Root‐knot nematodes exploit the catalase‐like effector to manipulate plant reactive oxygen species levels by directly degrading H2O2

Abstract Plants produce reactive oxygen species (ROS) upon infection, which typically trigger defence mechanisms and impede pathogen proliferation. Root‐knot nematodes (RKNs, Meloidogyne spp.) represent highly detrimental pathogens capable of parasitizing a broad spectrum of crops, resulting in substantial annual agricultural losses. The involvement of ROS in RKN parasitism is well acknowledged. In this study, we identified a novel effector from Meloidogyne incognita, named CATLe, that contains a conserved catalase domain, exhibiting potential functions in regulating host ROS levels. Phylogenetic analysis revealed that CATLe is conserved across RKNs. Temporal and spatial expression assays showed that the CATLe gene was specifically up‐regulated at the early infection stages and accumulated in the subventral oesophageal gland cells of M. incognita. Immunolocalization demonstrated that CATLe was secreted into the giant cells of the host plant during M. incognita parasitism. Transient expression of CATLe significantly dampened the flg22‐induced ROS production in Nicotiana benthamiana. In planta assays confirmed that M. incognita can exploit CATLe to manipulate host ROS levels by directly degrading H2O2. Additionally, interfering with expression of the CATLe gene through double‐stranded RNA soaking and host‐induced gene silencing significantly attenuated M. incognita parasitism, highlighting the important role of CATLe. Taken together, our results suggest that RKNs can directly degrade ROS products using a functional catalase, thereby manipulating host ROS levels and facilitating parasitism.

The infective second-stage juveniles (J2s) of RKNs use a hollow protrusive stylet to penetrate the plant root elongation zone and migrate intercellularly toward the vascular cylinder, where they establish feeding sites and become sedentary (Abad et al., 2008;Nguyen et al., 2018).The feeding sites, composed of five to seven large multinucleate cells known as giant cells, serve as the primary nutrient pool for RKN growth and reproduction (Favery et al., 2020).The establishment of feeding sites, coupled with the proliferation of plant tissue surrounding the giant cells, results in the formation of knot-like galls, impeding the transport of water and nutrients through the plant roots and ultimately leading to yield losses (Siddique & Grundler, 2018).
The oxidative burst, a hallmark of plant innate immunity characterized by the rapid, massive and transient production of reactive oxygen species (ROS) including H 2 O 2 and O 2 − , plays important roles in plant's resistance against pathogens (Ali et al., 2018;Langebartels et al., 2002;Wu & Ge, 2004;Zhou et al., 2018).ROS can cause DNA oxidative damage, trigger a hypersensitive response (HR) to block pathogen colonization and serve as signalling molecules in plant defence cascades (Wang et al., 2020).Notably, ROS have been identified to play important roles in plant resistance against RKNs; for instance, the induction of increased ROS accumulation in rice resulted in reduced infection by the RKN Meloidogyne graminicola (Chavan et al., 2022;Holbein et al., 2016).To withstand the host-derived ROS stress, RKNs have evolved sophisticated mechanisms to manipulate the ROS metabolism of host plants (Rutter et al., 2022).Previous research has confirmed that using effectors to suppress the host ROS production is an important strategy employed by RKNs.For instance, the effector Mg16820 from M. graminicola can interact with dehydration stress-inducible protein 1 of plants to suppress ROS production (Naalden et al., 2018), while the effector MiPDCD6 from Meloidogyne incognita can inhibit ROS accumulation by suppressing salicylic acid-mediated plant immunity (Kamaruzzaman et al., 2023).
Additionally, evidence indicates that the host ROS scavenging systems are important targets of RKNs.For example, the effector MgMO289 from M. graminicola can interact with rice copper metallochaperone heavy metal-associated plant protein 04 (OsHPP04) to activate the host O 2 •− -scavenging system (Song et al., 2021).Similarly, the effector MjTTL5 from Meloidogyne javanica can enhance plant ROS scavenging activities through interaction with ferredoxin of Arabidopsis (Lin et al., 2016).In addition, ROS-scavenging enzymes, such as peroxiredoxins in M. incognita, are localized within the nematode's hypodermis, suggesting a role in protecting the nematode's body against ROS (Dubreuil et al., 2011)

| Identification of the M. incognita catalase-like effector
In this study, we identified a protein named CATLe (Mi_08123.1)from the M. incognita genome (Genome_assembly_V1) (Dai et al., 2023) that contains a secretory signal peptide and a catalase domain, consists of 560 amino acids and has a calculated molecular weight of 61,803 Da (Figure 1a).Catalase is a core antioxidant enzyme in most organisms; it catalyses the decomposition of H 2 O 2 into water and molecular oxygen (Baker et al., 2023).Notably, H 2 O 2 is a major component of ROS, which inhibit pathogen infection and trigger local and systemic defence responses (Dutta et al., 2023).Thus, we speculated that CATLe may exhibit potential functions in regulating host ROS levels during M. incognita parasitism.
To  1c).These results suggest that CATLe is conserved among RKNs and may play an important role in the early stages of nematode parasitism.

