The TIR-NB-LRR pairDSC1andWRKY19contributes to basal immunity of Arabidopsis to the root-knot nematodeMeloidogyne incognita

Background

Root-knot nematodes transform vascular host cells into permanent feeding structures to withdraw nutrients from the host plant. Ecotypes ofArabidopsis thalianacan display large quantitative variation in susceptibility to the root-knot nematodeMeloidogyne incognita, which is thought to be independent of dominant major resistance genes. However, in an earlier genome-wide association study of the interaction between Arabidopsis andM. incognitawe identified a quantitative trait locus harboring homologs of dominant resistance genes but with minor effect on susceptibility to theM. incognitapopulation tested.

Results

Here, we report on the characterization of two of these genes encoding the TIR-NB-LRR immune receptor DSC1 (DOMINANT SUPPRESSOR OF Camta 3 NUMBER 1) and the TIR-NB-LRR-WRKY-MAPx protein WRKY19 in nematode-infected Arabidopsis roots. Nematode infection studies and whole transcriptome analyses using the Arabidopsis mutants showed thatDSC1andWRKY19co-regulate susceptibility of Arabidopsis toM. incognita．

Conclusion

Given the head-to-head orientation ofDSC1andWRKY19in the Arabidopsis genome our data suggests that both genes may function as a TIR-NB-LRR immune receptor pair. Unlike other TIR-NB-LRR pairs involved in dominant disease resistance in plants, DSC1 and WRKY19 most likely regulate basal levels of immunity to root-knot nematodes.

The root-knot nematodeMeloidogyne incognitais currently ranked as one of the most invasive plant disease-causing agents, having major impact on global agricultural productivity [1]. Infective second stage juveniles (J2) ofM. incognitapenetrate their host at the root elongation zone. Thereafter, they migrate through the cortex to the root tip and enter the vascular cylinder via the columella and quiescence center. Inside the differentiating vascular cylinder, the J2 carefully puncture the cell walls of several host cells with their stylet to initiate the formation of a permanent feeding site [2,3,4,5]. This permanent feeding site includes several giant cells, which are formed by major structural and metabolic changes in host cells, most likely in response to stylet secretions ofM. incognita[2,6]. Juveniles ofM. incognitatake up their nutrients from these giant cells during the course of several weeks while undergoing three molts to enter the adult stage. Adult females produce eggs, which are held together in a gelatinous matrix at the surface of the roots [2,3,4].

Plants have developed several lines of defense to protect themselves against attacks by parasitic nematodes [3]. The first line of defense is thought to be structural, where plants make use of rigid cell walls to prevent host invasion (i.e., by migratory ectoparasites). Next, plant cells carry surface-localized receptors to detect molecular patterns in the apoplast that are uniquely associated with host invasion by endoparasitic nematodes [4,7,8]. For example, root-knot nematodes release small glycolipids commonly referred to as ascarosides that are recognized as invasion-associated molecular patterns [9]. The exposure of Arabidopsis seedlings to these ascarosides activates basal plant defenses to a broad range of pathogens. Furthermore, Arabidopsis mutant analyses (includingBRASSINOSTEROID INSENSITIVE 1 (BRI1)-associated receptor kinase 1 (BAK1)) have shown that receptor-mediated basal immunity plays a significant role in the susceptibility of plants to root-knot nematodes [10]. Interestingly, root-knot nematodes have effectors capable of selectively suppressing responses activated by surface-localized immune receptors, which indicates adaptation to this line of defense [11,12,13,14].

At a later stage in the infection process, nematode resistant plants can counteract the establishment of a permanent feeding site with effector-triggered immunity, which is predominantly based on sensing nematode effectors by intracellular immune receptors [3,15]. Effector-triggered immunity to root-knot nematodes often involves a hypersensitive-response in- and around giant cells, which interrupts the flows of assimilates towards the feeding nematodes. As a consequence of insufficient supply of nutrients, this type of major resistance induces a developmental arrest in juveniles.

