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.

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.

Expression of two functionally distinct plant endo-beta-1,4-glucanases is essential for the compatible interaction between potato cyst nematode and its hosts.

For the proliferation of their feeding sites (syncytia), the potato cyst nematode Globodera rostochiensis is thought to recruit plant endo-beta-1,4-glucanases (EGases, EC. 3.2.1.4). Reverse-transcription polymerase chain reaction experiments on tomato (Solanum lycopersicum) indicated that the expression of two out of the at least eight EGases, namely Sl-cel7 and Sl-cel9C1, is specifically upregulated during syncytium formation. In situ hybridization and immunodetection studies demonstrated that both EGases are specifically expressed inside and adjacent to proliferating syncytia. To assess the importance of Sl-cel7 and Sl-cel9C1 for nematode development, we decided to knock them out individually. Sl-cel9C1 probably is the only class C EGase in tomato, and we were unable to regenerate Sl-cel9C1-silenced plants. Potato (S. tuberosum), a close relative of tomato, harbors at least two class C EGases, and St-cel7-or St-cel9C1-silenced potato plants showed no obvious aberrant phenotype. Infection with potato cyst nematodes resulted in a severe reduction of the number of adult females (up to 60%) and a sharp increase in the fraction of females without eggs (up to 89%). Hence, the recruitment of CEL7, an enzyme that uses xyloglucan and noncrystalline cellulose as natural substrates, and CEL9C1, an enzyme that uses crystalline cellulose, is essential for growth and development of potato cyst nematodes.

Two tomato alpha-expansins show distinct spatial and temporal expression patterns during development of nematode-induced syncytia.

Cyst nematodes induce specific syncytial feeding structures within the root which develop from an initial cell by successive incorporation of neighbouring cells through local cell wall dissolutions followed by hypertrophy of included cells. Expansins are known to induce cell wall relaxation and extension in acidic pH, and they are involved in many processes requiring wall modification from cell expansion to cell wall disassembly. We studied the expression pattern of tomato (Lycopersicon esculentum L., cv. Money Maker) expansins during development of syncytia induced by the potato cyst nematode (Globodera rostochiensis Woll.). Based on semi-quantitative reverse transcription-polymerase chain reaction, two expansin genes, LeEXPA4 and LeEXPA5, were selected for detailed examinations because their expression was either elevated in infected roots (LeEXPA4) or specifically induced in the root upon nematode infection (LeEXPA5). Both genes have distinct spatial and temporal expression patterns that may reflect their different roles in syncytium development. LeEXPA4 transcripts were localized predominantly in parenchymatous vascular cylinder cells surrounding syncytia. This finding suggests that LeEXPA4 might be involved in cell wall disassembly or relaxation, mediating syncytium expansion and/or development of conductive tissues. By contrast, LeEXPA5 transcripts were localized in enlarging syncytial elements. Similarly, in immunogold localization experiments, polyclonal antibodies localized the LeEXPA5 protein in cell walls of syncytial elements. This expression pattern suggests that LeEXPA5 gene is specifically involved in enlargement of cells incorporated into syncytium.

Expansins are among plant cell wall modifying agents specifically expressed during development of nematode-induced syncytia.

Cyst nematodes are economically important pests. As obligatory biotrophic endoparasites they invade host roots and induce formation of syncytia, structures that serve them as the only source of nutrients. During syncytium development, extensive cell wall modifications take place. Cell wall dissolution occurs during cell wall opening formation, cell walls expand during hypertrophy of syncytial elements and local cell wall synthesis leads to the thickening of syncytial cell wall and the formation of cell wall ingrowths. Numerous studies revealed that nematodes change expression of plant genes encoding cell wall modifying proteins including expansins. Expansins poses unique abilities to induce cell wall extension in acidic pH. Recently, we demonstrated that two alpha-expansin genes LeEXPA4 and LeEXPA5 are upregulated in tomato roots infected with potato cyst nematode (Globodera rostochiensis). In this addendum, we present the most recent results concerning involvement of plant cell wall modifying genes in syncytium development and discuss possible practical applications of this knowledge for developing plants with resistance against nematodes.