Syncytium Induced by Plant-Parasitic Nematodes.

Plant-parasitic nematodes from the genera Globodera, Heterodera (cyst-forming nematodes), and Meloidogyne (root-knot nematodes) are notorious and serious pests of crops. They cause tremendous economic losses between US $80 and 358 billion a year. Nematodes infect the roots of plants and induce the formation of specialised feeding structures (syncytium and giant cells, respectively) that nourish juveniles and adults of the nematodes. The specialised secretory glands enable nematodes to synthesise and secrete effectors that facilitate migration through root tissues and alter the morphogenetic programme of host cells. The formation of feeding sites is associated with the suppression of plant defence responses and deep reprogramming of the development and metabolism of plant cells.In this chapter, we focus on syncytia induced by the sedentary cyst-forming nematodes and provide an overview of ultrastructural changes that occur in the host roots during syncytium formation in conjunction with the most important molecular changes during compatible and incompatible plant responses to infection with nematodes.

The role of AtPP2-A3 and AtPP2-A8 genes encoding Nictaba-related lectin domains in the defense response of Arabidopsis thaliana to Heterodera schachtii.

MAIN CONCLUSION: Expression levels of AtPP2-A3 and AtPP2-A8 are reduced in syncytia induced by Heterodera schachtii and decline of their expression levels decreases host susceptibility, whereas their overexpression promotes susceptibility to parasite. Plant-parasitic nematodes cause huge crop losses worldwide. Heterodera schachtii is a sedentary cyst-forming nematode that induces a feeding site called a syncytium via the delivery of secreted chemical substances (effectors) to host cells, which modulate host genes expression and phytohormone regulation patterns. Genes encoding the Nictaba-related lectin domain have been found among the plant genes with downregulated expression during the development of syncytia induced by H. schachtii in Arabidopsis thaliana roots. To investigate the role of two selected Nictaba-related genes in the plant response to beet cyst nematode parasitism, mutants and plants overexpressing AtPP2-A3 or AtPP2-A8 were infected, and promoter activity and protein localization were analyzed. In wild-type plants, AtPP2-A3 and AtPP2-A8 were expressed only in roots, especially in the cortex and rhizodermis. After nematode infection, their expression was switched off in regions surrounding a developing syncytium. Astonishingly, plants overexpressing AtPP2-A3 or AtPP2-A8 were more susceptible to nematode infection than wild-type plants, whereas mutants were less susceptible. Based on these results and changes in AtPP2-A3 and AtPP2-A8 expression patterns after treatments with different stress phytohormones, we postulate that the AtPP2-A3 and AtPP2-A8 genes play important roles in the defense response to beet cyst nematode infection.

The genome and lifestage-specific transcriptomes of a plant-parasitic nematode and its host reveal susceptibility genes involved in trans-kingdom synthesis of vitamin B5.

Plant-parasitic nematodes are a major threat to crop production in all agricultural systems. The scarcity of classical resistance genes highlights a pressing need to find new ways to develop nematode-resistant germplasm. Here, we sequence and assemble a high-quality phased genome of the model cyst nematode Heterodera schachtii to provide a platform for the first system-wide dual analysis of host and parasite gene expression over time, covering all major parasitism stages. Analysis of the hologenome of the plant-nematode infection site identified metabolic pathways that were incomplete in the parasite but complemented by the host. Using a combination of bioinformatic, genetic, and biochemical approaches, we show that a highly atypical completion of vitamin B5 biosynthesis by the parasitic animal, putatively enabled by a horizontal gene transfer from a bacterium, is required for full pathogenicity. Knockout of either plant-encoded or now nematode-encoded steps in the pathway significantly reduces parasitic success. Our experiments establish a reference for cyst nematodes, further our understanding of the evolution of plant-parasitism by nematodes, and show that congruent differential expression of metabolic pathways in the infection hologenome represents a new way to find nematode susceptibility genes. The approach identifies genome-editing-amenable targets for future development of nematode-resistant crops.

Arabidopsis thaliana AtHRS1 gene is involved in the response to Heterodera schachtii infection and its overexpression hampers development of syncytia and involves a jasmonic acid-dependent mechanism.

