Three-dimensional imaging reveals that positions of cyst nematode feeding sites relative to xylem vessels differ between susceptible and resistant wheat.

KEY MESSAGE: Resistance conferred by the Cre8 locus of wheat prevents cereal cyst nematode feeding sites from reaching and invading root metaxylem vessels. Cyst nematodes develop syncytial feeding sites within plant roots. The success of these sites is affected by host plant resistance. In wheat (Triticum aestivum L.), 'Cre' loci affect resistance against the cereal cyst nematode (CCN) Heterodera avenae. To investigate how one of these loci (Cre8, on chromosome 6B) confers resistance, CCN-infected root tissue from susceptible (-Cre8) and resistant (+Cre8) wheat plants was examined using confocal microscopy and laser ablation tomography. Confocal analysis of transverse sections showed that feeding sites in the roots of -Cre8 plants were always adjacent to metaxylem vessels, contained many intricate 'web-like' cell walls, and sometimes 'invaded' metaxylem vessels. In contrast, feeding sites in the roots of +Cre8 plants were usually not directly adjacent to metaxylem vessels, had few inner cell walls and did not 'invade' metaxylem vessels. Models based on data from laser ablation tomography confirmed these observations. Confocal analysis of longitudinal sections revealed that CCN-induced xylem modification that had previously been reported for susceptible (-Cre8) wheat plants is less extreme in resistant (+Cre8) plants. Application of a lignin-specific stain revealed that secondary thickening around xylem vessels in CCN-infected roots was greater in +Cre8 plants than in -Cre8 plants. Collectively, these results indicate that Cre8 resistance in wheat acts by preventing cyst nematode feeding sites from reaching and invading root metaxylem vessels.

Defense Response in Chickpea Pod Wall due to Simulated Herbivory Unfolds Differential Proteome Profile.

The pod wall of legumes is known to protect the developing seeds from pests and pathogens. However, the mechanism of conferring defense against insects has not yet been deciphered. Here, we have utilized 2-dimensional gel electrophoresis (2D-GE) coupled with mass spectrometry (MS/MS) to identify over expressed proteins in the pod wall of two different cultivars (commercial cultivar: JG 11 and tolerant cultivar: ICC 506-EB) of chickpea after 12 h of application of Helicoverpa armigera oral secretions (simulated herbivory). The assays were performed with a view that larvae are a voracious feeder and cause substantial damage to the pod within 12 h. A total of 600 reproducible protein spots were detected on gels, and the comparative analysis helped identify 35 (12 up-regulated, 23 down-regulated) and 20 (10 up-regulated, 10 down-regulated) differentially expressed proteins in JG 11 and ICC 506-EB, respectively. Functional classification of protein spots of each cultivar after MS/MS indicated that the differentially expressed proteins were associated with various metabolic activities. Also, stress-related proteins such as mannitol dehydrogenase (MADH), disease resistance-like protein-CSA1, serine/threonine kinase (D6PKL2), endoglucanase-19 etc. were up-regulated due to simulated herbivory. The proteins identified with a possible role in defense were further analyzed using the STRING database to advance our knowledge on their interacting partners. It decoded the involvement of several reactive oxygen species (ROS) scavengers and other proteins involved in cell wall reinforcement. The biochemical analysis also confirmed the active role of ROS scavengers during simulated herbivory. Thus, our study provides valuable new insights on chickpea-H.armigera interactions at the protein level.

Root-knot nematodes modulate cell walls during root-knot formation in Arabidopsis roots.