| CATLe is expressed in the subventral oesophageal gland and can be secreted into giant cells
As the effectors of plant-parasitic nematodes are mainly secreted by subventral and the dorsal oesophageal gland cells, we performed in situ hybridization to investigate the localization of CATLe transcripts in M. incognita preparasitic J2s.A strong signal was detected in subventral oesophageal gland (SvG) cells with the digoxigenin-labelled CATLe probe (Figure 2a), while no signal was observed in preparasitic J2s with negative controls (Figure 2b).Additionally, we generated an antibody to CATLe to investigate the immunolocalization of CATLe in preparasitic J2s.The specificity of the antibody was confirmed through western blot analysis, demonstrating a clear band of the anticipated size in the total proteins extracted from preparasitic J2s (Figure S2).
As expected, immunolocalization showed that the CATLe was present in the SvG of preparasitic J2s (Figure 2c,d), which is consistent with the in situ hybridization result.These results suggest that CATLe could potentially act as an effector of M. incognita.
To examine whether CATLe is secreted into host plants during M.
incognita parasitism, the immunolocalization was performed on tomato gall sections.Western blot analysis confirmed the specificity of the antibody, as no signal was observed in samples from uninfected tomato plants (Figure S2).Immunohistochemistry conducted on sections of tomato root galls at 5 days post-inoculation (dpi) revealed the presence of CATLe within the giant cells (Figure 2e,f).As anticipated, no signal was detected in planta when employing pre-immune serum (Figure S3).These results confirm that CATLe is an effector that can be secreted into plant cells and is probably functional during M. incognita parasitism.

| CATLe exhibits catalase activity and can dampen plant ROS production
To examine whether CATLe is a functional catalase that can catalyse the decomposition of H 2 O 2 , the CATLe-coding sequence without the signal peptide was cloned and expressed in Escherichia coli Rosetta (DE3).Enzyme activity assays for purified CATLe demonstrated that CATLe had an enzyme activity of about 42,500 U/mg (Figure 3a).To examine its catalytic activity in vivo, CATLe (without signal peptide) was transiently expressed in Nicotiana benthamiana leaves.Subsequently, we detected the ROS production in N. benthamiana leaf discs under flg22 stimulation.A comparison with the negative control showed that ROS production was significantly decreased in N. benthamiana leaf discs under the expression of CATLe (Figure 3b).These results suggest that CATLe can efficiently dampen the flg22-induced ROS production.

| CATLe targets plant ROS products by directly degrading H 2 O 2
To | 5 of 11 increase in H 2 O 2 levels in tomato seed radicles infected by CATLesilenced nematodes compared to the negative control (Figure 4b).
These results confirm that M. incognita employs CATLe to directly degrade plant H 2 O 2 .

| CATLe silencing affects M. incognita parasitism
To investigate the role of CATLe in M. incognita parasitism, we inoculated the dsRNA (CATLe/GFP)-treated nematodes onto 4-week-old tomato seedlings and counted the galls at 20 dpi.We found that silencing the CATLe gene led to a significant reduction in both the