Sedentary plant parasitic nematodes have developed competences to reprogram host plant cell metabolism via sophisticated manipulation of gene expression, leading to the formation of permanent feeding sites for an unlimited source of food. Arabidopsis thaliana and the beet cyst nematode Heterodera schachtii is a good model for studying the mechanisms of compatible plant-nematode interactions and basic plant responses to nematode infection. Transcription factors are proteins that modulate plant reactions during regular development and under different biotic and abiotic stresses via direct binding to promoter regions of genes. Here, we report on the AtHRS1 gene encoding a MYB-related transcription factor belonging to the GARP family, whose expression is downregulated in syncytia, as confirmed by gene expression analysis. Constitutive overexpression of AtHRS1 disturbed the development of nematode-induced syncytia and led to a reduction in the number of developed females in transgenic A. thaliana roots. In contrast, the hrs1 mutant with decreased expression of AtHRS1 was more susceptible to cyst nematode infection. The influence of AtHRS1 on selected elements of the JA-dependent defence pathway suggests its mode of action in plant response to nematode attack. Based on these results, we suggest that the downregulation of AtHRS1 expression by nematode is important for its successful development.

Profiling the Proteome of Cyst Nematode-Induced Syncytia on Tomato Roots.

Cyst nematodes are important herbivorous pests in agriculture that obtain nutrients through specialized root structures termed syncytia. Syncytium initiation, development, and functioning are a research focus because syncytia are the primary interface for molecular interactions between the host plant and parasite. The small size and complex development (over approximately two weeks) of syncytia hinder precise analyses, therefore most studies have analyzed the transcriptome of infested whole-root systems or syncytia-containing root segments. Here, we describe an effective procedure to microdissect syncytia induced by Globodera rostochiensis from tomato roots and to analyze the syncytial proteome using mass spectrometry. As little as 15 mm2 of 10-µm-thick sections dissected from 30 syncytia enabled the identification of 100-200 proteins in each sample, indicating that mass-spectrometric methods currently in use achieved acceptable sensitivity for proteome profiling of microscopic samples of plant tissues (approximately 100 µg). Among the identified proteins, 48 were specifically detected in syncytia and 7 in uninfected roots. The occurrence of approximately 50% of these proteins in syncytia was not correlated with transcript abundance estimated by quantitative reverse-transcription PCR analysis. The functional categories of these proteins confirmed that protein turnover, stress responses, and intracellular trafficking are important components of the proteome dynamics of developing syncytia.

Arabidopsis thaliana Myb59 Gene Is Involved in the Response to Heterodera schachtii Infestation, and Its Overexpression Disturbs Regular Development of Nematode-Induced Syncytia.

Transcription factors are proteins that directly bind to regulatory sequences of genes to modulate and adjust plants' responses to different stimuli including biotic and abiotic stresses. Sedentary plant parasitic nematodes, such as beet cyst nematode, Heterodera schachtii, have developed molecular tools to reprogram plant cell metabolism via the sophisticated manipulation of genes expression, to allow root invasion and the induction of a sequence of structural and physiological changes in plant tissues, leading to the formation of permanent feeding sites composed of modified plant cells (commonly called a syncytium). Here, we report on the AtMYB59 gene encoding putative MYB transcription factor that is downregulated in syncytia, as confirmed by RT-PCR and a promoter pMyb59::GUS activity assays. The constitutive overexpression of AtMYB59 led to the reduction in A. thaliana susceptibility, as indicated by decreased numbers of developed females, and to the disturbed development of nematode-induced syncytia. In contrast, mutant lines with a silenced expression of AtMYB59 were more susceptible to this parasite. The involvement of ABA in the modulation of AtMYB59 gene transcription appears feasible by several ABA-responsive cis regulatory elements, which were identified in silico in the gene promoter sequence, and experimental assays showed the induction of AtMYB59 transcription after ABA treatment. Based on these results, we suggest that AtMYB59 plays an important role in the successful parasitism of H. schachtii on A. thaliana roots.

Plant parasitic cyst nematodes redirect host indole metabolism via NADPH oxidase-mediated ROS to promote infection.

