Tomato (Lycopersicon esculentum) cv Avigail 870 was used as the background line for the transformation in experiments involved tomato plants. For the transformation protocol, tomato seeds were treated with 1.6% NaOCl for 10 min with continuous shaking, washed with sterile water for 5 min, and placed on standard-strength Gambourg's B5 medium supplemented with 2% sucrose and 0.8% Gelrite agar (Duchefa, Haarlem, The Netherlands). The plates were kept in darkness at 26°C for 2 days, followed by 2 weeks under a 16/8-h photoperiod until cotyledons emerged. They then were used immediately for cocultivation as described by Chinnapandi et al. (2017). Similarly, tomato roots sections were subculture, by placing one root per Petri dish (Miniplast, Ein Shemer, Israel), containing B5 medium. Branching roots were used for inoculation as described below. For experiments conducted on Arabidopsis, A. thaliana WT and transgenic lines were grown in a growth chamber at 24°C on MS medium (Murashige and Skoog, 1962) or in pots filled with cocopeat. The plants were maintained under a 14 h/10 h day/night cycle at a light intensity of 50–60 μmol/m²s. Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used as the genetic background for all transgenic lines.

Meloidogyne javanica was propagated on greenhouse-grown tomato “Avigail” (870) plants. Nematode egg masses were extracted from roots with 0.05% (v/v) sodium hypochlorite followed by sucrose flotation (Hussey and Baker, 1973). For sterilization, eggs were placed on a cellulose–acetate filter membrane (Sartorius Stedim Biotech GmbH, Goettingen, Germany, pore size 5 μm) in a sterile Whatman® filter holder (Whatman International Ltd., Dassel, Germany). Eggs on the filter were exposed for 10 min to 0.01% (w/v) mercuric chloride (Sigma-Aldrich, St Louis, MO, USA), followed by 0.7% (v/v) streptomycin solution (Sigma-Aldrich), and three washing steps with 50 ml sterilized distilled water (Jansen van Vuuren and Woodward, 2001). The sterilized eggs were collected from the membrane and placed on 25-μm-pore sieves in 0.01 M 2-morpholinoethanesulfonic acid buffer (Sigma-Aldrich) under aseptic dark conditions for 3 days, allowing J2s to hatch. Freshly hatched preparasitic J2s were collected in a 50 ml falcon tube. For nematode infection, 1-week-old transgenic tomato root hairy root lines, growing on standard-strength Gamborg's B5 salt medium, were inoculated with 200 sterile freshly hatched M. javanica preparasitic J2s. Plates were left uncovered in a laminar flow hood until water had completely soaked into the medium (Sijmons et al., 1991). The inoculated and non-inoculated roots were incubated horizontally in the dark, and root samples were taken for GUS bioassay at the designated time points after wounding or inoculation. To test the response of transgenic Arabidopsis plants to nematode infection, three transgenic lines were used, 10 plants per line in each experiment. Seeds of WT, vector-only and transgenic Arabidopsis lines were surface sterilized and germinated on standard-strength GB media with 2% (w/v) sucrose and 0.8% (w/v) Gelrite as described previously by Joshi et al. (2019). One-week-old seedlings grown in vitro were gently transferred with a forceps to a petri dish containing GB media, one plant per dish. Each seedling was inoculated with 300 sterile freshly hatched M. javanica J2s applied near the roots; the plates were left open until the solution was completely absorbed into the media (Sijmons et al., 1991). The infected seedlings were kept vertically and root samples were gently harvested 28 days post-infection (dpi).

