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. 2018 Sep 28;19(11):2416–2430. doi: 10.1111/mpp.12719

The Meloidogyne graminicola effector Mg16820 is secreted in the apoplast and cytoplasm to suppress plant host defense responses

Diana Naalden 1, Annelies Haegeman 1,3, Janice de Almeida‐Engler 2, Firehiwot Birhane Eshetu 1,4, Lander Bauters 1, Godelieve Gheysen 1,✉
PMCID: PMC6638014  PMID: 30011122

SUMMARY

On invasion of roots, plant‐parasitic nematodes secrete effectors to manipulate the cellular regulation of the host to promote parasitism. The root‐knot nematode Meloidogyne graminicola is one of the most damaging nematodes of rice. Here, we identified a novel effector of this nematode, named Mg16820, expressed in the nematode subventral glands. We localized the Mg16820 effector in the apoplast during the migration phase of the second‐stage juvenile in rice roots. In addition, during early development of the feeding site, Mg16820 was localized in giant cells, where it accumulated in the cytoplasm and the nucleus. Using transient expression in Nicotiana benthamiana leaves, we demonstrated that Mg16820 directed to the apoplast was able to suppress flg22‐induced reactive oxygen species production. In addition, expression of Mg16820 in the cytoplasm resulted in the suppression of the R2/Avr2‐ and Mi‐1.2‐induced hypersensitive response. A potential target protein of Mg16820 identified with the yeast two‐hybrid system was the dehydration stress‐inducible protein 1 (DIP1). Bimolecular fluorescence complementation resulted in a strong signal in the nucleus. DIP1 has been described as an abscisic acid (ABA)‐responsive gene and ABA is involved in the biotic and abiotic stress response. Our results demonstrate that Mg16820 is able to act in two cellular compartments as an immune suppressor and targets a protein involved in the stress response, therefore indicating an important role for this effector in parasitism.

Keywords: effector‐triggered immunity, Nicotiana benthamiana, Oryza sativa, PAMP‐triggered immunity, parasitism, reactive oxygen species, root‐knot nematodes

INTRODUCTION

The root‐knot nematode (RKN) Meloidogyne graminicola is able to infect over 100 plant species, including cereals, grasses and dicots, although the number of known dicot hosts is limited (Bridge et al., 2005; Yik and Birchfield, 1979). Meloidogyne graminicola is best known as the most damaging RKN on the staple food rice and is responsible for yield losses of between 17% and 32% (Kyndt et al., 2014). Pre‐parasitic second‐stage juveniles (J2s) invade the root elongation region, migrate to the tip, turn and search for a suitable cell in the vascular cylinder to induce a specialized feeding site. This feeding site is formed by five to eight highly active giant cells that arise by repeated nuclear division without cytokinesis (Kavitha et al., 2016; Kyndt et al., 2014). When the nematode becomes sedentary, the parasitic J2 develops into a J3, J4 and, finally, an adult male or female with egg mass within 14–27 days, depending on the environmental conditions (Kyndt et al., 2014; Mantelin et al., 2017).

To colonize the host, plant‐parasitic nematodes (PPN) have developed sophisticated methods to invade roots and change host cell regulation to support parasitism. Effectors are secreted from the glands into the host plant tissue, either in the apoplast or the cytoplasm. Secreted in the cytoplasm, the effector can interact there with receptors or other proteins, or can be relocated to several other cell compartments, such as the nucleus or plasma membrane (Jaouannet and Rosso, 2013). Secreted in the apoplast, effectors may have a cell wall‐degrading function or may interact with a receptor in the plasma membrane (Jaouannet and Rosso, 2013). Effectors may play a role in many pathways to induce a feeding site and to suppress the immune system of the plant. The plant’s immune system is composed of two layers (Jones and Dangl, 2006). The basal defence response recognizes slowly evolving microbial‐ or pathogen‐associated molecular patterns (MAMPs or PAMPs), which are molecules common to many invaders. These molecules are recognized by surface pattern recognition receptors (PRRs) connected to the apoplast of plant cells, inducing a non‐specific defence response called PAMP‐triggered immunity (PTI). Nematode secretions and cuticle factors may be recognized as PAMPs (Abad et al., 2003; Manosalva et al., 2012). As a result of the PTI response, the plant becomes less susceptible, and therefore nematodes have evolved effectors to increase the susceptibility of the plant: effector‐triggered susceptibility (ETS). In the second layer of the immune system, the effector can be recognized by the plant cell, inducing effector‐triggered immunity (ETI). In response, nematodes have evolved effectors to suppress the second layer of defence as well. Finally, ETI can lead to local cell death, the hypersensitive response (HR), which stops the development or kills the nematode.

The first nematode effector described by Smant et al. (2012) was identified as a β‐1,4‐endoglucanase, used by the nematode for the degradation of cellulose of plant cell walls. Now, many more effectors of PPNs have been identified, but only a relatively small number have been functionally analysed (Ali et al., 2017). Several potential effectors have been found in the transcriptome of M. graminicola (Haegeman et al., 2013; Petitot et al., 2016). However, insight into their function is either based on predictions compared with homologues, or their function remains unclear because of a lack of known domains or well‐described homologues. Recently, the first effector of M. graminicola, MgGPP, has been functionally analysed by Chen et al. (2017). This effector targets the endoplasmic reticulum (ER) and, after N‐glycosylation and C‐terminal proteolysis, the effector is translocated to the nucleus. N‐Glycosylation of this effector is essential for the suppression of the Gpa2/RBP‐1‐induced HR.

Here, we present the functional analysis of the M. graminicola Mg16820 protein, which was identified as an effector candidate by analysis of the data from Haegeman et al. (2013). However, Mg16820 was not included in that article, and therefore this is the first time this effector is presented. The coding sequence contains a predicted signal peptide (SP), but no transmembrane domain, and no homologues in expressed sequence tag (EST) datasets of free‐living nematodes were found during that study. In addition, the gene showed strong expression in pre‐parasitic J2s. In this study, in situ hybridization was performed to analyse the spatial expression of Mg16820 in the nematode and the location of the effector was determined in planta. Finally, plant immune triggering assays were carried out and a yeast two‐hybrid (Y2H) assay was performed to identify potential target proteins in rice.

