The Meloidogyne javanica effector Mj2G02 interferes with jasmonic acid signalling to suppress cell death and promote parasitism in Arabidopsis.

Plant-parasitic nematodes can cause devastating damage to crops. These nematodes secrete effectors that suppress the host immune responses to enhance their survival. In this study, Mj2G02, an effector from Meloidogyne javanica, is described. In situ hybridization and transcriptional analysis showed that Mj2G02 was highly expressed in the early infection stages and exclusively expressed in the nematode subventral oesophageal gland cells. In planta RNA interference targeting Mj2G02 impaired M. javanica parasitism, and Mj2G02-transgenic Arabidopsis lines displayed more susceptibility to M. javanica. Using an Agrobacterium-mediated transient expression system and plant immune response assays, we demonstrated that Mj2G02 localized in the plant cell nuclei and could suppress Gpa2/RBP-1-induced cell death. Moreover, by RNA-Seq and quantitative reverse transcription PCR analyses, we showed that Mj2G02 was capable of interfering with the host jasmonic acid (JA) signalling pathway. Multiple jasmonate ZIM-domain (JAZ) genes were significantly upregulated, whereas the JAR1 gene and four JA-responsive genes, MYC3, UPI, THI2.1, and WRKY75, were significantly downregulated. In addition, HPLC analysis showed that the endogenous jasmonoyl-isoleucine (JA-Ile) level in Mj2G02-transgenic Arabidopsis lines was significantly decreased compared to that in wildtype plants. Our results indicate that the M. javanica effector Mj2G02 suppresses the plant immune response, therefore facilitating nematode parasitism. This process is probably mediated by a JA-Ile reduction and JAZ enhancement to repress JA-responsive genes.

INTRODUCTION

Plant‐parasitic nematodes (PPNs) are economically important pests as they cause more than $80 billion in losses in world agricultural yield every year (Mantelin et al., 2017). Root‐knot nematodes (RKNs), Meloidogyne spp., are among the most economically devastating PPNs, infecting more than 5,500 plant species in all continents (Blok et al., 2008). As a sedentary parasite, infective second‐stage juveniles of RKNs penetrate the host root elongation zone and enter the vascular tissues, where they induce the formation of multinucleated giant cells. These giant cells constitute a source of nutrients for the RKNs (Davis et al., 2004). RKNs rely on sustained biotrophic interactions with host plants to establish and maintain the feeding sites. To do so, effector proteins synthesized in the nematode oesophageal glands usually play a role in the interaction, commonly suppressing plant defence responses and manipulating plant signalling pathways (Gheysen & Mitchum, 2011; Huang et al., 2004; Jaouannet & Rosso, 2013).

Over the past two decades, an increasing number of effectors have been isolated from RKNs using various strategies. In earlier years, 37 candidate pioneer effectors expressed in the oesophageal gland cells and 486 proteins from the secretome of M. incognita were obtained by two independent studies performing cDNA library and mass spectrometry screenings, respectively (Bellafiore et al., 2008; Huang et al., 2003). In recent years and thanks to technological advances, studies have identified hundreds of novel potential RKN effectors based on transcriptomes and genomes (Danchin et al., 2013; Nguyen et al., 2018; Petitot et al., 2016; Rutter et al., 2014). However, the roles of most of these effectors are still largely unknown. Therefore, it is imperative to explore their functions to better understand the molecular mechanism of nematode parasitism to develop new pest control methods.

Nucleus‐targeted effectors often play critical roles during the plant–pathogen interaction as more and more evidence suggests that the cell nucleus is an essential target of numerous pathogen effectors (Mukhtar et al., 2011; Rivas, 2012; Rivas & Genin, 2011). It has been experimentally demonstrated by transient expression and immunolocalization assays that several effectors with predicted nuclear localization signals (NLSs), from both RKNs and cyst nematodes (CNs), are localized in plant nuclei (Hewezi et al., 2015; Lin et al., 2013; Quentin et al., 2013; Zhang et al., 2015). Notably, some of these effectors were shown to interfere with host cellular processes, such as gene expression, plant immunity, and plant hormone signal transduction (Hewezi et al., 2015; Mejias et al., 2020; Pogorelko et al., 2019; Verma et al., 2018). Several notable examples are the M. graminicola effector MgGPP, the M. incognita effector MiEFF18, and the Heterodera schachtii effector Hs30D08. Despite their similar localization, these effectors exert their action through different molecular mechanisms. MgGPP recruits host‐mediated glycosylation and proteolysis, and is then translocated to the nuclei to suppress plant defence responses (Chen et al., 2017), MiEFF18 is secreted into the nuclei of giant cells and targeted to the plant core spliceosomal protein SmD1, promoting the development of giant cells (Mejias et al., 2020), and Hs30D08 targets the plant nuclei and interacts with a host auxiliary spliceosomal protein SMU2, which leads to the alteration of the pre‐mRNA splicing and gene expression in feeding sites (Verma et al., 2018).

In 2003, a study by Huang and colleagues obtained a profile of 37 cDNA sequences, including 2G02 encoding a putative parasitism protein expressed in the oesophageal gland cells of M. incognita (Huang et al., 2003). In this study, we aim to characterize a novel nuclear effector, Mj2G02 (an ortholog of M. incognita 2G02), from M. javanica. Herein, we provide evidence that the effector Mj2G02 enhances M. javanica parasitism and can interfere with the jasmonic acid (JA) signalling pathway to suppress plant immunity.

