Annelies Haegeman

Functional roles of effectors of plant-parasitic nematodes

Plant pathogens have evolved a variety of different strategies that allow them to successfully infect their hosts. Plant-parasitic nematodes secrete numerous proteins into their hosts. These proteins, called effectors, have various functions in the plant cell. The most studied effectors to date are the plant cell wall degrading enzymes, which have an interesting evolutionary history since they are believed to have been acquired from bacteria or fungi by horizontal gene transfer. Extensive genome, transcriptome and proteome studies have shown that plant-parasitic nematodes secrete many additional effectors. The function of many of these is less clear although during the last decade, several research groups have determined the function of some of these effectors. Even though many effectors remain to be investigated, it has already become clear that they can have very diverse functions. Some are involved in suppression of plant defences, while others can specifically interact with plant signalling or hormone pathways to promote the formation of nematode feeding sites. In this review, the most recent progress in the understanding of the function of plant-parasitic nematode effectors is discussed.

Horizontal gene transfer in nematodes: a catalyst for plant parasitism?

The origin of plant parasitism within the phylum Nematoda is intriguing. The ability to parasitize plants has originated independently at least three times during nematode evolution and, as more molecular data has emerged, it has become clear that multiple instances of horizontal gene transfer (HGT) from bacteria and fungi have played a crucial role in the nematodes adaptation to this new lifestyle. The first reported HGT cases in plant-parasitic nematodes were genes encoding plant cell wall-degrading enzymes. Other putative examples of HGT were subsequently described, including genes that may be involved in the modulation of the plants defense system, the establishment of a nematode feeding site, and the synthesis or processing of nutrients. Although, in many cases, it is difficult to pinpoint the donor organism, candidate donors are usually soil dwelling and are either plant-pathogenic or plant-associated microorganisms, hence occupying the same ecological niche as the nematodes. The exact mechanisms of transfer are unknown, although close contacts with donor microorganisms, such as symbiotic or trophic interactions, are a possibility. The widespread occurrence of horizontally transferred genes in evolutionarily independent plant-parasitic nematode lineages suggests that HGT may be a prerequisite for successful plant parasitism in nematodes.

An endosymbiotic bacterium in a plant-parasitic nematode: member of a new Wolbachia supergroup.

Wolbachia is an endosymbiotic bacterium widely present in arthropods and animal-parasitic nematodes. Despite previous efforts, it has never been identified in plant-parasitic nematodes. Random sequencing of genes expressed by the burrowing nematode Radopholus similis resulted in several sequences with similarity to Wolbachia genes. The presence of a Wolbachia-like endosymbiont in this plant-parasitic nematode was investigated using both morphological and molecular approaches. Transmission electronmicroscopy, fluorescent immunolocalisation and staining with DAPI confirmed the presence of the endosymbiont within the reproductive tract of female adults. 16S rDNA, ftsZ and groEL gene sequences showed that the endosymbiont of R. similis is distantly related to the known Wolbachia supergroups. Finally, based on our initial success in finding sequences of this endosymbiont by screening an expressed sequence tag (EST) dataset, all nematode ESTs were mined for Wolbachia-like sequences. Although the retained sequences belonged to six different nematode species, R. similis was the only plant-parasitic nematode with traces of Wolbachia. Based on our phylogenetic study and the current literature we designate the endosymbiont of R. similis to a new supergroup (supergroup I) rather than considering it as a new species. Although its role remains unknown, the endosymbiont was found in all individuals tested, pointing towards an essential function of the bacteria.

Transcriptional reprogramming by root knot and migratory nematode infection in rice

Rice is one of the most important staple crops worldwide, but its yield is compromised by different pathogens, including plant-parasitic nematodes. In this study we have characterized specific and general responses of rice (Oryza sativa) roots challenged with two endoparasitic nematodes with very different modes of action. Local transcriptional changes in rice roots upon root knot (Meloidogyne graminicola) and root rot nematode (RRN, Hirschmanniella oryzae) infection were studied at two time points (3 and 7 d after infection, dai), using mRNA-seq. Our results confirm that root knot nematodes (RKNs), which feed as sedentary endoparasites, stimulate metabolic pathways in the root, and enhance nutrient transport towards the induced root gall. The migratory RRNs, on the other hand, induce programmed cell death and oxidative stress, and obstruct the normal metabolic activity of the root. While RRN infection causes up-regulation of biotic stress-related genes early in the infection, the sedentary RKNs suppress the local defense pathways (e.g. salicylic acid and ethylene pathways). Interestingly, hormone pathways mainly involved in plant development were strongly induced (gibberellin) or repressed (cytokinin) at 3 dai. These results uncover previously unrecognized nematode-induced expression profiles related to their specific infection strategy.