RESULTS

Mg16820 encodes a protein with a predicted size of 69 amino acids and is only found in root‐knot nematodes

The open reading frame (ORF) of Mg16820 contains 273 nucleotides (Fig. S1, see Supporting Information), encoding a protein of 90 amino acids (Fig. 1). The first 21 amino acids are predicted to function as an N‐terminal SP. When Mg16820 was blasted against the M. graminicola genome (MGRAMBASE), we were unable to retrieve an identical hit. However, a homologous sequence was retrieved with 72% protein identity. The major difference was detected in the N‐terminal region coding for the predicted SP, which was shorter than the cDNA sequence of Mg16820. No significant hits were found when Mg16820 was blasted against the non‐redundant protein and nucleotide databases for all organisms. The search for possible homologues was extended by performing a blast against fungal and oomycete genomes (FungiDB) and bacterial genomes (Microbial nucleotide blast on NCBI), but no hits were found. In addition, some other genomes of PPNs were queried for homologues (Bursaphelenchus xylophilus, Globodera pallida, M. incognita and M. hapla). Only the genome of M. incognita contained two possible homologues (Minc05708 and Minc03663). Both were predicted to have an N‐terminal SP as well. The alignment of all sequences showed that the 18 amino acids directly following the SP were quite well conserved with only some additional aspartate and glycine residues conserved throughout the sequence. Using Wolf PSort, all four sequences (two sequences from M. graminicola and two from M. incognita) were predicted to have a nuclear localization once the putative secretion signal was cleaved. Using LOCALIZER, which specifically predicts effector localizations (Sperschneider et al., 1998), only the DNA sequence obtained from MGRAMBASE and the sequence of Minc03663 predicted a nuclear localization. A predicted nuclear localization signal (NLS), 23RKKGVPQRA31, was found just behind the SP, with a relatively low activity (cut‐off value of 0.244). Both Minc03663 and Minc05708 contained an NLS with a higher activity than predicted for Mg16820 (0.900 and 0.667, respectively). In addition, the NLS was found at another location in the sequence: 37KRKAGRPVGSK47 (Minc03663) and 40KRKAGR45 (Minc05708). In the DNA sequence obtained from MGRAMBASE, an NLS was found at 19RAATDDLRDK28 with a cut‐off value of 0.827.

Figure 1.

Figure 1

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Alignment of the protein sequence of Mg16820 with the DNA sequence obtained from MGRAMBASE and possible homologues (Minc05708 and Minc03663) in Meloidogyne incognita. The green line indicates the predicted signal peptide of Mg16820. The red line indicates the predicted nuclear localization signal. [Colour figure can be viewed at wileyonlinelibrary.com]

Mg16820 is expressed in the subventral glands of the pre‐parasitic juveniles

To determine whether Mg16820 was expressed in the pharyngeal glands of pre‐parasitic J2s, in situ hybridization experiments were performed. A strong signal was visible in the subventral glands of the nematodes when hybridized with the antisense probe (Fig. 2A). As negative control for the hybridization, a sense probe against Mg9152 was used, which did not result in a signal (Fig. 2B).

Figure 2.

Figure 2

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In situ hybridization of Meloidogyne graminicola pre‐parasitic second‐stage juveniles. (A) Antisense probe of Mg16820. (B) Sense probe (negative control; putative effector Mg9152). [Colour figure can be viewed at wileyonlinelibrary.com]

Mg16820 is secreted during the migratory and early sedentary stages

To determine whether and where the effector Mg16820 was secreted in the host plant, immunolocalization was applied to rice gall sections containing nematodes at 1, 3, 5, 7 and 10 days after inoculation (dai). At 1 dai, nematodes were at the migratory stage in or near the root tip. Mg16820 was detected in the head region, the subventral pharyngeal glands of the nematode or the apoplast along the plant tissue at the anterior part of the nematode (Fig. 3A–F). At early gall development, Mg16820 was detected inside the young giant cells (3 and 5 dai) (Fig. 3G–N). The effector was found in the cytoplasm and in the nuclei, most probably being excluded from the nucleoli (Fig. 3I–N). At 7 dai, the Mg16820 signal was still observed in the galls, but, at 10 dai, Mg16820 was undetectable (Fig. S2, see Supporting Information). Sections incubated with the pre‐immune serum or with secondary antibody only did not show any green fluorescence, indicating that the antibody specifically detected the Mg16820 effector protein (Fig. S2).

Figure 3.

Figure 3

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Immunolocalization of Mg16820 secreted by Meloidogyne graminicola in rice. (A–F) Immunolocalization of Mg16820 during the migratory phase of the nematodes [1 day after inoculation (dai)]. (A, B) Secretion of Mg16820 (green fluorescence) by two nematodes with signal seen in the subventral glands in one juvenile nematode. (C, D) Migrating nematode with Mg16820 signal at the anterior part, along the body in the plant apoplasm and in the nematode body. (E, F) Signal at the anterior part and inside the nematode body. (G–J) Immunolocalization of Mg16820 in the feeding sites at 3 dai. (G, H) Secretion of Mg16820 in the plant tissue prior to giant cell development. (I, J) Young giant cells showing signal in the cytoplasm and in the nuclei, apparently excluded from the nucleoli. (K–N) Immunolocalization of Mg16820 during feeding site development (5 dai). (K, L) Accumulation of Mg16820 in the cytoplasm of giant cells. (M, N) Young giant cells showing signal in the cytoplasm and nucleus, apparently excluding the nucleoli. Grey images, differential interference contrast (DIC), 4’,6‐diamidino‐2‐phenylindole (DAPI) and green fluorescent protein (GFP) overlay; black images, DAPI and GFP overlay; n, nematode; asterisks, giant cells; red arrowheads, Mg16820; blue colour, nuclei; white arrow, nucleolus. Scale bars: (A–J) 10 μm; (K–N) 20 μm. [Colour figure can be viewed at wileyonlinelibrary.com]

Mg16820 is localized in the cytoplasm and nucleus when expressed in Nicotiana benthamiana leaves

The subcellular localization of Mg16820 was also determined by transient expression in N. benthamiana leaves. To mimic the secretion of Mg16820 in the cytoplasm, Mg16820 was expressed without signal peptide (SP). Several constructs were used with Mg16820 fused to enhanced green fluorescent protein (eGFP) and enhanced red fluorescent protein (eRFP), both N‐ and C‐terminally, giving similar results, namely Mg16820 was detected in the nucleus and in the cytoplasm (Fig. 4B). Mg16820 exclusion from the nucleoli, as seen during immunodetection assays, was not observed. To mimic the secretion of Mg16820 in the apoplast, Mg16820 was expressed with SP (SPMg16820::GFP). A relatively weak signal was observed in the cell surroundings. Although it could not be confirmed that Mg16820 was actually located in the apoplast, the lack of signal in the nucleus indicates that the nematode SP is functional in plant cells and Mg16820 is transported to the apoplast (Fig. 4C).

Figure 4.

Figure 4

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Subcellular localization of Mg16820 after transient expression in Nicotiana benthamiana leaves. (A) Localization of free red fluorescent protein (RFP) in the cytoplasm and nucleus. (B) Localization of Mg16820::RFP in the cytoplasm and nucleus. (C) Localization of SPMg16820::GFP. Scale bars: (A) 20 μm; (B) 10 μm; (C) 20 μm. [Colour figure can be viewed at wileyonlinelibrary.com]

Mg16820 directed into the apoplast suppresses the flg22‐induced reactive oxygen species (ROS) production

To investigate whether Mg16820 may play a role in the suppression of the early immune response of the plant, a luminol‐based assay was used to measure the effect of Mg16820 expression on ROS production after PTI induction by flg22. When Mg16820 was expressed without SP, and therefore the protein was present in the cytoplasm, the suppression was variable between the different assays. In some cases, no suppression was seen, whereas around one‐half of the assays indicated a weak, but significant, suppression (Fig. 5A). ROS production was dramatically decreased when Mg16820 was translocated to the apoplast of the cell (Fig. 5B). This suppression was consistent in all assays.