RESULTS

Sequence analysis of the Mj2G02 gene from M. javanica

First, we performed a sequence analysis of the full‐length cDNA sequence of Mj2G02 (GenBank accession MW767382). Mj2G02 contained an open reading frame of 633 bp, encoding a 210 amino acid protein with a predicted 18 amino acid N‐terminal signal peptide (Figure S1). According to InterProScan, a putative N‐terminal ShK toxin (ShKT) domain (33–68 amino acids) and a coiled‐coil region (123–143 amino acids) were present (Figure 1a). In addition, Mj2G02 contained two putative SV40‐like NLS motifs, 62PKKCKVC68 and 189KRKK192 (Figure 1b). According to PSORT II, Mj2G02 was predicted to localize to the nucleus (Table S1).

FIGURE 1

Primary structure analysis of Mj2G02. (a) Structure domains of Mj2G02: N‐terminal signal peptide (1–18 amino acids), ShKT motif (33–68 amino acids), and coiled‐coil region (123–143 amino acids). (b) Multiple sequence alignment of the predicted Mj2G02 with other homologs from plant‐parasitic nematodes. Mj, Meloidogyne javanica (MW767382); Mi, M. incognita (Minc3s00855g18130); Ma, M. arenaria (Scaff918g016851); Me, M. enterolobii (scaffold13260_cov183.g16249); Hg, Heterodera glycines (Hetgly.G000014452). SP, signal peptide; ShKT, ShK toxin; NLS, nuclear localization signal

When we performed a BLAST search against WormBase, we found that Mj2G02 shared a high degree of homology with proteins from various RKN species, including M. incognita (Minc3s00855g18130, 99.5%), M. arenaria (Scaff918g016851, 92.4%), M. floridensis (maker‐nMf.1.1.scaf23378‐augustus‐gene‐0.3, 89.0%), M. enterolobii (scaffold13260_cov183.g16249, 85.4%), M. hapla (MhA1_Contig2148.frz3.gene8, 75.8%), and M. graminicola (NXFT01000603.1.1405_g, 55.6%), but a low degree of homology with proteins from CNs, including Heterodera glycines (Hetgly.G000014452, 46.2%) and Globodera rostochiensis (GROS_g02633.t1, 38.6%). A multiple sequence alignment of the deduced Mj2G02 amino acid sequences with PPN homologs is presented in Figure 1b. When searching against other released PPN genomes, as well as bioinformatics resources of other animal‐parasitic and free‐living nematodes, no significant matches were found.

Mj2G02 is expressed in the subventral oesophageal glands and upregulated in the invasion and early parasitic stages of M. javanica

Tissue localization of Mj2G02 was investigated by in situ hybridization using digoxigenin (DIG)‐labelled Mj2G02 probes in preparasitic second‐stage juveniles (pre‐J2) of M. javanica. The result showed that Mj2G02 transcripts were specifically present in the subventral oesophageal gland cells after hybridization with the antisense probes. As expected, no signals were detected with the sense probes (Figure 2a).

FIGURE 2

Expression patterns of Mj2G02 in Meloidogyne javanica. (a) Localization of Mj2G02 in the subventral oesophageal gland of M. javanica preparasitic second‐stage juveniles (pre‐J2) by in situ hybridization. Fixed nematodes were hybridized with digoxigenin‐labelled antisense (left) and sense (right) cDNA probes from Mj2G02. Scale bars, 20 μm. S, stylet; M, median bulb; DG, dorsal oesophageal gland; SvG, subventral oesophageal glands. (b) Expression of Mj2G02 by quantitative reverse transcription PCR in seven different developmental stages of M. javanica. The fold change values were calculated using the 2−ΔΔ t method. Data are shown as mean of three repeats ± SD. Three independent experiments were performed with similar results, and three technical replicates for each reaction. dpi, days postinoculation

Next, the expression of Mj2G02 was analysed at different developmental stages using quantitative reverse transcription PCR (RT‐qPCR). The expression level of Mj2G02 at the egg stage was set to 1 to calculate the relative fold changes in other stages. As a result, the maximum expression level of Mj2G02 was detected in the pre‐J2 stage, at approximately 26‐fold compared with that in eggs. After the pre‐J2 stage, Mj2G02 transcripts decreased gradually but remained at a high level (15‐fold) at 2 days postinoculation (dpi) (Figure 2b). These findings suggest that Mj2G02 may play a role in the invasion and migration of M. javanica.

Mj2G02 is localized in the plant cell nucleus

Enhanced green fluorescent protein (eGFP) and Mj2G02Δsp:eGFP were transiently expressed in Arabidopsis protoplast cells. After 48 hr, the GFP fluorescent signal of Mj2G02Δsp:eGFP was observed in the nuclei of transformed cells. In the control cells transformed with eGFP alone, the fluorescence signal was found in the cytoplasm and nuclei (Figure 3a). Western blotting using an anti‐GFP antibody detected bands at approximately 49 and 27 kDa for Mj2G02Δsp‐eGFP and eGFP alone, respectively (Figure 3b), indicating that the Mj2G02Δsp‐eGFP fusion protein was intact. To further confirm these results in a different plant species, these fusion proteins were also transiently expressed in Nicotiana benthamiana leaves. Similarly, the GFP signal of Mj2G02Δsp:eGFP was observed in the cell nuclei, and the free eGFP was detected in both cytoplasm and nuclei of cells (Figure 3c).