| DISCUSS ION
As a principal ROS component, H 2 O 2 plays a critical role in counteracting pathogen infection by inducing plant-programmed cell death (Zhang et al., 2014).Moreover, H 2 O 2 can also mediate downstream immune signalling in plants: some key phytohormones involved in the regulation of plant stress response, including salicylic acid, jasmonates, abscisic acid and ethylene, employ H 2 O 2 in their signalling cascades in an either upstream or downstream manner (Saxena et al., 2016).In addition, H 2 O 2 is relatively stable and can diffuse among cellular compartments or cells, which facilitates its signalling functions (Bienert et al., 2007;Henzler & Steudle, 2000).Therefore, increasing attention has been paid to the role of H 2 O 2 in the interaction between pathogens and plants.For instance, it has been identified to play a critical role in the susceptibility of Brassica napus to Leptosphaeria maculans (Nováková et al., 2016).PsCAT1, a monofunctional heme-containing catalase secreted by Puccinia striiformis, acts as an important pathogenic factor to facilitate the infection of P. striiformis by scavenging host-derived H 2 O 2 (Yuan et al., 2021).
Similarly, Blumeria graminis can also secrete extracellular catalase to clear H 2 O 2 during infection of barley (Zhang et al., 2004).
In this study, through an investigation into effectors modulating plant ROS levels during the initial infection stages of M. incognita, we discovered a novel effector, CATLe, that exhibits catalase activity and is specifically up-regulated during the early infection stages (Figures 1c and 3a).In vitro and in planta assays confirmed the critical roles of CATLe in suppressing the plant ROS burst and regulating plant H 2 O 2 levels (Figures 3b and 4b).Interestingly, recent research identified a C-type lectin (CTL)-like effector from M.
incognita, named MiCTL1a, that has been shown to interact with the catalase of plants, inhibiting its activity (Zhao et al., 2021).Through expression pattern analysis and RT-qPCR validation, we showed that CATLe and MiCTL1a were simultaneously up-regulated during early infection stages (Figure 1c; Figure S4).Therefore, based on these as cell differentiation and proliferation (Holmström & Finkel, 2014;Huang et al., 2019).The H 2 O 2 levels have also been demonstrated to play important roles in meristem enlargement, cell expansion and cell wall extensibility (Liszkay et al., 2004;Tsukagoshi et al., 2010).
More importantly, previous research has indicated that H 2 O 2 is also accumulated in the cell wall of giant cells in feeding sites induced by RKNs (Melillo et al., 2006).The formation of feeding sites induced by RKNs involves complex manipulation of host signal transduction to activate the redifferentiation of root cells into hypertrophied and multinucleate giant cells (Medina et al., 2017;Mejias et al., 2021;Noureddine et al., 2022).In this study, silencing the CATLe of M.
incognita not only altered the plant H 2 O 2 levels but also resulted in a smaller gall size induced by M. incognita (Figures 4b and 5a).These results suggest that the regulation of plant H 2 O 2 levels is not only important for M. incognita to resist host-derived ROS stress but also for it to induce the giant cells.Together, our research sheds light on the molecular mechanisms in regulating ROS levels employed by M.
incognita and highlights the critical role of effector CATLe in promoting parasitism.
N. benthamiana was used to perform the ROS burst detection assay.
Nicotiana tabacum was used to generate the transgenic plants.

| RNA extraction and quantitative real-time PCR
The total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer's instructions.Reverse transcription was per- (Vazyme).The expression of genes was determined using the Hieff UNICON Universal Blue qPCR SYBR Green Master Mix (YEASEN) by RT-qPCR.The relative changes in gene expression were determined using the 2 −ΔΔCt method.

| In situ hybridization and immunolocalization of CATLe
With a simple modification of the previously described method (Jaouannet et al., 2018), the digoxigenin (DIG)-labelled specific probe was amplified with specific primers (Table S1).In situ hybridization (ISH) was performed on fresh-hatched preparasitic J2s of M.
incognita using the MyLab DIG Labeling and Hybridization Detection System-DIG DNA PCR Labeling Kit (MyLab Corp.) according to the description of the manufacturer.Briefly, the nematodes were fixed with 4% formaldehyde for 24 h and chopped on a glass slide.
Proteinase K (1 mg/mL) was used to digest the chopped nematodes for 2 h.The nematode was hybridized with a DIG-labelled specific probe at 40°C for 16 h and then the hybridization signal was detected with anti-DIG antibodies and observed under a microscope.
The polyclonal antibody against CATLe was produced by ABclonal Biotech Co. Ltd against a specific peptide (C-AEYWNELSPVDRQH).
Nematodes incubated with pre-immune serum served as a negative control.Results were imaged using confocal microscopy (FV1000; Olympus).To validate the accumulation of CATLe within the giant cells, the root galls were collected at 7 dpi and after paraffin embedding and sectioning, the paraffin was removed using a dewaxing solution (G1128; Wuhan Servicebio Technology).The gall sections were treated with ethanol solutions and incubated in a blocking solution of 1% bovine serum albumen for 30 min.Subsequently, the gall sections were incubated with anti-CATLe antibody (1:200) at 4°C overnight.
Then, after shaking and washing three times on a shaker for 5 min each time to destain them with phosphate-buffered saline (pH 7.4), the gall sections were treated with the Alexa Fluor 488 fluorescenceconjugated goat anti-rabbit antibody (1:400).Finally, shaking and washing three times again, using 4′,6-diamidino-2-phenylindole (DAPI) (Wuhan Servicebio Technology) to stain the cell nuclei and imaged using confocal microscopy.The gall sections incubated with preimmune serum served as a negative control.