Figure 5.

Figure 5

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Suppression assay of flg22‐induced reactive oxygen species (ROS) production in Nicotiana benthamiana with Mg16820. (A) Weak suppression of ROS with Mg16820 located in the cytoplasm compared with the non‐suppressing effector Mg03015. Mp10 was used as a positive control suppressing ROS. (B) Very strong suppression with Mg16820 localized in the apoplasm (SPMg16820). The effector Mg03015 with signal peptide (SP) was used as a negative control not suppressing ROS. Mp10 was used as a positive control. Bars indicate standard error. [Colour figure can be viewed at wileyonlinelibrary.com]

Mg16820 directed into the cytoplasm suppresses the R2/Avr2‐ and Mi‐1.2T557S‐induced HR

The ETI assays were performed by co‐infiltration of several combinations of avirulence and resistance genes to induce an HR. These constructs were combined with Mg16820, with or without SP, to investigate whether the effector could interfere with ETI. Mg16820 with SP did not result in suppression in any of the tested HR pairs. Mg16820 localized in the cytoplasm was able to suppress two different pathways: the pathway induced by the combination of Solanum demissum R2 and Phytophthora infestans Avr2 and by the auto‐activation of the tomato resistance gene Mi‐1.2T557S(Fig. 6). The suppression of the Mi‐1.2‐induced HR was stronger, more consistent and the percentage of the suppressed spots was greater relative to the R2/Avr2‐induced HR. Mg16820 was unable to suppress both pathways when tagged with either haemagglutinin (HA) or GFP. Several other ETI pairs were tested, but did not result in suppression of the HR (Table 1).

Figure 6.

Figure 6

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Effector‐triggered immunity (ETI) suppression assays in Nicotiana benthamiana with effector Mg16820 suppressing two ETI pathways. (A) Suppression of the Mi‐1.2T557S‐induced hypersensitive response (HR) by Mg16820 without signal peptide, targeted to the cytoplasm. The controls empty vector (EV), green fluorescent protein (GFP) and non‐suppressing effector Mg03015 of Meloidogyne graminicola did not result in suppression. (B) Suppression of the R2/Avr2‐induced HR by Mg16820 localized in the cytoplasm with the same controls as in (A). (C) Percentage of necrotic and suppressed spots with Mi‐1.2T557S and R2/Avr2 co‐infiltrated with Mg16820. [Colour figure can be viewed at wileyonlinelibrary.com]

A potential target protein of Mg16820 is dehydration stress‐inducible protein 1 (DIP1)

To identify interactors of Mg16820, a Y2H screen was performed with a cDNA library from infected rice tissues. This resulted in one potential interactor which was identified as DIP1 (LOC_Os05g34070.1). The interaction with the isolated GAL4‐DNA‐activation‐domain::DIP1 and GAL4‐DNA‐binding‐domain::Mg16820 resulted in a faint blue colouring in the X‐gal (5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐galactopyranoside) assay. Cell growth was seen on ‐TLH selection medium (medium lacking the auxotrophic marker histidine), but not on ‐TLU selection medium (medium lacking the auxotrophic marker uracil), and therefore this interaction was considered as weak. The same assay with the complete DIP1 fused N‐terminally to the GAL4 DNA activation domain did not result in blue colour or growth on ‐TLU selection medium (Fig. 7).

Figure 7.

Figure 7

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Yeast two‐hybrid (Y2H) co‐transformation assay with GAL4‐DNA‐binding‐domain::Mg16820 and GAL4‐DNA‐activation‐domain::DIP1. As a control for cell growth colonies were grown on medium lacking the autotrophic markers trypophan and leucine. Interaction was tested using medium lacking the auxotrophic markers tryptophan, leucine and histidine (‐TLH) or tryptophan, leucine and uracil (‐TLU). To test β‐galactosidase activity, the X‐gal (5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐galactopyranoside) substrate was used. The truncated dehydration stress‐inducible protein 1 (DIP1) isolated from the library showed a weak interaction with Mg16820, whereas the complete DIP1 sequence did not show interaction in the Y2H assay. [Colour figure can be viewed at wileyonlinelibrary.com]

To determine whether interaction between effector Mg16820 and the complete DIP1 protein could be proven in planta, a bimolecular fluorescence complementation assay was performed using transient expression in N. benthamiana leaves. As negative control, the putative effector Mg03015 was used with the same plant subcellular localization as Mg16820 and with similar size. Reverse transcription‐polymerase chain reaction (RT‐PCR) showed a similar expression for Mg16820 and Mg03015 (Fig. S3, see Supporting Information). A yellow fluorescent protein (YFP) signal was detected in leaves infiltrated with Mg16820 and DIP1 (Fig. 8B,C), mostly in the nucleus, but excluded from the nucleolus, whereas Mg03015 co‐infiltrated with DIP1 did not result in any signal (Fig. 8A).

Figure 8.

Figure 8

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Bimolecular fluorescence complementation assay with Mg16820 and dehydration stress‐inducible protein 1 (DIP1) in Nicotiana benthamiana leaves. Yellow fluorescent protein (YFP) signal was seen in the nucleus with exclusion of the nucleolus. (A) Negative control YFPc::Mg03015 and YFPn::DIP1 with infiltration at an optical density at 600 nm (OD600) of 0.3. (B) YFP signal with infiltration of YFPc::Mg16820 and YFPn::DIP1 at OD600 = 0.3. (C) YFP signal with infiltration of YFPc::Mg16820 and YFPn::DIP1 at OD600 = 0.1. Scale bars: (A) 40 μm; (B) 5 μm; (C) 10 μm. [Colour figure can be viewed at wileyonlinelibrary.com]

DISCUSSION

We performed a functional analysis of the M. graminicola effector Mg16820, which was identified by 454 sequencing of the transcriptome of pre‐parasitic J2s [contig 016820; method described by Haegeman et al. (2013)]. In this study, we provide evidence that Mg16820 can be considered as an effector. The coding sequence of Mg16820 includes a predicted SP. Remarkable is the lack of a complete SP in the genomic sequence found in the M. graminicola genomics resource database. Because the two potential homologues found in the genome database of M. incognita do have a predicted SP, the M. graminicola genomic sequence might contain an assembly error. Most probably, the conserved part plays a more important role in the function of the protein. In addition to the well‐conserved N‐terminal part, the C‐terminus only shows some conserved aspartate and glycine residues. Possibly, these residues are essential for the function and protein folding. No homologues were detected in either the genome or transcriptome of other PPNs besides M. graminicola and M. incognita. It is likely that the gene undergoes diversifying selection under pressure from the host immune system (Hewezi and Baum, 2013).

The expression of Mg16820 in the subventral glands of the nematode was confirmed by in situ hybridization. The subventral glands are mainly active during the pre‐parasitic and migratory life stages, in contrast with the dorsal gland which becomes active during sedentarization when the giant cells are induced (Davis et al., 2000). Mg16820 is highly expressed by the pre‐parasitic J2s (Petitot et al., 2016). In the migratory stage (2 dai), the gene is still expressed, but at four‐fold lower levels; expression is further decreased at 4 dai and, at 8 dai, expression is close to zero (Fig. S4, see Supporting Information; Petitot et al., 2016). This indicates that Mg16820 plays a role in the early stages of parasitism.