Reactive oxygen species (ROS) generated in response to infections often activate immune responses in eukaryotes including plants. In plants, ROS are primarily produced by plasma membrane-bound NADPH oxidases called respiratory burst oxidase homologue (Rboh). Surprisingly, Rbohs can also promote the infection of plants by certain pathogens, including plant parasitic cyst nematodes. The Arabidopsis genome contains 10 Rboh genes (RbohA-RbohJ). Previously, we showed that cyst nematode infection causes a localised ROS burst in roots, mediated primarily by RbohD and RbohF. We also found that plants deficient in RbohD and RbohF (rbohD/F) exhibit strongly decreased susceptibility to cyst nematodes, suggesting that Rboh-mediated ROS plays a role in promoting infection. However, little information is known of the mechanism by which Rbohs promote cyst nematode infection. Here, using detailed genetic and biochemical analyses, we identified WALLS ARE THIN1 (WAT1), an auxin transporter, as a downstream target of Rboh-mediated ROS during parasitic infections. We found that WAT1 is required to modulate the host's indole metabolism, including indole-3-acetic acid levels, in infected cells and that this reprogramming is necessary for successful establishment of the parasite. In conclusion, this work clarifies a unique mechanism that enables cyst nematodes to use the host's ROS for their own benefit.

The Genomic Impact of Selection for Virulence against Resistance in the Potato Cyst Nematode, Globodera pallida.

Although the use of natural resistance is the most effective management approach against the potato cyst nematode (PCN) Globodera pallida, the existence of pathotypes with different virulence characteristics constitutes a constraint towards this goal. Two resistance sources, GpaV (from Solanum vernei) and H3 from S. tuberosum ssp. andigena CPC2802 (from the Commonwealth Potato Collection) are widely used in potato breeding programmes in European potato industry. However, the use of resistant cultivars may drive strong selection towards virulence, which allows the increase in frequency of virulent alleles in the population and therefore, the emergence of highly virulent nematode lineages. This study aimed to identify Avirulence (Avr) genes in G. pallida populations selected for virulence on the above resistance sources, and the genomic impact of selection processes on the nematode. The selection drive in the populations was found to be specific to their genetic background. At the genomic level, 11 genes were found that represent candidate Avr genes. Most of the variant calls determining selection were associated with H3-selected populations, while many of them seem to be organised in genomic islands facilitating selection evolution. These phenotypic and genomic findings combined with histological studies performed revealed potential mechanisms underlying selection in G. pallida.

Host factors influence the sex of nematodes parasitizing roots of Arabidopsis thaliana.

Plant-parasitic cyst nematodes induce hypermetabolic syncytial nurse cells in the roots of their host plants. Syncytia are their only food source. Cyst nematodes are sexually dimorphic, with their differentiation into male or female strongly influenced by host environmental conditions. Under favourable conditions with plenty of nutrients, more females develop, whereas mainly male nematodes develop under adverse conditions such as in resistant plants. Here, we developed and validated a method to predict the sex of beet cyst nematode (Heterodera schachtii) during the early stages of its parasitism in the host plant Arabidopsis thaliana. We collected root segments containing male-associated syncytia (MAS) or female-associated syncytia (FAS), isolated syncytial cells by laser microdissection, and performed a comparative transcriptome analysis. Genes belonging to categories of defence, nutrient deficiency, and nutrient starvation were over-represented in MAS as compared with FAS. Conversely, gene categories related to metabolism, modification, and biosynthesis of cell walls were over-represented in FAS. We used ß-glucuronidase analysis, qRT-PCR, and loss-of-function mutants to characterize FAS- and MAS-specific candidate genes. Our results demonstrate that various plant-based factors, including immune response, nutrient availability, and structural modifications, influence the sexual fate of the cyst nematodes.

Activity profiling of barley vacuolar processing enzymes provides new insights into the plant and cyst nematode interaction.