The largest group of intracellular plant immune receptors is formed by the nucleotide-binding site leucine-rich repeat (NB-LRR) superfamily of immune receptors. These NB-LRRs consist of a central nucleotide-binding domain attached to a C-terminal leucine-rich repeat (LRR) domain and a variable N-terminal domain that can either be a coiled-coil (CC) or a Toll-interleukin-1 receptor (TIR)-like domain [4,16,17,18]. Three major resistance (R) genes encoding intracellular NB-LRR immune receptors conferring resistance toM. incognitaare cloned, one of which is encoding a CC-NB-LRR receptor (Mi-1.2fromSolanum peruvianum) and two encode TIR-NB-LRR proteins (MafromPrunus cerasiferaandPsoRPM2fromPrunus sogdiana) [19,20,21]. Most of the commercially grown tomato varieties (Solanum lycopersicum) carry introgressions of theMi-1.2塞弗基因,提供高水平的抵抗ral tropical root-knot nematode species (e.g.,M. incognita, M. javanicaandM. arenaria) [22,23]. Resistance based onMi-1.2目前由于我失去功效ts temperature sensitivity and because of natural selection of virulent nematode populations [19,24,25]. The breakdown ofMi-1.2resistance and the small number of major resistance genes currently available for root-knot nematodes has prompted a search for alternative strategies to develop durable nematode resistant crops.

Previously, we used genome-wide association mapping to identify less conducive allelic variants of genes that critically determine susceptibility of Arabidopsis toM. incognita(i.e.S-genes [26,27];). In total, we identified 19 QTL (quantitative trait loci) in the genome of Arabidopsis contributing to quantitative variation in reproductive success ofM. incognita．We selected Arabidopsis as a model host for our studies because it is thought to lack major resistance toM. incognita．However, one of the QTL with minor effect on susceptibility of Arabidopsis toM. incognitaharbors homologs of the TIR-NB-LRR class of resistance genes. Here, we report on the functional characterization of these TIR-NB-LRR genes that were previously annotated asDSC1andWRKY19．DSC1encodes a typical TIR-NB-LRR immune receptor [28], whereasWRKY19also includes a WRKY domain and MAPx domain at the C-terminus [29]. Our data from mutant analyses and whole transcriptome analyses suggest that the coordinated activity of DSC1 and WRKY19 as a receptor pair may be involved in regulating basal defense of Arabidopsis toM. incognita．

Multiple TIR-NB-LRR protein encoding genes in a QTL for susceptibility

In our previously published GWA (genome-wide association) study of the susceptibility of Arabidopsis toM. incognitawe identified a single nucleotide polymorphism (SNP) marker on chromosome 4, which was closely linked to two genes with similarity to TIR-NB-LRR-type immune receptors [26]. This SNP marker (138442) was located inside an exon ofBILE ACID TRANSPORTER 5(BAT5; At4G12030) and leads to a non-synonymous mutation (Val to Ala) close to the amino terminus of the protein (Fig.1a). However, located directly upstream ofBAT5areDOMINANT SUPPRESSOR of Camta3 NUMBER 1(DSC1; At4G12010) andMITOGEN-ACTIVATED PROTEIN KINASE-WRKY19(WRKY19; At4G12020), which both possess TIR, NB-ARC and LRR domains (Fig.1a and b).BAT5,DSC1andWRKY19are all within 10 kb of SNP marker 138,442 and could therefore be causal for the effect on susceptibility toM. incognitaassociated with this marker.