FIGURE 3

Subcellular localization of Mj2G02 in plant cells. (a) pCambia 1305:Mj2G02Δsp:eGFP and pCambia 1305:eGFP were transformed into Arabidopsis protoplasts. (b) Western blot analysis was used to confirm the expression of Mj2G02Δsp:eGFP and eGFP after transformation in Arabidopsis protoplasts, using anti‐GFP antibodies. (c) pCambia 1305:Mj2G02Δsp:eGFP and pCambia 1305:eGFP were transiently expressed in Nicotiana benthamiana leaves. The GFP fluorescent signal of Mj2G02Δsp:eGFP was observed in the plant nucleus. As a control, the transformed cells expressing eGFP alone showed the fluorescence signal in the cytoplasm and nucleus. Scale bar, 20 μm. eGFP, enhanced green fluorescent protein

In planta RNA interference of Mj2G02 impairs M. javanica parasitism

To investigate whether Mj2G02 can affect M. javanica parasitism, host‐mediated gene silencing was performed to silence Mj2G02 in the feeding nematodes. A β‐glucuronidase (GUS) intron fragment of the RNA interference (RNAi) cassette was detected in four transgenic lines by RT‐PCR (Figure S2a), suggesting that these transgenic lines may express Mj2G02 dsRNA. No GUS fragments were detected in wildtype (WT) plants and plants expressing empty vector (EV) as controls. The phenotype of transgenic lines had no apparent differences compared to control plants. Next, the transcription of Mj2G02 in nematodes was assessed by RT‐qPCR. We found that the transcription of Mj2G02 from RNAi lines at 3 dpi was significantly lower than in WT and EV control plants (Figure 4a), demonstrating that the host‐mediated gene silencing of Mj2G02 was effective. Four independent T2 generation transgenic RNAi lines and control lines were used for M. javanica infection assays. RT‐PCR confirmed the expression of the RNAi cassette in transgenic plants before inoculation (Figure S2b). The results showed that the number of adult females per root in the four independent RNAi lines at 30 dpi was significantly reduced (37.5%–47.1%) compared with those in control plants (Figure 4b).

FIGURE 4

Effect of in planta RNA interference (RNAi) of Mj2G02 on Meloidogyne javanica. (a) The expression levels of Mj2G02 in M. javanica collected from RNAi lines at 2 days postinoculation were significantly lower than those from the wildtype (WT) and empty vector (EV) controls by quantitative reverse transcription PCR. (b) Transgenic RNAi lines showed a significant decrease in the number of adult females compared to that in the WT and EV controls. Data are presented as the mean ± SD from nine plants. Three independent experiments were performed with similar results. *p < 0.05, Student's t test. RNAi‐1, 2, 3, and 4, different T2 generation transgenic RNAi lines

Mj2G02‐transgenic Arabidopsis plants exhibit enhanced susceptibility to M. javanica

To further verify the potential role of Mj2G02 in nematode parasitism, four independent T3 homozygous Mj2G02‐transgenic Arabidopsis lines were used for the nematode infection assay. RT‐qPCR was used to confirm the expression of Mj2G02 in the four transgenic lines (Figure 5a). The transgenic plants showed an increase in lateral root growth compared with the WT plants, but the average root weight did not change obviously (Figure S3). The average number of adult females per gram root and per root system was increased in Mj2G02‐transgenic lines compared with WT plants (Figure 5b,c). These results demonstrate that all four transgenic lines were significantly more susceptible to M. javanica infection than the control plants, suggesting a role of Mj2G02 in nematode parasitism.

FIGURE 5

Expression of Mj2G02 in Arabidopsis lines enhances plant susceptibility to Meloidogyne javanica. (a) Quantitative reverse transcription PCR was used to confirm Mj2G02 mRNA expression level in Mj2G02‐transgenic Arabidopsis lines. (b) Mj2G02‐transgenic Arabidopsis lines showed a significantly increased number of adult females in per gram root system than in wildtype (WT) plants. (c) Mj2G02‐transgenic Arabidopsis lines showed a significantly increased number of adult females in per root system than in WT plants. Data are presented as the mean ± SD from 10 plants. Three independent experiments were performed with similar results. *p < 0.05, **p < 0.01, Student's t test. 2G1, 2G5, 2G6, and 2G7, four independent Mj2G02‐transgenic Arabidopsis lines

Mj2G02 suppresses Gpa2/RBP‐1‐triggered cell death

For the cell death suppression assay, we employed a coinfiltration Agrobacterium‐mediated transient gene expression assay using N. benthamiana leaves. Agrobacterium cells carrying the Mj2G02Δsp construct were infiltrated into N. benthamiana leaves 24 hr prior to a second infiltration of Agrobacterium cells carrying Gpa2 and RBP‐1. As negative controls, leaves were infiltrated with the EV and buffer followed by Gpa2/RBP‐1. After 5 days, Mj2G02 and the positive control GrCEP12 displayed the ability to suppress cell death mediated by Gpa2/RBP‐1 (Figure 6a). The average percentages of necrosis were 12.5% and 8.3%, respectively, compared to 81.6%–94.1% in the negative controls (Figure 6b). Therefore, these results strongly suggest that Mj2G02 might play a role in suppressing plant cell death.

FIGURE 6

Suppression of Gpa2/RBP‐1‐triggered cell death in Nicotiana benthamiana by Mj2G02. (a) Agrobacterium cells carrying pCambia 1305:Mj2G02Δsp, pCambia 1305, and pCambia 1305:GrCEP12 or buffer were infiltrated into N. benthamiana leaves at 24 hr before infiltration with Agrobacterium cells carrying Gpa2/RBP‐1. The cell death phenotype was scored and photographs were taken 5 days after the last infiltration. (b) The average percentage of cell death in leaves expressing Mj2G02 and GrCEP12 was significantly decreased compared to the buffer and empty vector controls. Each bar represents the mean ± SD (n = 50). **p < 0.01, Student's t test. EV, empty vector pCambia 1305

Mj2G02 interferes with plant JA signalling and decreases JA‐Ile accumulation

Next, we analysed the differentially expressed genes (DEGs) in roots of WT and Mj2G02‐transgenic Arabidopsis by RNA‐Seq. A total of 19,535 genes with fragments per kilobase of transcript per million mapped reads (FPKM) ≥ 1 were identified (Table S2). Among these, 2,275 DEGs were upregulated and 1,398 DEGs were downregulated in the Mj2G02‐transgenic line compared with those in WT plants (Figure S4). The complete list of the DEGs is provided in Table S3.