Immunolocalization confirmed the expression and secretion of Mg16820 in the early stage of parasitism. During the migratory stage, M. graminicola juveniles move intercellularly (Mantelin et al., 2015) and secretion of Mg16820 was observed in the apoplast. In addition, during early giant cell development, Mg16820 was secreted in the cytoplasm and targeted to the nuclei as well. Most probably, the nematode injects Mg16820 directly into the plant cell when initiating giant cells, as our results showed a clear distinction of localization in either the apoplast or cytoplasm. Therefore, the role of Mg16820 during development of the feeding site is clearly located in the cytoplasm or nucleus. Localization in the nucleus might indicate a role in the transcriptional regulation in the plant cell (Jaouannet and Rosso, 2013).

However, the molecular size of Mg16820 (7.5 kDa without SP) is very small and, most probably, the effector is just diffusing towards the nucleus, as even molecules larger than 60 kDa can diffuse through the nuclear pore (Wang and Brattain, 1990). Even fused to eGFP, Mg16820 is smaller than 60 kDa and, although the effector was predicted to have a nuclear localization, the effector does not seem to be translocated effectively to the nucleus, as the signal is similar in strength in the cytoplasm. This was observed in both the immunolocalization and subcellular localization assay. However, some contradictory results were observed concerning Mg16820 localization in the nucleoli. With immunolocalization, Mg16820 was excluded from the nucleoli, whereas, with subcellular localization, the nucleoli could not be distinguished. The reason for this is unclear, but the subcellular localization might show an artefact, related to the GFP tag on Mg16820, which is further discussed later.

The ability of M. graminicola to strongly suppress rice defence has been shown previously by the analysis of defence gene expression (Ji et al., 2013). We tested whether Mg16820 plays a role in suppression of the plant immune system. The immune response assays in this study show the capacity of the effector as an immune suppressor in both the apoplast and cytoplasm. ROS production was measured to gain an insight into the effect of Mg16820 in the apoplast. ROS are signalling molecules triggering PTI and ETI responses, which subsequently result in more ROS production (Jwa and Hwang, 2017). ROS can negatively affect the pathogen because they can damage the nematode directly, strengthen the cell wall by cross‐linking cell wall polymers, induce defence signals and regulate cell death responses (Torres et al., 2000). The strong suppression of ROS production accomplished by this effector when it is present in the apoplast benefits the nematode. It remains unclear which mechanism is responsible for the Mg16820‐mediated suppression of flg22‐induced ROS production. Possibly, Mg16820 works on the receptors or enzymes embedded in the plasma membrane to reduce the ROS response (Jwa and Hwang, 2017). For example, respiratory burst oxidase homologues (RBOHs) are an important group of enzymes in plants, responsible for electron transfer from cytosolic NADPH or FAD to oxygen present in the apoplast to form O2 –, which is subsequently converted to H2O2 (Qi et al., 2016). Recently, the M. graminicola effector MgM0237, targeting the cytoplasm, has been shown to be able to suppress the PTI response in N. benthamiana (Chen et al., 2018). When Mg16820 was present in the cytoplasm, ROS was not consistently suppressed and, when suppression was observed, this was very weak. The assay is, in general, quite variable, as also observed with the positive control Mp10, which resulted in variable levels of suppression. Therefore, very weak suppression might not have been detected during each assay. However, variable cell breakdown by ROS may release Mg16820 into the apoplast, possibly resulting in weak suppression. Therefore, most probably, Mg16820 is not able to suppress the PTI response when it is present in the cytoplasm.

The ETI response can lead to an HR that causes rapid cell death of plant tissue around the pathogen, halting the development of the pathogen (Jones and Dangl, 2006). Therefore, nematodes need to suppress this secondary layer of plant defence. The role of several effectors in ETI suppression has been confirmed (Ali et al., 2017), including the M. graminicola effector MgGPP, which is able to suppress Gpa2/RBP‐1‐induced cell death (Chen et al., 2017). Based on our experiments, Mg16820 is able to interfere with at least two different ETI pathways when present in the cytoplasm. Mg16820 suppresses the HR pathway induced by Solanum demissum R2 and Phytophthora infestans Avr2 and the HR pathway induced by the auto‐activation of the tomato resistance gene Mi‐1.2T557S. The suppression of multiple ETI pathways by one effector has also been observed for other nematode effectors. Msp40 secreted by M. incognita was able to prevent Bax‐triggered and R3a/Avr3a‐induced cell death when transiently expressed in the cytoplasm of N. benthamiana leaves. In addition, Msp40 was able to suppress callose deposition and the expression of genes related to the elf18‐triggered PTI immune response. Like Mg16820, Msp40 was able to suppress both PTI and multiple ETI pathways (Niu et al., 2018). However, in contrast with Mg16820, the different activities of Msp40 appeared in the same cell compartment, the nucleus and cytoplasm, whereas Mg16820 works in the apoplast as well as in the cytoplasm. The Globodera rostochiensis effector GrUBCEP12, with and without SP, was able to suppress several elicitor‐induced cell death pathways (Ali et al., 2015). Although it was not confirmed by subcellular localization that the effector GrUBCEP12 was indeed transported to the apoplast, it was confirmed that this effector works on several HR pathways. In addition, the effector MiSGCR1 secreted by M. incognita is able to suppress the AvrPto (Pseudomonas syringae)‐ and NPP1 (Phytophthora parasitica)‐induced HR (Nguyen et al., 2017). Interestingly, similar to Mg16820, this effector was unable to suppress the HR when fused to GFP. It was hypothesized that this is probably an effect of misfolding of the fused protein or physical interference of the GFP protein with the binding of the effector to its target (Nguyen et al., 2017).

The Mi‐1.2T557S‐ and R2/Avr2‐induced HR pathways may overlap, and Mg16820 could interact with a target protein which is involved in both pathways. A Y2H assay was performed to find a target protein in rice, and our data suggest an interaction with DIP1. Although the complete DIP1 protein did not show interaction in the Y2H, we continued to perform a bimolecular fluorescence complementation assay because the truncated part isolated from the Y2H library comprises almost the complete sequence, except that the start codon is missing. We detected an interaction between Mg16820 and DIP1 in planta with bimolecular fluorescence complementation. This might be because the interaction was tested in plant cells instead of yeast and/or because of the difference in the fused tags which may not interfere in the bimolecular fluorescence complementation technique.

