A root-knot nematode-secreted protein is injected into giant cells and targeted to the nuclei

Root-knot nematodes (RKNs) are obligate endoparasites that maintain a biotrophic relationship with their hosts over a period of several weeks and induce the differentiation of root cells into specialized feeding cells. Nematode effectors synthesized in the oesophageal glands and injected into the plant tissue through the syringe-like stylet certainly play a central role in these processes. In a search for nematode effectors, we used comparative genomics on expressed sequence tag (EST) datasets to identify Meloidogyne incognita genes encoding proteins potentially secreted upon the early steps of infection. We identified three genes specifically expressed in the oesophageal glands of parasitic juveniles that encode predicted secreted proteins. One of these genes, Mi-EFF1 is a pioneer gene that has no similarity in databases and a predicted nuclear localization signal. We demonstrate that RKNs secrete Mi-EFF1 within the feeding site and show Mi-EFF1 targeting to the nuclei of the feeding cells. RKNs were previously shown to secrete proteins in the apoplasm of infected tissues. Our results show that nematodes sedentarily established at the feeding site also deliver proteins within plant cells through their stylet. The protein Mi-EFF1 injected within the feeding cells is targeted at the nuclei where it may manipulate nuclear functions of the host cell.     

Proteins secreted by root-knot nematodes accumulate in the extracellular compartment during root infection

Root-knot nematodes are biotrophic parasites that invade the root apex of host plants and migrate towards the vascular cylinder where they induce the differentiation of root cells into hypertrophied multinucleated giant cells. Giant cells are part of the permanent feeding site required for nematode development into the adult stage. To date, a repertoire of candidate effectors potentially secreted by the nematode into the plant tissues to promote infection has been identified. However, the precise role of these candidate effectors during root invasion or during giant cell induction and maintenance remains largely unknown. Primarily, the identification of the destination of nematode effectors within plant cell compartment(s) is crucial to decipher their actual functions. We analyzed the fine localization in root tissues of five nematode effectors throughout the migratory and sedentary phases of parasitism using an adapted immunocytochemical method that preserves host and pathogen tissues. We showed that secretion of effectors from the amphids or the oesophageal glands is tightly regulated during the course of infection. The analyzed effectors accumulated in the root tissues along the nematode migratory path and along the cell wall of giant cells, showing the apoplasm as an important destination compartment for these effectors during migration and feeding cell formation.Key words: plant pathogen, effector, immunocytochemistry, root-knot nematode, secretion, plant apoplasm     

Functional C‐TERMINALLY ENCODED PEPTIDE (CEP) plant hormone domains evolved de novo in the plant parasite Rotylenchulus reniformis

Sedentary plant‐parasitic nematodes (PPNs) induce and maintain an intimate relationship with their host, stimulating cells adjacent to root vascular tissue to re‐differentiate into unique and metabolically active ‘feeding sites’. The interaction between PPNs and their host is mediated by nematode effectors. We describe the discovery of a large and diverse family of effector genes, encoding C‐TERMINALLY ENCODED PEPTIDE (CEP) plant hormone mimics (RrCEPs), in the syncytia‐forming plant parasite Rotylenchulus reniformis. The particular attributes of RrCEPs distinguish them from all other CEPs, regardless of origin. Together with the distant phylogenetic relationship of R. reniformis to the only other CEP‐encoding nematode genus identified to date (Meloidogyne), this suggests that CEPs probably evolved de novo in R. reniformis. We have characterized the first member of this large gene family (RrCEP1), demonstrating its significant up‐regulation during the plant–nematode interaction and expression in the effector‐producing pharyngeal gland cell. All internal CEP domains of multi‐domain RrCEPs are followed by di‐basic residues, suggesting a mechanism for cleavage. A synthetic peptide corresponding to RrCEP1 domain 1 is biologically active and capable of up‐regulating plant nitrate transporter (AtNRT2.1) expression, whilst simultaneously reducing primary root elongation. When a non‐CEP‐containing, syncytia‐forming PPN species (Heterodera schachtii) infects Arabidopsis in a CEP‐rich environment, a smaller feeding site is produced. We hypothesize that CEPs of R. reniformis represent a two‐fold adaptation to sustained biotrophy in this species: (i) increasing host nitrate uptake, whilst (ii) limiting the size of the syncytial feeding site produced.     