BAT5 is not causally linked to susceptibility of Arabidopsis toM. incognita

Since SNP marker 138,442 is located inside the coding sequence ofBAT5, we first tested if this gene is required for susceptibility of Arabidopsis toM. incognita．Thereto, we inoculated the roots of in vitro grown plants of the homozygous Arabidopsis T-DNA insert linebat5–2with infective J2 ofM. incognita．Thebat5–2mutant harbors a T-DNA insertion in the second last exon, resulting only in a slight reduction of mRNA levels ofBAT5(Fig.1a; Additional file1)。However, as the insert inbat5–2disrupts the open reading frame inBAT5, mRNAs are probably not translated into a functional protein. Nonetheless, six weeks after inoculation the number of egg masses produced byM. incognitaper plant were not significantly different betweenbat5–2and wildtype Arabidopsis plants (Fig.2)。We also investigated the root architecture of thebat5–2突变体线的时候接种(dpi = 0)s this may affect the susceptibility score of Arabidopsis forM. incognita, but we observed no significant difference in the number of root tips per plant for thebat5–2mutant compared to the wildtype Arabidopsis control plant Col-0 (Additional file2)。Altogether, our results provide no evidence for a significant role for BAT5 in susceptibility of Arabidopsis toM. incognita．

DSC1andWRKY19may both regulate susceptibility of Arabidopsis toM. incognita

To study whetherDSC1andWRKY19were involved in susceptibility of Arabidopsis toM. incognita, we also tested the homozygous Arabidopsis T-DNA insertion linesdsc1–1andwrky19–1in nematode infection assays. The T-DNA insert indsc1–1is located in the last exon ofDSC1, which causes a complete knock-out of gene expression (Additional file1)。In contrast, the T-DNA insert inwrky19–1is located in the putative promotor regions of both genes, which leads to strong upregulation ofWRKY19expression and a small but significant down-regulation ofDSC1(Additional file1)。Susceptibility of the mutant and wildtype plants was tested at 7 days post inoculation in this study, which corresponds to an early stage in root-knot nematode parasitism that includes the successful invasion of the roots and the initiation of a proper feeding site for further development and reproduction (two hallmarks for host susceptibility to plant-parasitic nematodes). At seven days after inoculation withM. incognita, we observed a significantly higher number of juveniles inside roots of thedsc1–1mutant plants compared to the roots of wildtype Arabidopsis control plants (Fig.3a). The number of juveniles inside the roots of thewrky19–1overexpressing mutant was also slightly, but not significantly, higher as compared to wildtype Arabidopsis control plants (Fig.3e). However, it should be noted that we also observed a significant lower number of root tips per plant for both thedsc1–1andwrky19–1mutants compared to wildtype Arabidopsis plants at the time of inoculation (dpi = 0; Additional file3)。When we corrected our data for this difference in root architecture, the number of juveniles per root tip was significantly higher in roots of bothdsc1–1andwrky19–1mutant lines as compared to wildtype Arabidopsis control plants (Fig.3b and f). Likewise, at six weeks after inoculation the number of egg masses per plant root system and the number of egg masses per root tip was significantly higher in both thedsc1–1knock-out mutant line andwrky19–1overexpressing mutant line as compared to the wildtype Arabidopsis control plants (Fig.3c, d, g, and f).

Our observations on thewrky19–1mutant line suggest that quantitative differences in expression levels ofDSC1andWRKY19could influence both root development and susceptibility toM. incognitain opposite ways. We therefore investigated if the expression ofDSC1andWRKY19is indeed regulated during root development and nematode infections in wildtype Arabidopsis plants using quantitative reverse transcription PCR (qRT-PCR) with gene specific primers (Fig.4)。Interestingly, bothDSC1andWRKY19were upregulated in non-infected roots of Arabidopsis Col-0 when comparing 14 day old and 21 day old plants. This is consistent with a positive role for both genes in root development as suggested by the root phenotype of thedsc1-1andwrky19–1mutant lines. In contrast, we only observed a significant down-regulation ofWRKY19in nematode-infected roots at 7 days post inoculation withM. incognita．This is consistent with a role for WRKY19 as a negative regulator of defense responses toM. incognita, as suggested by the increased number of nematodes on thewrky19–1mutant line, which is overexpressingWRKY19．NeitherDSC1norBAT5were significantly regulated in the roots of wildtype Arabidopsis Col-0 plants at seven days post inoculation withM. incognita．The lack of change inDSC1gene expression seems inconsistent with the observed increase in nematode infection on thedsc1–1mutant plants. However, this could indicate that no de novo synthesis of DSC1 is required for a role in regulating nematode susceptibility, or that we were not able to detect a local change in gene expression at the infection site due to the use of whole roots as input for the qRT-PCR analysis.

