A Meloidogyne incognita effector Minc03329 suppresses plant immunity and promotes parasitism

ZHOU Jing-jing,ZHANG Xiao-ping,LlU Rui,LlNG Jian,Ll Yan,YANG Yu-hong,XlE Bing-yan,ZHAO Jian-long ,MAO Zhen-chuan

State Key Laboratory of Vegetable Biobreeding,Institute of Vegetables and Flowers,Chinese Academy of Agricultural Sciences,Beijing 100081,P.R.China

Abstract Meloidogyne incognita is a devastating plant-parasitic nematode. Effectors play important roles during the stages of nematodes infection and parasitism,but their molecular functions remain largely unknown. In this study,we characterized a new effector,Minc03329,which contains signal peptide for secretion and a C-type lectin domain. The yeast signal sequence trap experiments indicated that the signal peptide of Minc03329 is functional. In situ hybridization showed that Minc03329 was specifically expressed in the subventral esophageal gland. Real-time qPCR confirmed that the expression level of Minc03329 transcript was significantly increased in pre-parasitic and parasitic second-stage juveniles (pre-J2s and par-J2s). Tobacco rattle virus (TRV)-mediated gene silencing of Minc03329 in host plants largely reduced the pathogenicity of nematodes. On the contrary,ectopic expression of Minc03329 in Arabidopsis thaliana significantly increased plant susceptibility to nematodes. Transient expression of Minc03329 in Nicotiana benthamiana leaves suppressed the programmed cell death triggered by the pro-apoptotic protein BAX. Moreover,the transcriptome analysis of Minc03329-transgenic Arabidopsis and wild type revealed that many defense-related genes were significantly down-regulated. Interestingly,some different expressed genes were involved in the formation of nematode feeding sites.These results revealed that Minc03329 is an important effector for M.incognita,suppressing host defense response and promoting pathogenicity.

Keywords: Meloidogyne incognita,effector,C-type lectin,pathogenicity,plant immunity

1.lntroduction

Root-knot nematodes (RKNs,Meloidogynespp.) are important plant-parasitic nematodes,which infect thousands of plant species and result in devastating yield losses every year (Phaniet al.2021). Among RKNs,the most yield-limiting species areM.incognita,M.arenaria,M.javanica,M.haplaandM.enterolobii,which could infect almost all cultivated plants and are responsible for multibillion dollar annual losses every year (Castagnone-Serenoet al.2013;Joneset al.2013).As sedentary endoparasites,the second-stage juveniles(J2s) ofM.incognitainfect roots and establish permanent feeding sites in the differentiation zone of roots. This is accomplished by inducing nuclear division without cytokinesis,thus creating multinucleate “giant cells” that nurture the developing nematodes (Faveryet al.2016).RKNs infection is associated with the reprogramming of plant cell architecture rather than cell death (Caillaudet al.2008). Recent advances in plant-nematode interactions reported that RKNs secrete many substances(e.g.,active small molecule substance and effectors) that mainly produced from the nematode’s two subventral and one dorsal esophageal gland cells (Mejiaset al.2019;Vieira and Gleason 2019;Hewezi 2020). These effectors appear to play crucial roles in modulating plant immune responses and formation of feeding cells (Williamson and Gleason 2003). Effectors are defined as proteins and small molecules secreted by pathogens that alter hostcell structures and functions,and these alterations either facilitate infection by subjugate plant immunity (virulence factors and toxins) or trigger defense responses(avirulence factors and elicitors) or both. For instance,MjTTL5 effector was expressed in the subventral gland ofM.javanicaand interacted withArabidopsisAtFTRc to affect host antioxidant system (Linet al.2016). AM.enterolobiieffector MeTCTP was expressed in the dorsal gland and promoted parasitism by suppressing programmed cell death (Zhuoet al.2017). AM.incognitachorismate mutase effector,Mi-CM-3,expressed in the subventral esophageal glands and suppressed salicylic acid (SA) mediated plant immunity in the early stages of nematode parasitism (Wanget al.2018). MiPDI1 was expressed in the subventral esophageal glands ofM.incognitaand secreted into plant cells during parasitism. Evidence showed that MiPDI1 modulated plant immunity by interacting with stress-associated protein 12 (Zhaoet al.2020). Recently,researchers showed that theM.incognitaeffector MiEFF18 interacted with the plant core spliceosomal protein SmD1 and played vital roles in giant cell formation (Mejiaset al.2021).Dozens of plant-parasitic nematodes effectors were identified and characterized functions,but the essential roles of a large number of effectors remain unknown.