Gall-forming root-knot nematodes hijack key plant cellular functions to induce multinucleate and hypertrophied feeding cells

Among plant-parasitic nematodes, the root-knot nematodes (RKNs) of the Meloidogyne spp. are the most economically important genus. RKN are root parasitic worms able to infect nearly all crop species and have a wide geographic distribution. During infection, RKNs establish and maintain an intimate relationship with the host plant. This includes the creation of a specialized nutritional structure composed of multinucleate and hypertrophied giant cells, which result from the redifferentiation of vascular root cells. Giant cells constitute the sole source of nutrients for the nematode and are essential for growth and reproduction. Hyperplasia of surrounding root cells leads to the formation of the gall or root-knot, an easily recognized symptom of plant infection by RKNs. Secreted effectors produced in nematode salivary glands and injected into plant cells through a specialized feeding structure called the stylet play a critical role in the formation of giant cells. Here, we describe the complex interactions between RKNs and their host plants. We highlight progress in understanding host plant responses, focusing on how RKNs manipulate key plant processes and functions, including cell cycle, defence, hormones, cellular scaffold, metabolism and transport.     

Nuclear effectors of plant pathogens: Distinct strategies to be one step ahead

Nuclear effector proteins released by bacteria, oomycete, nematode, and fungi burden the global environment and crop yield. Microbial effectors are key weapons in the evolutionary arms race between plants and pathogens, vital in determining the success of pathogenic colonization. Secreted effectors undermine a multitude of host cellular processes depending on their target destination. Effectors are classified by their localization as either extracellular (apoplastic) or intracellular. Intracellular effectors can be further subclassified by their compartment such as the nucleus, cytoplasm or chloroplast. Nuclear effectors bring into question the role of the plant nucleus' intrinsic defence strategies and their vulnerability to effector-based manipulation. Nuclear effectors interfere with multiple nuclear processes including the epigenetic regulation of the host chromatin, the impairment of the trans-kingdom antifungal RNAi machinery, and diverse classes of immunity-associated host proteins. These effector-targeted pathways are widely conserved among different hosts and regulate a broad array of plant cellular processes. Thus, these nuclear sites constitute meaningful targets for effectors to subvert the plant defence system and acquire resources for pathogenic propagation. This review provides an extensive and comparative compilation of diverse plant microbe nuclear effector libraries, thereby highlighting the distinct and conserved mechanisms these effectors employ to modulate plant cellular processes for the pathogen's profit.     

Cell cycle activation by plant parasitic nematodes

Sedentary nematodes are important pests of crop plants. They are biotrophic parasites that can induce the (re)differentiation of either differentiated or undifferentiated plant cells into specialized feeding cells. This (re)differentiation includes the reactivation of the cell cycle in specific plant cells finally resulting in a transfer cell-like feeding site. For growth and development the nematodes fully depend on these cells. The mechanisms underlying the ability of these nematodes to manipulate a plant for its own benefit are unknown. Nematode secretions are thought to play a key role both in plant penetration and feeding cell induction. Research on plant-nematode interactions is hampered by the minute size of cyst and root knot nematodes, their obligatory biotrophic nature and their relatively long life cycle. Recently, insights into cell cycle control in Arabidopsis thaliana in combination with reporter gene technologies showed the differential activation of cell cycle gene promoters upon infection with cyst or root knot nematodes. In this review, we integrate the current views of plant cell fate manipulation by these sedentary nematodes and made an inventory of possible links between cell cycle activation and local, nematode-induced changes in auxin levels.     

Genomic characterisation of the effector complement of the potato cyst nematode Globodera pallida

Background

The potato cyst nematode Globodera pallida has biotrophic interactions with its host. The nematode induces a feeding structure – the syncytium – which it keeps alive for the duration of the life cycle and on which it depends for all nutrients required to develop to the adult stage. Interactions of G. pallida with the host are mediated by effectors, which are produced in two sets of gland cells. These effectors suppress host defences, facilitate migration and induce the formation of the syncytium.

Results

The recent completion of the G. pallida genome sequence has allowed us to identify the effector complement from this species. We identify 128 orthologues of effectors from other nematodes as well as 117 novel effector candidates. We have used in situ hybridisation to confirm gland cell expression of a subset of these effectors, demonstrating the validity of our effector identification approach. We have examined the expression profiles of all effector candidates using RNAseq; this analysis shows that the majority of effectors fall into one of three clusters of sequences showing conserved expression characteristics (invasive stage nematode only, parasitic stage only or invasive stage and adult male only). We demonstrate that further diversity in the effector pool is generated by alternative splicing. In addition, we show that effectors target a diverse range of structures in plant cells, including the peroxisome. This is the first identification of effectors from any plant pathogen that target this structure.

