Advances in Migratory Plant Endoparasitic Nematode Effectors

Unlike sedentary plant-parasitic nematodes, migratory plant endoparasitic nematodes (MPENs) are unable to establish permanent feeding sites, and all developmental stages (except eggs) can invade and feed on plant tissues and can be easily overlooked because of the unspecific symptoms. They cause numerous economic losses in agriculture, forestry, and horticulture. In order to understand the pathogenetic mechanism of MPENs, here we describe research on functions and host targets focused on currently identified effectors from six MPENs, namely Radopholus similis, Pratylenchus spp., Ditylenchus destructor, Bursaphelenchus xylophilus, Aphelenchoides besseyi, and Hirschmanniella oryzae. This information will provide valuable insights into understanding MPEN effectors and for future fostering advancements in plant protection.


Introduction
Plant-parasitic nematodes are one of the pathogens that cause plant infectious diseases, with estimated global economic losses reaching as high as $173 billion annually [1].The parasitic nematodes can be classified into ectoparasitic, semi-endoparasitic, and endoparasitic nematodes according to the mode of parasitism, of which the endoparasitic nematodes can be further divided into migratory and sedentary endoparasites [2].Migratory plant endoparasitic nematodes (MPENs) are incapable of inducing the formation of feeding sites within host plants, primarily changing their feeding locations by migrating between plant cells, with all life stages observable within host tissues [3].Among the top ten plantparasitic nematodes known to cause serious crop diseases, 50% of them are migratory parasitic nematodes encompassing Radopholus similis, Pratylenchus spp., Ditylenchus spp., Bursaphelenchus xylophilus, and Aphelenchoides besseyi [4].
The burrowing nematode, R. similis, is a significant quarantine pest worldwide, notorious for infesting the phloem and cortex of plant roots.It displays a wide range of host species susceptibility, affecting over 360 plant species, including economically important crops such as bananas, citrus fruits, and various ornamental plants.The economic impact of its infestation on bananas alone can be substantial, reaching up to $187 million [5][6][7].Pratylenchus spp., also known as root lesion nematodes, ranks as the second most formidable nematode pest of cultivated crops globally.All species within this genus have the potential to cause damage to host plants by feeding and moving within the root cortex, posing a significant threat to global agricultural economies [8][9][10].Ditylenchus destructor, a species within the Ditylenchus genus, is listed as an important quarantine organism globally, severely impacting the production of sweet potatoes and potatoes, causing yield losses ranging from 20% to 50% [11,12].B. xylophilus can induce devastating diseases in pine trees, often leading to the death of infected trees and resulting in irreparable economic losses annually.It has become one of the most serious coniferous diseases worldwide, affecting pine species extensively [13,14].A. besseyi is a prominent nematode pest in rice cultivation, capable of causing yield reductions ranging from 10% to 70% under severe infestation.
Plant-parasitic nematodes inflict damage on plants not only through mechanical means and nutrient absorption but also through the chemical potency of their secretions.Nematode effectors, including proteins and various small biological molecules, are excreted by esophageal glands, head sensilla, tail sensilla, and the body wall, among other structures.These effectors can alter the structure and function of host cells, playing a crucial role in the nematode's interaction with host plants.Typically, they possess signal peptides without transmembrane domains and can penetrate various parts of plant cells [35,36].Effector molecules of plant-parasitic nematodes have garnered significant attention from scholars worldwide in recent years.While substantial research has focused on elucidating the effectors of sedentary plant-parasitic nematodes, studies on the effectors of migratory plant endoparasitic nematodes remain relatively scarce [37][38][39][40][41].Although efforts have been made to identify the effectors of migratory plant endoparasitic nematodes through cloning endeavors, they have not received as much attention as their sedentary counterparts.
In this review, we primarily concentrate on six MPENs: R. similis, Pratylenchus spp., D. destructor, B. xylophilus, A. besseyi, and H. oryzae.We discuss the synthesis, secretion, functions, and interactions with host plants of effectors derived from migratory plant endoparasitic nematodes.This elucidation aims to foster a deeper understanding of these effectors and provide a basis for identifying potential targets for their control.