In maize, DIP1 is an important component in the rab17 pathway, induced by the phytohormone abscisic acid (ABA) under the influence of drought stress (Saleh et al., 2009). In rice, the homologue of rab17 is called rab21 (Vilardell et al., 2011). Although it is not known whether the rab21 pathway is involved in the HR pathways tested in this study, we can speculate that interaction between DIP1 and Mg16820 changes the expression of rab21, influencing the immune response. In galls, the expression of rab21 was down‐regulated at 3 dai, whereas, at 7 dai, the expression was up‐regulated. In giant cells, rab21 was up‐regulated at both 7 and 14 dai (Ji et al., 2013), but the expression level at 3 dai was not determined. An increased expression of DIP1 was observed in the gall tissue at 7 dai, but not at 3 dai. In the giant cells at 7 and 14 dai, the expression level was similar to that in non‐infected tissue. The plant response to pathogens is regulated by a complex network of phytohormone signalling, and pathogens attempt to interfere with these pathways (Denancé et al., 2013). ABA is related to abiotic stresses, but its role in biotic stresses, such as pathogen attack, is becoming more understood (Lievens et al., 2017). For example, it has been shown that ABA plays a direct role in the regulation of resistance protein activity (Denancé et al., 2013). However, the role of ABA differs in different plant–pathogen interactions (Asselbergh et al., 2008). In rice, galls induced by M. graminicola showed a two‐fold increased ABA level at 3 dai, whereas, at 7 dai, the ABA level was still increased, but lower than at 3 dai. The application of ABA, as well as ABA biosynthesis, promotes the susceptibility of rice to M. graminicola, indicating a complex role for ABA in this plant–nematode relation (Kyndt et al., 2017). The ABA level in the host during the migratory stage of the nematode is not known. We should note that DIP1 is not present in the apoplast, but moves during drought stress from the cytoplasm to the nucleus (Saleh et al., 2009). Possibly, Mg16820 is able to interfere with two different mechanisms to suppress the immune system of the plant. Alternatively, Mg16820 may work in the apoplast and in the cytoplasm by interacting with a component important in ROS defence signalling, which is part of both the PTI and ETI responses.

In summary, we have identified, localized and functionally analysed the effector Mg16820 secreted by the root‐knot nematode M. graminicola. To our knowledge, this is the first effector published which has been proven to be secreted in both the apoplast and the cytoplasm of giant cells during the early stages of parasitism, and has immune‐suppressing capacities in both cell compartments. The mechanism behind the suppression of both the PTI and ETI responses at two different subcellular localizations is not understood and requires further study. In addition, DIP1 was identified as a potential target protein and, as a result of its role in the ABA response, further studies on this interaction may provide further insights into the molecular mechanism of plant parasitism.

EXPERIMENTAL PROCEDURES

Nematode culture

Nematode cultures were obtained by the inoculation of pre‐parasitic J2s of M. graminicola (isolate from Batangas, Philippines) on rice plants or the grass Echinocloa crus‐galli grown in universal soil (Structural, type 1, Kaprijke, Belgium; mire, garden peat and mixed nutrients). These were then incubated for 3 months at 28 ºC with light/dark cycles of 16 h/8 h. To extract pre‐parasitic J2s, the infected roots were washed with tap water and cut into small pieces of c. 0.5 mm to open the galls. Nematodes were collected for 3–7 days using a modified Baermann method (Baermann, 1917).

Plant growth conditions

Nicotiana benthamiana seeds were germinated in moist universal soil (mire, garden peat and mixed nutrients) at 27 ºC, and 1‐week‐old individual plantlets were transferred to pots (diameter, 15 cm) with the same soil and provided once (directly after transfer) with a commercial plant nutrient solution (Substral, NUTRI+MAX universal, Scotts, Sint‐Niklaas, Belgium, 15 mL/10 L water). The plantlets were grown for 5–6 weeks at 27 ºC with light/dark cycles of 16 h/8 h. At least 1 day before infiltration, the plants were transferred to room temperature (RT) and exposed to natural light for acclimatization. After infiltration the plants were kept at RT.

Oryza sativa cv. Nipponbare seeds were germinated on wet filter paper in the dark for 5 days at 30 ºC and then transferred to SAP [mixture of 0.7 g superabsorbent polymer (Aquaperla), DCM, Grobbendonk, Belgium per 1 kg of white sand]‐filled PVC tubes (Reversat and Boyer, 2017). The plantlets were grown for another 9 days at 27 ºC with light/dark cycles of 16 h/8 h before infection. The plantlets were provided three times a week with 10 mL of Hoagland solution (Hoagland and Arnon, 1950) added directly to the tubes.

Sequence analysis

The effector Mg16820 was identified using 454 sequencing of mRNA of pre‐parasitic J2s [contig16820; method described in Haegeman et al. (2013), but Mg16820 was not described in that article]. Prediction of the signal peptide was performed using SignalP 4.0 (https://www.cbs.dtu.dk/services/SignalP/). Prediction of cellular localization was performed using Wolf PSort (https://wolfpsort.hgc.jp) and LOCALIZER (https://localizer.csiro.au; Sperschneider et al., 1998). Prediction of the presence and localization of the NLS was performed with SeqNLS (Lin et al., 2012). The coding region of Mg16820 was blasted using default parameters with a cut‐off value of 50 (bitscore) against the M. graminicola genomics resource database (MGRAMBASE; https://insilico.iari.res.in/mgram/), the non‐redundant protein and nucleotide databases for all organisms of the National Center for Biotechnology Information (NCBI), fungal and oomycete genomes (FungiDB; https://fungidb.org/fungidb/), bacterial genomes (Microbial nucleotide blast on NCBI), and the four genomic datasets of the plant‐parasitic nematodes Bursaphelenchus xylophilus, Globodera pallida, M. incognita and M. hapla. In addition, a BLAST was performed against the online Meloidogyne genomic resources database (INRA; https://meloidogyne.inra.fr/; Blanc‐Mathieu et al., 2017). The alignment of the predicted protein was made using the ClustalW multiple alignment algorithm in Bioedit sequence alignment editor.