Vacuolar processing enzymes (VPEs) play an important role during regular growth and development and defence responses. Despite substantial attempts to understand the molecular basis of plant-cyst nematode interaction, the mechanism of VPEs functioning during this interaction remains unknown. The second-stage Heterodera filipjevi juvenile penetrates host roots and induces the formation of a permanent feeding site called a syncytium. To investigate whether infection with H. filipjevi alters plant host VPEs, the studies were performed in Hordeum vulgare roots and leaves on the day of inoculation and at 7, 14 and 21 days post-inoculation (dpi). Implementing molecular, biochemical and microscopic methods we identified reasons for modulation of barley VPE activity during interaction with H. filipjevi. Heterodera filipjevi parasitism caused a general decrease of VPE activity in infected roots, but live imaging of VPEs showed that their activity is up-regulated in syncytia at 7 and 14 dpi and down-regulated at 21 dpi. These findings were accompanied by tissue-specific VPE gene expression patterns. Expression of the barley cystatin HvCPI-4 gene was stimulated in leaves but diminished in roots upon infestation. External application of cyclotides that can be produced naturally by VPEs elicits in pre-parasitic juveniles vesiculation of their body, enhanced formation of granules, induction of exploratory behaviour (stylet thrusts and head movements), production of reactive oxygen species (ROS) and final death by methuosis. Taken together, down-regulation of VPE activity through nematode effectors promotes the nematode invasion rates and leads to avoidance of the induction of the plant proteolytic response and death of the invading juveniles.

Root endodermal barrier system contributes to defence against plant-parasitic cyst and root-knot nematodes.

Plant-parasitic nematodes (PPNs) cause tremendous yield losses worldwide in almost all economically important crops. The agriculturally most important PPNs belong to a small group of root-infecting sedentary endoparasites that includes cyst and root-knot nematodes. Both cyst and root-knot nematodes induce specialized long-term feeding structures in root vasculature from which they obtain their nutrients. A specialized cell layer in roots called the endodermis, which has cell walls reinforced with suberin deposits and a lignin-based Casparian strip (CS), protects the vascular cylinder against abiotic and biotic threats. To date, the role of the endodermis, and especially of suberin and the CS, during plant-nematode interactions was largely unknown. Here, we analyzed the role of suberin and CS during interaction between Arabidopsis plants and two sedentary root-parasitic nematode species, the cyst nematode Heterodera schachtii and the root-knot nematode Meloidogyne incognita. We found that nematode infection damages the endodermis leading to the activation of suberin biosynthesis genes at nematode infection sites. Although feeding sites induced by both cyst and root-knot nematodes are surrounded by endodermis during early stages of infection, the endodermis is degraded during later stages of feeding site development, indicating periderm formation or ectopic suberization of adjacent tissue. Chemical suberin analysis showed a characteristic suberin composition resembling peridermal suberin in nematode-infected tissue. Notably, infection assays using Arabidopsis lines with CS defects and impaired compensatory suberization, revealed that the CS and suberization impact nematode infectivity and feeding site size. Taken together, our work establishes the role of the endodermal barrier system in defence against a soil-borne pathogen.

Arabidopsis tonoplast intrinsic protein and vacuolar H+-adenosinetriphosphatase reflect vacuole dynamics during development of syncytia induced by the beet cyst nematode Heterodera schachtii.

Plant parasitic cyst nematodes induce specific hypermetabolic syncytial nurse cell structures in host roots. A characteristic feature of syncytia is the lack of the central vacuole and the formation of numerous small and larger vesicles. We show that these structures are formed de novo via widening of ER cisternae during the entire development of syncytium, whereas in advanced stages of syncytium development, larger vacuoles are also formed via fusion of vesicles/tubules surrounding organelle-free pre-vacuole regions. Immunogold transmission electron microscopy of syncytia localised the vacuolar markers E subunit of vacuolar H+-adenosinetriphosphatase (V-ATPase) complex and tonoplast intrinsic protein (γ-TIP1;1) mostly in membranes surrounding syncytial vesicles, thus indicating that these structures are vacuoles and that some of them have a lytic character. To study the function of syncytial vacuoles, changes in expression of AtVHA-B1, AtVHA-B2 and AtVHA-B3 (coding for isoforms of subunit B of V-ATPase), and TIP1;1 and TIP1;2 (coding for γ-TIP proteins) genes were analysed. RT-qPCR revealed significant downregulation of AtVHA-B2, TIP1;1 and TIP1;2 at the examined stages of syncytium development compared to uninfected roots. Expression of VHA-B1 and VHA-B3 decreased at 3 dpi but reached the level of control at 7 dpi. These results were confirmed for TIP1;1 by monitoring At-γ-TIP-YFP reporter construct expression. Infection test conducted on tip1;1 mutant plants showed formation of larger syncytia and higher numbers of females in comparison to wild-type plants indicating that reduced levels or lack of TIP1;1 protein promote nematode development.