Gene Ontology (GO) analysis of the DEGs involved in biological processes in Arabidopsis was performed using Agrigo. The three most significantly enriched GO terms were secondary metabolic process, apoplast, and tetrapyrrole binding (Figure S5a). To further investigate the functional classification of the DEGs, the Kyoto Encyclopedia of Genes and Genomes (KEGG) ontology of DEGs was conducted using the KEGG automatic annotation server (KAAS). The result showed that these DEGs were primarily enriched in the metabolic pathways involving carbon metabolism, plant hormone signal transduction, and starch and sucrose metabolism (Figure S5b).

In the JA signalling pathway, multiple jasmonate ZIM‐domain (JAZ) genes, including JAZ1, JAZ2, JAZ5, JAZ6, JAZ7, JAZ8, and JAZ10, were found to be significantly upregulated, while the JASMONATE RESISTANT 1 (JAR1) gene encoding a JA amino acid synthetase that conjugates isoleucine to JA was downregulated in the Mj2G02‐transgenic plants compared with WT plants. Moreover, the JA‐responsive genes MYC3, UPI, THI2.1, and WRKY75 also showed a significant decrease (Table 1). The transcription of these DEGs was further validated by RT‐qPCR in three independent Mj2G02‐transgenic lines (Figure 7a–c). All these genes showed a similar trend of transcription expression, supporting the RNA‐Seq data.

TABLE 1

The relative expression levels of JAR1, JAZ, and JA‐responsive genes in RNA‐Seq data

Gene ID	Gene name	Gene description	log2FC	padj
838501	JAZ1	Jasmonate‐ZIM‐domain protein 1	1.349	8.14E−03
843834	TIFY10b/JAZ2	TIFY domain/divergent CCT motif family protein	2.222	4.50E−06
838310	JAZ5	Jasmonate‐ZIM‐domain protein 5	2.924	1.18E−08
843577	JAZ6	Jasmonate‐ZIM‐domain protein 6	1.343	1.53E−02
818025	JAZ7	Jasmonate‐ZIM‐domain protein 7	2.426	9.74E−06
839893	JAZ8	Jasmonate‐ZIM‐domain protein 8	1.641	4.51E−04
831162	JAZ10	Jasmonate‐ZIM‐domain protein 10	1.522	9.14E−03
819244	JAR1	Jasmonate resistant 1	−1.465	0.02
843558	THI2.1	Thionin 2.1	−3.951	9.81E−05
834719	MYC3	Basic helix‐loop‐helix (bHLH) DNA‐binding family protein	−1.234	0.01
834378	UPI	Serine protease inhibitor 2C potato inhibitor I‐type family protein	−3.527	5.42E−09
831147	WRKY75	WRKY DNA‐binding protein 75	−2.932	2.13E−11

The absolute value of log2FC shows the expression fold changes of these differentially expressed genes in Mj2G02‐transgenic Arabidopsis compared with wildtype plants. padj, adjusted or corrected p value.

FIGURE 7

Confirmation of differentially expressed genes related to the jasmonic acid pathway in Mj2G02‐transgenic Arabidopsis lines and wildtype (WT) plants by quantitative reverse transcription PCR. (a) The expression level of seven JAZ genes in Mj2G02‐transgenic Arabidopsis lines showed a significant increase compared to those in WT plants. (b) The JASMONATE RESISTANT 1 (JAR1) gene expression level in Mj2G02‐transgenic Arabidopsis lines showed a significant decrease compared to WT plants. (c) The mRNA expression level of four JA‐responsive genes (MYC3, UPI, THI2.1, and WRKY75) in Mj2G02‐transgenic Arabidopsis lines was significantly lower than those in WT plants. The AtActin gene (At1g49240) of Arabidopsis was used as the reference gene. Data are shown as the mean ± SD of three repeats. The experiments were performed two times with similar results, and three technical replicates for each reaction. *p < 0.05, **p < 0.01, Student's t test. 2G1, 2G5, and 2G6 are three independent Mj2G02‐transgenic Arabidopsis lines

The downregulation of JAR1 in the Mj2G02‐transgenic plants prompted us to investigate the endogenous hormone jasmonoyl‐isoleucine (JA‐Ile) in Arabidopsis roots by high performance liquid chromatography mass spectrometry (HPLC‐MS) analysis. The results indicated that the JA‐Ile concentration was significantly lower in the two Mj2G02‐transgenic Arabidopsis lines than in WT plants, with reductions of 41.8% and 41.5%, respectively (Table 2).

TABLE 2

Metabolic analysis of jasmonoyl‐isoleucine (JA‐Ile) in Arabidopsis

Arabidopsis line	JA‐Ile (ng/g FW root)
Wildtype	30.23 ± 0.93a
2G1	17.58 ± 0.98b
2G5	17.68 ± 1.88b

2G1 and 2G5, two independent Mj2G02‐transgenic Arabidopsis lines; FW, fresh weight. Data are presented as the mean ± SD (n = 3). Different lower case letters represent significant differences (Duncan's multiple range test, α = 0.01).