| Heterologous expression of CATLe and enzyme activity measurement
The CATLe-coding sequence without signal peptide (Mi_08123.1,Mi_assembly_v1) was cloned and inserted into the prokaryotic expression vector pET-15D.The recombined vector was then transformed into the expression strain E. coli Rosetta (DE3).
Protein production was induced by adding 0.2 mM isopropylβd- thiogalactopyranoside (IPTG) to the cell culture.After 16 h of expression at 16°C, the His-CATLe protein was purified using Ni-agarose column (CWBIO) according to the description of the manufacturer.
After purification, the eluted protein was concentrated and desalinized by a 30-kDa cut-off Centricon filter (Millipore).
To assess the enzyme activity of CATLe, the protein concentration of CATLe was measured firstly using the BCA Protein Assay Kit (Coolaber) following the instructions of the manufacturer.Then, the enzyme activity of CATLe was measured following the previous descriptions (Zhao et al., 2021) using the Catalase Activity Assay Kit (Beyotime).A catalase activity assay on the purified protein from the same expression strain transformed with the recombinant vector and the empty vector was performed to exclude the possibility of catalase originating from bacteria.

| ROS burst detection
The CATLe-coding sequence without signal peptide (Mi_08123.1,Mi_assembly_v1) was amplified and inserted into the pCAMBIA1300s vector to generate a recombinant vector and then transformed into

| CATLe silencing and measurement of plant H 2 O 2 content
The region (CDS 683-891bp , 208 bp) of CATLe-coding sequence (Mi_08123.1,Mi_assembly_v1) was selected as the interference target to knock down the expression of CATLe using dsRNA soaking for preparasitic J2s.Non-target green fluorescent protein coding sequence (gfp, 287 bp) was used as the negative control.The dsRNA was synthesized using T7 RNAi transcription kit (Vazyme) using specific primers (Table S1) and following the instructions of the manufacturer.A final concentration of dsRNA exceeding 1 μg/ μL was obtained.RNAi was performed by soaking about 20,000 preparasitic J2s in 250 μL RNase-free water containing 1 μg/μL dsRNA (CATLe/GFP) and 1% resorcinol.Three biological replicates were performed.After 24 h of soaking at 25°C, the soaking solution was removed and the dsRNA-treated preparasitic J2s was

