Unlocking the development- and physiology-altering ‘effector toolbox’ of plant-parasitic nematodes

Abstract

Plant-parasitic nematodes (PPNs) profoundly alter plant development, but there is a paucity of information about nematode effectors that alter development and physiology.The recent advances in gland-cell-specific transcriptomics have allowed us to access the full effector repertoire of PPNs.The development of a novel, reproducible, high-throughput screen for development- and physiology-altering effector function will be essential to accelerate the field. Amongst parasites and pathogens, those that infect plants appear to be the most masterful manipulators of development and physiology, due to the nature of their host: plant developmental plasticity (see Glossary) [1.de Jong M. Leyser O. Developmental plasticity in plants.Cold Spring Harb. Symp. Quant. Biol. 2012; 77: 63-73Crossref PubMed Google Scholar]. This article argues that our understanding of the development- and physiology-altering ‘toolbox’ of PPNs is an understudied, and yet objectively important, area of parasitology with potentially far-reaching consequences. In order to successfully colonise the plant, parasites and pathogens hijack many aspects of host biology. The extent and diversity of these alterations is bewildering: parasites and pathogens have evolved to suppress the plant immune system and to manipulate plant development, physiology, and cell biology to their benefit. To do this, they secrete effectors [2.Hogenhout S.A. et al.Emerging concepts in effector biology of plant-associated organisms.Mol. Plant-Microbe Interact. 2009; 22: 115-122Crossref PubMed Scopus (468) Google Scholar] (Box 1). Despite the fact that developmental and cellular changes are a hallmark of many important pathologies [3.Harris M.O. Pitzschke A. Plants make galls to accommodate foreigners: some are friends, most are foes.New Phytol. 2020; 225: 1852-1872Crossref PubMed Scopus (27) Google Scholar], the field has historically focused on those proteinaceous effectors that suppress immunity, likely due to the availability of tractable assays and genetic test systems. As a result, there is a relative paucity of knowledge about pathogen effectors that alter other plant processes and their mechanisms of action.Box 1The effector conceptAlthough definitions vary, effectors are generally described as pathogen-secreted molecules that aid a parasite or pathogen in establishing disease. Historically, the term effector referred only to pathogen-secreted proteins, but this has since been updated to include a broader range of molecules including – but not limited to – peptides [37.Mitchum M.G. Liu X. Peptide effectors in phytonematode parasitism and beyond.Annu. Rev. Phytopathol. 2022; 60: 97-119Crossref PubMed Scopus (2) Google Scholar], small RNAs [38.Wang M. et al.Pathogen small RNAs: a new class of effectors for pathogen attacks.Mol. Plant Pathol. 2015; 16: 219-223Crossref PubMed Scopus (33) Google Scholar] and secondary metabolites [39.Rangel L.I. Bolton M.D. The unsung roles of microbial secondary metabolite effectors in the plant disease cacophony.Curr. Opin. Plant Biol. 2022; 68102233Crossref PubMed Scopus (3) Google Scholar]. Here, we use the following inclusive definition: an effector (i) is secreted into the plant (apoplast or translocated inside host cells); (ii) alters one or several aspects of plant biology in a host; and (iii) has a virulence function in a host that benefits the parasite or pathogen [40.Zhang S. et al.Action mechanisms of effectors in plant–pathogen interaction.Int. J. Mol. Sci. 2022; 23: 6758Crossref PubMed Scopus (14) Google Scholar]. Although definitions vary, effectors are generally described as pathogen-secreted molecules that aid a parasite or pathogen in establishing disease. Historically, the term effector referred only to pathogen-secreted proteins, but this has since been updated to include a broader range of molecules including – but not limited to – peptides [37.Mitchum M.G. Liu X. Peptide effectors in phytonematode parasitism and beyond.Annu. Rev. Phytopathol. 2022; 60: 97-119Crossref PubMed Scopus (2) Google Scholar], small RNAs [38.Wang M. et al.Pathogen small RNAs: a new class of effectors for pathogen attacks.Mol. Plant Pathol. 2015; 16: 219-223Crossref PubMed Scopus (33) Google Scholar] and secondary metabolites [39.Rangel L.I. Bolton M.D. The unsung roles of microbial secondary metabolite effectors in the plant disease cacophony.Curr. Opin. Plant Biol. 2022; 68102233Crossref PubMed Scopus (3) Google Scholar]. Here, we use the following inclusive definition: an effector (i) is secreted into the plant (apoplast or translocated inside host cells); (ii) alters one or several aspects of plant biology in a host; and (iii) has a virulence function in a host that benefits the parasite or pathogen [40.Zhang S. et al.Action mechanisms of effectors in plant–pathogen interaction.Int. J. Mol. Sci. 2022; 23: 6758Crossref PubMed Scopus (14) Google Scholar]. One important crop parasite group that is capable