Lectins are wide spread in plants,animals,and microorganisms. They showed not only carbohydrate binding properties,but also involved in protein-protein interactions,which exhibited various roles in regulating immunity (Zelensky and Gready 2005;Harcuset al.2009). InCaenorhabditiselegans,there are 278 C-type lectin domain (CTLD) proteins,which are arranged in clusters in the genome and respond specifically to different pathogens to produce immune responses(Schulenburget al.2008). CTLD proteins were also reported in RKNs.Meloidogynegraminicolahad three C-type lectin sequences,which were located in the subventral esophageal glands,and their expression levels increased significantly in the early stages of infection,indicating their essential roles during the nematodeparasitic process (Haegemanet al.2013). A CTLD protein Mg01965 was expressed in the subventral esophageal glands ofM.graminicola,which promoted nematode parasitism by inhibiting plant reactive oxygen species (ROS) burst in apoplasts (Zhuoet al.2019). A genome-wide search for lectin-like domains was carried out in the phylum Nematoda. 57 CTLD proteins were identified in theM.incognita,containing 2-3 CTLDs (Bauterset al.2017). However,only one CTLD protein’s function was characterized. MiCTL1a was identified in the subventral esophageal glands ofM.incognita,which was secreted into plant cells,interacted with plant catalases CATs,and modulated H2O2homeostasis (Zhaoet al.2021). To fully understand how CTLD proteins involve in RKNs parasitism,further functional analysis of CTLD effectors is necessary.

In this study,we identified a novel CTLD effector,Minc03329,inM.incognitagenome and transcriptome.Minc03329 contained a N-terminal signal peptide for secretion and one CTLD domain. Evidence showed thatMinc03329transcript was specifically expressed in the subventral esophageal glands ofM.incognitapre-parasitic J2s and significantly up-regulated in parasitic J2s. Virusinduced gene silencing (VIGS) ofMinc03329inNicotiana benthamianaaffected nematode infection ability. On the contrary,Arabidopsisectopic expressionMinc03329significantly increased susceptibility toM.incognita.We showed that transient expression of Minc03329 inN.benthamianaleaves suppressed BAX-induced cell death. Meanwhile,transcriptomic profiling provided evidence that Minc03329 expressioninplantaaffected host defense-related genes expression. Altogether,our results highlight a novel effector,Minc03329,which suppresses plant basal immunity and promotesM.incognitaparasitism.

2.Materials and methods

2.1.Sequence analysis of Minc03329

Minc03329and its homologue sequences were explored fromMeloidogynegenomic resources (https://www6.inrae.fr/meloidogyne_incognita/),and we also screened sequences from WormBase (https://parasite.wormbase.org/index.html) to ensure its accuracy and reliability.Protein annotations are predicted by using the simple modular architecture research tool (http://smart.emblheidelberg.de/). The full genome sequences and gene annotation ofM.incognitawere downloaded from Wormbase (Abadet al.2008) as a reference genome.

2.2.Functional validation of Minc03329 signal peptide

The function of the predicted Minc03329 signal peptide was validated using the yeast secretion system(Jacobset al.1997). The system,based on vector pSUC2T7M13ORI (pSUC2),carries a truncated invertase gene,SUC2,lacking both the initiation Met and signal peptide. The construct pSUC2-Minc03329(primers were listed in Table 1),which was confirmed by sequencing,was transferred into the yeast SUC2-minus strain YTK12 using the Frozen-EZ Yeast Transformation IITMKit (Zymo Research,USA) following the manufacturer’s instructions.The oomycete (Phytophthorainfestans) effector Avr3a was used as a positive control,while the empty vector of pSUC2 was used as a negative control. Transformants were selected by CMD-W plates. To test whether the signal peptide was functional by yeast growth assays,single colonies were streaked onto raffinose containing YPRAA plates (Shiet al.2018).

