The corn smut fungus Ustilago maydis is a model organism for elucidating host colonization strategies of biotrophic fungi. Here we performed an in depth transcriptional profiling of the entire plant-associated development of U. maydis wild-type strains. In our analysis we focused on fungal metabolism, nutritional strategies, secreted effectors and regulatory networks. Secreted proteins were enriched in three distinct expression modules corresponding to stages on the plant surface, establishment of biotrophy and induction of tumors. These modules are likely the key determinants for U. maydis virulence. With respect to nutrient utilization, we observed that expression of several nutrient transporters was tied to these virulence modules rather than being controlled by nutrient availability. We show that oligopeptide transporters likely involved in nitrogen assimilation are important virulence factors. By measuring the intramodular connectivity of transcription factors, we identified the potential drivers for the virulence modules. While known components of the b-mating type cascade emerged as inducers for the plant surface and biotrophy module, we identified a set of yet uncharacterized transcription factors as likely responsible for expression of the tumor module. We demonstrate a crucial role for leaf tumor formation and effector gene expression for one of these transcription factors.

Fungi are major players in the carbon cycle in forest ecosystems due to the wide range of interactions they have with plants either through soil degradation processes by litter decayers or biotrophic interactions with pathogenic and ectomycorrhizal symbionts. Secretion of fungal proteins mediates these interactions by allowing the fungus to interact with its environment and/or host. Ectomycorrhizal (ECM) symbiosis independently appeared several times throughout evolution and involves approximately 80% of trees. Despite extensive physiological studies on ECM symbionts, little is known about the composition and specificities of their secretomes. In this study, we used a bioinformatics pipeline to predict and analyze the secretomes of 49 fungal species, including 11 ECM fungi, wood and soil decayers and pathogenic fungi to tackle the following questions: (1) Are there differences between the secretomes of saprophytic and ECM fungi? (2) Are small-secreted proteins (SSPs) more abundant in biotrophic fungi than in saprophytic fungi? and (3) Are there SSPs shared between ECM, saprotrophic and pathogenic fungi? We showed that the number of predicted secreted proteins is similar in the surveyed species, independently of their lifestyle. The secretome from ECM fungi is characterized by a restricted number of secreted CAZymes, but their repertoires of secreted proteases and lipases are similar to those of saprotrophic fungi. Focusing on SSPs, we showed that the secretome of ECM fungi is enriched in SSPs compared with other species. Most of the SSPs are coded by orphan genes with no known PFAM domain or similarities to known sequences in databases. Finally, based on the clustering analysis, we identified shared- and lifestyle-specific SSPs between saprotrophic and ECM fungi. The presence of SSPs is not limited to fungi interacting with living plants as the genome of saprotrophic fungi also code for numerous SSPs. ECM fungi shared lifestyle-specific SSPs likely involved in symbiosis that are good candidates for further functional analyses.

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.

Summary The ectomycorrhizal symbiosis is a predominant tree–microbe interaction in forest ecosystems sustaining tree growth and health. Its establishment and functioning implies a long‐term and intimate relationship between the soil‐borne fungi and the roots of trees. Mycorrhiza‐induced Small‐Secreted Proteins (MiSSPs) are hypothesized as keystone symbiotic proteins, required to set up the symbiosis by modifying the host metabolism and/or building the symbiotic interfaces. L. bicolor MiSSP8 is the third most highly induced MiSSPs in symbiotic tissues and it is also expressed in fruiting bodies. The MiSSP8‐RNAi knockdown mutants are strongly impaired in their mycorrhization ability with Populus, with the lack of fungal mantle and Hartig net development due to the lack of hyphal aggregation. MiSSP8 C‐terminus displays a repetitive motif containing a kexin cleavage site, recognized by KEX2 in vitro. This suggests MiSSP8 protein might be cleaved into small peptides. Moreover, the MiSSP8 repetitive motif is found in other proteins predicted secreted by both saprotrophic and ectomycorrhizal fungi. Thus, our data indicate that MiSSP8 is a small‐secreted protein involved at early stages of ectomycorrhizal symbiosis, likely by regulating hyphal aggregation and pseudoparenchyma formation.

Root-knot nematodes (genus Meloidogyne) are the major contributor to crop losses caused by nematodes. These nematodes secrete effector proteins into the plant, derived from two sets of pharyngeal gland cells, to manipulate host physiology and immunity. Successful completion of the life cycle, involving successive molts from egg to adult, covers morphologically and functionally distinct stages and will require precise control of gene expression, including effector genes. The details of how root-knot nematodes regulate transcription remain sparse. Here, we report a life stage-specific transcriptome of Meloidogyne incognita. Combined with an available annotated genome, we explore the spatio-temporal regulation of gene expression. We reveal gene expression clusters and predicted functions that accompany the major developmental transitions. Focusing on effectors, we identify a putative cis-regulatory motif associated with expression in the dorsal glands, providing an insight into effector regulation. We combine the presence of this motif with several other criteria to predict a novel set of putative dorsal gland effectors. Finally, we show this motif, and thereby its utility, is broadly conserved across the Meloidogyne genus, and we name it Mel-DOG. Taken together, we provide the first genome-wide analysis of spatio-temporal gene expression in a root-knot nematode and identify a new set of candidate effector genes that will guide future functional analyses.

Background The root lesion nematode Pratylenchus penetrans is a migratory plant-parasitic nematode responsible for economically important losses in a wide number of crops. Despite the importance of P. penetrans, the molecular mechanisms employed by this nematode to promote virulence remain largely unknown. Results Here we generated a new and comprehensive esophageal glands-specific transcriptome library for P. penetrans. In-depth analysis of this transcriptome enabled a robust identification of a catalogue of 30 new candidate effector genes, which were experimentally validated in the esophageal glands by in situ hybridization. We further validated the expression of a multifaceted network of candidate effectors during the interaction with different plants. To advance our understanding of the “effectorome” of P. penetrans, we adopted a phylogenetic approach and compared the expanded effector repertoire of P. penetrans to the genome/transcriptome of other nematode species with similar or contrasting parasitism strategies. Our data allowed us to infer plausible evolutionary histories that shaped the effector repertoire of P. penetrans, as well as other close and distant plant-parasitic nematodes. Two remarkable trends were apparent: 1) large scale effector birth in the Pratylenchidae in general and P. penetrans in particular, and 2) large scale effector death in sedentary (endo) plant-parasitic nematodes. Conclusions Our study doubles the number of validated Pratylenchus penetrans effectors reported in the literature. The dramatic effector gene gain in P. penetrans could be related to the remarkable ability of this nematode to parasitize a large number of plants. Our data provide valuable insights into nematode parasitism and contribute towards basic understating of the adaptation of P. penetrans and other root lesion nematodes to specific host plants.

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.

• MiSSP8 is a Laccaria-specific small-secreted protein required for early stages of ectomycorrhizal symbiosis. Our data suggest that MiSSP8 has a possible dual role: one in basic fungal biology regulating hyphal aggregation and another in planta, related to its plasmodesmata co-localization.