Conclusion

This is the first genome scale search for effectors, combined to a life-cycle expression analysis, for any plant-parasitic nematode. We show that, like other phylogenetically unrelated plant pathogens, plant parasitic nematodes deploy hundreds of effectors in order to parasitise plants, with different effectors required for different phases of the infection process.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-923) contains supplementary material, which is available to authorized users.     

植物重要功能基因研究进展及其应用

随着越来越多植物全基因组测序的完成,植物基因研究的重点将逐渐从基因的发现转移到对基因功能的研究上来。近年来,植物基因功能的阐述日益深入,尤其是与作物产量和抗性相关的重要农艺性状调控机理的研究更加引人注目,一些具有应用价值的功能基因相继被鉴定并得到功能注释。该文综述植物功能基因研究领域近年来的主要进展,着重介绍具有应用前景的重要功能基因的研究。同时,对目前利用基因工程、分子标记辅助选择等于段改良作物的现状及其前景进行讨论。     

Cellular Signaling Pathways and Posttranslational Modifications Mediated by Nematode Effector Proteins

Plant-parasitic cyst and root-knot nematodes synthesize and secrete a suite of effector proteins into infected host cells and tissues. These effectors are the major virulence determinants mediating the transformation of normal root cells into specialized feeding structures. Compelling evidence indicates that these effectors directly hijack or manipulate refined host physiological processes to promote the successful parasitism of host plants. Here, we provide an update on recent progress in elucidating the molecular functions of nematode effectors. In particular, we emphasize how nematode effectors modify plant cell wall structure, mimic the activity of host proteins, alter auxin signaling, and subvert defense signaling and immune responses. In addition, we discuss the emerging evidence suggesting that nematode effectors target and recruit various components of host posttranslational machinery in order to perturb the host signaling networks required for immunity and to regulate their own activity and subcellular localization.The root-knot (Meloidogyne spp.) and cyst (Globodera and Heterodera spp.) nematodes are sedentary endoparasites of the root system in a wide range of plant species. These obligate parasites engage in intricate relationships with their host plants that result in the transformation of normal root cells into specialized feeding sites, which provide the nematodes with all the nutrients required for their development. The initiation and maintenance of functional feeding cells by root-knot nematodes (giant cells) and cyst nematodes (syncytia) seems to be a dynamic process involving active dialogue between the nematodes and their host plants. The nematodes use their stylet, a needle-like apparatus, to deliver effector proteins into the host cells (Williamson and Hussey, 1996; Davis et al., 2004). These effector proteins are mainly synthesized in the nematode esophageal glands, which consist of one dorsal cell and two subventral cells. The activity of these glands is developmentally regulated, with secretions from the two subventral glands being most dynamic during the early stage of infection, consisting of root penetration, migration, and feeding site initiation. Secretions from the single dorsal cell seem to be more active during the sedentary stage of nematode feeding (Hussey and Mims, 1990).Recent progress in the functional characterization of effector proteins from a number of phytonematodes has elucidated diverse mechanisms through which these effectors facilitate the nematode parasitism of host plants. One such mechanism involves depolymerization of the main structural polysaccharide constituents of the plant cell wall by using a diverse collection of extracellular effector proteins (Davis et al., 2011; Wieczorek, 2015). Another mechanism includes the molecular mimicry of host proteins in both form and function (Gheysen and Mitchum, 2011). This strategy could be highly successful when the nematode-secreted effectors imitate host functions to subvert cellular processes in favor of nematodes while escaping the regulation of host cellular processes. Another mechanism of effector action is the modulation of central components of auxin signaling to apparently generate unique patterns of auxin-responsive gene expression, leading to numerous physiological and developmental changes required for feeding site formation and development (Cabrera et al., 2015). In addition, cyst and root-knot nematodes have evolved to efficiently suppress defense responses during their prolonged period of sedentary biotrophic interaction with their hosts. Accordingly, a large number of nematode effectors are engaged in suppressing host immune responses and defense signaling (Hewezi and Baum, 2013; Goverse and Smant, 2014). Finally, there is accumulating evidence that nematode effector proteins target and exploit the host posttranslational machinery to the parasite’s advantage. Posttranslational modifications (PTMs) are tightly controlled and highly specific processes that enable rapid cellular responses to specific stimuli without the requirement of new protein synthesis (Kwon et al., 2006). Phosphorylation, ubiquitination, and histone modifications, among others, have recently been identified as fundamental cellular processes controlling immune signaling pathways (Stulemeijer and Joosten, 2008; Howden and Huitema, 2012; Marino et al., 2012; Salomon and Orth, 2013). This finding underscores the importance of targeting and coopting host posttranslational machinery by pathogen effectors to exert their virulence functions. Here, we review recent progress in the functional characterization of nematode effector proteins and the parasitic strategies that involve modifications of the plant cell wall, molecular mimicry of host factors, alteration of auxin signaling, subversion of defense signaling, and targeting and utilizing the host posttranslational machinery.     