2.3.Fluorescence in situ hybridization

Fluorescenceinsituhybridization referred to the protocol in visualization of bacteria (Sundeet al.2003). A 24-bp specific cDNA region (5′-CCAAACACGGTTCATTCC AATCCA-3′) ofMinc03329was selected for hybridization probe labeling with fluorescein isothiocyanate (Sangon Biotech,China). Firstly,the collected pre-parastic J2 nematodes were disinfected with 0.1% (w/v) benzalkonium chlorides for 1 min,and washed with 0.85% (w/v) NaCl twice for 2 min,fixed in glacial acetic acid containing 50%(v/v) for 10 min,and dehydrated them in a graded ethanol series 2 min. Secondly,the pre-parasitic J2 nematodes were transferred in buffer consisting of a 1:1 mixture of methanol and PBT for 10 min,then re-suspended them in PBT with 1% (v/v) methanol for 5 min and washed with PBT twice for 2 min. Thirdly,the nematodes were suspended in phosphate-buffered saline (PBS) containing 0.3% (v/v) Triton-100,then treated with hybridization buffer for 10 min,and then 10 μmol L-1FITC-labeled probe was applied to nematodes at room temperature for 12 h. At last,the nematodes were washed with PBS containing 0.3% (v/v) Triton-100 to remove the excess probe,and observed under a confocal microscope (Zeiss LSM700,Germany).

2.4.Subcellular localization

The open reading frame (ORF) ofMinc03329without signal peptide sequence was cloned into the pCambia1302 vector with GFP fused at the C-terminal(Table 1).Nicotianabenthamianaplants were grown in a growth chamber under standard conditions with 16 h light and 50-60% humidity. Expanded leaves of 5 to 7-week-old plants were inoculated using a needleless syringe. Bacteria were centrifuged referring to Zhaoet al.(2021). Cells were pelleted as before and resuspended in infiltration buffer to a density of OD600=0.5. The cell suspension was incubated for 3 h at room temperature and then infiltrated into theN.benthamianaleaves.Sample collection began after 2 days. The subcellular localization of theMinc03329was visualized using confocal microscope (Wanget al.2013).

Table 1 The primers used for constructing pSUC2 vector,pCambia1302 vector,pEGAD vector and pGR107 vector

2.5.In planta virus induced gene silencing (VlGS)

Minc03329andGFPfragments were cloned into theBamHI-HindIII site of the pTV00 vector. Then they were transformed intoAgrobacteriumtumefaciensstrain GV3101 respectively. The transformant was cultured at 28°C,and re-suspended to an OD600=0.5 in buffer for 5 h,mixed at a 1:1 ratio withA.tumefacienscontaining the TRV1 vector (Maoet al.2015).

The mixture was infiltrated into cotyledons of 4-weekold pepper (CapsicumannuumL.cv.Qiemen) seedlingsusing a 1-mL needleless syringe. Infiltrations were performed at six sites on each leaf with three time repeats.Mock inoculations using distilled water were performed as controls. For each experiment treatment,10 plants were inoculated with three biological replicates. Ten days after infiltration (dai),200 pre-parasiticM.incognitaJ2s were inoculated to each plant. The plants were detected to verify the invasion of TRV. At 7 days post-inoculation (dpi),about 100 parasitic J2s were collected from the root to analyzed the expression level ofMinc03329and counted the numbers of galls and egg masses were counted at 8 weeks after nematode inoculation.

2.6.RNA isolation and RT-qPCR analysis

Total RNA samples were extracted with Trizol reagent(Invitrogen,US) from pre-parasitic J2s,parasitic J2 nematodes taken from inoculated root tissue at 1 and 3 dpi,with three biological replicates for each sample.The target genes and reference genes (18S ribosomalU81578,actin-2At3G18780) were listed in Table 2.The relative expression levels of candidate genes were normalized using the 2-ΔΔCTmethod. At least three independent replications were performed.

2.7.Programmed cell death (PCD) suppression assay

The CDS ofMinc03329without signal peptide sequences andGFPwere amplified and cloned into the potato virus X(PVX) vector pGR107. The constructs of pGR107-Minc03329 and pGR107-GFP were confirmed by sequencing and then transformed intoA.tumefaciensstrain GV3101. Recombinant strains (Minc03329/BAX,1:1,v/v,OD600=0.5) were then infiltrated into leaves ofN.benthamiana(7 to 8 weeks) by pressure infiltration(Zhaoet al.2019). Symptom was monitored from 4 to 8 dai,and photographs were taken after 5 dai. TheBAX,GFP,andMinc03329genes were infiltrated on the same leaf ofN.benthamiana. Each strain was infiltrated into at least three different leaves,with three replicates on different plants. The phenotypes of PCD triggered by BAX were observed at 5 dpi.