JAZ and JA‐responsive genes respond to M. javanica infection

To investigate whether the upregulated JAZ genes and downregulated JA‐responsive genes in Mj2G02‐transgenic Arabidopsis lines were related to nematode parasitism, the expression levels of the seven JAZ genes and four JA‐responsive genes were analysed by RT‐qPCR at 1, 2, and 5 days after infection (dai) by M. javanica. As a result, the expression levels of UPI and WRKY75 were significantly increased at all three time points, compared with uninfected roots, the expression level of MYC3 was significantly increased at 1 and 2 dai, but not at 5 dai, and the expression levels of six JAZ genes (JAZ1, JAZ5, JAZ6, JAZ7, JAZ8, and JAZ10) were significantly increased only at 1 dai, whereas JAZ2 and THI2.1 were significantly increased only at 2 dai (Figure 8). Our results suggest that all seven JAZ genes and four JA‐responsive genes might be related to M. javanica parasitism.

FIGURE 8

Expression patterns of seven JAZ genes and four JA‐responsive genes in Arabidopsis roots during Meloidogyne javanica (Mj) parasitism. The transcript abundances of the seven JAZ genes and four JA‐responsive genes were analysed by quantitative reverse transcription PCR in M. javanica‐infected roots at 1, 2, and 5 days after infection (dai), using uninfected Arabidopsis roots as controls. The Arabidopsis AtActin gene (At1g49240) was used as the reference gene. Data are shown as the mean ± SD of three repeats. The experiments were performed two times with similar results, and three technical replicates for each reaction. *p < 0.05, **p < 0.01, Student's t test. WT, wild type

DISCUSSION

In this study, we cloned the Mj2G02 gene from M. javanica and characterized its role in nematode parasitism. A BLAST search showed that various RKNs have genes with a high degree of homology to Mj2G02, while sequences with low homology occur in CNs and no homologs were found in other PPNs and free‐living nematodes, suggesting that this gene may be conserved in Meloidogyne species.

The present study also demonstrated that the Mj2G02 transcript is specifically present in the subventral oesophageal glands. It is believed that the two subventral gland cells of PPNs are essential secretory organs that participate in nematode penetration and migration in plant roots (Davis et al., 2000; Haegeman et al., 2012). Consistent with this notion, the Mj2G02 developmental expression profile demonstrated that the highest expression level occurred in the pre‐J2 stage and persisted at 2 dpi. This, together with the fact that Mj2G02 possesses an N‐terminal signal peptide that is considered to aid protein translocation into the endoplasmic reticulum for secretion (Elling et al., 2007; Petersen et al., 2011), suggests that Mj2G02 is probably secreted during the stages of nematode invasion and migration in the roots. More importantly, Mj2G02‐transgenic Arabidopsis plants were more susceptible to M. javanica infection than controls and, conversely, in planta RNAi silencing of Mj2G02 significantly increased the plant resistance to M. javanica. Similarly, fewer galls, females, and egg masses were observed in transgenic Arabidopsis lines expressing Mi‐msp2 (an Mj2G02 ortholog) dsRNA than control plants (Joshi et al., 2019). Taken together, these results strongly suggest that Mj2G02 plays a role in nematode invasion and the early stages of parasitism, promoting infection by M. javanica.

To resist the infection of pathogens, plants have evolved two layers of immunity: pathogen‐associated molecular pattern (PAMP)‐triggered immunity (PTI) and effector‐triggered immunity (ETI). In general, ETI is an accelerated and amplified PTI response, leading to an exacerbated cell death response at the infection site to prevent further development of the invading pathogen (Jones & Dangl, 2006). It is also suggested that the boundaries between PTI and ETI are not strictly distinct (Naveed et al., 2020; Thomma et al., 2011; Yuan et al., 2021). Some RKN effectors have been experimentally demonstrated to facilitate nematode parasitism by suppressing cell death (Chen et al., 2017; Niu et al., 2016; Shi et al., 2018; Zhuo et al., 2017). In the present study, using bioinformatic tools, we found that Mj2G02 has a predicted ShKT domain. Although the exact biological function of the ShKT domain remains unclear, previous reports have shown that this domain might be associated with immunosuppression. For example, Toxocara canis‐secreted mucins (Doedens et al., 2001; Loukas et al., 2000) and the M. incognita‐secreted effector Msp40 (Niu et al., 2016), both containing ShKT domains, are responsible for immune evasion and suppression of Bax‐triggered cell death, respectively. Accordingly, an assay of cell death suppression by Mj2G02 was performed in N. benthamiana to explore whether Mj2G02 can suppress plant immunity. Indeed, the result showed that Mj2G02 could inhibit Gpa2/RBP‐1‐triggered cell death. Mj2G02 is predicted to contain two NLS motifs, and the transient expression assay confirmed its nuclear localization in plant cells. It has been previously reported that some nucleus‐targeted effectors from PPNs, such as the M. graminicola‐secreted effector MgGPP, M. incognita‐secreted effector MiISE6, and H. avenae‐secreted effector HaVAP2, can inhibit cell death (Chen et al., 2017; Luo et al., 2019; Shi et al., 2018). Thus, the molecular mechanisms of cell death suppression by nucleus‐targeted effectors deserve further studies. Mi2G02, a homolog of Mj2G02, has been shown to be localized in the cytoplasm (Zhang et al., 2015); however, the Mi2G02 was fused to GUS protein, which might have resulted in the cytoplasmic localization. Taken together, these results suggest that secreted Mj2G02 might target the plant nucleus to suppress plant immunity and enhance M. javanica infection.