Cloning Mg16820 in different vectors

The full‐length coding sequence of effector Mg16820 was PCR amplified from M. graminicola J2 cDNA using gene‐specific primers (Mg16820‐F‐FL and Mg16820‐R‐FL; all primers are listed in Table S1, see Supporting Information) designed to the untranslated regions and ligated into the pGEM‐T easy vector (Promega Leiden, The Netherlands). PCR was performed using VWR® Taq DNA‐polymerase with 30 cycles at an annealing temperature of 58 ºC and an elongation time of 1 min. The PCR product was purified according to the manufacturer’s instructions (QIAquick PCR Purification Kit, Qiagen, Hilden, Germany) and ligated into the pGEM‐T vector according to the manufacturer’s protocol (Promega). The plasmid was transformed into Escherichia coli (DH5‐α or Top10) using heat shock treatment (42 s at 42 ºC, incubation for 2 min on ice and recovery for 1.5 h with shaking at 200 rpm at 37 ºC) and grown overnight at 37 ºC on solid Luria–Bertani (LB) broth medium with 25 μg/mL carbenicillin. Colony PCR was performed to confirm construct presence and the selected colonies were grown overnight in liquid LB with the appropriate antibiotics at 37 ºC. Plasmids were isolated (GeneJET Plasmid Miniprep Kit, Fermentas, Thermo Fisher Scientific, Waltham, Massachusetts, USA) and verified by sequencing. This construct was used as template to fuse the effector sequence to attb sites and to ligate in the Gateway® pDONRTM221 vector (Thermo Fisher Scientific Waltham, Massachusetts, USA). The effector was cloned with start and/or stop codon and with and without its native signal peptide using Mg16820‐attb1, Mg16820‐attb1+start, Mg16820‐SP‐attb1 or Mg16820‐SP‐attb1+start as forward primer and Mg16820‐attb2 or Mg16820‐attb2+stop as reverse primer. The PCR product was amplified in two subsequent PCRs. In the first step, gene‐specific primers were used in 20 μL of PCR mix with an annealing temperature of 45 ºC (five cycles), followed by 54 ºC (15 cycles). In the second step, 3 μL were taken from this PCR mix and used in 17 μL of fresh PCR mix employing the primers M‐13‐F and M‐13‐R using the same program as described above. Ligation reactions (LR Clonase™II, Thermo Fisher Scientific Waltham, Massachusetts, USA) were performed to bring the Mg16820 coding sequence to the appropriate destination vectors (pk7WG2, pk7FWG2, pk7WGF2, pH7RWG2, pH7WGR2, pDEST32, pDEST22 and pCL113). The same approach was followed for cloning of the negative control, the pioneer putative effector Mg03015 (contig03015; Haegeman et al., 2013). This putative effector was chosen as negative control because it has a similar protein size and subcellular localization in N. benthamiana to Mg16820, but is not related in protein sequence. In addition, Mg03015 does not play a role in immune suppression in the assays presented in this research, and therefore could be used as a negative control. Mg03015 was cloned into the pGEM‐T easy vector using the primer pair Mg03015‐F‐FL and Mg03015‐R‐FL. Mg03015 was cloned into pDONRTM221 with start and stop codon, and with and without its native signal peptide into the destination vector pk7WG2, using primers Mg03015‐attb1+start, Mg03015‐SP‐attb1 as forward primer and Mg03015‐attb2 or Mg03015‐attb2+stop as reverse primer. Mg03015 was cloned without native signal peptide and with stop codon into pDONRTM221 and, subsequently, into pCL113. All constructs were sequenced to check for mutations (LGC Genomics, Berlin, Germany) and destination vectors were screened for frameshifts before being used in the experiments (sequencing primers listed in Table S1). Constructs were either transformed into yeast strain MaV203 for the yeast two‐hybrid assay (protocol of Invitrogen, Carlsbad, California, USA) or into Agrobacterium strain GV3101 for agroinfiltration [freeze–thaw method of Holsters et al. (1978)].

In situ hybridization

A 316‐bp PCR product was obtained from the pGEM‐T‐Mg16820 construct using gene‐specific primers (Mg16820‐F‐ISH, Mg16820‐R‐ISH). Single‐strand DNA probes were amplified using the Mg16820‐R‐ISH primer in an asymmetric PCR with digoxigenin (DIG)‐labelled dNTPs (Roche, Mannheim, Germany). Because the in situ hybridization was part of a high‐throughput experiment, only one negative control was used for the reaction instead of for each effector separately. As a negative control for the in situ hybridization, a sense probe of the putative effector Mg9152 was used [Contig9152; database of Haegeman et al. (2013)]. A 333‐bp PCR product was obtained using the primer pair Mg9152‐F‐ISH, Mg9152‐R‐ISH with pGEMT‐Mg9152 as template. Single‐strand DNA probes using primer Mg9152‐F‐ISH were amplified as described above. Pre‐parasitic J2s were fixed in 2% paraformaldehyde overnight at 4 ºC, cut into pieces and permeabilized with proteinase K. Nematodes were hybridized with the DIG‐labelled sense or antisense probes, which were detected with an anti‐digoxigenin alkaline phosphatase‐conjugated antibody following the protocol of de Boer et al. (1998), with the hybridization temperature adapted to 47 ºC to increase probe binding to the template.

Peptide synthesis and antibody purification

Peptides for immunolocalization were designed according to the Thermo Fisher Scientific Antigen profiler. To avoid background, the designed peptides were blasted against the rice genome annotation project (https://rice.plantbiology.msu.edu) and NCBI Basic Local Alignment Search Tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to check for potential similarities between the selected peptide sequences and host plant proteins. Two rabbits were immunized with two synthetic peptides: KRHAQTDHGKDSDS and HIPREELDFQRMIGQ (Thermo Fisher Scientific). Primary antibodies were purified from the rabbit with the lowest background (Thermo Fisher Scientific).

Fixation and embedding of galls

Two‐week‐old rice plantlets in SAP were infected with freshly hatched pre‐parasitic J2s. At 24 h after inoculation, the plantlets were washed briefly and transferred to clean SAP to avoid nematode infection at later time points, thus synchronizing the infection. To obtain galls, the infected root systems were collected at 1, 2, 3, 5, 7 and 10 dai, washed thoroughly with tap water and galls were then dissected from the roots and placed in fixative [4% formaldehyde in 50 mm piperazine‐N,N′‐bis(ethanesulfonic acid) (PIPES) buffer, pH 7.2]. Galls were vacuum treated for 30 min to remove air from the tissue, transferred to fresh fixative in cell strainers and placed at 4 ºC, shaking gently for 2–10 days depending on the gall size. Fixation and embedding were further performed essentially as described by de Almeida Engler et al. (2004) and Kronenberger et al. (1993). Galls were then sectioned to 5 µm using an ultramicrotome and placed on poly‐l‐lysine‐coated slides as described by Vieira et al. (2006).

Immunolocalization of Mg16820

The immunolocalization was performed according to de Almeida Engler et al. (2004) and Vieira et al. (2006) with slight modifications as described below. Gall sections were incubated with blocking buffer [1% bovine serum albumin (BSA) in PIPES buffer, pH 6.9] at RT for 2 h. The Mg16820‐specific antibody was diluted to 1 : 100 in fresh blocking solution and 200 µL were placed on each slide. As a negative control, the pre‐immune serum was diluted 100 times in fresh blocking solution and 200 µL were added to each slide. Dilutions containing the antibody or pre‐immune serum were incubated for 1 h at 37 ºC and centrifuged for 5 min. As another negative control, slides were only treated with secondary antibody. All slides were incubated in a humid box at 4 ºC overnight and, prior to incubation with the secondary antibody, placed at 37 ºC for 1 h and washed for 30 min in 50 mm PIPES buffer. Subsequently, the slides were incubated with the secondary antibody (goat anti‐rabbit IgG (H+L) Alexa Fluor 488, Thermo Fisher Scientific), diluted 1 : 300 in blocking solution, and incubated at 37 ºC for 2 h. The slides were washed in PIPES buffer for 30 min. To stain the nuclei, the slides were incubated in 1.0 µg/mL 4’,6‐diamidino‐2‐phenylindole (DAPI) for 1 min at RT, rinsed in distilled water, mounted in 90% glycerol and coverslipped. Sections were observed with a fluorescence microscope (Zeiss, Oberkochen, Germany) and imaged. Auto‐fluorescence (reddish signal) and Alexa‐488 fluorescence (green) were distinguished using a double bandpass filter (Zeiss). In addition, differential interference contrast (DIC) transmission and DAPI‐stained DNA images were obtained to provide overlaid images.

