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Plant-parasitic nematodes are a major, and in some cases a dominant, threat to crop production in all agricultural systems. The relative scarcity of classical resistance genes highlights a pressing need to identify new ways to develop nematode-resistant germplasm. Here, we sequence and assemble a high-quality 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 stages of the interaction. This novel approach enabled the analysis of the hologenome of the infection site, to identify 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 the highly atypical completion of vitamin B5 biosynthesis by the parasitic animal, putatively enabled by a horizontal gene transfer from a bacterium, is critically important for parasitism. Knockout of either the plant-encoded or the now nematode-encoded steps in the pathway blocks parasitism. Our experiments establish a reference for cyst nematodes, use this platform to further our fundamental understanding of the evolution of plant-parasitism by nematodes, and show that understanding congruent differential expression of metabolic pathways represents a new way to find nematode susceptibility genes, and thereby, targets for future genome editing-mediated generation of nematode-resistant crops.

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.

Cyst and root-knot nematodes are obligate parasites of economic importance with a remarkable ability to reprogram root cells into unique metabolically active feeding sites. Previous studies have suggested a role for cytokinin in feeding site formation induced by these two types of nematodes, but the mechanistic details have not yet been described. Using Arabidopsis as a host plant species, we conducted a comparative analysis of cytokinin genes in response to the beet cyst nematode (BCN), Heterodera schachtii, and the root-knot nematode (RKN), Meloidogyne incognita. We identified distinct differences in the expression of cytokinin biosynthesis, catabolism and signaling genes in response to infection by BCN and RKN, suggesting differential manipulation of the cytokinin pathway by these two nematode species. Furthermore, we evaluated Arabidopsis histidine kinase receptor mutant lines ahk2/3, ahk2/4 and ahk3/4 in response to RKN infection. Similar to our previous studies with BCN, these lines were significantly less susceptible to RKN without compromising nematode penetration, suggesting a requirement of cytokinin signaling in RKN feeding site formation. Moreover, an analysis of ahk double mutants using CycB1;1:GUS/ahk introgressed lines revealed contrasting differences in the cytokinin receptors mediating cell cycle activation in feeding sites induced by BCN and RKN.

SummarySedentary plant-parasitic cyst nematodes are obligate biotrophs that infect the roots of their host plant. Their parasitism is based on 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 susceptibility to other pathogens or negatively affecting plant phenotype.

Plant-parasitic nematodes are destructive pests causing losses of billions of dollars annually. An effective plant defence against pathogens relies on the recognition of pathogen-associated molecular patterns (PAMPs) by surface-localised receptors leading to the activation of PAMP-triggered immunity (PTI). Extensive studies have been conducted to characterise the role of PTI in various models of plant-pathogen interactions. However, far less is known about the role of PTI in roots in general and in plant-nematode interactions in particular. Here we show that nematode-derived proteinaceous elicitor/s is/are capable of inducing PTI in Arabidopsis in a manner dependent on the common immune co-receptor BAK1. Consistent with the role played by BAK1, we identified a leucine-rich repeat receptor-like kinase, termed NILR1 that is specifically regulated upon infection by nematodes. We show that NILR1 is essential for PTI responses initiated by nematodes and nilr1 loss-of-function mutants are hypersusceptible to a broad category of nematodes. To our knowledge, NILR1 is the first example of an immune receptor that is involved in induction of basal immunity (PTI) in plants or in animals in response to nematodes. Manipulation of NILR1 will provide new options for nematode control in crop plants in future., Author summary Host perception of pathogens via receptors leads to the activation of antimicrobial defence responses in all multicellular organisms, including plants. Plant-parasitic nematodes cause significant yield losses in agriculture; therefore resistance is an important trait in crop breeding. However, not much is known about the perception of nematodes in plants. Here we identified an Arabidopsis leucine-rich repeat receptor-like kinase, NILR1 that is specifically activated upon nematode infection. We show that NILR1 is required for the induction of immune responses initiated by nematodes and nilr1 loss-of-function mutants are hypersusceptible to a broad category of nematodes. Manipulation of NILR1 will provide new options for nematode control in crop plants in the future.

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.

Plants and animals produce reactive oxygen species (ROS) in response to infection. In plants, ROS not only activate defense responses and promote cell death to limit the spread of pathogens but also restrict the amount of cell death in response to pathogen recognition. Plants also use hormones, such as salicylic acid, to mediate immune responses to infection. However, there are long-lasting biotrophic plant-pathogen interactions, such as the interaction between parasitic nematodes and plant roots during which defense responses are suppressed and root cells are reorganized to specific nurse cell systems. In plants, ROS are primarily generated by plasma membrane–localized NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidases, and loss of NADPH oxidase activity compromises immune responses and cell death. We found that infection of Arabidopsis thaliana by the parasitic nematode Heterodera schachtii activated the NADPH oxidases RbohD and RbohF to produce ROS, which was necessary to restrict infected plant cell death and promote nurse cell formation. RbohD- and RbohF-deficient plants exhibited larger regions of cell death in response to nematode infection, and nurse cell formation was greatly reduced. Genetic disruption of SID2 , which is required for salicylic acid accumulation and immune activation in nematode-infected plants, led to the increased size of nematodes in RbohD- and RbohF-deficient plants, but did not decrease plant cell death. Thus, by stimulating NADPH oxidase–generated ROS, parasitic nematodes fine-tune the pattern of plant cell death during the destructive root invasion and may antagonize salicylic acid–induced defense responses during biotrophic life stages.