Expression of LeDES in infected tomato roots (non-transgenic) at different time points was analyzed by qRT-PCR using gene-specific primers LeDes-rtF: 5′-CCGGATGAGTTTGTACCTGA-3′ and LeDes-rtR: 5′-ATCTTTGCCTGGACATTGCT-3′(López-Ráez et al., 2010). Ten root systems (100 mg) from each time point were pooled from uninfected and infected roots. Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and subjected to Turbo DNase (Ambion, Thermo Fisher Scientific, Waltham, MA, USA). One microgram of total RNA was reverse-transcribed to cDNA using the Verso cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's instructions; qRT-PCR was carried out in a StepOne Real Time PCR system (Applied Biosystems, Foster City, CA, USA) in 10 μl reaction mix comprised of 3.4 μl cDNA, SYBR-Green ROX Mix (Abgene, Portsmouth, NH, USA), 150 nM forward primer and 150 nM reverse primer as described in Chinnapandi et al. (2019). Reactions were performed in MicroAmp 96-well plates (Applied Biosystems) and PCR cycles consisted of initial denaturation at 95°C for 10 min; 40 cycles at 95°C for 15 s, 56°C for 20 s, and 72°C for 20 s, and a final extension at 72°C for 2 min. Reactions were conducted in triplicate, with a control with no template, and the presented results are the means of two independent biological experiments. The qRT-PCR results were analyzed and interpreted using the 2^−ΔΔCt method (Livak and Schmittgen, 2001) integrated into StepOne v2.3 of the StepOne Plus (Applied Biosystems) real-time PCR instrument. Tomato β-tubulin gene (GenBank accession number NM_001247878.1) was used as the internal reference to normalize the expression of LeDES.

Genomic DNA was isolated from soil-grown tomato seedlings, cv. Avigail 870, according to the cetyltrimethylammonium bromide (CTAB) method (Murray and Thompson, 1980). A promoter–GUS construct, to study the expression pattern of LeDES, was generated by Gateway Technology (Invitrogen). A 1,602-bp sequence upstream of the ATG start codon of LeDES was amplified with Platinum Taq DNA polymerase (Invitrogen) using the primers LeDESprom (attB1) 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCT-GTATAGTGTAGCTACGCGCTT-3′ and LeDESprom (attB2) 5′-GGGGACCACTTTGTACAAGAAAGCTGGGT-TTTTCTTAACAAGTTTTGG-3′ and tomato genomic DNA as the template. The purified product was first cloned into the pDONR221 vector (Invitrogen) by BP reaction, generating the entry vector. The entry vector was then cloned by LR Clonase II (Thermo Fisher Scientific) into the destination vector pKGWFS7 (VIB-Ugent Center for Plant System Biology, Ghent, Belgium) upstream of GFP::GUS-coding sequences, generating the pKGWFS7:prom-LeDES (pLeDES::GUS) construct. The resulting promoter construct was confirmed by sequencing prior to Rhizobium rhizogenes transformation. The pLeDES::GUS construct was mobilized to Rhizobium rhizogenes ATCC 15834 by freeze–thaw procedure as described by Xu and Li (2008). The transformed R. rhizogenes colonies were confirmed by colony PCR using LeDES promoter-specific primers (pLeDESF: 5′-GTATAGTGGAGCTCCGCGCTT-3′ and pLeDESR: 5′-CCCCCGGGTTTTCTTAACAAGTTTTGG-3′) and kanamycin primers (kanF: 5′-GCTCTTCGTCCAGATCATCC-3′ and kanR: 5′-GCGTTCAAAAGTCGCCTAAG-3′).

R. rhizogenes-mediated transformation of tomato cotyledons was carried out according to the protocol described by Chinnapandi et al. (2017). Two weeks after transformation, cotyledons with emerging kanamycin-resistant roots were transferred to Gamborg's B5 salts medium (GB; Duchefa Biochemie, Haarlem, The Netherlands) containing 0.8% (w/v) Gelrite + cefotaxime (200 mg/L) + kanamycin (50 mg/L). Roots were excised from the cotyledons once they were 1.0 cm long. The excised roots were transferred to individual GB medium plates (with 200 mg/L cefotaxime + 50 mg/L kanamycin). After two rounds of subculture, cefotaxime was eliminated from the media and transgenic roots were maintained on GB medium plates (with 50 mg/L kanamycin) for further analysis. The genomic DNA of the hairy roots was isolated and used as a template for identification of the positive transgenic lines with promoter-specific (pLeDESF and pLeDESR) and kanamycin (kanF and kanR) primers.

