Lander Bauters

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.

Identification of candidate effector genes in the transcriptome of the rice root knot nematode Meloidogyne graminicola.

Plant-parasitic nematodes secrete so-called effectors into their host plant which are able to suppress the plants defence responses, alter plant signalling pathways and, in the case of root knot nematodes, induce the formation of giant cells. Putative effectors have been successfully identified by genomics, transcriptomics and proteomics approaches. In this study, we investigated the transcriptome of the rice root knot nematode Meloidogyne graminicola by 454 sequencing of second-stage juveniles as well as mRNA-seq of rice infected tissue. Over 350 000 reads derived from M. graminicola preparasitic juveniles were assembled, annotated and checked for homologues in different databases. From infected rice tissue, 1.4% of all reads generated were identified as being derived from the nematode. Using multiple strategies, several putative effector genes were identified, both pioneer genes and genes corresponding to already known effectors. To check whether these genes could be involved in the interaction with the plant, in situ hybridization was performed on a selection of genes to localize their expression in the nematode. Most were expressed in the gland cells or amphids of the nematode, confirming possible secretion of the proteins and hence a role in infection. Other putative effectors showed a different expression pattern, potentially linked with the excretory/secretory system. This transcriptome study is a good starting point to functionally investigate novel effectors derived from M. graminicola. This will lead to better insights into the interaction between these nematodes and the model plant rice. Moreover, the transcriptome can be used to identify possible target genes for RNA interference (RNAi)-based control strategies. Four genes proved to be interesting targets by showing up to 40% higher mortality relative to the control treatment when soaked in gene-specific small interfering RNAs (siRNAs).

Analysis of the transcriptome of Hirschmanniella oryzae to explore potential survival strategies and host-nematode interactions

The rice root nematode Hirschmanniella oryzae is the most abundant plant-parasitic nematode in flooded rice fields and is distributed world-wide. Although it is economically less important than sedentary nematodes, it can cause severe yield reductions and economic losses in specific environmental conditions. No transcriptome data for this genus were available until now. We have performed 454 sequencing on a mixed life stages population to gain an insight into nematode-plant interactions and nematode survival strategies. The results of two assembly strategies were combined to reduce the redundancy of the data, generating a final dataset of 21 360 contigs. The data were screened for putative plant cell wall-modifying proteins, which facilitate nematode migration through host roots. A β-mannanase, previously not reported in nematodes, was detected in the dataset. The data were screened for putative effector proteins that may alter the host defence mechanism. Two enzymes, chorismate mutase and isochorismatase, thought to be involved in the salicyclic acid pathway, were identified. Experimental treatments of H. oryzae with artificial seawater showed that late embryogenesis abundant (LEA) proteins and SXP/RAL-2 are induced, suggesting that these proteins are involved in the process of anhydrobiosis. The newly generated data can highlight potential differences between sedentary and migratory nematodes, and will be useful in the further study of host-nematode interactions and the developmental biology of this nematode.

Transcriptome Analysis and Systemic RNAi Response in the African Sweetpotato Weevil (Cylas puncticollis, Coleoptera, Brentidae)

The African sweetpotato weevil (SPW) Cylas puncticollis Boheman is one of the most important constraints of sweetpotato production in Sub-Saharan Africa and yet is largely an uncharacterized insect pest. Here, we report on the transcriptome analysis of SPW generated using an Illumina platform. More than 213 million sequencing reads were obtained and assembled into 89,599 contigs. This assembly was followed by a gene ontology annotation. Subsequently, a transcriptome search showed that the necessary RNAi components relevant to the three major RNAi pathways, were found to be expressed in SPW. To address the functionality of the RNAi mechanism in this species, dsRNA was injected into second instar larvae targeting laccase2, a gene which encodes an enzyme involved in the sclerotization of insect exoskeleton. The body of treated insects showed inhibition of sclerotization, leading eventually to death. Quantitative Real Time PCR (qPCR) confirmed this phenotype to be the result of gene silencing. Together, our results provide valuable sequence data on this important insect pest and demonstrate that a functional RNAi pathway with a strong and systemic effect is present in SPW and can further be explored as a new strategy for controlling this important pest.

Systemic suppression of the shoot metabolism upon rice root nematode infection

Hirschmanniella oryzae is the most common plant-parasitic nematode in flooded rice cultivation systems. These migratory animals penetrate the plant roots and feed on the root cells, creating large cavities, extensive root necrosis and rotting. The objective of this study was to investigate the systemic response of the rice plant upon root infection by this nematode. RNA sequencing was applied on the above-ground parts of the rice plants at 3 and 7 days post inoculation. The data revealed significant modifications in the primary metabolism of the plant shoot, with a general suppression of for instance chlorophyll biosynthesis, the brassinosteroid pathway, and amino acid production. In the secondary metabolism, we detected a repression of the isoprenoid and shikimate pathways. These molecular changes can have dramatic consequences for the growth and yield of the rice plants, and could potentially change their susceptibility to above-ground pathogens and pests.

The distribution of lectins across the phylum Nematoda : a genome-wide search

Nematodes are a very diverse phylum that has adapted to nearly every ecosystem. They have developed specialized lifestyles, dividing the phylum into free-living, animal, and plant parasitic species. Their sheer abundance in numbers and presence in nearly every ecosystem make them the most prevalent animals on earth. In this research nematode-specific profiles were designed to retrieve predicted lectin-like domains from the sequence data of nematode genomes and transcriptomes. Lectins are carbohydrate-binding proteins that play numerous roles inside and outside the cell depending on their sugar specificity and associated protein domains. The sugar-binding properties of the retrieved lectin-like proteins were predicted in silico. Although most research has focused on C-type lectin-like, galectin-like, and calreticulin-like proteins in nematodes, we show that the lectin-like repertoire in nematodes is far more diverse. We focused on C-type lectins, which are abundantly present in all investigated nematode species, but seem to be far more abundant in free-living species. Although C-type lectin-like proteins are omnipresent in nematodes, we have shown that only a small part possesses the residues that are thought to be essential for carbohydrate binding. Curiously, hevein, a typical plant lectin domain not reported in animals before, was found in some nematode species.