Phytoparasitic nematodes parasitize many species of rooting plants to take up nutrients, thus causing severe growth defects in the host plants. During infection, root-knot nematodes induce the formation of a characteristic hyperplastic structure called a root-knot or gall on the roots of host plants. Although many previous studies addressed this abnormal morphogenesis, the underlying mechanisms remain uncharacterized. To analyze the plant-microorganism interaction at the molecular level, we established an in vitro infection assay system using the nematode Meloidogyne incognita and the model plant Arabidopsis thaliana. Time-course mRNA-seq analyses indicated the increased levels of procambium-associated genes in the galls, suggesting that vascular stem cells play important roles in the gall formation. Conversely, genes involved in the formation of secondary cell walls were decreased in galls. A neutral sugar analysis indicated that the level of xylan, which is one of the major secondary cell wall components, was dramatically reduced in the galls. These observations were consistent with the hypothesis of a decrease in the number of highly differentiated cells and an increase in the density of undifferentiated cells lead to gall formation. Our findings suggest that phytoparasitic nematodes modulate the developmental mechanisms of the host to modify various aspects of plant physiological processes and establish a feeding site.

Cell Wall Modifications in Giant Cells Induced by the Plant Parasitic Nematode Meloidogyne incognita in Wild-Type (Col-0) and the fra2 Arabidopsis thaliana Katanin Mutant.

Meloidogyne incognita is a root knot nematode (RKN) species which is among the most notoriously unmanageable crop pests with a wide host range. It inhabits plants and induces unique feeding site structures within host roots, known as giant cells (GCs). The cell walls of the GCs undergo the process of both thickening and loosening to allow expansion and finally support nutrient uptake by the nematode. In this study, a comparative in situ analysis of cell wall polysaccharides in the GCs of wild-type Col-0 and the microtubule-defective fra2 katanin mutant, both infected with M. incognita has been carried out. The fra2 mutant had an increased infection rate. Moreover, fra2 roots exhibited a differential pectin and hemicellulose distribution when compared to Col-0 probably mirroring the fra2 root developmental defects. Features of fra2 GC walls include the presence of high-esterified pectic homogalacturonan and pectic arabinan, possibly to compensate for the reduced levels of callose, which was omnipresent in GCs of Col-0. Katanin severing of microtubules seems important in plant defense against M. incognita, with the nematode, however, to be nonchalant about this "katanin deficiency" and eventually induce the necessary GC cell wall modifications to establish a feeding site.

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.

A Combinational Approach of Enhanced Methanol Production and Double Bt Genes for Broad Spectrum Insect Resistance in Transgenic Cotton.

The prevalence of insect resistance against Bt toxins has led to the idea of enhancing demethylation from cell wall pectin by pectin methylesterase enzyme for overproduction of methanol which is toxic to insects pests. The AtPME and AnPME fragments ligated into pCAMBIA1301 vector were confirmed through restriction digestion with EcoR1 and BamH1. Excision of 3363 bp fragment from 11,850 bp vector confirmed the ligation of both fragments into pCAMBIA1301 vector. Transformation of pectin methylesterase-producing genes, i.e., AtPME and AnPME from Arabidopsis thaliana and Aspergillus niger cloned in plant expression vector pCAMBIA1301 under 35S promoter into cotton variety CEMB-33 harboring two Bt genes Cry1Ac and Cry2A, respectively, was done by using shoot apex-cut Agrobacterium-mediated transformation method. The plantlets were screened on MS medium supplemented with hygromycin on initial basis. Amplification of 412 and 543 bp, respectively, through gene-specific primer has been obtained which confirmed the successful introduction of pCAMBIA AtPME and AnPME genes into cotton variety CEMB 33. Relative expression of AtPME and AnPME genes through real-time PCR determined the expression level of both gene ranges between 3- and 3.5-fold in different transgenic cotton lines along with quantity of methanol ranging from 0.8 to 0.9% of maximum while 0.5% to 0.6% of minimum but no expression was obtained in negative non-transgenic control cotton plant with least quantity of methanol, i.e., 0.1%. Almost 100% mortality was observed in insect bioassay for Helicoverpa armigera on detached leaves bioassay and 63% for Pink Bollworm (Pectinophora gossypiella) on growing transgenic cotton bolls as compared to positive control transgenic cotton with double Bt genes where mortality was found to be 82% for H. armigera and 50% for P. gossypiella while 0% in negative control non-transgenic plants.

Characterization of the gut microbiota of invasive Agrilus mali Matsumara (Coleoptera: Buprestidae) using high-throughput sequencing: uncovering plant cell-wall degrading bacteria.