The Synthetic Sites of Effectors in Migratory Plant Endoparasitic Nematodes
Effectors originating from the esophageal glands of plant-parasitic nematodes are primarily associated with pathogenicity, exerting control over the host's immune responses and assuming pivotal roles in parasitic interactions, whereas effectors derived from alternate sites may incite physiological reactions within plants [42].Some MPEN effectors display specific localization within the esophageal glands, such as the five pectate lyase genes (Rs-Pel-1, Rs-Pel-2, Rs-Pel-3, Rs-Pel-4, Rs-Pel-5) of R. similis [43], the serine carboxypeptidase gene AbSCP1 of A. besseyi [44], the cellulase gene Bx-eng-1 of B. xylophilus [45], the venom allergen-like gene DdVAP2 of D. destructor [46], and the pectin methylesterase gene Pp-pme of P. penetrans [47], among others.However, besides the esophageal glands, effectors are also secreted in other parts of the nematode's body.For example, the effector BxSCD1 of B. xylophilus is not only localized in the esophageal glands but also detected in the intestine with hybridization signals [48].Additionally, the cellulase gene Pv-eng-5 of P. vulnus exhibits hybridization signals in the intestine [49].Ding et al. demonstrated the immunolocalization of the fatty acid and retinol-binding protein gene Ab-FAR-1 in the body wall of A. besseyi [50].The gene Dd-flp-1 is expressed in the circumpharyngeal nerve ring, retrovesicular ganglion, and ventral cord of D. destructor [51].Some effectors are expressed differently at different development stages of plant-parasitic nematodes, which could be linked to their characteristics and biological functions.For instance, the localization of serine carboxypeptidase Rs-cps in the esophageal gland of R. similis may be related to destroying the host immune response and promoting the establishment of parasitic relationship, while the localization of Rs-cps in eggs and reproductive system may indicate that it may play an important role in the development and reproduction of nematodes [52].

Localization of Migratory Plant Endoparasitic Nematode Effectors in Plant Cells
The targeting efficacy of plant nematode effectors within plant cells underscores their function [53], emphasizing the crucial significance of understanding their localization within plant cells when investigating effector function.Subcellular localization and histological sectioning are the primary methods for observing the action sites of effector proteins within plant tissues [54].Additionally, bioinformatics can be utilized to analyze and predict the localization of effector proteins in plants, although validation through corresponding experiments remains necessary.Numerous studies have demonstrated that effectors from MPENs primarily target the nucleus and cytoplasm of plant cells.The protein AbSCP1 acts on the nucleus of host plants, which may be related to its acceleration of protein degradation of host plants, thus promoting the feeding and parasitism of A. besseyi [44].The protein BxSCD1 is localized within the nucleus and cytoplasm, with its cytoplasmic presence being essential for B. xylophilus to suppress programmed cell death induced by PsXEG1 [48].PSORT II software predicted that Ab-FAR-1 was located in the plant nucleus with 82.6% possibility.Further subcellular localization experiments showed that fluorescence signals were found in the nucleus and cytoplasm of Arabidopsis thaliana and tobacco leaves, and its localization was closely related to the host's defense response [50].Furthermore, the effector Ppen10370 of Pratylenchus penetrans exhibited fluorescence signals on the endoplasmic reticulum and nuclear membrane of Nicotiana benthamiana [55].

The Functions of Effectors in Migratory Plant Endoparasitic Nematodes
The functions of plant-parasitic nematode effectors can be summarized into four major categories: (a) effectors involved in the degradation and modification of plant cell walls; (b) effectors influencing host immune responses; (c) effectors promoting nematode reproduction; (d) effectors inducing and maintaining nematode feeding sites by modulating host plant developmental pathways.Root-knot nematodes (RKN, Meloidogyne spp.) and cyst nematodes (CNs, Globodera and Heterodera spp.) have the ability to modulate the developmental pathways of host plants, thereby eliciting the formation of feeding sites conducive to their growth and development, such as giant cells and syncytium formation [56,57].Distinguished from sedentary plant-parasitic nematodes like RKN and CNs, MPENs do not establish stationary feeding sites, and their effector functionalities predominantly encompass the top three categories.It remains uncertain whether there exist effectors analogous to those instigating and perpetuating nematode feeding sites through the modulation of host plant developmental pathways.Reny et al. compared certain effectors inferred from R. similis with those of other plant-parasitic nematodes, revealing an absence of effectors linked to the formation and development of feeding sites in the genome of R. similis [58].