For the wounding treatment, transgenic roots were subcultured in GB media for 1 week. Roots were mechanically wounded at several points across their length using sterile forceps. Wounded roots were kept on the GB plates in the dark and collected after 6 and 24 h. GUS activity was assessed histochemically by incubating root samples in GUS staining buffer: 50 mM sodium phosphate (pH 7.0), 10 mM EDTA, 5 mM K₄[Fe₂(CN)₆], 5 mM K₃[Fe₂(CN)₆], 0.2% (v/v) Triton X-100, and 2 mM 5-bromo-4-chloro-3-indolyl ß- D-glucuronide (X-Gluc) overnight at 37°C as described previously (Iberkleid et al., 2015). The stained roots were washed twice with distilled water, and then suspended in water in petri plates or mounted on slides and observed under a Leica DMLB light microscope (Leica Microsystems GmbH, Wetzlar, Germany) and photographed with a Nikon Eclipse 90i (Nikon Corporation, Tokyo, Japan), or observed under a stereomicroscope (Leica MZFLIII, Leica Microsystems GmbH) equipped with a Nikon DS-Fi1 camera.

To study the effect of hormones on the expression of LeDES, 1-week-old pLeDES::GUS roots were subcultured on a GB plate supplemented with indoleacetic acid (IAA; 1 and 5 μM), indolebutyric acid (IBA; 1 and 10 μM), methyl jasmonate (Me-JA; 0.01 and 0.1 mM), or methyl salicylate (Me-SA; 1 and 5 mM) for 16 h. One-week-old roots grown on GB medium were used as a control. Treated roots were harvested and analyzed by GUS staining as described above.

Prior to the infection assay with M. javanica J2s, the established root cultures were transferred to antibiotic-free GB media for 1 week. Control and pLeDES::GUS root cultures were inoculated with 300 J2s per culture previously sterilized as described above. Non-infected transgenic roots served as the control. The inoculated and non-inoculated transgenic roots were placed in the dark, and the roots were gently harvested for analysis of histochemical GUS activity at designated time points post-inoculation.

For tissue localization of GUS signal within galls, stained galls were dissected, fixed in 1% (w/v) glutaraldehyde and 4% (v/v) formaldehyde in 50 mM sodium phosphate buffer pH 7.2, dehydrated, and embedded in Technovit 7100 (Heraeus Kulzer, Wehrheim, Germany) according to the manufacturer's instructions. Semi-thin sections (3 μm) were cut, mounted in DePeX (Sigma-Aldrich) and observed under a Nikon DS Ri2-equipped microscope with dark-field illumination (Leica Microsystems GmbH).

For amplification of LeDES, PCR was performed with cDNA from 1-month-old tomato plants as the template. The full-length LeDES (1,437 bp) coding sequence was amplified using gene-specific primers (LeDESF 5′-GTATGGGTACCATGGATACAAA CTTGG-3′ and LeDESR 5′-CCTAGAAGCTTTTATATAATTTTTTGCATTTGA-3′), with KpnI and HindIII restriction enzyme cutting sites in the forward and reverse primers, respectively. The PCR products were digested with KpnI/HindIII and ligated into the pHANNIBAL vector. The expression cassette (3,700 bp) containing LeDES flanked by the CaMV35S promoter and OCS terminator was obtained as fallout after digestion with NotI restriction enzyme, which was subsequently cloned into the pART27 binary vector to generate expression plasmid pART27::LeDES. The identity, orientation, and vector-insert junctions for pART27::LeDES were checked by restriction enzyme digestion and sequencing. The pART27::LeDES and pART27 empty vector were transferred into Agrobacterium tumefaciens strain GV3101 by freeze–thaw method as described in Xu and Li (2008). Positive Agrobacterium transformants containing pART27::LeDES and pART27 were transformed into Arabidopsis Col-0 plants (5-week-old flowering plants) by floral dipping method (Clough and Bent, 1998). The T₁ transformants were screened on agar plates containing half-strength MS medium supplemented with 50 mg/L kanamycin. Segregation ratios of T₂ lines with kanamycin resistance were calculated and lines showing an insertional event were selected. Independent T₃ plants homozygous for kanamycin resistance were used for the infection experiments. The presence of LeDES was confirmed by PCR on genomic DNA isolated from the transgenic lines. Expression of LeDES for each homozygous line was determined by RT-PCR using cDNA.