The phytohormone signalling network is required for plant immunity (Kazan & Lyons, 2014; Mine et al., 2018; Robert‐Seilaniantz et al., 2011). JA and salicylic acid are considered the principal plant defence hormones and play an important role in fighting against pathogens, including RKNs (Gheysen & Mitchum, 2019; Gutjahr & Paszkowski, 2009; Molinari et al., 2014; Mur et al., 2006). Other phytohormones, such as abscisic acid and gibberellin, also play a role against RKNs, but mainly through crosstalk with the JA signalling pathway (Kyndt et al., 2017; Yimer et al., 2018). The present study showed that plant hormone signal transduction pathways including the JA signalling pathway were significantly enriched in Mj2G02‐transgenic plants. Transcriptome analysis and RT‐qPCR showed that multiple JAZ genes were clearly upregulated, whereas JAR1 was significantly downregulated. It is believed that the upregulation of JAZ and the downregulation of JAR1 in the plant are beneficial for invading pathogens. For instance, previous studies showed that overexpression of JAZ7 enhanced the susceptibility of Arabidopsis to Pseudomonas syringae (Zhang et al., 2018), and the jar1‐1 mutation increased the plant susceptibility to various pathogens (Berrocal‐Lobo & Molina, 2004; Clarke et al., 2000; Ryu et al., 2004; Staswick et al., 1998). Evidence has emerged that the JA signalling is involved in resistance to RKNs in monocotyledons and dicotyledons (Fujimoto et al., 2011; Mendy et al., 2017; Nahar et al., 2011). For example, the expression of the M. incognita‐secreted effector MiISE6 in Arabidopsis resulted in the upregulation of some JAZ genes and the downregulation of several JA‐responsive marker genes, facilitating nematode parasitism in Arabidopsis (Shi et al., 2018); the tomato COP9 signalosome CSN4 and CSN5 interacted with JAZ2, a signalling component of the JA pathway, and played critical roles in JA‐dependent basal defence against M. incognita (Shang et al., 2019); and M. graminicola infection in rice roots inhibited the accumulation of JA and JA‐Ile, and foliar spraying of the strigolactone analog GR24 reduced JA and JA‐Ile accumulation in rice roots, leading to a higher nematode infection (Lahari et al., 2019).

JAR1 is involved in the conjugation of JA to isoleucine for the synthesis of JA‐Ile, which is considered to be the endogenous bioactive form of JA perceived by plants (Staswick & Tiryaki, 2004; Thines et al., 2007). Additionally, JAZ proteins can repress JA signalling via a regulatory negative feedback loop involving the transcription factor MYC2 (Chini et al., 2007; Thines et al., 2007). When the level of JA‐Ile is low, the JAZ proteins recruit the NINJA/TOPLESS complex to repress the MYC2 activity, then repress in turn JA‐responsive genes (Chico et al., 2008; Chini et al., 2007; Thines et al., 2007). Therefore, based on current knowledge, we speculate that Mj2G02 promotes nematode parasitism, probably modulating the JA signalling pathway via JAZ upregulation and JAR1 downregulation. This regulation results in a decrease of JA‐Ile concentrations and suppression of JA‐responsive genes. Consistent with this speculation, we found that the concentrations of JA‐Ile were significantly lower in the Mj2G02‐transgenic Arabidopsis lines than in WT plants. Moreover, the expression levels of four JA‐responsive genes, MYC3, UPI, THI2.1, and WRKY75, which have been reported to be involved in plant defence against plant pathogens (Chen et al., 2020; Fernandez‐Calvo et al., 2011; Kammerhofer et al., 2015; Laluk & Mengiste, 2011), were also significantly reduced in the Mj2G02‐transgenic Arabidopsis lines.

In summary, we described a novel effector protein, Mj2G02, from M. javanica and investigated its role in Arabidopsis infection. Our experimental evidence suggests that M. javanica might secrete Mj2G02 into the plant cell nuclei during the early infection stages, reducing JA‐Ile and promoting JAZ proteins to repress JA‐responsive genes. JA‐responsive gene repression impairs the plant immunity, increasing the plant susceptibility to M. javanica infection. Further studies of the interaction between the Mj2G02 effector and its receptor in plant cells may reveal the molecular mechanism controlling suppression of the JA signalling.

EXPERIMENTAL PROCEDURES

Nematode and plant materials

M. javanica was collected from towel gourd (Luffa sp.) in Guangxi, China, using a single egg mass, and maintained on tomato plants (Solanum lycopersicum) in a greenhouse at 25 ℃ under 16 hr light/8 hr dark (16/8 LD) conditions. Egg masses, pre‐J2s, and parasitic stage nematodes were collected as previously described (Ding et al., 1998; Huang et al., 2005). Arabidopsis thaliana and N. benthamiana were cultivated at 25 ℃ in 16/8 LD cycles in a glasshouse.

Gene cloning and sequence analysis

Total RNA was isolated from freshly hatched pre‐J2s using the RNAprep Pure Micro Kit (TianGen Biotech). The cDNA was synthesized using the TransScript One‐Step gDNA Removal and the cDNA Synthesis SuperMix kits (Transgen Biotech). The full‐length cDNA sequence of Mj2G02 was amplified using the Mj2G02F and Mj2G02R primers designed based on the Mi2G02 sequence of M. incognita (Huang et al., 2003). All primers used in this study were synthesized by Tianyi Huiyuan Biotech and the full sequences are disclosed in Table S4.

The sequence of the predicted protein was used to identify homologous sequences by searches in the National Center for Biotechnology Information (NCBI) database, the WormBase database, and Nematode.net. The signal peptide was predicted using SignalP v. 4.0. Molecular mass was analysed using ProtParam. InterProScan was used to predict the putative conserved domains. The subcellular localization of effectors was predicted using the website PSORTII (http://psort.hgc.jp/form2.html) (Nakai & Horton, 1999).

In situ hybridization

Approximately 10,000 pre‐J2s of M. javanica were collected as described above. The 2G‐ISHF and 2G‐ISHR primers were employed to synthesize digoxygenin (DIG)‐labelled sense and antisense cDNA probes based on the Mj2G02 fragment of 360–537 bp using a PCR DIG Probe Synthesis Kit (Roche Applied Science). The nematode sections were hybridized as previously described (Jaouannet et al., 2012) and the signals were detected by microscopy using an ECLIPSE Ni microscope (Nikon).