Effectors Involved in the Degradation and Modification of Plant Cell Walls
The cell wall serves as the primary barrier in plants against pathogenic infections.Throughout the extensive co-evolutionary history between plants and pathogens, the latter have developed the capability of secreting cell wall degrading enzymes (CWDEs) for various polysaccharide components in plant cell walls, such as pectinase, cellulase, hemicellulase, lignin degrading enzymes, and so on [59].CWDEs play a key role in the successful infection of plants by plant-parasitic nematodes, with their coding genes being prevalent across the genomes of such nematodes [60].In 1998, the β-1, 4-glucan endonuclease gene of cellulase of animal origin was first found in the esophageal gland of the cyst nematodes [61].At present, horizontal gene transfer (HGT) stands as the primary mechanism through which plant-parasitic nematodes acquire cell wall degrading enzymes [62].B. xylophilus can be cultivated by fungi, and the resemblance between its cellulase and fungal cellulase is more pronounced, suggesting potential acquisition through HGT from fungal sources [45].Nicol et al. disclosed that a gene encoding a plant cell wall degrading enzyme in Pratylenchus spp.might have been obtained by HGT [63].The evolutionary analysis of Rs-eng-1 indicates its association with the cellulase secreted by the bacterial species Bacillus subtilis and Erwinia carotovora, thereby possibly indicating a bacterial origin [64].With the profound investigation into nematode effectors, an increasing array of genes encoding cell wall degrading enzymes from MPENs have been reported.Lander et al. identified a novel cell wall degrading enzyme, β-glucuronidase, in the ESTs of H. oryzae [65].The cellulase Pv-eng-1 from P. vulnus and two pectate lyases, Bx-pel-1 and Bx-pel-2, from B. xylophilus can degrade the cell walls of host plants, facilitating nematode invasion and migration [49,66].Some effectors with the function of modifying plant cell walls also play an important role in the infection and parasitism of plant-parasitic nematodes.For example, expansin can destroy the covalent bond of plant cell walls, thus relaxing plant cell walls, which is more conducive to the infection of nematodes [67].Expansin-like proteins have been reported on two MPENs, namely B. xylophilus (Bx-EXPB1, Bx-EXPB2, and Bx-EXPB3) [68] and D. destructor (DD-EXP-1 and DD-EXP-2) [69].

Effectors Influencing Host Immune Responses
Two-layer immune systems of plants, PAMP-triggered immunity response (PTI) and effector-triggered immunity (ETI), are the main means for plants to resist pathogen infection [70].Thus far, our understanding of plant-parasitic nematode-associated molecular patterns and the signaling pathways triggered by PAMPs from plant-parasitic nematodes remains limited [71].The role of MPENs in modulating plant immune responses has garnered increasing attention in recent studies.Indeed, the impact of plant-parasitic nematode effectors on Reactive Oxygen Species (ROS) levels, callose deposition, plant defense gene expression, and the formation of host necrotic spots serves as a significant indicator for assessing whether these effectors promote or inhibit the immune response of plants [72].Many effectors of MPENs have been proved to effectively suppress the immune response of plants, including the venom allergen-like protein RsVAP [73] and the chorismate mutase RsCM [74] of R. simili; effectors BxSCD1 [48], BxSCD3 [75], BxSCD5 [76], BxICD1 [77], and BxLip-3 [78] of B. xylophilus; the fatty acid and vitamin A binding protein Ab-FAD-1 [50] of A. besseyi, and various genes encoding tissue proteases such as Pc-CZ, Pc-CD, Pc-CB, and Pc-CL, along with membrane-associated protein genes PC-NEX-1 and PC-NEX-2 from P. coffeae [79][80][81], among others.Research has revealed that the inhibitory effect of effector proteins on plant immune responses requires reaching a certain concentration threshold.Li et al. found that the effector protein Bx-FAR-1 from B. xylophilus could only suppress Nicotiana benthamiana necrosis induced by INF1 when its concentration reached 100 nmol/L [82].Plant-parasitic nematodes not only have the ability to suppress the host's immune responses but also induce immune responses in the host.Reported effectors capable of inducing host immune responses from migratory plant endoparasitic nematodes include the apoptotic protein BxCDP1 [83], the venom allergen-like protein Bx-vap-1 [84], and the sphingolipid activator protein BxSapB1 [85] from B. xylophilus, as well as the ATP synthase Ab-atps from A. besseyi [86].