2.8.Minc03329 ectopic expression Arabidopsis generation and transcriptome analysis

The coding sequence ofMinc03329without signal peptide was cloned into the vector pEGAD (Table 1).And then transformed into theArabidopsisthalianaecotype Columbia-0 (Col-0) using the floral dip method.The transgenic T1plants were sown on soil and were screened by spraying the herbicide BASTA (Niuet al.2016). The spraying was repeated three times. To test the susceptibility of transgenic plants,three homozygous transgenic lines (T3-1,T3-2,and T3-3) were inoculated with 200 pre-parasitic J2s,and 10 plants were inoculated for each line. At 35 dpi,the galls and egg masses were analyzed.

Table 2 The primers used for RT-qPCR

About 20 μL (100 ng μL-1) total RNA samples of transgenic and wild typeArabidopsiswere extracted.RNA-seq was performed with Illumina technology at Berry Genomics (Beijing,China),and had three independent replicates respectively. For the clean date,Tophat2 tools were used to map mRNA to theArabidopsisgenome(Langdon 2015),the expected FPKM was calculated with htseq-count (Anderset al.2015),differentially expressed genes (DEGs) of transgenic and wild typeArabidopsiswere identified with a threshold criterion of FDR＜0.05 with the R Statistical Programming Package (https://www.r-project.org). MapMan (version 3.6) were used to generate a representative overview of genes identified DEGs (Vermaet al.2018).

3.Results

3.1.Min03329 was expressed in the subventral gland and was up-regulated during parasitic stages of Meloidogyne incognita

In a recent study,we showed that there were 59 proteins containing the C-type lectin domain (CTLD) inM.incognita.Among them,Minc03329 contained a 21 amino acids signal peptide sequence for secretion at the N-terminus,and one CTLD. To determine the localization ofMinc03329expression in the nematode,fluorescenceinsituhybridization was performed on the freshly hatched pre-parasitic second-stage juveniles (J2s) nematodes usingMinc03329antisense cDNA probes. A strong fluorescence signal was observed on the subventral esophageal glands of nematodes,indicating that the FITC-labeledMinc03329probe was hybridized exclusively to transcripts within the subventral esophageal glands(Fig.1-A).

Expression profile ofMinc03329was examined by RT-qPCR using different developmental stages ofM.incognita.Resultsshowed thatMinc03329expression peak was at 2 dpi,whenM.incognitamigrated inside roots and established feeding sites.Minc03329expression was also significantly up-regulated at 5 dpi,when giant cells were forming. Subsequently,the transcript level ofMinc03329was dramatically reduced at later parasitic stages (Fig.1-B). These results suggest thatMinc03329could play an essential role in the early stages ofM.incognitaparasitism.

Fig.1 Localization and expression pattern of Minc03329 in nematode. A,FITC-labeled in situ mRNA hybridization indicated Minc03329 was expressed within the subventral esophageal gland (SvG). The scale bar is 10 μm. B,expression profile of Minc03329 in different parasitic stages of Meloidogyne incognita. The relative expression level of Minc03329 was normalized by RT-qPCR,using the 2-ΔΔCT method. J2,pre-parasitic second-stage juveniles;dpi,days post-infection. Bars were mean±SE (n=4).

3.2.Minc03329 was a secreted protein

To verify whether Minc03329 was a secreted protein,the yeast signal sequence trap assay was conducted,which the yeast cells for invertase secretion was grown on sucrose or raffinose media as the sole carbon source.The predicted signal peptide sequence ofMinc03329was fused in frame to the mature sequence of yeast invertase in the vector pSUC2. The constructs pSUC2-Minc03329and positive control pSUC2-Avr3a(+) enabled the invertase mutant yeast strain YTK12 to grow on yeast peptone raffinose antimycin Agar (YPRAA) medium,with raffinose instead of sucrose,which growth can only occur when invertase was secreted. In contrast,the negative control pSUC2 empty vector yeast strains did not grow on YPRAA (Fig.2). These results indicated that the signal peptide of Minc03329 was functional,suggesting Minc03329 effector was likely secreted fromM.incognita.