DSC1andWRKY19regulate gene expression duringM. incognitainfection

To gain more insight in the possible role ofDSC1in susceptibility of Arabidopsis toM. incognita, we analyzed the transcriptome of whole roots of thedsc1–1mutant line and wildtype Arabidopsis control seven days after inoculation withM. incognitausing Arabidopsis gene expression microarrays. As expected, in non-infected roots of thedsc1–1mutant expression ofDSC1was absent, but – to our surprise – no other genes were differentially expressed in comparison with non-infected roots of wildtype Arabidopsis plants of the same age (at -log₁₀(P) > 3.5). However, we observed the differential expression of 221 genes in a comparison between nematode-infected roots of thedsc1–1突变体和野生型拟南芥控制植物seven days after inoculation (Fig.5a). To determine which genes were strongly affected by the mutation indsc1–1, we focused on genes that were either standing out because of a large change in expression level (i.e., mean normalized log₂-change in probe intensities < − 0.3 or > 0.3), because of high statistical support of the changes (−log₁₀(P) > 6.5)), or both (Fig.5a). Applying these criteria resulted in a list of twelve differentially expressed genes in the absence ofDSC1, which included several genes –likeDSC1– with a link to abiotic- and biotic stress responses (Table1, Fig.5a). Three genes were selected for testing by qRT-PCR (Additional file6) to confirm the observed changes in gene expression in the microarray analysis. Similar expression patterns were observed (Fig.6), supporting the up- or downregulation of the selected genes inM. incognita-infected roots of thedsc1–1mutant at 7 dpi as observed in the microarray analysis.

To identify genes regulated in association withWRKY19, we also analyzed the transcriptome of whole roots of thewrky19–1mutant in non-infected andM. incognitainfected plants. When comparing non-infected roots of thewrky19–1mutant and wildtype Arabidopsis control plants, no differentially expressed genes were observed (threshold for significance -log10(P) > 3.5). As expected, the expression ofWRKY19was slightly – but not significantly – upregulated in thewrky19–1mutant line as compared to wildtype Arabidopsis (significance -log10(P) = 1.67; relative expression 0.22). However, in nematode-infected roots we detected 1710 differently expressed genes in a comparison betweenwrky19–1and wildtype Arabidopsis plants at 7 days after inoculation (Fig.5b). Notably, the expression of DSC1 was significantly reduced in thewrky19–1mutant (significance -log10(P) = 5.2; relative expression − 0.31). To determine which genes are greatly affected inwrky19–1,we focused on genes that were above the threshold of significance of -log10(P) > 6.5 or had a relatively large change in relative expression (relative expression < − 0.3 or > 0.3). In total, 253 differentially expressed genes matched these criteria (Additional file4)。It was noted that 13 out of the 15 most significantly regulated genes - or those with largest relative expression ofwrky19–1– contain a W-box motif ((T/A) TGAC(T/A)) in the corresponding promotor region (Table1) consistent with a regulatory role for WRKY19 during nematode infection. Three genes from each category in Table1were selected for testing by qRT-PCR to confirm the observed changes in gene expression using the Arabidopsis microarray. Similar expression patterns were observed (Fig.6), supporting the up or downregulation of the selected genes inM. incognita-infected roots of thewrky19–1mutant at 7 dpi. Most of the genes in this subset have been linked to biotic and abiotic stresses like observed for thedsc1–1mutant. For instance, the gene with by far the highest relative expression inwrky19–1isILITHYIA(ILA, At1G64790), which encodes a HEAT-repeat protein required for basal defense toPseudomonas syringea[41].