of eliciting profound changes to host development and physiology is the PPNs. Globally, PPNs pose a considerable threat to food security; every major crop species can be parasitised by at least one species of nematode, with collective annual damage estimated at over $100 billion [4.Nicol J.M. et al.Current nematode threats to world agriculture.in: Jones J. Genomics and Molecular Genetics of Plant–Nematode Interactions. Springer, 2011: 21-43Crossref Google Scholar]. The most damaging, and hence the most widely studied are also those that appear to have the most substantial development- and physiology-altering ‘toolbox’: the root-knot and cyst nematodes [5.Jones J.T. et al.Top 10 plant-parasitic nematodes in molecular plant pathology.Mol. Plant Pathol. 2013; 14: 946-961Crossref PubMed Scopus (1118) Google Scholar]. These nematodes are master biotrophs, capable of drastically altering plant biology to form intimate, long-term relationships with the host plant. Infection is characterised by extreme changes in host plant development to form a feeding site, a unique organ from which the nematode draws nutrition for the remainder of its life cycle [6.Jones M.G.K. Host cell responses to endoparasitic nematode attack: structure and function of giant cells and syncytia.Ann. Appl. Biol. 1981; 97: 353-372Crossref Google Scholar]. In this way, the nematode deprives the host plant of essential water and nutrients. Like other pathosystems, nematodes deploy effectors to establish parasitism [7.Eves-van den Akker S. et al.Identification and characterisation of a hyper-variable apoplastic effector gene family of the potato cyst nematodes.PLoS Pathog. 2014; 10e1004391Crossref PubMed Scopus (56) Google Scholar]. Consistent with overarching trends in plant pathology, the majority of nematode effectors are unknown, and those that alter development and physiology remain almost totally undescribed. The interaction between nematode and plant is remarkable in that, during infection, a cross-kingdom dialogue between the parasite and the host determines the developmental outcomes of both parties. Upon perception of host-specific diffusates in the soil, nematodes hatch as sexless second-stage juveniles (J2s) which migrate towards the growing root [8.Wyss U. Zunke U. Observations on the behaviour of second stage juveniles of Heteroder schachtii inside host roots.Rev. Nématol. 1986; 9: 153-165Google Scholar]. To penetrate host cells, infective juveniles secrete effectors which allow them to degrade the plant cell wall and move to the vascular system [9.Popeijus H. et al.Degradation of plant cell walls by a nematode.Nature. 2000; 406: 36-37Crossref PubMed Scopus (0) Google Scholar]. Here, the nematodes initiate their association with the plant by delivering a mixture of effector proteins and metabolites into host cells using a needle-like structure called a stylet. Crucially, these effectors are secreted from two sets of specialised pharyngeal gland cells: the subventral glands and the dorsal gland [10.Hussey R.S. Mims C.W. Ultrastructure of esophageal glands and their secretory granules in the root-knot nematode Meloidogyne incognita.Protoplasma. 1990; 156: 9-18Crossref Scopus (0) Google Scholar]. It is thought that the subventral gland is primarily active during the early life-stages, whereas the dorsal gland is primary active during the sedentary phase [11.Davis E.L. et al.Parasitism proteins in nematode-plant interactions.Curr. Opin. Plant Biol. 2008; 11: 360-366Crossref PubMed Scopus (178) Google Scholar]. Upon interaction with the cell, nematodes evoke a major reprogramming of host biology to establish the feeding site. The specific morphological and subcellular changes elicited by the nematode depend on species. Cyst nematodes (Heterodera spp. and Globodera spp.) induce the formation of a feeding structure called a syncytium [12.Grundler F.M.W. et al.Formation of wall openings in root cells of Arabidopsis thaliana following infection by the plant-parasitic nematode Heterodera schachtii.Eur. J. Plant Pathol. 1998; 104: 545-551Crossref Scopus (68) Google Scholar]. They do so by injecting effectors into a single initial syncytial cell concurrent with numerous changes in subcellular architecture. The initial cell is arrested in G2 stage of the cell cycle, the nucleus enlarges, the vacuole reduces in size, and there is extensive proliferation of subcellular organelles including the smooth endoplasmic reticulum (ER), ribosomes, mitochondria, and plastids. Plasmodesmata between adjacent cells enlarge, the middle lamella is digested, and protoplasts are incorporated into the syncytium via plasma membrane fusion [12.Grundler F.M.W. et al.Formation of wall openings in root cells of Arabidopsis thaliana following infection by the plant-parasitic nematode Heterodera schachtii.Eur. J. Plant Pathol. 1998; 104: 545-551Crossref Scopus (68) Google Scholar,13.Golinowski W. et al.Changes in the structure of Arabidopsis thaliana during female development of the plant-parasitic nematode Heterodera schachtii.Protoplasma. 