Nucleus- and plastid-targeted annexin 5 promotes reproductive development in Arabidopsis and is essential for pollen and embryo formation.

BACKGROUND: Pollen development is a strictly controlled post-meiotic process during which microspores differentiate into microgametophytes and profound structural and functional changes occur in organelles. Annexin 5 is a calcium- and lipid-binding protein that is highly expressed in pollen grains and regulates pollen development and physiology. To gain further insights into the role of ANN5 in Arabidopsis development, we performed detailed phenotypic characterization of Arabidopsis plants with modified ANN5 levels. In addition, interaction partners and subcellular localization of ANN5 were analyzed to investigate potential functions of ANN5 at cellular level. RESULTS: Here, we report that RNAi-mediated suppression of ANN5 results in formation of smaller pollen grains, enhanced pollen lethality, and delayed pollen tube growth. ANN5 RNAi knockdown plants also displayed aberrant development during the transition from the vegetative to generative phase and during embryogenesis, reflected by delayed bolting time and reduced embryo size, respectively. At the subcellular level, ANN5 was delivered to the nucleus, nucleolus, and cytoplasm, and was frequently localized in plastid nucleoids, suggesting a likely role in interorganellar communication. Furthermore, ANN5-YFP co-immunoprecipitated with RABE1b, a putative GTPase, and interaction in planta was confirmed in plastidial nucleoids using FLIM-FRET analysis. CONCLUSIONS: Our findings let us to propose that ANN5 influences basal cell homeostasis via modulation of plastid activity during pollen maturation. We hypothesize that the role of ANN5 is to orchestrate the plastidial and nuclear genome activities via protein-protein interactions however not only in maturing pollen but also during the transition from the vegetative to the generative growth and seed development.

Expression of both Arabidopsis γ-tubulin genes is essential for development of a functional syncytium induced by Heterodera schachtii.

KEY MESSAGE: After initial up-regulation, expression of TUBG1 and TUBG2 is significantly down-regulated in mature syncytia, but lack of expression of either of γ-tubulin genes reduces numbers of nematode infections and developing females. Infective second stage juveniles of sedentary plant parasitic nematode Heterodera schachtii invade the root vascular tissue and induce a feeding site, named syncytium, formed as a result of cell hypertrophy and partial cell wall dissolution leading to a multinucleate state. Syncytium formation and maintenance involves a molecular interplay between the plant host and the developing juveniles leading to rearrangements and fragmentation of the plant cytoskeleton. In this study, we investigated the role of two Arabidopsis γ-tubulin genes (TUBG1 and TUBG2), involved in MTs nucleation during syncytium development. Expression analysis revealed that both γ-tubulin's transcript levels changed during syncytium development and after initial up-regulation (1-3 dpi) they were significantly down-regulated in 7, 10 and 15 dpi syncytia. Moreover, TUBG1 and TUBG2 showed distinct immunolocalization patterns in uninfected roots and syncytia. Although no severe changes in syncytium anatomy and ultrastructure in tubg1-1 and tubg2-1 mutants were observed compared to syncytia induced in wild-type plants, nematode infection assays revealed reduced numbers of infecting juveniles and developed female nematodes in mutant lines. Our results indicate that the expression of both TUBG1 and TUBG2 genes, although generally down-regulated in mature syncytia, is essential for successful root infection, development of functional syncytium and nematode maturation.

The Role of Programmed Cell Death Regulator LSD1 in Nematode-Induced Syncytium Formation.

