 and WRKY31-OE-E6) overexpressing WRKY31, showing significant transcript levels (Fig. S2b), were also chosen for subsequent analysis.Notably, the transgenic roots did not exhibit any visible phenotypic differences compared to the control roots (Fig. S3a and b).These selected lines were then subjected nematode-inoculation assays to   www.nature.com/scientificreports/determine their degree of susceptibility to M. javanica.A 3-week-old root culture was inoculated with infective J2 nematodes, and the number of galls and eggs per gram root was determined 28 dpi and used to evaluate the involvement of WRKY16 and WRKY31 in regulating plant susceptibility.Notably, the SlWRKY16-overexpressing lines were hyper-susceptible to M. javanica as indicated by increased reproduction on the respective roots as measured by egg counting, resulting in a 96 to 113% for WRKY16::OE-E2 and WRKY16::OE-E5, respectively compared to control (empty vector) roots (Fig. 6a,c).Similarly, gall numbers increased by 41 to 69% for WRKY16-OE-E2 and WRKY16-OE-E5, respectively compared to control roots (Fig. 6b,c).SlWRKY31-overexpressing lines also showed hyper-susceptibility to M. javanica as indicated by an increase in reproduction by143 to 385% for WRKY31-OE-E1 and WRKY31-OE-E6, respectively compared to control (empty vector) roots (Fig. 7a,c).Similarly, gall counts showed an 80 to 131% increment for WRKY31-OE-E1 and WRKY31-OE-E6, respectively compared to control roots (Fig. 7b,c).This hyper-susceptible phenotype suggests that increased expression of WRKY16 and WRKY31 plays an important role in mediating plant susceptibility to M. javanica infection.

SlWRKY16-and SlWRKY31-overexpressing root lines show alterations in hormone-responsive gene expression.
To assess the contribution of phytohormone-JA, SA and cytokinin-biosynthesis and regulation to the observed increase in susceptibility of roots overexpressing WRKY16 (WRKY16-OE-E5) and WRKY31 (WRKY31-OE-E6), the expression levels of gene markers related to these pathways were investigated using qRT-PCR.The expression of genes commonly used as molecular markers for the activation of the SA signaling pathway was assessed, including pathogenesis-related 1 (PR-1; accession number M69247) and phenylalanine ammonia lyase 5 (PAL5; accession number M90692.1).WRKY16-overexpressing lines demonstrated a significant decrease in SlPR-1 (0.19-fold) compared to wild-type (WT) control roots in both noninoculated (onefold) and inoculated SlWRKY16-overexpressing roots (Fig. 8).Expression of the SA-related marker PAL5 was significantly reduced (0.84-fold) in noninoculated SlWRK16-overexpressing root lines compared to the noninoculated WT control roots (onefold) (Fig. 8).This suggested negative regulation of the SA pathway by SlWRKY16.Expression of the JA-biosynthesis gene SlOPR3 (accession number A1486 721) significantly increased (3.1-fold) in inoculated SlWRKY16-overexpressing roots, whereas the JA marker gene proteinase inhibitor (PI; accession number L21194 ) showed a significant increase (2.4-fold)only in the noninoculated SlWRKY16-overexpressing line compared to the noninoculated WT control (Fig. 8).Subsequently, we examined the expression levels of cytokinin response factors (CRFs), which are known to be transcriptionally induced by cytokinin and participate in the cytokinin signal-transduction pathway.In the case of SlWRKY16-overexpressing roots, both CRF1 (accession number NM_ 00124 7062.2) and CRF6 (accession number XM_ 00424 1080.4)exhibited downregulation with fold changes of 0.38 and 0.19, respectively; in response to RKN inoculation compared to the WT inoculated controls.These findings suggest a suppression of the cytokinin signaling pathway in the SlWRKY16-overexpressing line following RKN inoculation (Fig. 8).In the noninoculated WRKY31-overexpressing line, both SA-related genes-SlPR-1 and SlPAL5-showed significantly increased expression with fold changes of 6.5-and 1.4-fold, respectively related to the noninoculated control (Fig. 9).Infected WRKY31 overexpressing line showed reduced SIPR-1 gene transcript significantly in compare to WT uninoculated control roots.Regarding JA-related genes, inoculated WRKY31-overexpressing lines showed strong repression of OPR3 (0.56-fold) and PI (0.2-fold) compared to control inoculated WT roots.For the cytokinin-responsive genes, a significant reduction (0.58-to 0.61-fold) in CRF1 expression was observed in both noninoculated and inoculated SlWRKY31-overexpressing root lines, along with a significant reduction (0.87-fold) of CRF6 in the inoculated SlWRKY31-overexpressing line compared to control inoculated WT roots (Fig. 9).Taken together, these results indicate that overexpression of WRKY16 and WRKY31 manipulates the signaling pathways of hormones which have been implicated in determining plant immunity during nematode infection.

