The ectomycorrhizal fungus Pisolithus microcarpus encodes a microRNA involved in cross-kingdom gene silencing during symbiosis

Small RNAs (sRNAs) are known to regulate pathogenic plant–microbe interactions. Emerging evidence from the study of these model systems suggests that microRNAs (miRNAs) can be translocated between microbes and plants to facilitate symbiosis. The roles of sRNAs in mutualistic mycorrhizal fungal interactions, however, are largely unknown. In this study, we characterized miRNAs encoded by the ectomycorrhizal fungus Pisolithus microcarpus and investigated their expression during mutualistic interaction with Eucalyptus grandis. Using sRNA sequencing data and in situ miRNA detection, a novel fungal miRNA, Pmic_miR-8, was found to be transported into E. grandis roots after interaction with P. microcarpus. Further characterization experiments demonstrate that inhibition of Pmic_miR-8 negatively impacts the maintenance of mycorrhizal roots in E. grandis, while supplementation of Pmic_miR-8 led to deeper integration of the fungus into plant tissues. Target prediction and experimental testing suggest that Pmic_miR-8 may target the host NB-ARC domain containing transcripts, suggesting a potential role for this miRNA in subverting host signaling to stabilize the symbiotic interaction. Altogether, we provide evidence of previously undescribed cross-kingdom sRNA transfer from ectomycorrhizal fungi to plant roots, shedding light onto the involvement of miRNAs during the developmental process of mutualistic symbioses.

Exploiting Structural Modelling Tools to Explore Host-Translocated Effector Proteins

Oomycete and fungal interactions with plants can be neutral, symbiotic or pathogenic with different impact on plant health and fitness. Both fungi and oomycetes can generate so-called effector proteins in order to successfully colonize the host plant. These proteins modify stress pathways, developmental processes and the innate immune system to the microbes’ benefit, with a very different outcome for the plant. Investigating the biological and functional roles of effectors during plant–microbe interactions are accessible through bioinformatics and experimental approaches. The next generation protein modeling software RoseTTafold and AlphaFold2 have made significant progress in defining the 3D-structure of proteins by utilizing novel machine-learning algorithms using amino acid sequences as their only input. As these two methods rely on super computers, Google Colabfold alternatives have received significant attention, making the approaches more accessible to users. Here, we focus on current structural biology, sequence motif and domain knowledge of effector proteins from filamentous microbes and discuss the broader use of novel modelling strategies, namely AlphaFold2 and RoseTTafold, in the field of effector biology. Finally, we compare the original programs and their Colab versions to assess current strengths, ease of access, limitations and future applications.

Heterospecific Neighbor Plants Impact Root Microbiome Diversity and Molecular Function of Root Fungi

Within the forest community, competition and facilitation between adjacent-growing conspecific and heterospecific plants are mediated by interactions involving common mycorrhizal networks. The ability of plants to alter their neighbor’s microbiome is well documented, but the molecular biology of plant-fungal interactions during competition and facilitation has not been previously examined. We used a common soil-plant bioassay experiment to study molecular plant-microbial interactions among rhizosphere communities associated with Pinus taeda (native host) and Populus trichocarpa (non-native host). Gene expression of interacting fungal and bacterial rhizosphere communities was compared among three plant-pairs: Populus growing with Populus, Populus with Pinus, and Pinus with Pinus. Our results demonstrate that heterospecific plant partners affect the assembly of root microbiomes, including the changes in the structure of host specific community. Comparative metatranscriptomics reveals that several species of ectomycorrhizal fungi (EMF) and saprotrophic fungi exhibit different patterns of functional and regulatory gene expression with these two plant hosts. Heterospecific plants affect the transcriptional expression pattern of EMF host-specialists (e.g., Pinus-associated Suillus spp.) on both plant species, mainly including the genes involved in the transportation of amino acids, carbohydrates, and inorganic ions. Alteration of root microbiome by neighboring plants may help regulate basic plant physiological processes via modulation of molecular functions in the root microbiome.

Methods for Studying Bacterial–Fungal Interactions in the Microenvironments of Soil

Due to their small size, microorganisms directly experience only a tiny portion of the environmental heterogeneity manifested in the soil. The microscale variations in soil properties constrain the distribution of fungi and bacteria, and the extent to which they can interact with each other, thereby directly influencing their behavior and ecological roles. Thus, to obtain a realistic understanding of bacterial–fungal interactions, the spatiotemporal complexity of their microenvironments must be accounted for. The objective of this review is to further raise awareness of this important aspect and to discuss an overview of possible methodologies, some of easier applicability than others, that can be implemented in the experimental design in this field of research. The experimental design can be rationalized in three different scales, namely reconstructing the physicochemical complexity of the soil matrix, identifying and locating fungi and bacteria to depict their physical interactions, and, lastly, analyzing their molecular environment to describe their activity. In the long term, only relevant experimental data at the cell-to-cell level can provide the base for any solid theory or model that may serve for accurate functional prediction at the ecosystem level. The way to this level of application is still long, but we should all start small.