Fig.2 Functional validation of the signal peptide of Minc03329 was performed using the yeast signal sequence trap assay.Yeast YTK12 strain carrying the Minc03329 signal peptide fragment fused in frame to the invertase gene in the pSUC2 vector are able to grow in both the CMD-W,YPDA and YPRAA media. The pSUC2-Avr3a(+) was a positive control,and the pSUC2-empty(-) was a negative control.

3.3.Minc03329 was involved in M.incognita pathogenicity

To investigate the role ofMinc03329inM.incognitaparasitism,virus-induced gene silencing (VIGS)experiment was performed on pepper (C.annuumL.cv.Qiemen) to silenceMinc03329expression of feeding nematodes. For the VIGS test,the qPCR results showed that the expression levels ofMinc03329were significantly decreased in the parasitic second-stage juveniles (par-J2s) of plant infiltrated with pTV-Minc03329compared with the control plants at 1 and 3 dpi (Fig.3-A). Eight weeks after nematodes inoculation,the average number of galls and egg masses was significantly reduced(P＜0.001) in the VIGS-treated plants (pTV:Minc03329),as 8.2±0.8 galls and 3.3±0.5 egg masses per plant,but 40.3±1.6 galls and 30.7±1.5 egg masses for pTV00 empty vector treatments,41.4±1.9 galls and 31.2±2.2 egg masses for mock controls (Fig.3-B). These results indicated that the silencing ofMinc03329expression affects the pathogenicity ofM.incognitato plant hosts.

To further determine the function ofMinc03329on plant phenotypes,three independent homozygousArabidopsislines expressingMinc03329were generated (T3-1,T3-2,and T3-3),which driven by the CaMV35S promoter.RT-PCR was conducted to confirm the expression ofMinc03329in all transgenic lines,and the qPCR results showed that the expression folds ofMinc03329were significantly increased in the transgenic plants (4.7 to 5.6 folds),while there was no expression in wild type control(Fig.3-C). These threeMinc03329transgenic lines were subjected to inoculate withM.incognitato test the susceptibility. After 35 days,the average galls of three transgenic lines T3-1,T3-2,and T3-3 were 24.1±1.4,24.5±1.6,and 25.6±1.6 per plant,respectively. Compared with the wild type (17.9±1.0),there was a significant increase in the number of galls (P＜0.05) (Fig.3-D). These results indicated that the ectopic expression ofMinc03329inArabidopsiscould increase the pathogenicity ofM.incognita. Taken together,nematodes infection test results showed that Minc03329 was a virulence factor duringM.incognitaparasitism.

Fig.3 Minc03329 is involved in Meloidogyne incognita parasitism. A,the effect of virus-induced gene silencing (VIGS) of Minc03329 in pepper (Capsicum annuum L.cv.Qiemen). Changes in transcript abundance of Minc03329 expression levels after VIGS treatment. B,VIGS treatment of MiCTL1 in pepper reduced the pathogenicity of M.incognita. The number of galls and egg masses in each plant was counted at 50 day post-infection (dpi). Data are presented as mean±SD (n≥15). C,RT-qPCR analysis of Minc03329 expression in transgenic Arabidopsis lines. Actin-2 (At3G18780) was used as internal control. Data shown are mean±SD (n=4). WT,wild-type Arabidopsis (Col-0);T3-1,T3-2,and T3-3,three independent Minc03329 transgenic Arabidopsis lines,respectively. D,Minc03329 expression in Arabidopsis increased susceptibility to M.incognita. The number of galls and egg masses was counted at 35 dpi. Data are presented as mean±SD (n＞20). The data were analyzed using one-way ANOVA following Dunnett’s post hoc test (＊,P＜0.05;＊＊＊,P＜0.001) using GraphPad Software.

3.4.Minc03329 was localized in the plant cell and suppressed BAX-induced cell death

Previous evidence suggested that effectors of plant-parasitic nematodes targeted various cellular compartments to manipulate plant functions and promote infection. To investigate the subcellular localization of Minc03329 in plant cells,the Minc03329 fragment without signal peptide sequence was fused withGFPgene,and was transiently expressed inN.benthamianaleaf cells.The results showed that Minc03329 was mainly localized in the cell membrane and cytoplasm (Fig.4-A).