Previously, we mapped a QTL on chromosome 4 of Arabidopsis linked to reproductive success ofM. incognitaharboring two genes encoding TIR-NB-LRR proteins [26]. Although the SNP marker identified at this locus is located inBAT5, we did not find further evidence that allelic variation in this gene can be causal for variation in the number ofM. incognitaegg masses per plant at six weeks after inoculation (Fig.2)。Others have shown that BAT5 is associated with jasmonic acid-dependent signaling and wound responses [48], which are also relevant processes in the context ofM. incognitainfections [49,50,51]. Nevertheless, our data of the bioassays with thebat5–2knock-out mutant makes it unlikely that BAT5 plays a significant role in regulating the susceptibility of Arabidopsis toM. incognita．

After eliminatingBAT5as a candidate susceptibility factor forM. incognitainfections in Arabidopsis, we focused on the TIR-NB-LRR-encoding genesDSC1andWRKY19to explain the phenotypic variation associated with this locus. We show that the loss ofDSC1expression in thedsc1–1Arabidopsis mutant significantly increases the number of juveniles per plant shortly after inoculation and the number of egg masses at the end of the life cycle ofM. incognita(Fig.3)。This demonstrates that allelic variation linked toDSC1may indeed contribute to the phenotypic variation in the susceptibility of Arabidopsis toM. incognita[26].

Less straightforward is the interpretation of the data from our nematode infections assays with thewrky19–1Arabidopsis mutant. The T-DNA insert inwrky19–1is located 191 base pairs upstream of the transcription start site ofWRKY19and 71 base pairs upstream of the transcription start site of the reversely orientedDSC1gene．Our qRT-PCR study suggested that thewrky19–1 T-DNA insert decreases expression ofDSC1but increases expression ofWRKY19(Fig. S1)。The microarray analysis shows also thatDSC1expression is significantly reduced in roots of thewrky19–1mutant, but relative weakly. The expression ofWRKY19is also increased in this mutant as expected, but not significantly (Fig.5b). Although the transcriptional effects on eitherDSC1,WRKY19or both are mild inwrky19–1, we observe a significant phenotype in root architecture and susceptibility toM. incognitaindicating that this mutation and the subsequent changes inWRKY19andDSC1expression has an impact on the condition of the plant.

We used transcriptome analyses by microarray to further investigate possible regulatory networks underlying the enhanced susceptibility of bothdsc1–1andwrky19–1Arabidopsis mutants toM. incognita．Overall, we observe only a small overlap (17 genes) in the sets of differentially expressed genes in nematode-infected roots ofdsc1–1andwrky19–1(Fig.5c). Despite this small overlap in commonly regulated genes, we note that both sets are enriched for genes with a regulatory W-box in their putative promoter sequences (Table1)。W-box被认为的共识DNA binding site of WRKY domains of WRKY transcription factors [52]. The overrepresentation of the W-box could indicate that the regulation of these genes is indeed under control of the WRKY domain present in WRKY19. However, as we did not observe a major change inWRKY19expression in nematode-infected roots of thewrky19–1mutant as compared to wildtype Arabidopsis, this needs further investigation.

Another striking observation is the number of differentially expressed genes in nematode-infected roots of bothdsc1–1andwrky19–1related to responses to abiotic stress (Table1)。This is in line with data from our earlier multi-trait genome wide association study showing that the susceptibility of Arabidopsis toM. incognitacross-correlates with responses to osmotic stress [53]. Likewise, we have recently shown that the transcription factorERF6,which functions as a mediator of abiotic stress, is also required for susceptibility of Arabidopsis toM. incognita[26]. Altogether, these findings suggest that the ability to mitigate abiotic stress is one of the key regulating factors in susceptibility of the Arabidopsis toM. incognita．

The fact that many of the genes differentially regulated in thedsc1–1andwrky19–1mutants uponM. incognitainfection have been linked to plant defense and responses to abiotic stress before might not be surprising. It is clear that nematode-infections are likely to cause stress in Arabidopsis roots. Furthermore, it has already been shown that DSC1 functions in plant immunity [28]. Nevertheless, to the best of our knowledge this is the first time that DSC1 can be linked to immunity to plant parasitic nematodes. DSC1 is a dominant suppressor of the calmodulin-binding transcription activator CAMTA3, which regulates resistance to various pathogens [54,55]. The loss of DSC1 could increase the activity of CAMTA3 in nematode-infected roots of thedsc1–1突变,导致抑制的防御反应s. However, no changes inCAMTA3expression was detected in the transcriptome data to support this model (data not shown). How DSC1 contributes to nematode defense needs further investigations.