Metabolic Profiling and Metabolite Correlation Network Analysis Reveal That Fusarium solani Induces Differential Metabolic Responses in Lotus japonicus and Lotus tenuis against Severe Phosphate Starvation

Root fungal endophytes are essential mediators of plant nutrition under mild stress conditions. However, variations in the rhizosphere environment, such as nutrient depletion, could result in a stressful situation for both partners, shifting mutualistic to nonconvenient interactions. Mycorrhizal fungi and dark septate endophytes (DSEs) have demonstrated their ability to facilitate phosphate (Pi) acquisition. However, few studies have investigated other plant–fungal interactions that take place in the root environment with regard to phosphate nutrition. In the present research work, we aimed to analyze the effect of extreme Pi starvation and the fungal endophyte Fusarium solani on the model Lotus japonicus and the crop L. tenuis. We conducted metabolomics analysis based on gas chromatography-mass spectrometry (GC-MS) on plant tissues under optimal conditions, severe Pi starvation and F.solani presence. By combining statistical and correlation network analysis strategies, we demonstrated the differential outcomes of the two plant species against the combination of treatments. The combination of nutritional stress and Fusarium presence activated significant modifications in the metabolism of L. japonicus affecting the levels of sugars, polyols and some amino acids. Our results display potential markers for further inspection of the factors related to plant nutrition and plant–fungal interactions.

Succession and potential role of bacterial communities during Pleurotus ostreatus production

ABSTRACT There is an increasing interest in studying bacterial-fungal interactions (BFIs), also the interactions of Pleurotus ostreatus, a model white-rot fungus and important cultivated mushroom. In Europe, P. ostreatus is produced on a wheat straw-based substrate with a characteristic bacterial community, where P. ostreatus is exposed to the microbiome during substrate colonisation. This study investigated how the bacterial community structure was affected by the introduction of P. ostreatus into the mature substrate. Based on the results obtained, the effect of the presence and absence of this microbiome on P. ostreatus production in an experimental cultivation setup was determined. 16S rRNA gene-based terminal restriction fragment length polymorphism (T-RFLP) and amplicon sequencing revealed a definite succession of the microbiome during substrate colonisation and fruiting body production: a sharp decrease in relative abundance of Thermus spp. and Actinobacteria, and the increasing dominance of Bacillales and Halomonas spp. The introduced experimental cultivation setup proved the protective role of the microbial community against competing fungi without affecting P. ostreatus growth. We could also demonstrate that this effect could be attributed to both living microbes and their secreted metabolites. These findings highlight the importance of bacterial-fungal interactions during mushroom production.

Conserved secreted effectors determine endophytic growth and multi-host plant compatibility in a vascular wilt fungus

Fungal interactions with plant roots, either beneficial or detrimental, have a major impact on agriculture and ecosystems. The soil inhabiting ascomycete Fusarium oxysporum (Fo) constitutes a species complex of worldwide distribution causing vascular wilt in more than a hundred different crops. Individual isolates of the fungus exhibit host-specific pathogenicity, determined by proteinaceous effectors termed secreted in xylem (SIX). However, such isolates can also colonize roots of non-host plants asymptomatically as endophytes, or even protect them against pathogenic isolates. The molecular determinants of multi-host plant colonization are currently unknown. Here, we identified a set of fungal effectors termed ERCs (Early Root Compatibility effectors), which are secreted during early biotrophic growth of Fo on both host and non-host plants. In contrast to the strain-specific SIX effectors, which are encoded on accessory genomic regions, ERCs are encoded on core regions and are found across the entire Fo species complex as well as in other phytopathogens, suggesting a conserved role in fungus-plant associations. Targeted deletion of ERC genes in a pathogenic Fo isolate resulted in reduced virulence on the host plant and rapid activation of plant immune responses, while in a non-pathogenic isolate it led to impaired root colonization and loss of biocontrol ability. Strikingly, some ERCs also contribute to Fo infection on the non-vascular land plant Marchantia polymorpha. Our results reveal an evolutionarily conserved mechanism for multi-host colonization by root infecting fungi.

Fungal Pathogens in Grasslands

Grasslands are major primary producers and function as major components of important watersheds. Although a concise definition of grasslands cannot be given using a physiognomic or structural approach, grasslands can be described as vegetation communities experiencing periodical droughts and with canopies dominated by grasses and grass-like plants. Grasslands have a cosmopolitan distribution except for the Antarctic region. Fungal interactions with grasses can be pathogenic or symbiotic. Herbivorous mammals, insects, other grassland animals, and fungal pathogens are known to play important roles in maintaining the biomass and biodiversity of grasslands. Although most pathogenicity studies on the members of Poaceae have been focused on economically important crops, the plant-fungal pathogenic interactions involved can extend to the full range of ecological circumstances that exist in nature. Hence, it is important to delineate the fungal pathogen communities and their interactions in man-made monoculture systems and highly diverse natural ecosystems. A better understanding of the key fungal players can be achieved by combining modern techniques such as next-generation sequencing (NGS) together with studies involving classic phytopathology, taxonomy, and phylogeny. It is of utmost importance to develop experimental designs that account for the ecological complexity of the relationships between grasses and fungi, both above and below ground. In grasslands, loss in species diversity increases interactions such as herbivory, mutualism, predation or infectious disease transmission. Host species density and the presence of heterospecific host species, also affect the disease dynamics in grasslands. Many studies have shown that lower species diversity increases the severity as well as the transmission rate of fungal diseases. Moreover, communities that were once highly diverse but have experienced decreased species richness and dominancy have also shown higher pathogenicity load due to the relaxed competition, although this effect is lower in natural communities. This review addresses the taxonomy, phylogeny, and ecology of grassland fungal pathogens and their interactions in grassland ecosystems.