As the expression ofMinc03329inArabidopsisenhanced susceptibility toM.incognitainfection,we investigated whetherMinc03329could affect plant immune responses. The pro-apoptotic mouse protein BAX was widely used in triggering programmed cell death (PCD)inN.benthamiana. PVX expression system,previously used as an initial screen for pathogen candidate effectors capable of suppressing defense-associated PCD. In this study,infiltration withA.tumefacienscarryingMinc03329did not induce necrosis,nor did the negative control,strain carrying an enhanced green fluorescent protein (eGFP),while the leaf tissues infiltrated with aBAX-carrying strain exhibited complete necrosis. Further,BAX-induced PCD was completely suppressed when the leaves were coinfiltrated with Minc03329,whereas necrosis was not inhibited at the co-expression ofBAXandeGFP(Fig.4-B).These results indicated that Minc03329 could suppress BAX-induced cell death.

Fig.4 Subcellular localization of Minc03329 in plant cell and suppression of BAX-triggered cell death. A,Agrobacterium cells carrying the 35S-Minc03329-GFP and 35S-GFP recombinant vectors were transiently expressed in Nicotiana benthamiana cells. GFP signals were observed at 2 days after infiltration(dai) using confocal microscope. Scale bars,50 μm. B,the typical symptoms of N.benthamiana leaves infiltrated with Agrobacterium tumefaciens cells carrying the gene Minc03329,BAX and eGFP,respectively. Symptoms of the leaf infiltrated with the A.tumefaciens mixture of Minc03329 and BAX. The cell death phenotypes were observed at 5 dpi (7 plants and infiltrate 3 leaves of each plant,n=21).

3.5.Minc03329 modulated expression of plant genes involved in immune responses

We showed thatMinc03329was involved inM.incognitaparasitism and played roles in suppressing BAX-induced cell death,but the working mechanism of Minc03329 effector was unclear. To further analyze the potential roles of Minc03329 effector in modulating gene expression and signaling pathways,RNA-seq was performed using wild typeArabidopsisandMinc03329transgenic lines.The comparing transcriptomes analysis showed that 256 differentially expressed genes (DEGs) were identified inMinc03329ectopic expressing lines,among them 112 were upregulated and 144 were downregulated (Appendix A). The functional enrichment analysis of DEGs was performed by MapMan analysis,of which 253 genes have function annotations,and the results of pathway enrichment analysis indicate that these DEGs were mainly classified into 9 categories,including regulation,biotic stress,secondary metabolism,receptor like kinases,cell function,and cellar response. The functional classes of DEGs were found to be significantly enriched in regulation(78 genes) and biotic stress (77 genes). The regulation dataset involved in transcription factor (41 genes),protein modification and degradation (14 genes),IAA(11 genes),and receptor kinase (4 genes). Interestingly,the biotic stress dataset showed a compound regulation relation,including signal (23 genes),cell wall (17 genes),transcription factors (14 genes),abiotic stress (6 genes),defense and secondary metabolites (Appendix B).

In the “plant-pathogen interaction” pathway analysis,the significant enrichment of DEGs was clustered in signaling and transcription factors. And Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis showed that the salicylic acid (SA) pathway and defense responses were significantly suppressed,which genes involved in the biotic stress signaling (At1G33960),in the salicylic acid hormone signaling (At5G55250),MAPK (At5G11620),peroxidases (At1G44970),redox(At3G09940),PR protein (At5G44420,At2G26010,andAt3G59930),leucine-rich repeat transmembrane protein kinase (At1G66830),protein kinase-related (At3G46280),and receptor serine/threonine kinase (At4G18250),MYB domain transcription factor (At3G04030) and bZIP transcription factor (At1G13600) were significantly downregulated. Interestingly,some genes might involve in nematode feeding sites development were changed,such as the auxin-responsive family protein (At3G09870andAt3G25880) and cell wall proteins (At1G62440andAt3G59190). In addition,some secondary metabolism pathway were changed,which genes involved in the phenylpropanoids (At1G62940) and flavonoids(At4G34850andAt3G55970) were down-regulated.Further,the expression patterns of these genes were verified by qPCR,and the results are similar to the transcriptome analysis (Fig.5). These results suggested thatMinc03329-transgenic plants regulated immune responses by modulating SA signaling pathway. Based on the transcriptome data and experiment results,the functions of Minc03329 effector expressinginplantawere proposed by interplaying with potential DEGs involving in modulating host defense responses (Fig.6).