Discussion
We explored the involvement of tomato TFs SlWRKY16 and SlWRKY31 during M. javanica RKN infection.By utilizing promoter-GUS fusions and qRT-PCR, we demonstrated that the expression of these transcription factors is primarily induced during the first half of the M. javanica infection process, at the critical time when nematodes are embedded in the colonized roots engaging in feeding sites construction.During the later stage of the infection (28 dpi), the expression of both SlWRKY16 and SlWRKY31 gradually diminishes, suggesting a plausible function of these transcription factors at the onset of the infection.Accumulating transcriptomic and proteomic data have revealed upregulation of tomato WRKYs upon nematode infection [37][38][39][40] .Upregulation of WRKY TFs in response to nematode infection is believed to contribute to the plant's defense against these pathogens, the precise roles of the majority of the 83 WRKYs identified in the tomato genome 14 in regulating nematode infection remain unclear.
RKN-induced genes SlWRKY16 and SlWRKY31 are members of the IIc subgroup of WRKY TFs.WRKY16 and WRKY31 were classified as members of the IIc subgroup of WRKY TFs, which comprises 16 members in tomato according to Huang et al. 21.Previous studies have attributed diverse functions to members of this subgroup [59][60][61][62][63] .However, recent research has revealed that WRKY TFs within the same subfamily often share similar functions in regulating the expression of common downstream genes.For instance, in Arabidopsis, WRKY8, WRKY28, and WRKY71 from the IIc subgroup redundantly contribute to the acceleration of flowering by directly activating the transcription of FLOWERING LOCUS T and LEAFY 64,65 .Studies have indicated that the group III WRKYs in Arabidopsis are involved in different plant defense signaling pathways 66 , whereas our knowledge regarding the role of the relatively large IIc subgroup in disease regulation is limited.An example is AtWRKY12, a member of the IIc subgroup in Arabidopsis, which acts in phenylpropanoid pathways by regulating lignin deposition 67 .Mutant plants expressing an atwrky12 mutation, similar to its ortholog in www.nature.com/scientificreports/Medicago truncatula, exhibit thickened secondary walls in pith cells and a significant increase in stem biomass.AtWRKY12 negatively regulates downstream NAC and CCCH-type zinc finger TFs that control the formation of lignin, xylan, and cellulose 67 .Likewise, suppression of AtWRKY12 orthologs in switchgrass and maize leads to abnormal deposition of secondary cell walls 68 .These findings suggest that the IIc WRKY TF AtWRKY12 has conserved roles across different plant species 69 .Another IIc subgroup WRKY protein, AtWRKY23, participates in root development by controlling auxin distribution 70 .AtWRKY23 was found to be inducible by the plantparasitic cyst nematode H. schachtii 1 , especially during the initial stages of nematode feeding-site establishment, and suppression of AtWRKY23 reduces susceptibility to the parasitic nematode 1 .More recently, group IIc WRKY TFs were identified as key regulators in the response of cotton (Gossypium hirsutum) to Fusarium oxysporum.Group IIc WRKY TFs directly bind to the W-box element in the GhMKK2 promoter and regulate the transcription of GhMKK2 in response to F. oxysporum, thus influencing cotton's response to Fusarium 71 .In conjunction with the findings presented here, it appears that members of the IIc subgroup play a significant role in regulating the plant's response to pathogens, among other functions.