Cyst-forming plant-parasitic nematodes are common pests of many crops. They inject secretions into host cells to induce the developmental and metabolic reprogramming that leads to the formation of a syncytium, which is the sole food source for growing nematodes. As in other host-parasite models, avirulence leads to rapid and local programmed cell death (PCD) known as the hypersensitive response (HR), whereas in the case of virulence, PCD is still observed but is limited to only some cells. Several regulators of PCD were analyzed to understand the role of PCD in compatible plant-nematode interactions. Thus, Arabidopsis plants carrying recessive mutations in LESION SIMULATING DISEASE1 (LSD1) family genes were subjected to nematode infection assays with juveniles of Heterodera schachtii. LSD1 is a negative and conditional regulator of PCD, and fewer and smaller syncytia were induced in the roots of lsd1 mutants than in wild-type Col-0 plants. Mutation in LSD ONE LIKE2 (LOL2) revealed a pattern of susceptibility to H. schachtii antagonistic to lsd1. Syncytia induced on lsd1 roots compared to Col0 showed significantly retarded growth, modified cell wall structure, increased vesiculation, and some myelin-like bodies present at 7 and 12 days post-infection. To place these data in a wider context, RNA-sequencing analysis of infected and uninfected roots was conducted. During nematode infection, the number of transcripts with changed expression in lsd1 was approximately three times smaller than in wild-type plants (1440 vs. 4206 differentially expressed genes, respectively). LSD1-dependent PCD in roots is thus a highly regulated process in compatible plant-nematode interactions. Two genes identified in this analysis, coding for AUTOPHAGY-RELATED PROTEIN 8F and 8H were down-regulated in syncytia in the presence of LSD1 and showed an increased susceptibility to nematode infection contrasting with lsd1 phenotype. Our data indicate that molecular regulators belonging to the LSD1 family play an important role in precise balancing of diverse PCD players during syncytium development required for successful nematode parasitism.

A cryotechnique-based method for low abundance protein immunolocalization in tomato (Solanum lycopersicum) roots infected with a nematode, Globodera rostochiensis.

Plant-parasitic cyst forming nematodes induce in host roots a specific feeding site called a syncytium. Modifications induced by the pathogen in cells incorporated into syncytium include their hypertrophy and changes in apoplast caused by over-expression of plant proteins, e.g. cellulases. As a result cell wall openings between syncytial elements are formed. The major aim of our investigation was to immunolocalize cellulases involved in these cell-wall modifications. Experiments were conducted on tomato (Solanum lycopersicum cv. "Money Maker") infected with Globodera rostochiensis. Root segments containing syncytia were processed using two techniques: conventional method of embedding in LR-White resin and cryotechnique of progressive lowering of temperature (PLT). It is believed that the latter is superior to other techniques in keeping in place cell components and preserving antigenicity of macromolecules. It is especially useful when low abundance proteins have to be immunodetected at their place of action. The main principle of the PLT technique is a stepwise lowering of temperature throughout probe dehydration, infiltration and embedding in an appropriate resin. Two-step immunolocalization and visualization using fluorochrome (FITC) at light microscopy level or colloidal gold particles at transmission electron microscopy level was performed in this study. The labeling of cellulase 7 protein at both microscopy levels was more intensive and specific on PLT-treated sections as compared to sections obtained from the classical method. Our results confirm the usefulness of the PLT cryotechnique for plant immunocytochemistry and indicate that in nematode-infected roots cellulase 7 is predominantly present in the syncytia.

Arabidopsis HIPP27 is a host susceptibility gene for the beet cyst nematode Heterodera schachtii.

Sedentary plant-parasitic cyst nematodes are obligate biotrophs that infect the roots of their host plant. Their parasitism is based on the modification of root cells to form a hypermetabolic syncytium from which the nematodes draw their nutrients. The aim of this study was to identify nematode susceptibility genes in Arabidopsis thaliana and to characterize their roles in supporting the parasitism of Heterodera schachtii. By selecting genes that were most strongly upregulated in response to cyst nematode infection, we identified HIPP27 (HEAVY METAL-ASSOCIATED ISOPRENYLATED PLANT PROTEIN 27) as a host susceptibility factor required for beet cyst nematode infection and development. Detailed expression analysis revealed that HIPP27 is a cytoplasmic protein and that HIPP27 is strongly expressed in leaves, young roots and nematode-induced syncytia. Loss-of-function Arabidopsis hipp27 mutants exhibited severely reduced susceptibility to H. schachtii and abnormal starch accumulation in syncytial and peridermal plastids. Our results suggest that HIPP27 is a susceptibility gene in Arabidopsis whose loss of function reduces plant susceptibility to cyst nematode infection without increasing the susceptibility to other pathogens or negatively affecting the plant phenotype.

Systemic changes in photosynthesis and reactive oxygen species homeostasis in shoots of Arabidopsis thaliana infected with the beet cyst nematode Heterodera schachtii.