Fig.5 RT-qPCR verification of candidate differentially expressed genes (DEGs) between wild-type and Minc03329 transgenic Arabidopsis lines. Relative expression ratio of DEGs was normalized using Actin-2 (At3G18780) as the internal control and the data shown are mean±SD (n=4). The experiments were performed twice.

Fig.6 The putative functions involved in biotic stress pathways mediated by expressing Minc03329 effector in plants. The color bars represent the expression level (log2FC) and number of differentially expressed genes (DEGs). The words in the box showed the rectangle or ellipse function categories of DEGs. The arrows indicate the physical interaction among different function categories.

4.Discussion

Root-knot nematodes (RKNs) have complex molecular dialogues with their host. Evidence confirmed that RKNs secrete effectors to interfere with plant immune responses,promoting nematodes parasitism (Bellafiore and Briggs 2010). The C-type lectin domain like (CTLD)effector proteins have been found abundantly in parasitic nematodes and free living nematodes (Brownet al.2007;Schulenburget al.2008). InCaenorhabditiselegans,278 CTL genes were found,among them 61 genes were induced expression by pathogens,and 45 of which had a signal sequence. These secreted pathogeninduced CTLD proteins contributed to pathogen-specific antimicrobial activity. RKNs establish a biotrophic parasite relationship with plant hosts,which needs to fine-tune plant immunity (Eves-Van Den Akker 2021).The majority of signal pathways involved in innate immune responses inMeloidogyneare similar toC.elegans,suggesting that the underlying mechanisms of nematode-pathogen interactions are highly conserved(Davies and Curtis 2011). However,unlike free-living nematodes,as sedentary endoparasites,RKNs spend majority of their life cycles inside plant root tissues. As a consequence,the RKNs may not be exposed to the diversity of microbes,making many of the antibacterial and antifungalCTLgenes unnecessary (Liet al.2015).Based on transcriptomic analysis,only 11CTLgenes ofRotylenchulusreniformiswere found (Ganjiet al.2014).Moreover,manyCTLgenes were found in clusters within the genome,and showed closely phylogenetic relationships. In this study,a typical CTLD effector ofM.incognita,Minc03329,was characterized,which was a homologue of Mg01965.

The secretion of RKNs effectors supports their functional characterization,insituhybridization showed thatMinc03329was expressed specifically within the subventral glands. Furthermore,with the validation of the signal peptide activity,it was confirmed thatMinc03329encoded a secretory protein. Previous report showed that RKNs effectors targeted different subcellular compartments when secreted into plant cells(Jaouannetet al.2013). Here,we showed that transient expression ofMinc03329inN.benthamianaleaves was located in whole cell. It is possible that Minc03329 has an interaction with host cells to its function as a putative effector protein.

There are two layers of plant immunity,which are pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). PTI is initiated by plant cell surfacelocalized pattern-recognition receptors (PRRs),which leads to a number of downstream reactions,such as callose deposition,reactive oxygen species (ROS) burst,intracellular and extracellular calcium flux,transcriptional reprograming of defense-related genes and phytohormone signaling (Ngouet al.2021;Yuanet al.2021). Recent advances in functional analysis of effectors showed that some RKNs effectors recruited plant targeting proteins to interfere with PTI immune responses,such as Mi-CRT (Jaouannetet al.2013),MjTTL5 (Linet al.2016),MiMIF-2 (Zhaoet al.2019),and MiPDI1 (Zhaoet al.2020). While a few RKNs effectors and peptides not only modulated plant immune responses,but also involved in giant cell development,such as MiRALFs (Zhanget al.2020) and MiEFF18 (Mejiaset al.2021). Also,some effectors could promote RKNs parasitism by directly suppressing plant defenses PTI reactions and/or ETI reactions,such as MeTCTP (Zhuoet al.2017),MgGPP(Chenet al.2017),Mg16820 (Naaldenet al.2018),and