Both SlWRKY16 and SlWRKY31 negatively regulate root resistance to the RKN M. javanica.Since wounding and pathogens both trigger a defense response, transcripts of many genes have been shown to increase after both of these events.It is hypothesized that as nematodes migrate through the root and establish feeding sites, they cause some wounding of the plant tissue 72 .However, several studies have shown that many transcripts induced by both processes might have a different role within host cells, as shown for extensin transcripts induced following wounding as compared to RKN infection.Wounding experiments on WRKY16:GUS tomato roots enabled exploring wounding-mediated activation of WRKY TFs 20 as part of the general wound-response mechanism intended to enhance tolerance to wounding.Previous studies have indicated that JA biosynthesis contributes, partially to substantially, to the upregulation of most WRKY genes upon wounding 73,74 .However, their suppression by SA is also observed, because JA and SA have largely antagonistic functions [75][76][77] .While wound-induced suppression of SlWRKY16 expression was observed, a slight increase in SlWRKY31 expression was also noted following the wound, although it was not significant.Paradoxical to the expected function of WRKY proteins in plant defense 20,78 , throughout our study, both WRKY16-and WRKY31overexpressing tomato roots showed increased susceptibility to M. javanica infection.Figure 8.Effect of WRKY16 overexpression on manipulation of hormonal pathway responses.Expression level of defense-related target genes in roots of WRKY16-overexpressing line OE-E5 compared to the control prior to and 1 dpi with M. javanica.Total RNA was prepared from infected and noninfected WT control roots and infected and noninfected roots overexpressing SlWRKY16.The graph displays the mean and SEM representing the relative transcript levels of these genes in SlWRKY16-overexpressing roots (OE-E5) compared to control roots grown under identical conditions.The control expression level was set to zero.All target genes were normalized using the normalization factor, which was calculated as the geometric mean of the expression levels of tomato β-tubulin.Each reaction was performed in triplicate, and the results represent the mean of three independent biological replicates.The statistical significance of the differences between OE-E5 and control roots was assessed using the Tukey-Kramer multiple comparison test, and significant differences in expression (P < 0. This paradoxical observation could be attributed to two potential scenarios.Firstly, it is possible that both WRKY proteins function as negative regulators of basal defense.Negative regulators are known to modulate the plant's response to nematode infection by suppressing or attenuating the plant's defense mechanisms.It is important for plants to regulate their response to nematode infection, as an overactive defense response can lead to excessive cell death and tissue damage, while an insufficient defense response can result in increased susceptibility to infection.Thus, one option is that upon RKN infection, during migration and feeding-site establishment, WRKY16 and WRKY31 operate as negative regulators of plant defense responses.This hypothesis might also be supported by the expression profiling of marker genes responsive to the defense-associated phytohormones SA, JA and cytokinin, indicating that in infected SlWRKY16-overexpressing lines, decreased expression of PR-1, CRF1 and CRF6, along with induced expression of OPR3, is observed.Similarly, SlWRKY31overexpressing lines show suppressed expression of the SA signaling marker genes PR-1 and PAL5, and the JA-related genes PI and OPR3, as well as the cytokinin-responsive gene CRF1 following inoculation.These results suggest that overexpression of SlWRKY16 and SlWRKY31 leads to manipulation of the plant's phytohormone regulation upon RKN inoculation. Enhanced disease resistance in plants is often associated with the upregulation of PR-1 gene transcripts, which play a crucial role in the SA-mediated defense pathway.In our study, we investigated the impact of WRKY16 and WRKY31 genes on PR-1 gene expression in transgenic tomato roots susceptible to nematode infection.Our findings demonstrated a significant decrease in SlPR1 marker transcript levels in susceptible transgenic roots overexpressing WRKY16 and WRKY31.This suggests that WRKY16 and WRKY31 act as negative regulators of pathogen-induced PR gene expression.These results align with a previous study conducted by Xing et al. 28 , which also reported similar results in WRKY48 overexpressing plants.In their study, the overexpression of WRKY48 resulted in enhanced susceptibility to P. syringae and a concomitant reduction in the expression of the SA-regulated PR1 gene.Importantly, the reduction in PR-1 gene expression did not compromise the accumulation of SA in the plants, highlighting the specificity of the regulatory mechanism.Moreover, another report also indicated a reduction in PR-1 transcript levels in plants overexpressing WRKY7 79 , rendering them susceptible to