Although, we cannot directly pinpoint the probable cause for the enhanced susceptibility of thewrky19–1mutant toM. incognita, our analyses of the transcriptome of nematode-infected roots of this mutant reveal a remarkably strong upregulation of the defense related geneILITYHIA(ILA)。ILAencodes a HEAT repeat protein, which is required for basal defense, resistance mediated by a subset of CC- and TIR-NB-LRR proteins, and systemic acquired resistance againstPseudomonas syringae[41].ILAhas not been linked to susceptibility of Arabidopsis to nematodes before, but the relative expression level of this gene in the microarray analyses is so high (relative expression of 1.19) that it could in fact be causal to the increased susceptibility of thewrky19–1mutant toM. incognita．

TIR-NB-LRR encoding genes can confer dominant disease resistance to Arabidopsis [56], but our data on the role ofDSC1andWRKY19in nematode-infected roots does not point into that direction. First of all, the relatively low level of significance and small effect size of SNP marker 138,442 do not fit the typical dominant phenotype of a major resistance based onTIR-NB-LRRtypeRgenes. Second, the differences in reproductive success ofM. incognitaon thedsc1–1knock-out mutant and thewrky19–1overexpressing mutant as compared to wildtype Arabidopsis plants were significant, but relatively small, and unlike major disease resistance responses conferred byRgenes. We therefore conclude thatDSC1andWRKY19, either separately or together as a pair, do not confer major resistance againstM. incognitain Arabidopsis to the population tested. Instead, they are most likely involved in basal immunity to root-knot nematodes during early stages of the compatible interaction with Arabidopsis as a host plant.

A role for DSC1 and WRKY19 in basal immunity is consistent with observations by others thatDSC1transcript levels increase upon application of SA (salicylic acid) or flg22 (flagellin 22) [57] and thatWRKY19is thought to be an early component in regulatory networks of PTI [58]. Likewise, other TIR-NB-LRR proteins have been found to contribute to basal defense in Arabidopsis againstPseudomonas syringae(TN13) and the hemibiotrophic fungusLeptosphaeria maculans(RLM3) [59,60]. Furthermore, the head-to-head genomic orientation ofDSC1andWKRY19could indicate that they form an immune receptor pair [56,57,61]. So far, otherR-gene pairs have been identified in Arabidopsis consisting of two tightly linked NB-LRR coding genes located in a similar head-to-head tandem arrangement [61]. For instance, the genomic organization ofDSC1andWRKY19pair shows much similarity with that of the resistance toRalstonia solanacearum RRS1and the resistance gene toPseudomonassyringae RPS4suggesting that they may act as a TIR-NB-LRR pair in plant immunity [56,57,61]. This is further supported by the similarities in protein architecture including the presence of functional domains required for immune receptor activity like the NB-ARC and LRR domain [62].

Here, we provide first evidence for a functional role ofDCS1andWRKY19in basal plant immunity to a plant pathogen as aTIR-NB-LRR基因的一对。这将是有趣的调查hether DSC1 and WRKY19 form indeed a functional protein complex and how this complex contributes to basal immunity in plants to root-knot nematodes.

Plant material

The following homozygous Arabidopsis T-DNA insertion mutant lines were obtained from the NottinghamArabidopsisStock Centre [63]: SALK_145043 with T-DNA insert in At4G12010 (dsc1–1); SALK_014300 with T-DNA in At4G12020 (wrky19–1); SALK_201408 with T-DNA in At4G12030 (bat5–2)。The T-DNA insert lines were all generated in the background of Columbia-0 (Col-0 N60000), which was used as wildtype Arabidopsis throughout this study.

The presence and homozygosity of the T-DNA insert in the mutant lines was verified with PCR on genomic DNA isolated from leaf material of twelve seedlings [27]. The detection of the wild type allele or the T-DNA insert by PCR was performed as previous described [26] with different combination of primers for each line (Additional file5) and the T-DNA-insert specific Lbl3.1 primer [63].