Subcellular localization

Agrobacterium tumefaciens strain GV3101, carrying the construct of Mg16820 fused to the eGFP tag C‐ or N‐terminally, or to the eRFP tag C‐ or N‐terminally, was suspended in infiltration buffer (IB) to an optical density at 600 nm (OD600) of 0.01 and used for infiltration in N. benthamiana leaves as described above. The same was carried out for Mg16820 fused to its native signal peptide and the eGFP tag N‐terminally. As a control, a pK7WG2 derivative for the expression of eGFP or eRFP was used. Infiltrated plants were kept in daylight at RT and, 48 h after inoculation, the infiltrated spots were imaged using a confocal laser‐scanning microscope (Nikon Instruments Inc., Tokyo, Japan). eGFP was excited with a wavelength of 488 nm and emission was detected at 495–530 nm; eRFP was excited with a wavelength of 561 nm and emission was detected at 592–632 nm. Auto‐fluorescence of chlorophyll was collected at 657–737 nm.

Assay for ROS produced after flg22 incubation

Agrobacterium carrying Mg16820, with or without its native SP and under control of the 35S promoter (vector pK7WG2), was grown in the dark for 2 days at 28 ºC in LB medium with 25 μg/mL rifampicin, 25 μg/mL gentamicin and 50 μg/mL spectinomycin. As a negative control, a plasmid with either a non‐suppressing putative effector [Mg03015; contig03015 in Haegeman et al. (2013), no suppression of flg22‐induced ROS production in more than three assays, unpublished data; D. Naalden] with or without signal peptide was used. The aphid (Myzus persicae) effector Mp10 was used as positive control (Bos et al., 2010). Before infiltration, the agrobacteria were washed and diluted in IB to an OD600 of 0.3 and incubated overnight with slow shaking in the dark at RT.

Nicotiana benthamiana leaves of 5–6‐week‐old plants were infiltrated with agrobacteria using a needleless syringe. Prior to infiltration, a small hole was made on the abaxial side of the leaf with a needle. The agrobacteria holding the above‐mentioned constructs were infiltrated at different spots on the same leaf. For each assay, one leaf of eight plants was used with two replicates per spot. Approximately 30 h after infiltration, 16‐mm2 leaf discs were collected from the infiltrated areas with a cork borer and transferred to 96‐well plates and floated overnight on 190 μL filter‐sterilized ultrapure water for recovery. After 48 h, the water was removed and replaced by a mixture of 100 nm flg22 (QRLSSGLRINSAKDDAAGLAIS; Felix et al., 1999), 0.5 mm luminol probe 8‐amino‐5‐chloro‐7‐phenyl‐pyrido[3,4‐d]pyridazine‐1,4(2H,3H)dione (L‐012) (Wako Chemicals, Richmond, Virginia, USA) and 20 μg/mL horseradish peroxidase (Sigma, Saint Louis, Missouri, Verenigde Staten). ROS production was measured by a luminol‐based assay (Keppler et al., 1989) over 60 min with measurements every 46 s with integration at 750 ms. For Mg16820, both with (SPMg Mg16820) or without (Mg Mg16820) the predicted signal peptide, the assay was repeated more than three times. Student’s t‐test was used to determine significant differences per time point.

ETI assay

ETI assays with several combinations of resistance/avirulence (R/Avr) genes were co‐infiltrated with Mg16820, either with (SPMg16820) or without (Mg16820) the predicted signal peptide, to test for its capacity to suppress the HR. The R/Avr pairs used for the induction of an HR were R2/Avr2 (Saunders et al., 2006), R3a/Avr3aKI 32 (Armstrong et al., 2005), Gpa2/RBP‐1 (Sacco et al., 1999) Cf‐4/Avr4 and Cf‐9/Avr9 (Thomas et al., 2017). Furthermore, Mi‐1.2T557S, the auto‐active form of Mi‐1.2 (Gabriëls et al., 2007), and the PAMP elicitor INF1 (Kamoun et al., 2003) were included. Agrobacterium tumefaciens strain GV3101 was used for transformation as described above. Two negative controls were included in the suppression assay: GV3101 without construct or with pK7WG2‐GFP. Agrobacteria carrying a plasmid for Mg16820 expression or for expression of R/Avr genes were grown for 2–3 days in 10 mL of LB medium with the appropriate antibiotics. Before infiltration, the cells were pelleted, washed and resuspended in IB, and incubated for at least 3 h at RT. Depending on the combination of constructs, the final concentration in the mixtures was adjusted to an OD600 of 0.5 or 0.6 for Mg16820 and 0.5 or 0.4 for the R and Avr genes. The mixtures were spot infiltrated into N. benthamiana leaves of 5–6‐week‐old plants, as described before, with negative controls on the same leaf as the tested effector. For each plant, two leaves were infiltrated and 20 plants were used per assay. When an HR started to appear, the response was recorded for 2 or 3 days until almost all control spots resulted in an HR. HR on a spot was considered to be suppressed when less than 50% of that spot showed cell death following the method of Gilroy et al. (2011). Fisher’s exact test was used to statistically analyse the results. Each assay was performed at least twice and combinations showing suppression were repeated at least three times.

Yeast two‐hybrid screening

A Y2H screen was performed using the ProQuest system (Invitrogen). pDEST32‐Mg16820 and empty pDEST22 were co‐infiltrated in the yeast strain MaV103 to test auto‐activation on SC‐Leu‐Trp‐His (‐TLH) medium supplemented with 10 mm 3‐amino‐1,2,4‐triazole to screen for HIS3 expression, and SC‐Leu‐Trp‐Ura medium for URA3 expression (‐TLU) and for expression of the lacZ reporter gene (X‐gal). A commercially synthesized cDNA library of pathogen‐infected rice tissue was cloned into vector pDEST22 (Invitrogen). The rice tissue was a collection of root tissue infected with M. graminicola and Hirschmanniella oryzae at several time points, as well as root and leaf tissue infected with fungi. The screening was repeated three times resulting in at least 1 000 000 transformants in each screen. Plasmids were extracted from colonies obtained on ‐TLH selection medium and cloned into E. coli. Plasmid preparations were sequenced (LGC Genomics) and one prey was found in frame. This prey was co‐transformed with Mg16820 in pDEST32 to confirm the positive result. The complete coding sequence of the host protein was picked up from rice leaf tissue cDNA using gene‐specific primers (DIP1‐FL‐F and DIP1‐FL‐R). DIP1 without start codon was cloned as described above, ligated into the pDEST22 and pDEST32 vectors and co‐transformed with Mg16820 in pDEST32 and pDEST22, respectively. Colonies obtained from SC‐Leu‐Trp (–TL) plates were further analysed relative to the auto‐activation test.

Bimolecular fluorescence complementation

Mg16820 was cloned into vector pCL113 (YFPc::Mg16820) and the gene for the potential target protein DIP1 was cloned into pCL112 (YFPn::DIP1). As a negative control, a putative effector (Mg03015, described above) with a similar protein size and subcellular localization was cloned into vector pCL113. Constructs were transformed into A. tumefaciens strain GV3101 and complementary constructs were co‐infiltrated into N. benthamiana leaves at OD600 in four different final concentrations: 0.01, 0.03, 0.1 and 0.3. The YFP signal was analysed 48 h after infiltration. YFP was excited with a wavelength of 514 nm and emission was collected at 530–575 nm using confocal microscopy (Nikon Instruments Inc.).