Analysis for the presence of CnA and CA in transgenic Arabidopsis roots was performed according to method described by Kuroda with minor modifications (Kuroda et al., 2005). Production of CnA and CA from (9S)-hydroperoxy linoleic acid (9-HPODE; Cayman Chemicals, Ann Arbor, MI, USA) by root lysate of transgenic Arabidopsis lines (D1, D2, and D3) and control lines was measured by LC–MS as follows. Arabidopsis root samples were harvested 2 weeks after germination and homogenized in Bead Ruptor Elite (OMNI International, Kennesaw, GA, USA) and 500 μl of 50 mM sodium phosphate buffer pH 7. Samples were centrifuged at 15,000 g and the supernatant was collected and incubated with 5 μg/ml 9-HPODE at 30°C overnight. The reaction was terminated by adding an equal amount of 1 ppm 2-hydroxydecanoic acid (Cayman Chemicals) in ethanol. LC–MS analyses were conducted using a UPLC-Triple Quadrupole mass spectrometer (Waters Xevo TQ MS, Waters Corp., Milford, MA, USA). Separation was performed on a Waters Acquity UPLC BEH C18 1.7 μm, 2.1 × 100 mm column with a VanGuard precolumn (BEH C18 1.7 μm, 2.1 × 5 mm). Chromatographic and MS parameters were as follows: the mobile phase consisted of water (phase A) and acetonitrile (phase B), both containing 0.1% (v/v) formic acid in gradient elution mode. The flow rate was 0.3 ml/min, and the column temperature was kept at 35 °C. LC–MS analysis was performed using the ESI source in positive ion mode for CA and CnA and in negative ion mode for 2-hydroxydecanoic acid with the following settings: capillary voltage 3.1 kV, cone voltage 30 V, desolvation temperature 350°C, desolvation gas flow 650 L/h, source temperature 150°C. Quantification was performed using MRM acquisition by monitoring the 293/275, 293/93 [retention time (RT) = 9.50, dwell time of 100 ms for each transition] for CnA, 295/81, 295/277 (RT = 10.08, dwell time of 100 ms) for CA, 187/141, 187/169 (RT = 6.51, dwell time of 161 ms) for 2-hydroxydecanoic acid. Acquisition of LC–MS data was performed using MassLynx v4.1 software (Waters).

To study the extent of nematode infection in WT, vector-only, and transgenic Arabidopsis lines, infected roots grown in monoxenic culture were collected at 28 dpi and analyzed following the procedure describe by Joshi et al. (2019, 2020) with minor modifications. Roots were gently harvested from the plates, agar was removed, and they were weighed. After cleaning, the roots were stained with acid fuchsin (Sigma-Aldrich) solution (17.5 mg acid fuchsin, 500 ml ethanol, 500 ml acetic acid) overnight. Stained roots were washed three times with distilled water, then mounted in water, and roots and galls were manually dissected under a stereomicroscope (Olympus SZX12, Tokyo, Japan). Different nematode development stages (J3/J4 and adult females) embedded in the roots and galls were manually dissected and counted for each root. Each treatment (Arabidopsis line) included 10 individual plates and the infection assays were repeated three times. Infection assay data were fitted with general linear mixed model in SPSS, by keeping samples as fixed factors and repeated assays as random factor. Further, Tukey post-hoc range test was applied to check significant differences (p < 0.05) among different samples.

Full-length LeDES cDNA was cloned into the yeast expression vector pFL61 under the control of the constitutive yeast phosphoglycerate kinase (PGK) promoter (Minet et al., 1992). To generate yeast expression of the construct, LeDES cDNA was amplified from pART27::LeDES using NotI-containing primers (DesNotF: 5′-TTGCGGCCGCATGTCTTCTTATTCAGAG-3′ and DesNotR: 5′-AAGCGGCCGCCTATTTACTTGCTTTAGT-3′) and cloned into the pFL61 vector. Saccharomyces cerevisiae INVSc1 (MATa his3Δ1 leu2 trp1-289 ura3-52/MATα his3Δ1 leu2 trp1-289 ura3-52), procured from Invitrogen, was used as the host strain for yeast transformation. S. cerevisiae was transformed with the empty pFL61 vector (negative control) and with the pFL61::LeDES construct using the lithium acetate/polyethylene glycol transformation method (Sanadhya et al., 2015). Positive colonies were selected on synthetic dropout (SD) plates with synthetic defined medium lacking uracil (SD/-Ura; Clontech, Mountain View, CA, USA), and verified by PCR using gene-specific primers. Expression of LeDES in transgenic yeast clones was further confirmed by RT-PCR using cDNA.