1996; 194: 103-116Crossref Scopus (152) Google Scholar] (Figure 1). These changes are essential for nematode infection as, in this way, hundreds of cells form a nutrient sink capable of sustaining the nematode as it matures and reproduces. Comparatively, root-knot nematodes (Meloidogyne spp.) select several feeding site-progenitor cells, which all undergo successive rounds of mitosis without cytokinesis, resulting in a group of giant, multinucleate cells up to 100 times larger than their neighbours. As these giant cells develop, the cells surrounding them proliferate, forming a gall within which the nematode resides [14.Jones M.G. Payne H.L. Early stages of nematode-induced giant-cell formation in roots of Impatiens balsamina.J. Nematol. 1978; 10: 70-84PubMed Google Scholar]. Whilst nematodes alter host development, the host also profoundly alters nematode development. Once feeding commences, around 48 h post-infection, J2s develop into J3s. The sex of the J3 is determined post-embryonically during parasitism. Where conditions for the parasite are unfavourable (e.g., on a resistant host plant), proportionally more males are produced [15.Anjam M.S. et al.Host factors influence the sex of nematodes parasitizing roots of Arabidopsis thaliana.Plant Cell Environ. 2020; 43: 1160-1174Crossref PubMed Scopus (11) Google Scholar,16.Ellenby C. Environmental determination of the sex ratio of a plant parasitic nematode.Nature. 1954; 174: 1016-1017Crossref Scopus (23) Google Scholar]. The female cyst nematode is fertilised by the mobile adult male, and subsequently develops into a cyst containing fertilised eggs, whereas the female root-knot nematode deposits eggs into a gelatinous matrix. Despite the obvious importance of the feeding site as the sole source of nutrients for the developing nematode, and a key factor in nematode sex determination, very little is known about the mechanisms governing its establishment. Only a handful of PPN effectors have been characterised. Consistent with progress in other pathosystems, there is a conspicuous absence of development- and physiology-altering effectors amongst the small proportion of effectors which have been characterised to date (Figure 2; see also Table S1 in the supplemental information online). Yet, taking into account the dramatic changes involved in feeding site establishment, it is entirely possible that such effectors might dominate the PPN effector repertoire. Historically, functional characterisation of effectors has focused on those that suppress immunity, in part due to the availability of well-established, high-throughput screens for immune suppression [17.Pogorelko G. et al.Screening soybean cyst nematode effectors for their ability to suppress plant immunity.Mol. Plant Pathol. 2020; 21: 1240-1247Crossref PubMed Scopus (14) Google Scholar,18.Derevnina L. et al.Plant pathogens convergently evolved to counteract redundant nodes of an NLR immune receptor network.PLoS Biol. 2021; 19e3001136Crossref PubMed Scopus (8) Google Scholar]. The lack of early established screens for nonimmune effector functions may have contributed to the concomitant lack of momentum in this area. Whilst it is true that parasites and pathogens must ubiquitously evade the immune system, many important plant pathologies are characterised by developmental changes, including fungi (e.g., smuts), insects (e.g., galling aphids), and PPNs – principally, although by no means exclusively, the cyst and root-knot nematodes [3.Harris M.O. Pitzschke A. Plants make galls to accommodate foreigners: some are friends, most are foes.New Phytol. 2020; 225: 1852-1872Crossref PubMed Scopus (27) Google Scholar]. Whilst it is true that few physiology- and development-altering effectors are known, by far the best characterised example of development-altering PPN effectors are the CLAVATA3/EMBRYO SURROUND REGION-RELATED (CLE)-like peptide effectors in cyst nematodes [19.Wang X. et al.A parasitism gene from a plant-parasitic nematode with function similar to CLAVATA3/ESR (CLE) of Arabidopsis thaliana.Mol. Plant Pathol. 2005; 6: 187-191Crossref PubMed Google Scholar]. CLE-like peptide effectors mimic plant CLE peptide hormones, ligands involved in the CLE-receptor-WUS-homeobox containing (WOX) transcription factor signalling system, which functions to maintain stem cell populations to facilitate post-embryonic organogenesis in the shoot, root, and vascular meristems [20.Yamaguchi Y.L. et al.CLE peptides and their signalling pathways in plant development.J. Exp. Bot. 2016; 67: 4813-4826Crossref PubMed Scopus (0) Google Scholar]. Remarkably, CLE mimics are redirected to the apoplast by a previously undescribed trafficking pathway in plants. CLE mimics are delivered into the host cytoplasm as propeptides, containing a specific motif which is recognised by host plant machinery, and allows the effectors to hijack a host mechanism for post-translational trafficking into the ER [21.Wang J. et al.Phytonematode peptide effectors exploit a host post-translational trafficking mechanism to the ER using a novel translocation signal.New Phytol. 