RNA extraction from leaves

To compare the expression of Mg16820 and the negative control putative effector Mg03015 used in the bimolecular fluorescence complementation assay, RNA extraction was performed by grinding 0.2 g of infiltrated N. benthamiana leaf tissue in liquid nitrogen with extraction following the protocol of the RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA was treated with DNAseI, checked for the presence of gDNA and then used as template for cDNA synthesis (3 µg in 36 µL of reaction mix) (tetro cDNA synthesis kit, Bioline, London, UK). Semi‐quantitative PCR was performed with primers targeting cDNA of the housekeeping gene elongation factor 1a (EF1α‐F and EF1 α‐R) (supplementary data Table S1; Liu et al., 2012) to normalize the cDNA template. Primers 52FNew and 52RNew and, for the negative control, 50FNew and 50RNew were used for detection of the effector mRNA (supplementary data Table S1). As PCR template, 5 µL of cDNA mix were used in 40 µL PCR mix. At 25, 30 and 35 PCR cycles, 10 µL of PCR mix were withdrawn from the PCR mix and loaded onto the gel to analyse the results. Thirty‐five PCR cycles were repeated in 30 µL PCR mix and loaded onto the gel to normalize Mg03015/Mg16820 expression to elongation factor 1a expression.

Table 1.

Effector‐triggered immunity suppression assays in Nicotiana benthamiana with effector Mg16820 with and without signal peptide.

Mg16820 SPMg16820 EV GFP
Cf‐4/Avr4 – – – –
Cf‐9/Avr9 – – – –
INF1 – – – –
Mi‐1.2TSS7S + – – –
Gpa2/RBP‐1 – nt – –
R2/Avr2 + nt – –
R3a/Avr3a – nt – –
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EV, empty vector; GFP, green fluorescent protein; –, no suppression; +, suppression; nt, not tested. Each negative assay was performed at least twice with 19–20 plants with two leaves per plant; each positive assay was performed at least three times with 19–20 plants with two leaves per plant.

Supporting information

Fig. S1 cDNA and protein sequence of Mg16820. Start and stop codons are shown in bold. The predicted signal peptide is underlined.

Click here for additional data file. (177.8KB, jpg)

Fig. S2 Immunodetection of the effector Mg16820 in sectioned rice galls. (A–D) Migratory nematodes incubated with pre‐immune serum, showing no signal. (E–H) Galls and nematode at 5 days post‐inoculation (dai) incubated with pre‐immune serum, showing no signal. (I–L) Galls at 7 dai incubated with primary and secondary antibody, showing signal in the cytoplasm. (M–P) Galls at 10 dai incubated with primary and secondary antibody, showing no signal. (A, E, I, M) Detection of Alexa Fluor 488‐conjugated secondary antibody. (B, F, J, N) Detection of 4’,6‐diamidino‐2‐phenylindole (DAPI)‐stained nuclei. (C, G, K, O) Images of differential interference contrast (DIC). (D, H, L, P) DIC, DAPI and green fluorescent protein (GFP) overlay. n, nematode; asterisks, giant cells. Scale bars: (A–D) 10 μm; (E–P) 20 μm.

Click here for additional data file. (1.6MB, jpg)

Fig. S3 Reverse transcription‐polymerase chain reaction (RTPCR) to compare the expression of Mg16820 (YFPc::Mg16820) and Mg03015 (YFPc::Mg03015) in Nicotiana benthamiana leaves with the housekeeping gene elongation factor 1α(EF1α) as control. (A) Expression of EF1α in Mg03015 sample. (B) Expression of EF1α in Mg16820 sample (182 bp). Expression of Mg03015 in (C) is similar to expression of Mg16820 (198 bp) in (D). Result after 35 PCR cycles.

Click here for additional data file. (20.2KB, jpg)

Fig. S4 Expression of Mg16820 in pre‐parasitic second‐stage juveniles (J2s) and at different time points in root tissue. RNA sequencing results in reads per kilobase million (RPKM). Results present the average of two replicates.

Click here for additional data file. (92.1KB, jpg)

Table S1 List of primers used and their purpose in this study.

Click here for additional data file. (16.3KB, docx)

ACKNOWLEDGEMENTS

The authors would like to acknowledge the Research Foundation Flanders FWO for financial support (FWO grants G010712N and 1502312N) and the Special Research Fund of Ghent University for the BOF13/GOA/030 project.

The authors would also like to thank Lien De Smet for technical support, Dr John Jones for hosting Dr Annelies Haegeman at the James Hutton Institute (Dundee, UK), and Dr Miles Armstrong for teaching the yeast two‐hybrid protocol during that stay, funded by COST Action 872 (COST‐STSM‐872‐6657). The visit to INRA to perform the immunolocalization was funded by COST Action SUSTAIN FA1208.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1 cDNA and protein sequence of Mg16820. Start and stop codons are shown in bold. The predicted signal peptide is underlined.

Click here for additional data file. (177.8KB, jpg)

Fig. S2 Immunodetection of the effector Mg16820 in sectioned rice galls. (A–D) Migratory nematodes incubated with pre‐immune serum, showing no signal. (E–H) Galls and nematode at 5 days post‐inoculation (dai) incubated with pre‐immune serum, showing no signal. (I–L) Galls at 7 dai incubated with primary and secondary antibody, showing signal in the cytoplasm. (M–P) Galls at 10 dai incubated with primary and secondary antibody, showing no signal. (A, E, I, M) Detection of Alexa Fluor 488‐conjugated secondary antibody. (B, F, J, N) Detection of 4’,6‐diamidino‐2‐phenylindole (DAPI)‐stained nuclei. (C, G, K, O) Images of differential interference contrast (DIC). (D, H, L, P) DIC, DAPI and green fluorescent protein (GFP) overlay. n, nematode; asterisks, giant cells. Scale bars: (A–D) 10 μm; (E–P) 20 μm.

Click here for additional data file. (1.6MB, jpg)

Fig. S3 Reverse transcription‐polymerase chain reaction (RTPCR) to compare the expression of Mg16820 (YFPc::Mg16820) and Mg03015 (YFPc::Mg03015) in Nicotiana benthamiana leaves with the housekeeping gene elongation factor 1α(EF1α) as control. (A) Expression of EF1α in Mg03015 sample. (B) Expression of EF1α in Mg16820 sample (182 bp). Expression of Mg03015 in (C) is similar to expression of Mg16820 (198 bp) in (D). Result after 35 PCR cycles.

Click here for additional data file. (20.2KB, jpg)

Fig. S4 Expression of Mg16820 in pre‐parasitic second‐stage juveniles (J2s) and at different time points in root tissue. RNA sequencing results in reads per kilobase million (RPKM). Results present the average of two replicates.

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Table S1 List of primers used and their purpose in this study.

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