Transcriptional analysis through RNA sequencing of giant cells induced by Meloidogyne graminicola in rice roots

One of the reasons for the progressive yield decline observed in aerobic rice production is the rapid build-up of populations of the rice root knot nematode Meloidogyne graminicola. These nematodes induce specialized feeding cells inside root tissue, called giant cells. By injecting effectors in and sipping metabolites out of these cells, they reprogramme normal cell development and deprive the plant of its nutrients. In this research we have studied the transcriptome of giant cells in rice, after isolation of these cells by laser-capture microdissection. The expression profiles revealed a general induction of primary metabolism inside the giant cells. Although the roots were shielded from light induction, we detected a remarkable induction of genes involved in chloroplast biogenesis and tetrapyrrole synthesis. The presence of chloroplast-like structures inside these dark-grown cells was confirmed by confocal microscopy. On the other hand, genes involved in secondary metabolism and more specifically, the majority of defence-related genes were strongly suppressed in the giant cells. In addition, significant induction of transcripts involved in epigenetic processes was detected inside these cells 7 days after infection.

Analysis of the transcriptome of the root lesion nematode Pratylenchus coffeae generated by 454 sequencing technology

To study interactions between plants and plant-parasitic nematodes, several omics studies have nowadays become extremely useful. Since most data available so far is derived from sedentary nematodes, we decided to improve the knowledge on migratory nematodes by studying the transcriptome of the nematode Pratylenchus coffeae through generating expressed sequence tags (ESTs) on a 454 sequencing platform. In this manuscript we present the generation, assembly and annotation of over 325,000 reads from P. coffeae. After assembling these reads, 56,325 contigs and singletons with an average length of 353bp were selected for further analyses. Homology searches revealed that 25% of these sequences had significant matches to the Swiss-prot/trEMBL database and 29% had significant matches in nematode ESTs. Over 10,000 sequences were successfully annotated, corresponding to over 6000 unique Gene Ontology identifiers and 5000 KEGG orthologues. Different approaches led to the identification of different sequences putatively involved in the parasitism process. Several plant cell wall modifying enzymes were identified, including an arabinogalactan galactosidase, so far identified in cyst nematodes only. Additionally, some new putative cell wall modifying enzymes are present belonging to GHF5 and GHF16, although further functional studies are needed to determine the true role of these proteins. Furthermore, a homologue to a chorismate mutase was found, suggesting that this parasitism gene has a wider occurrence in plant-parasitic nematodes than previously assumed. Finally, the dataset was searched for orthologues against the Meloidogyne genomes and genes involved in the RNAi pathway. In conclusion, the generated transcriptome data of P. coffeae will be very useful in the future for several projects: (1) evolutionary studies of specific gene families, such as the plant cell wall modifying enzymes, (2) the identification and functional analysis of candidate effector genes, (3) the development of new control strategies, e.g. by finding new targets for RNAi and (4) the annotation of the upcoming genome sequence.

Expressed sequence tags of the peanut pod nematode Ditylenchus africanus: The first transcriptome analysis of an Anguinid nematode

In this study, 4847 expressed sequenced tags (ESTs) from mixed stages of the migratory plant-parasitic nematode Ditylenchus africanus (peanut pod nematode) were investigated. It is the first molecular survey of a nematode which belongs to the family of the Anguinidae (order Rhabditida, superfamily Sphaerularioidea). The sequences were clustered into 2596 unigenes, of which 43% did not show any homology to known protein, nucleotide, nematode EST or plant-parasitic nematode genome sequences. Gene ontology mapping revealed that most putative proteins are involved in developmental and reproductive processes. In addition unigenes involved in oxidative stress as well as in anhydrobiosis, such as LEA (late embryogenesis abundant protein) and trehalose-6-phosphate synthase were identified. Other tags showed homology to genes previously described as being involved in parasitism (expansin, SEC-2, calreticulin, 14-3-3b and various allergen proteins). In situ hybridization revealed that the expression of a putative expansin and a venom allergen protein was restricted to the gland cell area of the nematode, being in agreement with their presumed role in parasitism. Furthermore, seven putative novel candidate parasitism genes were identified based on the prediction of a signal peptide in the corresponding protein sequence and homologous ESTs exclusively in parasitic nematodes. These genes are interesting for further research and functional characterization. Finally, 34 unigenes were retained as good target candidates for future RNAi experiments, because of their nematode specific nature and observed lethal phenotypes of Caenorhabditis elegans homologs.