F
Characterization of the Meloidogyne incognita catalase-like effector.(a) Primary structure of CATLe.(b) Phylogenetic tree of proteins homologous to CATLe based on the maximum-likelihood method.The grey shapes represent the collapsed tree branches.(c) Reverse transcription-quantitative PCR validation of relative expression level of CATLe in M. incognita at different infection stages.The foldchange values were analysed by the 2 −ΔΔCt method.The GAPDH gene of M. incognita was used as a reference.***p < 0.001, Student's t test.Bars represent mean ± SD.
further confirm the role of CATLe in regulating host H 2 O 2 levels, we silenced the expression of the CATLe gene in M. incognita preparasitic J2s through soaking with double-stranded (ds) RNA.A 208 bp fragment of the CATLe-coding sequence was selected as the interference target and the non-target green fluorescent protein gene (gfp, 287 bp) was used as the negative control.After soaking M. incognita preparasitic J2s in dsRNA (CATLe/GFP), RT-qPCR analysis showed a significant decrease in CATLe gene expression compared to the negative control, indicating effective silencing (Figure 4a).Subsequently, we inoculated the dsRNA (CATLe/GFP)-treated nematodes onto tomato seed radicles.At 24 hpi, we measured the H 2 O 2 levels in the infected radicles.Our results revealed a significant F I G U R E 2 Localization of CATLe.(a) In situ hybridization (ISH) of digoxigenin-labelled antisense CATLe probe to preparasitic Meloidogyne incognita second-stage juveniles (J2s) indicates the transcription of CATLe in the subventral oesophageal gland (SvG).Bar = 20 μm.(b) No ISH signal was observed in the CATLe sense probe (right).Bar = 20 μm.(c-e) Immunolocalization with the anti-CATLe antibody showed that the CATLe protein was present in the SvG of preparasitic M. incognita J2s.Bar = 20 μm.(e, f) Immunolocalization of the secreted CATLe protein in tomato tissues.CATLe accumulated in the giant cell.The images are shown by merging the differential interference contrast (DIC), DAPIstained nuclei and Alexa Fluor 488 fluorescence.N, nematodes; m, metacorpus; *, giant cells, bar = 20 μm.
number and size of galls induced by M. incognita infection compared to the negative control (Figure5a,b).Additionally, we explored the role of CATLe in M. incognita parasitism using host-induced gene silencing (HIGS).Transgenic Nicotiana tabacum plants expressing CATLe hairpin dsRNA were generated, with transgenic plants expressing GFP hairpin dsRNA serving as negative controls.After inoculating transgenic plants with nematodes, RT-qPCR analysis at 5 dpi showed a significant decrease in CATLe expression in M. incognita compared to negative controls, indicating effective silencing of CATLe (Figure5c).Moreover, there was a notable decrease in the number of galls induced by M. incognita (at 20 dpi) in the transgenic plants (Figure5d).Overall, these results indicate that CATLe plays a crucial role in facilitating M. incognita parasitism.F I G U R E 3 CATLe exhibits catalase activity and suppresses reactive oxygen species (ROS) burst.(a) Catalase activity assay for purified CATLe.The Escherichia coli expression strain transformed with the empty vector served as a negative control.Bars represent mean ± SD.(b) CATLe suppressed the flg22-induced ROS production in Nicotiana benthamiana leaves.Agrobacterium tumefaciens GV3101 carrying CATLecoding sequence and red fluorescent protein (mCherry) was infiltrated into 4-week-old leaves of N. benthamiana.Leaf discs (diameter 4 mm) were collected and incubated with 100 nM flg22 after 48 h infiltration to perform the ROS assay.The curve was drawn by the average values of the relative luminescence unit (RLU).Bars represent mean ± SD.Control represents N. benthamiana with mCherry infiltration.F I G U R E 4 CATLe targets plant reactive oxygen species products by directly degrading H 2 O 2 .(a) Interference efficiency detection of CATLe gene by reverse transcription-quantitative PCR.The fold-change values were analysed by the 2 −ΔΔCt method.The GAPDH gene of Meloidogyne incognita was used as a reference, ***p < 0.001.(b) CATLe silencing leads to an increased in host H 2 O 2 accumulation.The H 2 O 2 content in tomato seed radicles was measured at 24 h post-inoculation, ***p < 0.001.

F
CATLe silencing affects Meloidogyne incognita parasitism.(a) CATLe silencing leads to a decrease in gall size induced by M. incognita infection.(b) CATLe silencing leads to a decrease in gall number induced by M. incognita infection.Four-week-old tomato seedlings were inoculated with 300 dsRNA (GFP/CATLe)-treated preparasitic J2s and galls were counted at 20 days post-inoculation (dpi).(c) Expression level of CATLe gene detected by reverse transcription-quantitative PCR (RT-qPCR).Transgenic Nicotiana benthamiana lines expressing hairpin dsRNA (GFP/CATLe) were inoculated with 2000 preparasitic J2s.The infected roots were collected at 5 dpi and the RNA was extracted for RT-qPCR.(d) Host-induced gene silencing (HIGS) for CATLe decreased the gall number induced by M. incognita infection in N. benthamiana.The transgenic lines expressing hairpin dsRNA (GFP/CATLe) were inoculated with 200 fresh-hatched preparasitic J2s.The galls were counted at 20 dpi.**p < 0.01, ***p < 0.001, Student's t test.Bars represent mean ± SD.
formed using HiScript III All-in-one RT SuperMix Perfect for qPCR F I G U R E 6 Schematic illustration depicts how Meloidogyne incognita uses effectors to manipulate the plant H 2 O 2 levels during early infection.M. incognita secretes MiCTL1a to interact with plant catalase, suppressing its activity and preventing H 2 O 2 decomposition by plants.M. incognita also secretes CATLe to catalyse H 2 O 2 decomposition, potentially enabling nematodes to autonomously regulate the H 2 O 2 levels of plants.
Agrobacterium tumefaciens GV3101.The pCAMBIA1300s vector with mCherry was used as the control.The leaves of 4-week-old N. benthamiana were infiltrated with recombinant A. tumefaciens GV3101.The leaf discs (diameter 4 mm) expressing CATLe and mCherry were collected after 48 h of infiltration and incubated with 200 μL sterile water in 96-well plates in the dark for 16 h.The ROS production of the leaf discs was measured in 100 μL detection buffer (34 μg luminol, 20 μg horseradish peroxidase and 100 nM flg22) using a microplate reader (Victor NIVO 3S; PerkinElmer).All experiments were performed three times and 16 leaf discs were analysed per treatment.