Gibberellin reduces the susceptibility of rice, Oryza sativa, to the migratory nematode Hirschmanniella oryzae

Upon pathogen attack, the plant defence response is mediated by a set of connected signal transduction pathways, guided by several classes of plant hormones. In this study, experiments were conducted to observe the role of the plant hormone gibberellic acid in the response of rice to infection by the migratory root-rot nematode Hirschmanniella oryzae. Foliar treatments with gibberellic acid showed a negative effect on H. oryzae infection in the roots. Analyses of mutant rice lines impaired in the production or signalling of gibberellic acid confirmed the effect of the plant hormone on H. oryzae infection. Taken together, the results clearly indicate that gibberellic acid has a positive effect on the capability of the rice plant to fend off an infection by the migratory nematode H. oryzae.

A Meloidogyne graminicola C-type lectin, Mg01965 is secreted into the host apoplast to suppress plant defense and promote parasitism

Summary C‐type lectins (CTLs), a class of multifunctional proteins, are numerous in nematodes. One CTL gene, Mg01965, shown to be expressed in the subventral glands, especially in the second‐stage juveniles of the root‐knot nematode Meloidogyne graminicola, was further analysed in this study. In vitro RNA interference targeting Mg01965 in the preparasitic juveniles significantly reduced their ability to infect host plant roots. Immunolocalizations showed that Mg01965 is secreted by M. graminicola into the roots during the early parasitic stages and accumulates in the apoplast. Transient expression of Mg01965 in Nicotiana benthamiana and targeting it to the apoplast suppressed the burst of reactive oxygen species triggered by flg22. The CTL Mg01965 suppresses plant innate immunity in the host apoplast, promoting nematode parasitism in the early infection stages.

The Meloidogyne graminicola effector Mg16820 is secreted in the apoplast and cytoplasm to suppress plant host defense responses: Meloidogyne graminicola effector Mg16820

On invasion of roots, plant-parasitic nematodes secrete effectors to manipulate the cellular regulation of the host to promote parasitism. The root-knot nematode Meloidogyne graminicola is one of the most damaging nematodes of rice. Here, we identified a novel effector of this nematode, named Mg16820, expressed in the nematode subventral glands. We localized the Mg16820 effector in the apoplast during the migration phase of the second-stage juvenile in rice roots. In addition, during early development of the feeding site, Mg16820 was localized in giant cells, where it accumulated in the cytoplasm and the nucleus. Using transient expression in Nicotiana benthamiana leaves, we demonstrated that Mg16820 directed to the apoplast was able to suppress flg22-induced reactive oxygen species production. In addition, expression of Mg16820 in the cytoplasm resulted in the suppression of the R2/Avr2- and Mi-1.2-induced hypersensitive response. A potential target protein of Mg16820 identified with the yeast two-hybrid system was the dehydration stress-inducible protein 1 (DIP1). Bimolecular fluorescence complementation resulted in a strong signal in the nucleus. DIP1 has been described as an abscisic acid (ABA)-responsive gene and ABA is involved in the biotic and abiotic stress response. Our results demonstrate that Mg16820 is able to act in two cellular compartments as an immune suppressor and targets a protein involved in the stress response, therefore indicating an important role for this effector in parasitism.

The Globodera pallida SPRYSEC effector GpSPRY-414-2 that suppresses plant defenses targets a regulatory component of the dynamic microtubule network

The white potato cyst nematode, Globodera pallida, is an obligate biotrophic pathogen of a limited number of Solanaceous plants. Like other plant pathogens, G. pallida deploys effectors into its host that manipulate the plant to the benefit of the nematode. Genome analysis has led to the identification of large numbers of candidate effectors from this nematode, including the cyst nematode-specific SPRYSEC proteins. These are a secreted subset of a hugely expanded gene family encoding SPRY domain-containing proteins, many of which remain to be characterized. We investigated the function of one of these SPRYSEC effector candidates, GpSPRY-414-2. Expression of the gene encoding GpSPRY-414-2 is restricted to the dorsal pharyngeal gland cell and reducing its expression in G. pallida infective second stage juveniles using RNA interference causes a reduction in parasitic success on potato. Transient expression assays in Nicotiana benthamiana indicated that GpSPRY-414-2 disrupts plant defenses. It specifically suppresses effector-triggered immunity (ETI) induced by co-expression of the Gpa2 resistance gene and its cognate avirulence factor RBP-1. It also causes a reduction in the production of reactive oxygen species triggered by exposure of plants to the bacterial flagellin epitope flg22. Yeast two-hybrid screening identified a potato cytoplasmic linker protein (CLIP)-associated protein (StCLASP) as a host target of GpSPRY-414-2. The two proteins co-localize in planta at the microtubules. CLASPs are members of a conserved class of microtubule-associated proteins that contribute to microtubule stability and growth. However, disruption of the microtubule network does not prevent suppression of ETI by GpSPRY-414-2 nor the interaction of the effector with its host target. Besides, GpSPRY-414-2 stabilizes its target while effector dimerization and the formation of high molecular weight protein complexes including GpSPRY-414-2 are prompted in the presence of the StCLASP. These data indicate that the nematode effector GpSPRY-414-2 targets the microtubules to facilitate infection.