2021; 229: 563-574Crossref PubMed Scopus (7) Google Scholar]. Nematode CLEs provide an excellent case study to justify the importance of investigating development- and physiology-altering PPN effectors for two reasons: (i) they highlight the abilities of development-altering effectors to utilise endogenous machinery to co-opt pathways involved in plant developmental plasticity; and (ii) an understanding of nematode CLE biology has allowed the discovery of a novel pathway for trafficking to the apoplast in planta, exemplifying how effectors can provide valuable tools for understanding fundamental plant processes. The discovery of CLEs also clearly demonstrates the current limitations of PPN biology – when it comes to PPN effectors the low hanging fruit has been picked. CLE peptides were identified in nematodes because they possess sequence similarity (in some cases identity) with CLE peptides in plants [19.Wang X. et al.A parasitism gene from a plant-parasitic nematode with function similar to CLAVATA3/ESR (CLE) of Arabidopsis thaliana.Mol. Plant Pathol. 2005; 6: 187-191Crossref PubMed Google Scholar]. This is also the case for a number of plant peptide-hormone mimics in root-knot nematodes. These include peptides containing CLE motifs, C-terminally encoded peptides (CEPs), rapid alkalization factors (RALFs), INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) peptides and, most recently, nematode mimics of plant peptides containing sulfated tyrosine (PSYs), which promote cell expansion and proliferation in root growth [22.Zhang X. et al.Nematode-encoded RALF peptide mimics facilitate parasitism of plants through the FERONIA receptor kinase.Mol. Plant. 2020; 13: 1434-1454Abstract PubMed Scopus Google gene a peptide with similarity to the plant peptide is most in the root-knot nematode (Meloidogyne root 2013; PubMed Scopus (8) Google et of the plant peptide 2013; PubMed Scopus Google J. et root-knot nematode Meloidogyne a functional mimic of the Arabidopsis INFLORESCENCE DEFICIENT IN ABSCISSION Exp. Bot. PubMed Scopus (11) Google et of the Meloidogyne family plant 2014; 104: PubMed Scopus Google Scholar]. In most PPN effectors have no known of PPN which for their discovery and functional These are by the absence of established to screen for effector the of proteins that function as effectors a recent advances in gland-cell-specific transcriptomics have us the ability to the effector repertoire in full et of esophageal gland cells from plant-parasitic nematodes for and effector Plant-Microbe Interact. 2013; PubMed Scopus Google Scholar]. with these new we can to the full of PPN effector Nematode effector number in the hundreds S. et and of a plant-parasitic nematode and its host involved in of 2022; 13: PubMed Scopus Google Scholar]. It is that the current of investigating effector is to be we to the PPN The study of effector functions has been by the availability of high-throughput immune suppression assays for effector These assays are on of parasite or pathogen effectors in by and the of their ability to the immune [17.Pogorelko G. et al.Screening soybean cyst nematode effectors for their ability to suppress plant immunity.Mol. Plant Pathol. 2020; 21: 1240-1247Crossref PubMed Scopus (14) Google Scholar,18.Derevnina L. et al.Plant pathogens convergently evolved to counteract redundant nodes of an NLR immune receptor network.PLoS Biol. 2021; 19e3001136Crossref PubMed Scopus (8) Google Scholar]. the field is reproducible, high-throughput screens for development- and physiology-altering effector The of a high-throughput screen for development- and physiology-altering the field the of development- and physiology-altering PPN their and their molecular mechanisms in planta, may novel for PPN to this, as in the case of nematode CLE the remarkable abilities of PPNs to plant developmental and utilise host to a new organ has the to about the of plant developmental processes and most knowledge of effector function also has the for and advances in biology den Akker S. the effector for plant-parasitic cyst nematode Plant. 2016; 9: PubMed Scopus Google Scholar]. is a for the use of effectors in for in A. plant and et using a 2012; PubMed Scopus Google a for of Arabidopsis thaliana.Plant J. 1998; 16: PubMed Google Scholar]. 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PubMed Scopus Google Scholar], there is no for high-throughput of cellular and subcellular changes in to parasite and pathogen The range of changes about by nematode infection is and for these changes the of numerous subcellular screens be on the of effector in and the of cellular and subcellular changes in plants for including mitochondria, smooth and The and of cells and also be To all of these in a reproducible, high-throughput a development- and physiology-altering screen likely and et al.A and to and of cyst nematode infection assays for Arabidopsis thaliana.Plant 2022; PubMed Scopus (0) Google Scholar]. 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