P. syringae.Similarly, Yokotani et al. demonstrated that the overexpression of OsWRKY76 in rice plants resulted in a significant increase in susceptibility to M. oryzae fungi 80 .To gain insights into the underlying mechanisms, microarray analysis was employed.The analysis provided valuable insights, indicating that the overexpression of OsWRKY76 suppresses the induction of PR1, PR10b, and PR15 genes following inoculation Figure 9.Effect of WRKY31 overexpression on manipulation of hormonal pathway responses.Expression level of defense-related target genes in roots of WRKY31-overexpressing line OE-E6 compared to the control prior to and 1 dpi with M. javanica.Total RNA was prepared from infected and noninfected WT control roots and infected and noninfected roots overexpressing SlWRKY31.The graph displays the mean and SEM representing the relative transcript levels of these genes in SlWRKY31-overexpressing roots (OE-E6) compared to control roots grown under the same conditions.The control expression level was set to zero.All target genes were normalized using the normalization factor, which was calculated as the geometric mean of the expression levels of tomato β-tubulin.Each reaction was performed in triplicate, and the results represent the mean of three independent biological replicates.The statistical significance of the differences between OE-E6 and control roots was determined using the Tukey-Kramer multiple comparison test, and significant differences in expression (P < 0. www.nature.com/scientificreports/with the blast fungus 80 .Overall, our study and the aforementioned studies shed light on the regulatory role of WRKY genes, such as WRKY16, WRKY31, WRKY48, WRKY7 79 , and OsWRKY76, in the modulation of PR gene expression and plant susceptibility to various pathogen.The expression of PAL (Phenylalanine Ammonia-Lyase) plays a positive role in the activation of SA signaling pathways.This activation leads to the production of antimicrobial secondary metabolites that protect the plant from pathogens.In our study, we observed that SIPAL5, a marker for SA signaling, was significantly downregulated in WRKY16 and WRKY31 overexpressing lines that showed susceptibility to nematode.Similar findings were reported in CaWRKY70 overexpressing chickpea shoots, where the transcripts of CaPAL were downregulated following Fusarium oxysporum infection 81 .Furthermore, a recent study demonstrated that the overexpression of SlWRKY46 in tomato plants increased their susceptibility to Botrytis cinerea, and was associated with a reduction in PAL 82 .This suggests that the overexpression of WRKY16 and WRKY31 negatively affects PAL expression and, subsequently, the JA signaling pathway.
Protease Inhibitor (PI) is a key gene in the jasmonic acid (JA) pathway.In our study, we found that WRKY31overexpression roots displayed increased susceptibility to nematodes, which was accompanied by a reduction in PI gene transcript levels.Similarly, as previously reported, over-expression of SlWRKY46 resulted in a lower level of PI and a more severe phenotype after Botrytis cinerea infection compared to wild-type plants 82 .Our results are consistent with the findings of the aforementioned studies and confirm that WRKY31 may negatively regulate the resistance of tomato roots to nematodes by affecting the jasmonic acid pathway.Regards alteration in other hormone regulating marker genes OPR3, CRF1, CRF6, no indication for WRKYs implication was indicated so far, however their differential expression in overexpressing lines might support their regulation by the respective SlWRKY.
Several other WRKY genes have been identified with a similar function as negative regulators of basal defense responses in plants.Notably, mutations in Arabidopsis WRKY7, WRKY8, WRKY11, and WRKY17 have been shown to enhance basal resistance to virulent strains of the bacterial pathogen P. syringae 79,[83][84][85] .The Arabidopsis homologs AtWRKY71, AtWRKY48, and AtWRKY23 negatively regulate the plant's immunity to P. syringae and cyst nematodes 28 .In recent study, it has been demonstrated that WRKY48, along with two closely related proteins, WRKY38 and WRKY62, act in an additive manner as negative regulators of basal defense against P. syringae 27,28 .Interestingly, our phylogenetic analysis indicated high similarity of SlWRKY16 to AtWRKY48 (40.62%) and of SlWRKY31 to AtWRKY48 (43.06%), emphasizing their likelihood of also functioning as negative regulators of basal defenses.
The notion that WRKY16 and WRKY31 are subject to hormonal regulation is further supported by the promoter motif analysis, which indicated the presence of a cis-acting element involved in SA responsiveness (CCA TCT TTTT), a W-box recognized specifically by SA-induced WRKY DNA-binding protein (TTG ACC ), and a pathogenesis-related element (TGTCA) in both promoter sequences.Similarly, decreased expression of cytokinin-related genes CRF1 and CRF6 could be explained by gibberellin hormone regulatory elements found on WRKY16 and WRKY31 as a mutually antagonistic interaction between gibberellin and cytokinin in tomato has been suggested 86 .
