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Funding: The work at University of Bonn was supported by the Federal Ministry of Education and Research, Germany (BMBF) (Grant 031A326B to FMWG) and by the German Research Foundation (DFG) (Grant SI1739/3–1 and SI1739/5-1 to S.S.). M.S.H. was supported by a fellowship from German Academic Exchange Service (DAAD; Grant 91525252)). The work at University of California Davis was supported by the National Science Foundation (NSF) (Grant IOS-1954929) and National Institute of Food and Agriculture (NIFA) (Grant 20-3994). Work on plant-parasitic nematodes at the University of Cambridge is supported by DEFRA license 125034/359149/3, and funded by BBSRC grants BB/R011311/1, BB/N021908/1, and BB/S006397/1. C.P. received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 882941. P.T. was supported by the University of St Andrews Bioinformatics Unit, funded by Wellcome Trust ISSF awards 105621/Z/14/Z and 204821/Z/16/Z. Work at Iowa State University was supported by Hatch and State of Iowa funds and a grant from the North Central Soybean Research Program. J.L.L.-T. was supported by an NWO domain Applied and Engineering Sciences VENI grant (14250) and a Wageningen University & Research Experimental Plant Sciences strategic funds grant. M.G.S. was supported by NWO domain Applied and Engineering Sciences VENI grant (17282). 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. Publisher PDF

Cyst nematodes (CNs) are an important group of root-infecting sedentary endoparasites that severely damage many crop plants worldwide. An infective CN juvenile enters the host's roots and migrates towards the vascular cylinder, where it induces the formation of syncytial feeding cells, which nourish the CN throughout its parasitic stages. Here, we examined the role of glutathione (l-γ-glutamyl-l-cysteinyl-glycine) in Arabidopsis thaliana on infection with the CN Heterodera schachtii. Arabidopsis lines with mutations pad2, cad2, or zir1 in the glutamate-cysteine ligase (GSH1) gene, which encodes the first enzyme in the glutathione biosynthetic pathway, displayed enhanced CN susceptibility, but susceptibility was reduced for rax1, another GSH1 allele. Biochemical analysis revealed differentially altered thiol levels in these mutants that was independent of nematode infection. All glutathione-deficient mutants exhibited impaired activation of defence marker genes as well as genes for biosynthesis of the antimicrobial compound camalexin early in infection. Further analysis revealed a link between glutathione-mediated plant resistance to CN infection and the production of camalexin on nematode infection. These results suggest that glutathione levels affect plant resistance to CN by fine-tuning the balance between the cellular redox environment and the production of compounds related to defence against infection.

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.

Cyst nematodes (CNs) are an important group of root-infecting sedentary endoparasites that severely damage many crop plants worldwide. An infective CN juvenile enters the host’s roots and migrates towards the vascular cylinder, where it induces the formation of syncytial feeding cells, which nourish the CN throughout its parasitic stages. Here, we examined the role of glutathione (L-γ-glutamyl-L-cysteinylglycine, GSH) in Arabidopsis thaliana upon infection with the CN Heterodera schachtii. Arabidopsis lines with mutations pad2, cad2, or zir1 in the glutamate–cysteine ligase (GSH1) gene, which encodes the first enzyme in the glutathione biosynthetic pathway, displayed enhanced CN susceptibility, but susceptibility was reduced for rax1, another GSH1 allele. Biochemical analysis revealed differentially altered thiol levels in these mutants that was independent of nematode infection. All GSH-deficient mutants exhibited impaired activation of defense marker genes as well as genes for biosynthesis of the antimicrobial compound camalexin early in infection. Further analysis revealed a link between glutathione-mediated plant susceptibility to CN infection and the production of camalexin upon nematode infection. These results suggest that GSH levels affects plant susceptibility to CN by fine-tuning the balance between the cellular redox environment and the production of compounds related to defense against 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.

Competitive binding and complex formation of MBW proteins has a functional relevance for anthocyanidin production and trichome development across a range of different species, which can be explained by changes in one amino acid., A protein complex consisting of a MYB, basic Helix-Loop-Helix, and a WDR protein, the MBW complex, regulates five traits, namely the production of anthocyanidin, proanthocyanidin, and seed-coat mucilage, and the development of trichomes and root hairs. For complexes involved in trichome and root hair development it has been shown that the interaction of two MBW proteins can be counteracted by the respective third protein (called competitive complex formation). We examined competitive complex formation for selected MBW proteins from Arabidopsis thaliana, Arabis alpina, Gossypium hirsutum, Petunia hybrida, and Zea mays. Quantitative analyses of the competitive binding of MYBs and WDRs to bHLHs were done by pull-down assays using ProtA- and luciferase-tagged proteins expressed in human HEC cells. We found that some bHLHs show competitive complex formation whilst others do not. Competitive complex formation strongly correlated with a phylogenetic tree constructed with the bHLH proteins under investigation, suggesting a functional relevance. We demonstrate that this different behavior can be explained by changes in one amino acid and that this position is functionally relevant in trichome development but not in anthocyanidin regulation.

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.

Background In Arabidopsis thaliana (A. thaliana) the WD40 protein TRANSPARENT TESTA GLABRA1 (TTG1) controls five traits relevant for the adaptation of plants to environmental changes including the production of proanthocyanidin, anthocyanidin, seed coat mucilage, trichomes and root hairs. The analysis of different Brassicaceae species suggests that the function of TTG1 is conserved within the family. Results In this work, we studied the function of TTG1 in Arabis alpina (A. alpina). A comparison of wild type and two Aattg1 alleles revealed that AaTTG1 is involved in the regulation of all five traits. A detailed analysis of the five traits showed striking phenotypic differences between A. alpina and A. thaliana such that trichome formation occurs also at later stages of leaf development and that root hairs form at non-root hair positions. Conclusions The evolutionary conservation of the regulation of the five traits by TTG1 on the one hand and the striking phenotypic differences make A. alpina a very interesting genetic model system to study the evolution of TTG1-dependent gene regulatory networks at a functional level.