The genus Agrilus comprises diverse exotic and agriculturally important wood-boring insects that have evolved efficient digestive systems. Agrilus mali Matsumara, an invasive insect, is causing extensive mortality to endangered wild apple trees in Tianshan. In this study, we present an in-depth characterization of the gut microbiota of A. mali based on high-throughput sequencing of the 16S rRNA gene and report the presence of lignocellulose-degrading bacteria. Thirty-nine operational taxonomic units (OTUs) were characterized from the larval gut. OTUs represented 6 phyla, 10 classes, 16 orders, 20 families, and 20 genera. The majority of bacterial OTUs belonged to the order Enterobacteriales which was the most abundant taxa in the larval gut. Cultivable bacteria revealed 9 OTUs that all belonged to Gammaproteobacteria. Subsequently, we examined the breakdown of plant cell-wall compounds by bacterial isolates. Among the isolates, the highest efficiency was observed in Pantoea sp., which was able to synthesize four out of the six enzymes (cellulase, cellobiase, ß-xylanase, and ß-gluconase) responsible for plant-cell wall degradation. One isolate identified as Pseudomonas orientalis exhibited lignin peroxidase activity. Our study provides the first characterization of the gut microbial diversity of A. mali larvae and shows that some cultivable bacteria play a significant role in the digestive tracts of larvae by providing nutritional needs.

Host-specific signatures of the cell wall changes induced by the plant parasitic nematode, Meloidogyne incognita.

Root-knot nematodes (Meloidogyne spp.) are an important group of plant parasitic nematodes that induce within host plant roots unique feeding site structures, termed giant cells, which supply nutrient flow to the nematode. A comparative in situ analysis of cell wall polysaccharides in the giant cells of three host species (Arabidopsis, maize and aduki bean) infected with Meloidogyne incognita has been carried out. Features common to giant cell walls of all three species include the presence of high-esterified pectic homogalacturonan, xyloglucan and pectic arabinan. The species-specific presence of xylan and mixed-linkage glucan (MLG) epitopes in giant cell walls of maize reflected that host's taxonomic group. The LM5 galactan and LM21 mannan epitopes were not detected in the giant cell walls of aduki bean but were detected in Arabidopsis and maize giant cell walls. The LM2 arabinogalactan-protein epitope was notable for its apparent global variations in root cell walls as a response to infection across the three host species. Additionally, a set of Arabidopsis cell wall mutants were used to determine any impacts of altered cell wall structures on M. incognita infection. Disruption of the arabinogalactan-protein 8 gene had the greatest impact and resulted in an increased infection rate.

Spatiotemporal deep imaging of syncytium induced by the soybean cyst nematode Heterodera glycines.

Parasite infections cause dramatic anatomical and ultrastructural changes in host plants. Cyst nematodes are parasites that invade host roots and induce a specific feeding structure called a syncytium. A syncytium is a large multinucleate cell formed by cell wall dissolution-mediated cell fusion. The soybean cyst nematode (SCN), Heterodera glycines, is a major soybean pathogen. To investigate SCN infection and the syncytium structure, we established an in planta deep imaging system using a clearing solution ClearSee and two-photon excitation microscopy (2PEM). Using this system, we found that several cells were incorporated into the syncytium; the nuclei increased in size and the cell wall openings began to be visible at 2 days after inoculation (DAI). Moreover, at 14 DAI, in the syncytium developed in the cortex, there were thickened concave cell wall pillars that resembled "Parthenon pillars." In contrast, there were many thick board-like cell walls and rarely Parthenon pillars in the syncytium developed in the stele. We revealed that the syncytia were classified into two types based on the pattern of the cell wall structures, which appeared to be determined by the position of the syncytium inside roots. Our results provide new insights into the developmental process of syncytium induced by cyst nematode and a better understanding of the three-dimensional structure of the syncytium in host roots.

Identifying Virulence-Associated Genes Using Transcriptomic and Proteomic Association Analyses of the Plant Parasitic Nematode Bursaphelenchus mucronatus.