The method used in this study to analyze the impact of the transgenic yeast harboring LeDES was adapted from Naor et al. (2018) with some minor modifications. S. cerevisiae cells transformed with pFL61 and pFL61::LeDES were grown overnight at 28°C from individual colonies in 5 ml of SD/-Ura medium. The precultured yeast cells were pelleted by centrifugation and resuspended in 10 ml of induction medium: SD/-Ura with or without 9-HPODE or 13-HPODE to a final optical density at 600 nm of 2.0. After overnight incubation at 28°C with the respective oxylipin, 300 sterile J2s suspended in 0.01 M MES buffer were added to the cultures and incubated for additional 24 h at 25°C with shaking at 30 rpm. After 24 h incubation, the J2s in the suspensions were collected and subjected to 30-μm filtering (AD Sinun Technologies, Petach Tikvah, Israel) for 2 h (Sanadhya et al., 2018). J2s that actively passed through the filter were counted with nematode-counting slides (Chalex LLC, Portland, OR, USA) under the microscope (Wilovert Standard microscope, Helmut Hund GmbH, Wetzlar, Germany). To measure % motility, J2s counting in treatments included pFL61 or pFL61::LeDES plasmids and the oxylipins 9-HPODE or 13-HPODE were divided by J2s counts following incubation with respective plasmids but without the respective oxylipins. Thus, the pure nematotoxic activity of LeDES could be assessed. Ten replicates were used for each treatment and experiments were performed three times independently. A general linear mixed model ANOVA test was applied to the data with similar results. Different letters above the columns indicate significant difference (P ≤ 0.05) among the different treatments analyzed by Tukey post-hoc range test.

colnelenic acid (CnA) and colneleic acid (CA) were produced from 9-HPODE by yeast lysate of transgenic and control yeast lines according to the method described by Kuroda with minor modifications (Kuroda et al., 2005). Yeast strains carrying empty vector and LeDES were precultured in SD/-Ura liquid medium at 28°C. After 16 h, the starter culture was added to a 250-ml flask containing 50 ml SD/-Ura liquid medium and incubated at 28°C for 24 h. The yeast cultures were centrifuged at 2,744 g for 10 min, and the pellet was weighed and then homogenized in 500 μl 50 mM phosphate buffer pH 7 by Bead Ruptor Elite (OMNI International, Kennesaw, GA, USA) and centrifuged. The lysate was collected and incubated with 5 μg/ml 9-HPODE at 30°C overnight. The reaction was terminated by adding an equal amount of 1 ppm 2-hydroxydecanoic acid in ethanol. LC–MS analyses of the lysates were conducted as described above.

To study the expression of LeDES in roots of tomato challenged with M. javanica J2s, qRT-PCR was performed with cDNA from root tissues collected 1, 2, 3, 10, 15, and 28 dpi. The nematodes induced activation of LeDES, as shown by a moderate course of increase in expression at all-time points upon inoculation (Figure 1). The relative fold changes in LeDES transcript in infected roots at 1, 2, 3, and 10 dpi were approximately 1.9, 5.3, 3.6, and 4.3, respectively, compared to non-inoculated roots. Transcript abundance of LeDES was dramatically induced at 15 dpi (17.9-fold), and was the highest for all-time points considered in this study (Figure 1). LeDES expression levels declined at 28 dpi, suggesting that LeDES has a role in the early defense mechanism induced by nematode penetration and migration, with expression peaking when the young developing galls form.