Evolution of GHF5 endoglucanase gene structure in plant-parasitic nematodes: no evidence for an early domain shuffling event

BackgroundEndo-1,4-beta-glucanases or cellulases from the glycosyl hydrolase family 5 (GHF5) have been found in numerous bacteria and fungi, and recently also in higher eukaryotes, particularly in plant-parasitic nematodes (PPN). The origin of these genes has been attributed to horizontal gene transfer from bacteria, although there still is a lot of uncertainty about the origin and structure of the ancestral GHF5 PPN endoglucanase. It is not clear whether this ancestral endoglucanase consisted of the whole gene cassette, containing a catalytic domain and a carbohydrate-binding module (CBM, type 2 in PPN and bacteria) or only of the catalytic domain while the CBM2 was retrieved by domain shuffling later in evolution. Previous studies on the evolution of these genes have focused primarily on data of sedentary nematodes, while in this study, extra data from migratory nematodes were included.ResultsTwo new endoglucanases from the migratory nematodes Pratylenchus coffeae and Ditylenchus africanus were included in this study. The latter one is the first gene isolated from a PPN of a different superfamily (Sphaerularioidea); all previously known nematode endoglucanases belong to the superfamily Tylenchoidea (order Rhabditida). Phylogenetic analyses were conducted with the PPN GHF5 endoglucanases and homologous endoglucanases from bacterial and other eukaryotic lineages such as beetles, fungi and plants. No statistical incongruence between the phylogenetic trees deduced from the catalytic domain and the CBM2 was found, which could suggest that both domains have evolved together. Furthermore, based on gene structure data, we inferred a model for the evolution of the GHF5 endoglucanase gene structure in plant-parasitic nematodes. Our data confirm a close relationship between Pratylenchus spp. and the root knot nematodes, while some Radopholus similis endoglucanases are more similar to cyst nematode genes.ConclusionWe conclude that the ancestral PPN GHF5 endoglucanase gene most probably consisted of the whole gene cassette, i.e. the GHF5 catalytic domain and the CBM2, rather than that it evolved by domain shuffling. Our evolutionary model for the gene structure in PPN GHF5 endoglucanases implies the occurrence of an early duplication event, and more recent gene duplications at genus or species level.

Characterization of a putative endoxylanase in the migratory plant-parasitic nematode Radopholus similis

Plant-parasitic nematodes have developed an arsenal of enzymes to degrade the rigid plant cell wall. In this article, we report the presence of a putative endoxylanase in the migratory endoparasitic nematode Radopholus similis. This enzyme is thought to facilitate the migration of the nematode, as it breaks down xylan, the major component of hemicellulose. The corresponding gene (Rs-xyl1) was cloned and the sequence revealed three small introns. Interestingly, the position of all three introns was conserved in a putative endoxylanase from Meloidogyne hapla, and the position of one intron was conserved in two endoxylanases from Meloidogyne incognita, which suggests a common ancestral gene. The spatial and temporal expression of the Rs-xyl1 gene was examined by in situ hybridization and semi-quantitative reverse transcriptase-polymerase chain reaction. The putative protein consists of a signal peptide, a catalytic domain and a carbohydrate-binding module (CBM). The catalytic domain showed similarity to both glycosyl hydrolase family 5 (GHF5) and GHF30 enzymes. Using Hidden Markov Model profiles and phylogenetic analysis, we were able to show that Rs-XYL1 and its closest homologues are not members of GHF5, as suggested previously, but rather form a subclass within GHF30. Silencing the putative endoxylanase by double-stranded RNA targeting of the CBM region resulted in an average decrease in infection of 60%, indicating that the gene is important for the nematode to complete its life cycle.

Comparing systemic defence‐related gene expression changes upon migratory and sedentary nematode attack in rice

Complex defence signalling pathways, controlled by different hormones, are known to be involved in the reaction of plants to a wide range of biotic and abiotic stress factors. Here, we studied the differential expression of genes involved in stress and defence responses in systemic tissue of rice infected with the root knot nematode (RKN) Meloidogyne graminicola and the migratory root rot nematode Hirschmanniella oryzae, two agronomically important rice pathogens with very different lifestyles. qRT-PCR revealed that all investigated systemic tissues had significantly lower expression of isochorismate synthase, a key enzyme for salicylic acid production involved in basal defence and systemic acquired resistance. The systemic defence response upon migratory nematode infection was remarkably similar to fungal rice blast infection. Almost all investigated defence-related genes were up-regulated in rice shoots 3 days after root rot nematode attack, including the phenylpropanoid pathway, ethylene pathway and PR genes, but many of which were suppressed at 7 dpi. Systemic shoot tissue of RKN-infected plants showed similar attenuation of expression of almost all studied genes already at 3 dpi, with clear attenuation of the ethylene pathway and methyl jasmonate biosynthesis. These results provide an interesting starting point for further studies to elucidate how nematodes are able to suppress systemic plant defence mechanisms and the effect in multitrophic interactions.