Bursaphelenchus mucronatus (B. mucronatus) isolates that originate from different regions may vary in their virulence, but their virulence-associated genes and proteins are poorly understood. Thus, we conducted an integrated study coupling RNA-Seq and isobaric tags for relative and absolute quantitation (iTRAQ) to analyse transcriptomic and proteomic data of highly and weakly virulent B. mucronatus isolates during the pathogenic processes. Approximately 40,000 annotated unigenes and 5000 proteins were gained from the isolates. When we matched all of the proteins with their detected transcripts, a low correlation coefficient of r = 0.138 was found, indicating probable post-transcriptional gene regulation involved in the pathogenic processes. A functional analysis showed that five differentially expressed proteins which were all highly expressed in the highly virulent isolate were involved in the pathogenic processes of nematodes. Peroxiredoxin, fatty acid- and retinol-binding protein, and glutathione peroxidase relate to resistance against plant defence responses, while ß-1,4-endoglucanase and expansin are associated with the breakdown of plant cell walls. Thus, the pathogenesis of B. mucronatus depends on its successful survival in host plants. Our work adds to the understanding of B. mucronatus' pathogenesis, and will aid in controlling B. mucronatus and other pinewood nematode species complexes in the future.

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.

Covalent cross-linking of cell-wall polysaccharides through esterified diferulates as a maize resistance mechanism against corn borers.

There is strong evidence to suggest that cross-linking of cell-wall polymers through ester-linked diferulates has a key role in plant resistance to pests; however, direct experimentation to provide conclusive proof is lacking. This study presents an evaluation of the damage caused by two corn borer species on six maize populations particularly selected for divergent diferulate concentrations in pith stem tissues. Maize populations selected for high total diferulate concentration had 31% higher diferulates than those selected for low diferulates. Stem tunneling by corn borer species was 29% greater in the population with the lowest diferulates than in the population with the highest diferulates (31.7 versus 22.6 cm), whereas total diferulate concentration was negatively correlated with stem tunneling by corn borers. Moreover, orthogonal contrasts between groups of populations evaluated showed that larvae fed in laboratory bioassays on pith stem tissues from maize populations with higher diferulates had 30-40% lower weight than larvae fed on the same tissues from maize populations with lower diferulates. This is the first report that shows a direct relationship between diferulate deposition in maize cell walls and corn borer resistance. Current findings will help to develop adapted maize varieties with an acceptable level of resistance against borers and be useful in special kinds of agriculture, such as organic farming.

Specifically expressed genes of the nematode Bursaphelenchus xylophilus involved with early interactions with pine trees.

As the causal agent of pine wilt disease (PWD), the pine wood nematode (PWN), Bursaphelenchus xylophilus, causes huge economic losses by devastating pine forests worldwide. However, the pathogenesis-related genes of B. xylophilus are not well characterized. Thus, DNA microarrays were used to investigate differential gene expression in PWN where Pinus thunbergii was inoculated with nematodes, compared with those cultured on Botrytis cinerea. The microarrays comprised 31121 probes, 1310 (4.2%) of which were differentially regulated (changes of >2-fold, P < 0.01) in the two growth conditions. Of these 1310 genes, 633 genes were upregulated, whereas 677 genes were downregulated. Gene Ontology (GO) categories were assigned to the classes Cellular Component, Molecular Function, and Biological Process. The comparative gene expression analysis showed that a large number of the pathogenesis-related genes of B. xylophilus, such as pectate lyase genes, cytochrome P450s, UGTs, and ABC transporter genes, were highly expressed when B. xylophilus infected P. thunbergii. Annotation analysis indicated that these genes contributed to cell wall degradation, detoxification, and the reproduction process. The microarray results were validated using quantitative RT-PCR (qRT-PCR). The microarray data confirmed the specific expression of B. xylophilus genes during infection of P. thunbergii, which provides basic information that facilitates a better understanding of the molecular mechanism of PWD.

crw1--A novel maize mutant highly susceptible to foliar damage by the western corn rootworm beetle.