Developmental expression analysis

Total RNA was isolated from approximately 200 M. javanica nematodes at different life stages as described above. The cDNA was then synthesized using the TransScript One‐Step gDNA Removal and the cDNA Synthesis SuperMix kits (Transgen Biotech). RT‐qPCR was performed using the qPCR2GF/qPCR2GR and qMj‐ACT2‐F/qMj‐ACT2‐R primer pairs for amplifying the Mj2G02 gene and the endogenous reference gene Mj‐β‐actin (accession no. AF532605), respectively. RT‐qPCR was performed using the TransStart Tip Green qPCR SuperMix kit (Transgen Biotech) on a Dice Real‐Time System thermal cycler (Takara). These experiments were repeated three times, with three technical replicates for each reaction. The relative changes in gene expression were calculated using the 2−ΔΔ t method (Livak & Schmittgen, 2001).

Subcellular localization analysis

To construct the Mj2G02Δsp:eGFP plasmid, the sequence of Mj2G02Δsp was fused to the N‐terminus of eGFP and cloned into pCambia 1305.1. eGFP alone was used as a control, and an auxin response factor 19 (ARF19) fused to the N‐terminus of red fluorescent protein (RFP) was used as a nuclear marker. The constructs were purified using the HighPure Maxi Plasmid Kit following the manufacturer's instructions (TianGen Biotech). The Arabidopsis root protoplast isolation and transformation were carried out as described previously (Yoo et al., 2007). Protoplasts were incubated in the dark at room temperature for c.48 hr and examined under an ECLIPSE Ni microscope. To verify the intact Mj2G02Δsp‐eGFP fusion protein, western blot was performed with anti‐GFP antibody (Transgen Biotech) as described previously (Zhuo et al., 2017). The proteins were visualized using the Immobilon Western Chemiluminescent system with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific). Additionally, Mj2G02Δsp:eGFP and eGFP plasmids were also transiently expressed in N. benthamiana leaves to analyse the subcellular localization as described previously (Chen et al., 2017).

In planta RNAi

For the Mj2G02 silencing construct, the fragment of 109 to 405 bp within the Mj2G02 sequence was selected as the RNAi target. This sequence was confirmed to have no contiguous 21‐nucleotide identical hits in other genes. Mj2G02 109‐405 was inserted into pMD‐18T (Takara) in both sense and antisense orientations separated by a GUS intron. Then, the entire RNAi structure was inserted into pCambia 1305.1 to generate the plasmid expressing the hairpin dsRNA. The recombinant plasmid was transformed into Agrobacterium tumefaciens EHA105, and an empty vector (EV) was used as the negative control. Next, the transgenic N. benthamiana plants were generated using the routine leaf disc method as described previously (Horsch et al., 1985). The RNAi transgenic lines were confirmed by RT‐PCR using the GUS intron fragment as a target. To investigate RNAi efficiency, RNA was purified from 200 parasitic‐stage nematodes collected from roots of 10 plants at 3 dpi. RT‐qPCR was performed to determine the expression level of Mj2G02. Independent RT‐qPCR experiments were performed three times.

Generation of transgenic Arabidopsis plants

The coding sequence of Mj2G02 without the signal peptide was cloned into pCambia 1305.1 to generate the plasmid pCambia 1305:Mj2G02Δsp. The overexpression plasmids were transformed into A. tumefaciens EHA105. Next, the transgenic Arabidopsis plants were generated using the floral dip method as previously described (Zhang et al., 2006). Transformants were selected by hygromycin B on Murashige and Skoog (MS) medium, and T3 generation homozygous seeds were collected from T2 generation plants after selection by hygromycin B and were used in this study. The expression level of Mj2G02 in each transgenic line was determined by RT‐qPCR. The Arabidopsis AtActin gene (At1g49240) was selected as an endogenous reference (Lin et al., 2016). The relative changes in gene expression were calculated using the 2−ΔΔ t method. Independent RT‐qPCR experiments were performed three times.

Infection assay

Fourteen‐day‐old Arabidopsis and tobacco plants were each inoculated with 150 M. javanica pre‐J2s. At 30 dpi the roots were collected, washed, and stained by acid fuchsin (Naalden et al., 2018), and the number of M. javanica females was counted. Each experiment was performed three independent times. Statistical differences between treatments were calculated by Student's t test using SAS v. 9.2 (SAS Institute).

Cell death suppression

The coding sequence of Mj2G02 was cloned into pCambia 1305.1 to generate the pCambia 1305:Mj2G02Δsp construct. The EV pCambia 1305 and pCambia 1305:GrCEP12 (Chronis et al., 2013) were used as the negative and positive control, respectively. The resistance/avirulence gene pair Gpa2/RBP‐1 was used to induce cell death (Sacco et al., 2009). These constructs were separately transformed into A. tumefaciens EHA105 and then suspended in a buffer containing 10 mM MES (pH 5.5) and 200 μM acetosyringone. Subsequently, Agrobacterium cells carrying these constructs (OD600 = 0.5) were infiltrated into N. benthamiana leaves as described previously (Zhuo et al., 2017). After 24 hr, the same infiltration sites were injected with Agrobacterium cells carrying the constructs pCambia 1305:Gpa2 and pCambia 1305:RBP‐1. The phenotypes of infiltrated N. benthamiana were photographed at 5 days after the last infiltration. The cell death phenotype was scored by an average necrosis percentage (Gilroy et al., 2011). This experiment was performed in triplicate.