In order to investigate the spatiotemporal activity of LeDES promoter, the 1,602-bp sequence upstream of the ATG start site of LeDES was fused to the GUS reporter gene, generating a pLeDES::GUS construct. The pLeDES::GUS reporter construct was then transformed into tomato via R. rhizogenes-mediated root transformation. Transgenic hairy roots that emerged from the cotyledons were confirmed to be carrying the pLeDES::GUS construct. pLeDES-driven GUS expression was monitored following wounding, exogenous application of defense-related signaling molecules, and in response to nematode infection conducted at specific time points after induction, in transgenic hairy roots by histochemical staining. In the absence of any stimulus, very intense GUS staining was typically observed in the root maturation zone (Figures 2A,D), whereas no GUS activity was found in the elongation zone (Figures 2A,C); notably, a very faint GUS signal could be observed in the root apex (Figure 2B). Strong GUS activity was also associated with the lateral root primordium of the non-inoculated roots, suggesting a role for LeDES in genetic regulation of root growth and development (Figures 2E,F).

Mechanical damage by wounding activates defense signaling pathways and alters hormonal levels in plants, which in turn protects the plant against injury and pathogen attack (Savatin et al., 2014). LeDES expression upon wounding was profiled by analyzing GUS expression in pLeDES::GUS roots of wounded and non-wounded roots 6 and 24 h after mechanical wounding. Whereas, a typical signal was observed along the root maturation zone (Figure 2G), no LeDES promoter activity was observed localized at the wound site at either 6 h (Figure 2H) or 24 h (Figure 2I) after wounding. It is clearly observed that a complete loss of GUS staining characterize the specific site of mechanical damage as observed (arrows in Figures 2H,I) 6 and 24 h after wounding, respectively. These results suggest wound-induced local suppression of LeDES at the site of the mechanical damage (Figure 2).

To assess whether LeDES promoter is activated through phytohormone signaling, we used the phytohormone signaling molecules IAA, IBA, Me-JA, and Me-SA in an LeDES::GUS promoter bioassay (Figure 3). GUS staining revealed high GUS expression in the root tips 16 h after treatment with the auxins (IAA and IBA) (Figures 3B,C,E,F) compared to the negative control (Figures 3A,D), and no activation of LeDES promoter by Me-JA (Figures 3H,I). Notably, strong induction of the GUS reporter gene was evident after Me-SA treatment, as observed in the root tip and elongation zone (Figures 3K,L). Our results suggest that various phytohormones coregulate LeDES expression, highlighting its possible role in mediating defense responses triggered by biotic stress.

Next, transcriptional activation of the GUS reporter gene driven by the LeDES promoter in the transgenic hairy roots of tomato was analyzed in a time-course analysis at 2, 3, 10, 15 and 28 dpi representing J2 (2 and 3 days), J3 and J4 (10 and 15 days) and female (28 days) stages of nematode development. Very strong staining was evident 2 and 3 dpi in the root elongation zone, the preferred site for nematode penetration, along with swelling and intensive root hair growth resulting from nematode penetration compared with non inoculated roots on the left panel (Figures 4A–D). An intense GUS signal was observed in the bulging root tissue at the initial nematode penetration site, which is part of the developing gall (Figure 4B). High promoter activity continued to be observed in the maturing galls compared with their respective controls (Figures 4E–H) at 10 (Figure 4F) and 15 (Figure 4H) dpi, mainly confined to the deformed vascular bundles (Figure 4H). Notably, a significant reduction in promoter activity was evident at 28 dpi compared with the control roots (Figures 4I,J), whereas a very mild GUS signal associated with the vascular system connected to the developed female could be observed (Figure 4J).

To investigate the spatial expression of LeDES at nematode feeding sites, thin sections of galls induced by M. javanica, 15 and 28 dpi, were prepared and analyzed. At 15 dpi, strong GUS staining showed a scattered signal distributed within the deformed stele harboring the feeding site and bounded by the endodermis cell layer, as observed under light and dark field, respectively (Figures 5A,B). At that time point, GUS signal was clearly visible in the giant cells, as well as in the surrounding xylem and phloem parenchyma cells. As infection progressed, GUS signal was significantly quenched, although it was predominantly retained within the developing giant cells (Figures 5C,D). However, as the female matured and the feeding site reached its maximum size, at 28 dpi, no GUS signal was observed in the adjacent parenchyma cells with only a very faint GUS signal in the feeding site (Figures 5E,F).