The molecular bases of plant resistance and defense responses to aphid feeding: current status

Plant genes participating in the recognition of aphid herbivory in concert with plant genes involved in defense against herbivores mediate plant resistance to aphids. Several such genes involved in plant disease and nematode resistance have been characterized in detail, but their existence has only recently begun to be determined for arthropod resistance. Hundreds of different genes are typically involved and the disruption of plant cell wall tissues during aphid feeding has been shown to induce defense responses in Arabidopsis, Triticum, Sorghum, and Nicotiana species. Mi‐1.2, a tomato gene for resistance to the potato aphid, Macrosiphum euphorbiae (Thomas), is a member of the nucleotide‐binding site and leucine‐rich region Class II family of disease, nematode, and arthropod resistance genes. Recent studies into the differential expression of Pto‐ and Pti1‐like kinase genes in wheat plants resistant to the Russian wheat aphid, Diuraphis noxia (Mordvilko), provide evidence of the involvement of the Pto class of resistance genes in arthropod resistance. An analysis of available data suggests that aphid feeding may trigger multiple signaling pathways in plants. Early signaling includes gene‐for‐gene recognition and defense signaling in aphid‐resistant plants, and recognition of aphid‐inflicted cell damage in both resistant and susceptible plants. Furthermore, signaling is mediated by several compounds, including jasmonic acid, salicylic acid, ethylene, abscisic acid, giberellic acid, nitric oxide, and auxin. These signals lead to the development of direct chemical defenses against aphids and general stress‐related responses that are well characterized for a number of abiotic and biotic stresses. In spite of major plant taxonomic differences, similarities exist in the types of plant genes expressed in response to feeding by different species of aphids. However, numerous differences in plant signaling and defense responses unique to specific aphid–plant interactions have been identified and warrant further investigation.     

Screening soybean cyst nematode effectors for their ability to suppress plant immunity

The soybean cyst nematode (SCN), Heterodera glycines, is one of the most destructive pathogens of soybeans. SCN is an obligate and sedentary parasite that transforms host plant root cells into an elaborate permanent feeding site, a syncytium. Formation and maintenance of a viable syncytium is an absolute requirement for nematode growth and reproduction. In turn, sensing pathogen attack, plants activate defence responses and may trigger programmed cell death at the sites of infection. For successful parasitism, H. glycines must suppress these host defence responses to establish and maintain viable syncytia. Similar to other pathogens, H. glycines engages in these molecular interactions with its host via effector proteins. The goal of this study was to conduct a comprehensive screen to identify H. glycines effectors that interfere with plant immune responses. We used Nicotiana benthamiana plants infected by Pseudomonas syringae and Pseudomonas fluorescens strains. Using these pathosystems, we screened 51 H. glycines effectors to identify candidates that could inhibit effector-triggered immunity (ETI) and/or pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). We identified three effectors as ETI suppressors and seven effectors as PTI suppressors. We also assessed expression modulation of plant immune marker genes as a function of these suppressors.     

New approaches to control plant parasitic nematodes

Plant parasitic nematodes are a serious threat for crop production worldwide. This review summarizes our understanding of plant nematode interactions and presents new alternatives for nematode control in the field. Breeding for resistance has been a major goal for many important crop species like soybean, potato, tomato and sugar-beet. As a result numerous nematode-resistance genes have been identified, two of which have been cloned recently, Hs1 ^pro-1 from sugar-beet, giving resistance to the beet cyst nematode Heterodera schachtii, and Mi from tomato, giving resistance to the root-knot nematode Meloidogyne incognita. Also artificial resistance genes, coding for nematotoxic proteins or causing rapid death of feeding cells, have been elucidated. In the future, genetic engineering of nematode resistance will become more and more important for plant breeding. Transformation techniques will allow genes to be quickly introduced into susceptible breeding lines and then combined with each other to produce plant varieties with durable resistance. Received: 26 August 1998 / Received revision: 16 December 1998 / Accepted: 21 December 1998