RNA‐Seq

The Arabidopsis roots of 14‐day‐old WT plants and homozygous Mj2G02‐transgenic plants (line 2G1) were used for the RNA‐Seq analysis. The sequencing was performed using the Illumina HiSeq2500 system according to the manufacturer's instructions, using two biological replicates. The raw reads from the sequencing run were filtered to remove adapter sequences, reads with more than 10% of unknown bases (N), and low‐quality sequences to generate the final clean reads. The clean reads were mapped to the reference genome of Arabidopsis and subsequent analyses were performed.

The FPKM value of each gene was calculated based on the gene length and read counts mapped to this gene. Differential expression analysis was performed using the DESeq2 R v. 1.16.1 package. DEGs were selected based on criteria of an adjusted p value (padj) < 0.05 and absolute values of log2 fold change (log2FC) ≥ 1. GO annotation analysis and functional classification of DEGs were implemented by the GOseq R package (Young et al., 2010). GO terms with padj < 0.05 were considered significantly enriched in DEGs. KEGG significant enrichment analyses from the KEGG database were conducted to identify the biological functions and related metabolic pathways in which these genes participate (Kanehisa & Goto, 2000).

To validate the RNA‐Seq results, RT‐qPCR assays were performed using independently collected samples in the same developmental stage as those used for the RNA‐Seq analysis.

Arabidopsis gene expression analyses

For the Arabidopsis gene expression assays during a compatible interaction of M. javanica and Arabidopsis, seedlings were inoculated by M. javanica as described above. Arabidopsis roots infected by M. javanica and uninfected roots were sampled at 1, 2, and 5 dai. The total RNA of each sample was extracted and converted to cDNA as described above. RT‐qPCR was performed to determine the expression changes of seven JAZ genes (JAZ1, JAZ2, JAZ5, JAZ6, JAZ7, JAZ8, and JAZ10) and four JA‐responsive genes (MYC3, UPI, THI2.1, and WRKY75). The Arabidopsis AtActin gene (At1g49240) was selected as an endogenous reference (Lin et al., 2016). Independent RT‐qPCR experiments were performed two times with three technical replicates. The gene expression levels of uninfected Arabidopsis roots were set to 1 to calculate the relative fold changes in M. javanica‐infected roots. The relative changes in gene expression were calculated using the 2−ΔΔ t method (Livak & Schmittgen, 2001).

Quantification of JA‐Ile

For the analysis of the JA‐Ile concentration, HPLC was performed on an EXIONLC System (SCIEX) as previously described (Šimura et al., 2018). WT and Mj2G02‐transgenic Arabidopsis were grown on 1/2 × MS medium, and root samples were collected from 14‐day‐old plants. A total of three biological replicates per treatment was used. The quantification of JA‐Ile was confirmed by analysing serial dilutions of a standard mixture with multilevel calibration curves (R 2 > 0.99). The metabolite concentrations were determined relative to the corresponding internal standard.

CONFLICT OF INTEREST

The authors declare that they have no competing interests.

FIGURE S1 Sequence analysis of Mj2G02. (a) cDNA sequence of Mj2G02. The predicted start and stop codons are shown in red. (b) Putative amino acid sequence of Mj2G02. The predicted signal peptide is underlined

Click here for additional data file.

FIGURE S2 Detection of RNAi transgenic lines of Mj2G02. (a) Reverse transcription (RT) PCR analysis to detect the GUS intron fragment of four independent T0 RNAi transgenic lines (RNAi‐1, 2, 3, and 4). WT, wild type; EV, empty vector. (b) RT‐PCR analysis to detect the GUS intron fragment of four independent T2 RNAi transgenic lines (RNAi‐1, 2, 3, and 4) used for Meloidogyne javanica infection assays. The numbers 1–14 represent different plants of each T2 generation transgenic RNAi line

Click here for additional data file.

FIGURE S3 Phenotype analysis of Mj2G02‐transgenic Arabidopsis lines. (a) T3 homozygous independent transgenic lines and wildtype (WT) plants were cultured in MS medium after 14 days. (b) The Mj2G02‐transgenic Arabidopsis lines have significantly more lateral roots than WT plants. Data are presented as the mean ± SD (n = 12). (c) The average root weight of Mj2G02‐transgenic Arabidopsis lines. *p < .05, Student’s t test. 2G1, 2G5, 2G6, 2G7, four Mj2G02‐transgenic Arabidopsis lines

Click here for additional data file.

FIGURE S4 Statistical analysis of differentially expressed genes (DEGs). Volcano map shows the DEGs in the Mj2G02‐transgenic Arabidopsis lines compared to wildtype Arabidopsis, in which 2,275 genes were upregulated and 1,398 genes were downregulated (padj < 0.05, |log2FC| ≥ 1). The abscissa is the log2FC value and the ordinate is the −log10 padj value

Click here for additional data file.

FIGURE S5 Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed genes (DEGs) in Mj2G02‐transgenic Arabidopsis lines compared to wildtype plants. (a) The 10 most significantly enriched GO terms in the three‐function classification are shown (padj < 0.05). BP, biological process; CC, cellular component; MF, molecular function. (b) The top 20 enriched KEGG pathways are shown (padj < 0.05)

Click here for additional data file.

TABLE S1 Prediction of subcellular localization of Mj2G02

Click here for additional data file.

TABLE S2 List of 19,535 genes identified in Mj2G02‐transgenic Arabidopsis lines and wild‐type plants, with fragments per kilobase of transcript per million mapped reads (FPKM) ≥ 1

Click here for additional data file.

TABLE S3 List of differentially expressed genes identified between Mj2G02‐transgenic Arabidopsis lines and wild‐type plants, with padj < 0.05 and |log2FC| ≥ 1

Click here for additional data file.

TABLE S4 Full nucleotide sequence of the primers used in the study

Click here for additional data file.