Several other WRKY genes have been identified with a similar function as negative regulators of basal defense responses in plants.Notably, mutations in Arabidopsis WRKY7, WRKY8, WRKY11, and WRKY17 have been shown to enhance basal resistance to virulent strains of the bacterial pathogen P. syringae 79,[83][84][85] .The Arabidopsis homologs AtWRKY71, AtWRKY48, and AtWRKY23 negatively regulate the plant's immunity to P. syringae and cyst nematodes 28 .In recent study, it has been demonstrated that WRKY48, along with two closely related proteins, WRKY38 and WRKY62, act in an additive manner as negative regulators of basal defense against P. syringae 27,28 .Interestingly, our phylogenetic analysis indicated high similarity of SlWRKY16 to AtWRKY48 (40.62%) and of SlWRKY31 to AtWRKY48 (43.06%), emphasizing their likelihood of also functioning as negative regulators of basal defenses.
The second scenario that might explain the enhanced susceptibility of SlWRKY16 and SlWRKY31 lines to M. javanica infection is hijacking of both WRKYs by the invading nematode.It has been suggested that plantparasitic nematodes exploit the developmental processes of their host plants to establish their nematode feeding sites (NFS) 87 .These nematodes manipulate the expression of plant genes for their own advantage.Given that both WRKY16 and WRKY31 play a role in regulating hormone-related genes, it might be that their expression supports the hormonal conditions that underlie feeding-site expansion and nematode development.Considering the dependence of plant-parasitic nematodes on nutrient acquisition from their host plants, it is tempting to speculate that the SA signaling pathway in the hosts would undergo constant activation.
The results here, where overexpression of both SlWRKY16 and SlWRKY31 suppressed SA marker genes and resulted in increased susceptibility is in agreement with those of Wubben et al. 88 who showed enhanced susceptibility in SA-deficient mutants infected with H. schachtii.This suggests a possible explanation that the nematode can evade plant defense signals by activating both WRKYs, thereby creating a hormone environment conducive to nematode development within the roots.
In conclusion, WRKY TFs play a crucial role in the intricate regulation and precise coordination of signaling and transcriptional networks that govern plant responses to wounding and RKN infection.Our results place SlWRKY16 and SlWRKY31 as negative regulators of plant defense, because overexpression of both TFs affected the expression of defense-related genes, enhancing susceptibility to nematode infection.Further investigation into the regulatory mechanisms underlying SlWRKY16 and SlWRKY31 overexpression may uncover a pivotal point of convergence in the regulatory pathways involved in the plant's responses to both wounding and pathogen infection.These findings highlight the importance of WRKYs in regulating RKN-induced responses.

Figure 1 .
Figure 1.Phylogenetic tree of SlWRKY16 and SlWRKY31 generated using the neighbor-joining method with bootstrap value of 1,000 in MEGA (version 6) software.Phylogenetic analysis was carried out on WRKY genes from Arabidopsis (AtWRKY), rice (Oryza sativa-OsWRKY) and tomato (SlWRKY).Phylogenetic tree representing homology among WRKY genes from tomato, Arabidopsis and rice.*Negative regulator of plant immunity.

Figure 4 .
Figure 4. Expression of tomato WRKY genes in response to M. javanica infection.Expression of SlWRKY16 (a) and SlWRKY31 (b) genes in tomato roots was determined by qRT-PCR at 1, 2, 3, 10 and 15 dpi and in uninfected control root segments (0 dpi).Data include three independent biological and three technical replicates.WRKY16 and WRKY31 expression values are relative to uninfected control roots and were normalized using tomato β-tubulin as a reference gene.Bars indicate SEM.Different letters above the bars indicate statistically significant differences based on Tukey's HSD test (P < 0.05).

Figure 5 .
Figure 5. Expression patterns of WRKY16 and WRKY31 in transgenic lines under basal and wound-induced conditions.Histochemical GUS staining of WRKY16::GUS in control unwounded roots (a) and in roots 9 h (b) and 24 h (c) after mechanical wounding.Histochemical GUS staining of WRKY31::GUS in unwounded control roots (d) and in roots 9 h (e) and 24 h (f) after mechanical wounding.Scale bar = 1 mm.Red arrows indicate the location of mechanical wounding.