Photosynthetic efficiency and redox homeostasis are important for plant physiological processes during regular development as well as defence responses. The second-stage juveniles of Heterodera schachtii induce syncytial feeding sites in host roots. To ascertain whether the development of syncytia alters photosynthesis and the metabolism of reactive oxygen species (ROS), chlorophyll a fluorescence measurements and antioxidant responses were studied in Arabidopsis thaliana shoots on the day of inoculation and at 3, 7 and 15 days post-inoculation (dpi). Nematode parasitism caused an accumulation of superoxide and hydrogen peroxide molecules in the shoots of infected plants at 3 dpi, probably as a result of the observed down-regulation of antioxidant enzymes. These changes were accompanied by an increase in RNA and lipid oxidation markers. The activities of antioxidant enzymes were found to be enhanced on infection at 7 and 15 dpi, and the content of anthocyanins was elevated from 3 dpi. The fluorescence parameter Rfd , defining plant vitality and the photosynthetic capacity of leaves, decreased by 11% only at 7 dpi, and non-photochemical quenching (NPQ), indicating the effectiveness of photoprotection mechanisms, was about 16% lower at 3 and 7 dpi. As a result of infection, the ultrastructure of chloroplasts was changed (large starch grains and plastoglobules), and more numerous and larger peroxisomes were observed in the mesophyll cells of leaves. We postulate that the joint action of antioxidant enzymes/molecules and photochemical mechanisms leading to the maintenance of photosynthetic efficiency promotes the fine-tuning of the infected plants to oxidative stress induced by parasitic cyst nematodes.

Protease activity and phytocystatin expression in Arabidopsis thaliana upon Heterodera schachtii infection.

The activity of plant proteases is important for amino acids recycling, removal of damaged proteins as well as defence responses. The second-stage juvenile of the beet cyst nematode Heterodera schachtii penetrates host roots and induces the feeding site called a syncytium. To determine whether infection by H. schachtii affects proteolysis, the protease activity was studied in Arabidopsis roots and shoots at the day of inoculation and 3, 7 and 15 days post inoculation (dpi). Nematode infection caused a decrease of protease activities in infected roots over the entire examination period at all studied pH values. In contrast, the activities of the low molecular mass as well as Ca2+-dependent cysteine proteases were found to be stimulated. In shoots of infected plants, the protease activity was diminished only at 15 dpi at all tested pH values. It was accompanied by changes in total soluble protein content, a higher protein carbonylation and a total polyphenol content. To go deeper into proteolysis regulation, the expression of phytocystatin genes, endogenous inhibitors of cysteine proteases, was examined in syncytia, roots and shoots. Expression of AtCYS1, AtCYS5 and AtCYS6 genes was enhanced upon cyst nematode infection. Our results suggest that changes in protease activities in roots and shoots and altered cystatin expression patterns in syncytia, roots and shoots are important for protein metabolism during cyst nematode infection.

A parasitic nematode releases cytokinin that controls cell division and orchestrates feeding site formation in host plants.

Sedentary plant-parasitic cyst nematodes are biotrophs that cause significant losses in agriculture. Parasitism is based on modifications of host root cells that lead to the formation of a hypermetabolic feeding site (a syncytium) from which nematodes withdraw nutrients. The host cell cycle is activated in an initial cell selected by the nematode for feeding, followed by activation of neighboring cells and subsequent expansion of feeding site through fusion of hundreds of cells. It is generally assumed that nematodes manipulate production and signaling of the plant hormone cytokinin to activate cell division. In fact, nematodes have been shown to produce cytokinin in vitro; however, whether the hormone is secreted into host plants and plays a role in parasitism remained unknown. Here, we analyzed the spatiotemporal activation of cytokinin signaling during interaction between the cyst nematode, Heterodera schachtii, and Arabidopsis using cytokinin-responsive promoter:reporter lines. Our results showed that cytokinin signaling is activated not only in the syncytium but also in neighboring cells to be incorporated into syncytium. An analysis of nematode infection on mutants that are deficient in cytokinin or cytokinin signaling revealed a significant decrease in susceptibility of these plants to nematodes. Further, we identified a cytokinin-synthesizing isopentenyltransferase gene in H. schachtii and show that silencing of this gene in nematodes leads to a significant decrease in virulence due to a reduced expansion of feeding sites. Our findings demonstrate the ability of a plant-parasitic nematode to synthesize a functional plant hormone to manipulate the host system and establish a long-term parasitic interaction.