Western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is the most destructive insect pest of corn (Zea mays L.) in the United States. The adult WCR beetles derive their nourishment from multiple sources including corn pollen and silks as well as the pollen of alternate hosts. Conversely, the corn foliage is largely neglected as a food source by WCR beetles, leading to a perception of a passive interaction between the two. We report here a novel recessive mutation of corn that was identified and named after its foliar susceptibility to corn rootworm beetles (crw1). The crw1 mutant under field conditions was exceptionally susceptible to foliar damage by WCR beetles in an age-specific manner. It exhibits pleiotropic defects on cell wall biochemistry, morphology of leaf epidermal cells and lower structural integrity via differential accumulation of cell wall bound phenolic acids. These findings indicate that crw1 is perturbed in a pathway that was not previously ascribed to WCR susceptibility, as well as implying the presence of an active mechanism(s) deterring WCR beetles from devouring corn foliage. The discovery and characterization of this mutant provides a unique opportunity for genetic analysis of interactions between maize and adult WCR beetles and identify new strategies to control the spread and invasion of this destructive pest.

Adição da parede celular de levedura (Saccharomyces cerevisiae) na dieta para frangos de corte / Addition of the yeast cell wall (Saccharomyces cerevisiae) in the diet for broilers chickens

Os efeitos da parede celular de Saccharomyces cerevisiae (PCSc) na dieta para frangos foram avaliados com base nos tratamentos: dieta referência com avilamicina (AV); dieta referência (DR); DR com 0,1% de PCSc (PCSc0,1); DR com 0,2% de PCSc (PCSc0,2); DR com 0,3% de PCSc (PCSc0,3). O delineamento foi o de blocos ao acaso, com cinco tratamentos e seis repetições de 10 aves, totalizando 300 frangos, machos. No período de nove a 21 dias de idade, o tratamento PCSc0,3 influenciou negativamente no ganho de peso (631g), e no período de 34 a 39 dias, o tratamento PCSc0,1 proporcionou melhor ganho de peso (461g) em relação ao tratamento com o antimicrobiano. O maior peso vivo ao abate, 2571g, foi obtido no tratamento PCSc0,1. Os pesos absolutos de sobrecoxa e coxa+sobrecoxa foram maiores nos tratamentos PCSc0,1 (290g e 560g) e PCSc0,2 (292g e 561g) e menores no tratamento PCSc0,3 (263g e 515g). Não houve influência na metabolizabilidade da ração e na contagem total de coliformes totais no íleo. Dietas com inclusão de até 0,2% de PCSc resultaram em índices produtivos adequados, podendo ser utilizadas como aditivo em dietas livres de antimicrobiano melhorador de desempenho sem o comprometimento do desempenho, características de carcaça, metabolizabilidade da ração e da contagem total de coliformes totais do íleo.

The effects of the yeast cell wall (Saccharomyces cerevisiae) (CWSc) in the diet of broiler chickens were evaluated through the following treatments: reference diet with avilamicin (AV); reference diet (RD); RD with 0.1% CWSc (CWSc0,1); RD with 0.2% CWSc (CWSc0,2); RD with 0.3% CWSc (CWSc0,3). The study design was randomized blocks, with five treatments and six replicates of 10 birds, totaling 300 male broilers. In the period from 9 to 21 days treatment CWSc0.3 had a negative influence on weight gain (631g) and from 34 to 39 days CWSc0.1 provided better weight gain (461g) compared to the treatment with the antimicrobial. The highest live weight value at slaughter, 2,571 grams, was obtained in treatment CWSc0.1. The absolute weights of drumstick and thigh+drumstick were higher in treatments CWSc0.1 (290g and 560g) and CWSc0.2 (292g and 561g) and were lower for CWSc0.3 (263g and 515g). There was no influence of the treatments on both the metabolizability and the total count of coliforms in the ileum. Diets until 0.2% of PCSc inclusion resulted in correct productive rates and they can be used as a ration growth promoter additive in antimicrobial free diets without damage on performance, carcass characteristics, organ weight, metabolizability of the diets and total coliform count in the ileum.