Figure 6 .
Figure 6.SlWRKY16 overexpression in tomato hairy roots enhances M. javanica nematode infestation.Increased susceptibility of tomato hairy roots overexpressing SlWRKY16 (WRKY16-OE-E2 and WRKY16-OE-E5) expressed as increased infection reflected by number of (a) eggs and (b) galls compared to the control line (empty vector).(c) Susceptibility of tomato roots overexpressing SlWRKY16 to nematodes compared to control line.All root lines were subjected to inoculation with 500 sterile preparasitic J2s, and the infected roots were examined 28 days post-infection (dpi) to evaluate gall and egg development using a dissecting microscope.It is worth noting the significant (P < 0.05) increase in the percentage of eggs and galls in the WRKY16-OE-E2 and WRKY16-OE-E5 root lines compared to the control group.The data are presented as the means of 15 plants from each line, and the experiment was repeated three times, yielding consistent outcomes.The SEM represents the percentage of each developmental stage.Different letters above the bars indicate significant differences (P < 0.05, ANOVA) among the hairy root lines, as determined by Tukey-Kramer multiple comparison tests.

Figure 7 .
Figure 7. SlWRKY31 overexpression in tomato hairy roots enhances M. javanica nematode infestation.Increased susceptibility of tomato hairy roots overexpressing SlWRKY31 (WRKY31-OE-E1 and WRKY31-OE-E6) expressed as increased infection reflected by number of (a) eggs and (b) galls compared to the control line (empty vector).(c) Susceptibility of tomato roots overexpressing SlWRKY31 compared to control line.All root lines were subjected to inoculation with 200 sterile preparasitic J2s, and the infected roots were examined 28 days post-infection (dpi) to evaluate gall and egg development using a dissecting microscope.It is important to note the significant (P < 0.05) increase in the percentage of eggs and galls observed in the WRKY31-OE-E1 and WRKY31-OE-E6 root lines compared to the control roots.The data are presented as the means of 15 plants from each line, and the experiment was repeated three times, yielding consistent results.The SEM represents the percentage of each developmental stage.Different letters above the bars indicate significant differences (P < 0.05) among the hairy root lines as determined by Tukey-Kramer multiple comparison tests.
Figure 8.Effect of WRKY16 overexpression on manipulation of hormonal pathway responses.Expression level of defense-related target genes in roots of WRKY16-overexpressing line OE-E5 compared to the control prior to and 1 dpi with M. javanica.Total RNA was prepared from infected and noninfected WT control roots and infected and noninfected roots overexpressing SlWRKY16.The graph displays the mean and SEM representing the relative transcript levels of these genes in SlWRKY16-overexpressing roots (OE-E5) compared to control roots grown under identical conditions.The control expression level was set to zero.All target genes were normalized using the normalization factor, which was calculated as the geometric mean of the expression levels of tomato β-tubulin.Each reaction was performed in triplicate, and the results represent the mean of three independent biological replicates.The statistical significance of the differences between OE-E5 and control roots was assessed using the Tukey-Kramer multiple comparison test, and significant differences in expression (P < 0.05) are indicated by different letters above the bars.
Figure 9.Effect of WRKY31 overexpression on manipulation of hormonal pathway responses.Expression level of defense-related target genes in roots of WRKY31-overexpressing line OE-E6 compared to the control prior to and 1 dpi with M. javanica.Total RNA was prepared from infected and noninfected WT control roots and infected and noninfected roots overexpressing SlWRKY31.The graph displays the mean and SEM representing the relative transcript levels of these genes in SlWRKY31-overexpressing roots (OE-E6) compared to control roots grown under the same conditions.The control expression level was set to zero.All target genes were normalized using the normalization factor, which was calculated as the geometric mean of the expression levels of tomato β-tubulin.Each reaction was performed in triplicate, and the results represent the mean of three independent biological replicates.The statistical significance of the differences between OE-E6 and control roots was determined using the Tukey-Kramer multiple comparison test, and significant differences in expression (P < 0.05) are indicated by different letters above the bars.

Table 1 .
Identification of hormone-specific cis-regulatory elements in WRKY16 and WRKY31 promoter regions using PLACE and PlantCARE databases.Element repeats are noted according to their occurrence.