Mj2G02 (Songet al.2021). Although RKNs effectors involved different regulation mechanisms,plant immunity eventually converged to be destructed,albeit with distinct strength and amplitudes. In this study,we verified that the nematode secretory protein genes (Minc03329),encoding a C-type lectin protein,belong to the CTL superfamily,homologous withAncylostomaceylanicumhypothetical protein ANCCEY-13254 (EPB67657.1) andM.graminicolaeffector protein Mg01965 (Haegemanet al.2013;Zhuoet al.2019). CTLD proteins are an important component of the innate immune system that are intimately involved in mediating the host-parasite interaction (Rozeet al.2007). CTLs were also related to overcoming host defenses,as reported in animaland plant-parasitic nematodes (Urwinet al.2002;Zhuoet al.2019). The C-type lectin (AAO33474) found inHeteroderaavenaewas putatively involved in overcoming host defenses (Zhenget al.2015). Here,we showed that Minc03329 could suppress programmed cell death triggered by BAX inN.benthamiana,suggesting potential roles of Minc03329 in modulating host immune responses duringM.incognitaparasitism.

The VIGS test revealed that knockdown ofMinc03329expression significantly reduced the infection rate of nematodes. Ectopic expression ofMinc03329increased the susceptibility ofA.thalianatoM.incognita. Salicylic acid (SA),jasmonic acid (JA) and ethylene signal transduction were important layers in induced defense of the plant against pathogens (Gutjahr and Paszkowski 2009). Some nematode effectors such asH.glycineseffector 10A06 andM.incognitaeffector MiISE6 are relative with SA-and JA-induced defense (Heweziet al.2010).RAP2.3is an AP2/EREBP family gene induced by ethylene,regulates plant defense response to the nematode as a mean to avoid plant defenses (Hermsmeieret al.2000). Based on the transcriptome data from theMinc03329-transgenicArabidopsis,SA signaling pathway was suppressed,and the ethylene signaling pathway was changed. In addition,some transcript factors such as bZIP transcription factor,MYB-related transcription factor were down-regulated,which played essential roles in plant defense responses (Osorioet al.2012;Wieczoreket al.2014). Feeding sites formation of PPNs is the key process for successful parasitism,PPNs can manipulate plant hormone to facilitate successful invasion or establish feeding cells,which involve in auxin transport,plant cell differentiation,and cell modification(Leeet al.2011). Auxin is known to participate in cell expansion and cell wall breakdown,and auxin induction is a trigger for root gall formation caused by RKNs and associates with the activation of the flavonoid pathway(Hutanguraet al.1999). InMinc03329-transgenic plants,the auxin-responsive family protein genes,secondary metabolize flavonoid pathway genes were significantly changed,indicating the development of root cells were altered. Meanwhile,the expression levels of some auxinresponsive genes (At5G20820,At3G03820,At5G18020,andAt5G18030) were suppressed,and some cell wall proteins,such as pectinesterase,extensin (At2G45220andAt1G76930) were also down-regulated,these genes were reported beneficial to nematode parasitism(Cosgrove 2000). Taken together,our results speculated that Minc03329 effector could regulate the SA signaling pathway to suppress host defenses,and might involve in the development of RKNs feeding sites.

5.Conclusion

Minc03329 exhibited typical characteristics of a secreted protein,which was expressed in the subventral glands,up-regulated during nematode parasitism stages,and suppressed programmed cell death.Inplanta,the silencing ofMinc03329decreased pathogenicity ofM.incognita. Minc03329 expression inArabidopsisregulated the plant defense by modulating SA signaling pathway and promotedM.incognitaparasitism. The functional characterization of Minc03329 effector provides essential experimental results to understand the molecular mechanism ofM.incognitain overcoming host resistance. Plant-parasitic nematodes can regulate the host immune mechanism,which not only helps to deepen the basic understanding of the interaction between plants and nematodes,but also provides new methods and theoretical guidance for plant immunity against nematode invasion and even disease-resistant breeding.

Acknowledgements

This work was funded by the National Natural Science Foundation of China (31672010 and 31871942),the Beijing Natural Science Foundation,China (6222054),the China Agriculture Research System (CARS-23),and the Natural Science Foundation of Inner Mongolia Autonomous Region of China (2018MS03083).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on http://www.ChinaAgriSci.com/V2/En/appendix.htm