Effectors Promoting Nematode Reproduction
In migratory parasitic nematodes, various effectors have been shown to be closely associated with nematode reproduction, primarily focusing on peptidases and calcium-binding proteins as the two principal effector categories.The serine carboxypeptidases Rs-scp-1 and AbSCP1 may potentially facilitate nematode degradation of host plant proteins and nutrient acquisition, thereby fostering their reproduction and parasitism [28,44,52].Ab-CB-1 possesses proteolytic properties, enabling the degradation of carrot callus tissue to provide sustenance for its growth and reproduction, as evidenced in the proliferation of A. besseyi [87].AbCRT affects the feeding and reproduction of A. besseyi, and downregulating the expression of this gene can reduce the frequency of nematode feeding, consequently decreasing their reproductive capacity [88].Similar effectors include Bx-CRT [34], Bx-cpl [89], and Rs-CRT [90].Furthermore, cytochrome P450 is also crucial for the reproduction of plant-parasitic nematodes, as evidenced by a significant reduction in the reproductive capacity of pine wood nematodes upon silencing the Bxcyp-33C9 gene [91].
The tissue localization of identified MPEN effectors involved in this paper is summarized in Table 1.

The Interaction between Effectors of Migratory Plant Endoparasitic Nematodes and Host Plants
In the theoretical investigation of the interaction mechanisms between plant pathogens and host plants, a plethora of models and hypotheses have emerged, including the genefor-gene hypothesis, the pattern-triggered immunity hypothesis, the Zigzag model, and the decoy model [70,[95][96][97].Among these, the "zig-zag model" presently occupies a central position in research, with a multitude of studies attesting to its relevance in elucidating the interaction dynamics between plant-parasitic nematodes and host plants [98].Pattern recognition receptors (PRRs) in plants recognize pathogen-associated molecular patterns (PAMPs) and activate the basic immune response PTI.Pathogens enhance their virulence by secreting effectors to interfere with PTI, which leads to effector-triggered susceptibility (ETS).At the same time, plants sense the effector effects of pathogens through nucleotide binding and leucine-rich repeat protein (NB-LRR protein) and start a specific immune response ETI.The interaction among PTI, ETS, and ETI is summarized as the zig-zag model [70].The intricate interaction between the MPEN effectors and host plants is crucial in meticulously orchestrating the infection process and in the fine-tuning of the host's physiological responses.Exploring the target proteins of plant-parasitic nematode effectors in the host will facilitate the elucidation of the pathogenic mechanisms of plant-parasitic nematodes and the formulation of more targeted control strategies [99][100][101].Currently, yeast two-hybrid (Y2H) assays serve as the primary method for screening plant-parasitic nematode effectors interacting with receptor proteins within the host.To mitigate the risk of false positives in receptor protein identification, additional techniques such as bimolecular fluorescent complementation (BiFC) or co-immunoprecipitation (Co-IP) are often employed for validation [54].Among plant-parasitic nematodes investigations into the interaction proteins of effectors in host plants predominantly focus on sedentary parasitic nematodes [102], while studies on migratory parasitic nematodes remain relatively sparse.
The cellular microfilament component of the cytoskeleton exerts a significant influence on the mediation of plant immune signaling pathways.In recent years, progress has been made in understanding plant cytoskeleton-pathogen interactions, and some effectors can regulate plant immune responses by influencing the cellular microfilament skeleton [103,104].Ding et al. reported that A. besseyi can interact with the AtADF3 of Arabidopsis thaliana by secreting Ab-FAR-1, disrupting the actin-depolymerizing function of AtADF3, thereby inhibiting plant PTI.This study reveals, for the first time in plant-parasitic nematodes, a novel mechanism by which effectors suppress plant disease resistance by influencing the microfilament skeleton, providing fresh insights into the interaction between plant-parasitic nematode effectors and host plants [50].The effector of plant-parasitic nematodes interacts with host plant defense-related proteins, subsequently influencing the expression of host plant defense-related genes, thereby modulating plant defense responses.Zhang et al. conducted a screening of the interaction protein PtCyP1 from B. xylophilus BxML1 in Pinus thunbergii through yeast two-hybrid and immunoprecipitation techniques.They elucidated that this interaction suppressed the plant defense response by modulating the expression of PtCyP1.Notably, PtCyP1 exhibited substantial expression levels during B. xylophilus infection in pine trees, whereas BxML1 repressed the expression of PtCyP1 [92].Additionally, the effector Ab-atps, secreted by A. besseyi, interacts with the rice defense gene OsRLK3, eliciting the self-defense response of rice.Subsequently, the secretion of OsRLK3 is suppressed by Ab-atps in the later stages.Further investigation is required to ascertain whether Ab-atps inhibits or evades the defense response mediated by OsRLK3 [86].Some effectors of plant-parasitic nematodes can regulate the metabolic pathway of plants and interact with host plants by simulating the host plant protein [105].Wen et al. discovered for the first time that PtACO1, an ethylene-forming enzyme in pine trees, can interact with BxSCD1, an effector of B. xylophilus, and speculated that its interaction interfered with ethylene biosynthesis [48].After secreting RsCM into the plant cytoplasm, R. similis may competitively inhibit the synthesis of salicylic acid (SA) in plant cells by vying for chorismic acid with chorismate mutase of the host plant, thereby suppressing plant defense responses [73].HoCM and HoICM of H. oryzae may interfere with the synthesis of salicylic acid in the host plant, thus reducing the secondary metabolism level of the plant and inhibiting the plant immune response [106].It is noteworthy that the ubiquitin pathway is integral to the interaction between plant-parasitic nematodes and host plants.There are some effectors in plant-parasitic nematodes that can hijack the ubiquitination system of the host or have ubiquitinated ligase activity in other ways, thus regulating the expression of defense-related factors of the host [107].Paulo et al. identified a significant upregulation of ubiquitin-related genes and the distinct localization of the Pratylenchus penetrans effector Ppen10370 within the endoplasmic reticulum in plants expressing this effector.They postulated its potential involvement in the host's ubiquitination pathway and its impact on the plant's immune response, although the precise mechanism warrants further investigation [55].Hu et al. demonstrated the interaction between BxCDP1 and Pinus thunbergii protein PtRHF1, potentially functioning as a plant E3 ligase and engaging in the ubiquitination process of BxCDP1 within plants, thus stimulating plant immunity [108].
The similar effector between different plant-parasitic nematodes may have high similarity in function, but it shows diversity in the mechanism of regulating host defense response.Li et al. identified the protein RsVAP of R. similis, which interacted with LeRabAld in the tomato, and postulated that their interaction may interfere with the role of LeRabAld in vesicle transport.Additionally, they also discovered that RsVAP from R. similis and sedentary plant-parasitic nematodes can suppress the plant's defense response in function; however, these proteins had different interaction host targets [73].As an elicitor, Bx-vap-1 induces the host's defense response and plays a crucial role in the interaction between B. xylophilus and its host pine tree [84].Both B. xylophilus and R. similis belong to migratory endoparasitic nematodes, yet their VAPs exhibit diametrically opposed functions in the host's defense response.BxKU1 and BxKU2 are Kunitz-type protease inhibitors identified in B. xylophilus.The former has been demonstrated to interact with PtCel2 and TLP4 of Pinus thunbergii, potentially disrupting the SA pathway, while the latter interacts with an extensin-like protein associated with PR10 of P. thunbergii.Through the distinct mechanisms of action of these two effectors, B. xylophilus inhibits the defense response of host plants [93].MPENs have evolved a variety of strategies to overcome plant immune response to establish parasitic relationship, which just indicates the complexity of the interaction between MPENs and host plants.
The effectors with host targets are listed in Table 2.