Nematode feeding sites: unique organs in plant roots.

Although generally unnoticed, nearly all crop plants have one or more species of nematodes that feed on their roots, frequently causing tremendous yield losses. The group of sedentary nematodes, which are among the most damaging plant-parasitic nematodes, cause the formation of special organs called nematode feeding sites (NFS) in the root tissue. In this review we discuss key metabolic and cellular changes correlated with NFS development, and similarities and discrepancies between different types of NFS are highlighted.

The cell walls of syncytia formed by Heterodera schachtii in Arabidopsis thaliana are abundant in methyl-esterified pectin.

Plant-parasitic cyst nematodes form a specialized feeding site, termed a syncytium, in the roots of host plants. Monoclonal antibodies to defined glycans, in addition to a cellulose-binding module, were used to characterize the cell walls of a functioning syncytia in situ. Cell walls of syncytia were found to contain cellulose, xyloglucan and mannan. Analysis of the pectin network revealed syncytial cell walls are abundant in homogalacturonan, which was heavily methyl-esterified. Arabinan was also detected and the results suggest the cell walls of syncytia are highly flexible.

The novel cyst nematode effector protein 19C07 interacts with the Arabidopsis auxin influx transporter LAX3 to control feeding site development.

Plant-parasitic cyst nematodes penetrate plant roots and transform cells near the vasculature into specialized feeding sites called syncytia. Syncytia form by incorporating neighboring cells into a single fused cell by cell wall dissolution. This process is initiated via injection of esophageal gland cell effector proteins from the nematode stylet into the host cell. Once inside the cell, these proteins may interact with host proteins that regulate the phytohormone auxin, as cellular concentrations of auxin increase in developing syncytia. Soybean cyst nematode (Heterodera glycines) Hg19C07 is a novel effector protein expressed specifically in the dorsal gland cell during nematode parasitism. Here, we describe its ortholog in the beet cyst nematode (Heterodera schachtii), Hs19C07. We demonstrate that Hs19C07 interacts with the Arabidopsis (Arabidopsis thaliana) auxin influx transporter LAX3. LAX3 is expressed in cells overlying lateral root primordia, providing auxin signaling that triggers the expression of cell wall-modifying enzymes, allowing lateral roots to emerge. We found that LAX3 and polygalacturonase, a LAX3-induced cell wall-modifying enzyme, are expressed in the developing syncytium and in cells to be incorporated into the syncytium. We observed no decrease in H. schachtii infectivity in aux1 and lax3 single mutants. However, a decrease was observed in both the aux1lax3 double mutant and the aux1lax1lax2lax3 quadruple mutant. In addition, ectopic expression of 19C07 was found to speed up lateral root emergence. We propose that Hs19C07 most likely increases LAX3-mediated auxin influx and may provide a mechanism for cyst nematodes to modulate auxin flow into root cells, stimulating cell wall hydrolysis for syncytium development.

The plant apoplasm is an important recipient compartment for nematode secreted proteins.

Similarly to microbial pathogens, plant-parasitic nematodes secrete into their host plants proteins that are essential to establish a functional interaction. Identifying the destination of nematode secreted proteins within plant cell compartment(s) will provide compelling clues on their molecular functions. Here the fine localization of five nematode secreted proteins was analysed throughout parasitism in Arabidopsis thaliana. An immunocytochemical method was developed that preserves both the host and the pathogen tissues, allowing the localization of nematode secreted proteins within both organisms. One secreted protein from the amphids and three secreted proteins from the subventral oesophageal glands involved in protein degradation and cell wall modification were secreted in the apoplasm during intercellular migration and to a lower extent by early sedentary stages during giant cell formation. Conversely, another protein produced by both subventral and dorsal oesophageal glands in parasitic stages accumulated profusely at the cell wall of young and mature giant cells. In addition, secretion of cell wall-modifying proteins by the vulva of adult females suggested a role in egg laying. The study shows that the plant apoplasm acts as an important destination compartment for proteins secreted during migration and during sedentary stages of the nematode.