To further explore the role of LeDES in regulating plant responses to nematode infection, a plant binary vector containing a 1,437-bp LeDES ORF under the control of the CaMV 35S promoter was introduced into A. thaliana by floral-dip method for Agrobacterium-mediated transformation. From all of the independent kanamycin-resistant transgenic lines harboring the 35S:LeDES construct, three homozygous lines (D1, D2, and D3) were validated by RT-PCR for LeDES heterologous expression, and used for nematode infection (Figure 6A). The transgenic plants did not show any phenotypic change in root or shoot growth compared to the WT Col-0 line (Figure 6B). To check for the presence of CnA and CA, indicative of active DES in the transgenic Arabidopsis plants, root lysates of transgenic and control plants were analyzed by LC–MS (Figure 6C). The root homogenates of transgenic and control plants were incubated with 9-HPODE and analyzed by LC–MS for the production of CnA and CA. No CnA was observed in the control plants, but it was detected in the D1, D2, and D3 transgenic lines (Figure 7C). No accumulation of CA was identified through LC–MS in either the transgenic or control WT lines. To determine the effect of LeDES overexpression on disease development in Arabidopsis transgenic lines, 10 plants each from WT, vector-only, and three transgenic lines were inoculated with 300 J2s, and the nematode developmental stages (J3/J4 and females) were monitored at 28 dpi (Figure 6D). It is commonly observed that at 28 dai, analyzed infected roots, harbor mainly J3/J4, and mature female stages with or without egg masses. While, at this late time point after infection, J2s which were suppressed or arrested at the earlier time points, will be starved to death by then, and thus barely could be visible, at 28 dai. Disease development, as indicated by number of developmental stages (J3/J4 and females) on D1, D2, and D3 and control lines (WT and vector-only) indicated that LeDES overexpression restricts nematode infection, and results in significantly less nematodes molting into J3/J4 stages, as observed in LeDES transgenic lines compared to control lines at 28 dpi. Similarly, a decrease in the number of mature females was observed in transgenic lines overexpressing LeDES (Figure 6D). These results indicate that heterologous expression of LeDES in Arabidopsis plants results in attenuation of nematode disease development in the roots. Infection assay data were fitted with general linear mixed model in SPSS, by keeping samples as fixed factors and repeated assays as random factor (Supplementary Table 1). Further, Tukey post-hoc range test was applied to check significant differences (p < 0.05) among different samples.

To characterize the direct activity of DVEs against M. javanica J2s, LeDES cDNA was cloned into the yeast expression vector pFL61, under the control of the constitutive PGK promoter of S. cerevisiae to obtain the pFL::LeDES construct. Then, following yeast transformation, pFL::LeDES was expressed in S. cerevisiae strain INVSc1. To confirm the presence and expression of LeDES, transgenic yeast lines were analyzed by RT-PCR, revealing accumulation of DES transcripts exclusively in the positive transformed line L1 (Figure 7A). Then, CnA production by the recombinant yeast was determined by incubating transgenic yeast with 9-HPODE as a substrate. LC–MS was used to detect the presence of CnA. Accumulation of 3.37 ± 0.060 ng/g CnA was observed for the L1 recombinant yeast strain, whereas no accumulation was detected in the reaction containing extract from yeast cells with pFL61 vector (Figure 7B).

Hence, recombinant yeast clones transformed with the pFL::LeDES construct and a negative control, the void pFL61, were used to study the direct nematotoxicfunction of DVEs against M. javanica J2s. Following 24 h incubation of recombinant yeast strains expressing either pFL::LeDES or the empty vector pFL61 with 9-HPODE or 13-HPODE M. javanica J2s were added to the yeast reaction for an additional 12 h, then J2 viability/motility was evaluated through sieving followed by microscopy observation. There was a significant difference in percent motility of J2s following incubation with recombinant strain pFL::LeDES and 9-HPODE (7%), compared to that with the negative control yeast carrying the empty vector pFL61 and 9-HPODE (37%) (Figure 7C). These differences in J2 motility were not present when 13-HPODE was used as the substrate for either of the yeast strains.