The expression of the T-DNA insertion affected gene was checked with reverse transcription quantitative PCR (qRT-PCR), the RNA was isolated as previous described [26]. To quantify the expression level for the gene of interest we used a gene specific forward and reverse primer (Table S1). For the qRT-PCR we used conditions as described below for gene expression analysis. Relative expression ratio between the gene of interest and the reference gene was calculated as described elsewhere [64].

Nematode bioassay

Eggs ofMeloidogyne incognitawere obtained by treating tomato roots infected withM. incognita(strain ‘Morelos’ from INRA, Sophia Antipolis, France) as described [27].

To test the susceptibility of Arabidopsis seedlings, seeds were vapour sterilized and grown as described previously [27]. Individual seedlings were inoculated with 180 infective J2s ofM. incognitaper plant and incubated at 24 °C in the dark. The number of egg masses per plant were counted six weeks after inoculation by visually inspecting the roots with a dissection microscope. Each Arabidopsis genotype was tested in at least three independent experiments and 18 replicates per experiment. The obtained values were batch-corrected using the following equation: variable corrected = variable + (total mean (variable) - batch mean (variable)). The differences in counts per plants were statistically analysed using two-way analysis of variance (ANOVA) and post-hoc Tukey HSD test in R (version 3.0.2,www.r-project.org)。

To collect roots of infected and non-infected Arabidopsis seedlings for gene expression analyses with microarrays and qRT-PCR, seeds were treated as described above for the susceptibility test. Seedlings were grown and inoculated and sampled as described [26].

Root phenotype

The root phenotype of Arabidopsis seedlings was determined as described [26]. Differences in the total root length per seedling in cm and number of root tips were statistically analysed with a two-way ANOVA and post-hoc Tukey HSD test in R (p < 0.05).

Gene expression analysis

Expression analysis for genes of interest was performed on the stored root samples produced during the nematode infection study. Whole root systems were cut from aerial parts of the seedlings and snap frozen in liquid nitrogen. Total RNA was isolated from whole roots of twelve 14-days-old plants ofdsc1–1,wrky19–1,bat5–2and Col-0 wildtype. The RNA isolation and cDNA synthesis for quantitative reverse transcription PCR (qRT-PCR) from the roots was performed as described [26]. cDNA matchingArabidopsis thalianaelongation factor 1 alpha was amplified as a reference for constitutive expression using primers as indicated in Table S1 [65]. To quantify the expression level for the gene of interest specific gene primers were used (Table S1 & S2). The efficiencies of these primer sets were tested prior to the qRT-PCR analysis. For the qRT-PCR 5 ng cDNA was used with the following conditions: 15 min at 95 °C, forty cycles of 30 s at 95 °C, 30 s at 62 °C, and 30 s at 72 °C, and a final incubation of 5 min at 72 °C. Relative expression ratio between the gene of interest and the reference gene was calculated as described elsewhere [64]. Relative expression ratio was statistically analysed for significance and compared with a student t-test (P-value< 0.05).

For microarray analysis, approximately 200 ng of total RNA of each sample of Col-0,dsc1–1andwrky19–1were used for gene expression analysis on anArabidopsisV4 Gene Expression Microarray (4x44K, Agilent Technologies). The microarray was performed as described i [26]. Two sets of data were generated: a set for comparison between Col-0 anddsc1–1and a set for the comparison between Col-0 andwrky19–1．Col-0包含不同的不同experimental samples. Each sample had at least three biological replicates.

Microarray analysis

After scanning, the spot intensities of the microarrays were not background corrected [66]. Gene expression profiles were normalized using the Loess (within array normalization) and the quantile method (between array normalization) [67] in the Bioconductor Limma package [68]. The normalized intensities were log2-transformed for further analysis.