Outlook
In the realm of plant-parasitic nematodes, most research efforts have been focused on sedentary plant-parasitic nematodes such as root-knot nematodes and cyst nematodes, leaving the MPENS investigation relatively scant, although to some extent, they exert a greater impact on crop quality and yield than some sedentary plant-parasitic nematodes.Therefore, identifying targets for their control has become one of the key directions in research.In recent years, both domestic and international scholars have made headway through transcriptomic analyses of MPENS, unveiling numerous effector transcripts.However, compared to sedentary parasitic nematodes, comprehensive investigations remain sparse.Presently, only a handful of effectors have been elucidated in terms of functionality, with a considerably limited understanding of their roles during the invasion of host plants by MPENs.Thus, many effectors await identification and further scrutiny.Furthermore, research on MPEN effectors have largely focused on the exploration and functional analysis of homologous effectors, with minimal attention devoted to the discovery of specific effectors dissimilar to known ones.Moving forward, there is a pressing need to intensify efforts in studying MPEN effectors, striving to unearth additional candidate effectors and conducting pertinent identification and functional analyses.Effectors have long been a focal point in research on MPENs, particularly concerning the identification of their targets and interactions within plants.These findings serve as foundational knowledge for a deeper understanding of the molecular mechanisms underlying the interaction between MPENs and plants, thereby facilitating the elucidation of the pathogenic mechanisms of these nematodes.Currently, our exploration and investigation of interacting proteins of MPEN effectors remain insufficient.This area warrants significant advancement in future research efforts focused on MPENs.

Table 1 .
The localization of migratory plant endoparasitic nematode effectors.

Table 2 .
Host targets of effectors in migratory plant endoparasitic nematodes.