A linear model was used to identify differentially expressed genes in a side-by-side comparison. The following treatments were compared: day-0 control Col-0 versus mutant, day 7 control Col-0 versus mutant, day 7 infected Col-0 versus infected mutant, and day 7 control Col-0 versus infected Col-0. Each treatment consisted of three biological replicates. We used the linear model

where the log2-normalized expression (E) of spot i (i in 1, 2, ..., 45,220) was explained over treatment (T). Afterwards, the obtained significances were corrected for multiple testing using the FDR procedure in p.adjust [69].

All microarray data were deposited in the ArrayExpress database at EMBL-EBI (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-6897. All other data generated during the current study are available from the corresponding author on a reasonable request.

ANOVA:

Analysis of variance

BAK1:

BRASSINOSTEROID INSENSITIVE 1 (BRI1)-associated receptor kinase 1

BAT5:

BILE ACID TRANSPORTER 5

CAMTA3:

Calmodulin-binding transcription activator number 3

CC:

Coiled-coil domain

DSC1:

DOMINANT SUPPRESSOR OF Camta 3 NUMBER 1 immune receptor

flg22:

Flagellin 22

GWA:

Genome-wide association

HEAT repeat:

Acronym for a structural motif present in the four proteins Huntingtin, elongation factor 3 (EF3), protein phosphatase 2A (PP2A), and the yeast kinase TOR1

ILA:

HEAT repeat protein ILITYHIA (after the Greek goddess of childbirth)

MAPx:

MITOGEN-ACTIVATED PROTEIN KINASE

Mi-1.2gene:

Meloidogyne incognitaresistance gene number 1.2

NB-LRR:

Nucleotide-binding site (NB) leucine-rich repeat (LRR) immune receptor

PCR:

Polymerase Chain Reaction

qRT-PCR:

Quantitative Reverse Transcription PCR

QTL:

Quantitative Trait Locus

Rgene:

Resistance gene

RLM3:

Resistance toLeptosphaeria maculans3

RPS4:

Resistance toPseudomonas syringae4

RRS1:

Resistance toRalstonia solanacearum1

SA:

Salicylic acid

S-gene:

Susceptibility gene

SNP:

Single Nucleotide Polymorphism

T-DNA:

Transfer DNA

TIR:

Toll-Interleukin-1 receptor (TIR)-like domain

TN13:

TIR-NBS protein number 13

W-box:

WRKY binding motif

WRKY:

Conserved WRKY amino acid signature sequence

WRKY19:

TIR-NB-LRR-WRKY-MAPx domain containing protein number 19

The PhD thesis by Sonja Warmerdam entitled “Susceptibility genes, a novel strategy to improve resistance against the root-knot nematodeMeloidogyne incognita” (2019) of the Wageningen University under supervision of prof. dr. J. Bakker (promoter) and dr. G. Smant and dr. A. Goverse (co-promoters) - ISBN 9789463435949 - p161 includes a chapter which was used as a draft of this paper and as such, submitted as a slightly modified version to BMC Plant Biology for publication.

This study was financially supported by the Netherlands Organization for Scientific Research (NWO, Perspectives Programme ‘Learning from Nature to Protect Crops’; STW grant 10997).

Affiliations

1. Laboratory of Nematology, Wageningen University, Droevendaalsesteeg 1, 6708, PB, Wageningen, The Netherlands

   Sonja Warmerdam, Mark G. Sterken, Octavina C. A. Sukarta, Casper C. van Schaik, Jose L. Lozano-Torres, Jaap Bakker, Geert Smant & Aska Goverse

2. Laboratory of Plant breeding, Wageningen University, Droevendaalsesteeg 1, 6708, PB, Wageningen, The Netherlands

   Marian E. P. Oortwijn

Contributions

J.B., G.S., A.G. and S.W. conceived and designed the experiments. S.W., M.S., O.C.A.S., M.E.P.O., C.C.v S. and J.L.L.T. performed the experiments. S.W., M.S., O.C.A.S., J.L.L.T., A.G., J.B. and G.S analyzed the data. S.W., M.S., O.C.A.S., J.B., G.S. and A.G. wrote the article. All authors edited and approved the final article.

Corresponding author

Correspondence toAska Goverse．

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Competing interests

The authors declare that they have no competing interests.

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