RNA silencing is a key regulatory mechanism in the biocontrol fungus Clonostachys rosea-wheat interactions.

BACKGROUND: Small RNA (sRNAs)- mediated RNA silencing is emerging as a key player in host-microbe interactions. However, its role in fungus-plant interactions relevant to biocontrol of plant diseases is yet to be explored. This study aimed to investigate Dicer (DCL)-mediated endogenous and cross-kingdom gene expression regulation in the biocontrol fungus Clonostachys rosea and wheat roots during interactions. RESULTS: C. rosea Δdcl2 strain exhibited significantly higher root colonization than the WT, whereas no significant differences were observed for Δdcl1 strains. Dual RNA-seq revealed the upregulation of CAZymes, membrane transporters, and effector coding genes in C. rosea, whereas wheat roots responded with the upregulation of stress-related genes and the downregulation of growth-related genes. The expression of many of these genes was downregulated in wheat during the interaction with DCL deletion strains, underscoring the influence of fungal DCL genes on wheat defense response. sRNA sequencing identified 18 wheat miRNAs responsive to C. rosea, and three were predicted to target the C. rosea polyketide synthase gene pks29. Two of these miRNAs (mir_17532_x1 and mir_12061_x13) were observed to enter C. rosea from wheat roots with fluorescence analyses and to downregulate the expression of pks29, showing plausible cross-kingdom RNA silencing of the C. rosea gene by wheat miRNAs. CONCLUSIONS: We provide insights into the mechanisms underlying the interaction between biocontrol fungi and plant roots. Moreover, the study sheds light on the role of sRNA-mediated gene expression regulation in C. rosea-wheat interactions and provides preliminary evidence of cross-kingdom RNA silencing between plants and biocontrol fungi.

Transcriptomic analysis identifies candidate genes for Aphanomyces root rot disease resistance in pea.

BACKGROUND: Aphanomyces euteiches is a soil-borne oomycete that causes root rot in pea and other legume species. Symptoms of Aphanomyces root rot (ARR) include root discoloration and wilting, leading to significant yield losses in pea production. Resistance to ARR is known to be polygenic but the roles of single genes in the pea immune response are still poorly understood. This study uses transcriptomics to elucidate the immune response of two pea genotypes varying in their levels of resistance to A. euteiches. RESULTS: In this study, we inoculated roots of the pea (P. sativum L.) genotypes 'Linnea' (susceptible) and 'PI180693' (resistant) with two different A. euteiches strains varying in levels of virulence. The roots were harvested at 6 h post-inoculation (hpi), 20 hpi and 48 hpi, followed by differential gene expression analysis. Our results showed a time- and genotype-dependent immune response towards A. euteiches infection, involving several WRKY and MYB-like transcription factors, along with genes associated with jasmonic acid (JA) and abscisic acid (ABA) signaling. By cross-referencing with genes segregating with partial resistance to ARR, we identified 39 candidate disease resistance genes at the later stage of infection. Among the genes solely upregulated in the resistant genotype 'PI180693', Psat7g091800.1 was polymorphic between the pea genotypes and encoded a Leucine-rich repeat receptor-like kinase reminiscent of the Arabidopsis thaliana FLAGELLIN-SENSITIVE 2 receptor. CONCLUSIONS: This study provides new insights into the gene expression dynamics controlling the immune response of resistant and susceptible pea genotypes to A. euteiches infection. We present a set of 39 candidate disease resistance genes for ARR in pea, including the putative immune receptor Psat7g091800.1, for future functional validation.

Several secondary metabolite gene clusters in the genomes of ten Penicillium spp. raise the risk of multiple mycotoxin occurrence in chestnuts.

Penicillium spp. produce a great variety of secondary metabolites, including several mycotoxins, on food substrates. Chestnuts represent a favorable substrate for Penicillium spp. development. In this study, the genomes of ten Penicillium species, virulent on chestnuts, were sequenced and annotated: P. bialowiezense. P. pancosmium, P. manginii, P. discolor, P. crustosum, P. palitans, P. viridicatum, P. glandicola, P. taurinense and P. terrarumae. Assembly size ranges from 27.5 to 36.8 Mb and the number of encoded genes ranges from 9,867 to 12,520. The total number of predicted biosynthetic gene clusters (BGCs) in the ten species is 551. The most represented families of BGCs are non ribosomal peptide synthase (191) and polyketide synthase (175), followed by terpene synthases (87). Genome-wide collections of gene phylogenies (phylomes) were reconstructed for each of the newly sequenced Penicillium species allowing for the prediction of orthologous relationships among our species, as well as other 20 annotated Penicillium species available in the public domain. We investigated in silico the presence of BGCs for 10 secondary metabolites, including 5 mycotoxins, whose production was validated in vivo through chemical analyses. Among the clusters present in this set of species we found andrastin A and its related cluster atlantinone A, mycophenolic acid, patulin, penitrem A and the cluster responsible for the synthesis of roquefortine C/glandicoline A/glandicoline B/meleagrin. We confirmed the presence of these clusters in several of the Penicillium species conforming our dataset and verified their capacity to synthesize them in a chestnut-based medium with chemical analysis. Interestingly, we identified mycotoxin clusters in some species for the first time, such as the andrastin A cluster in P. flavigenum and P. taurinense, and the roquefortine C cluster in P. nalgiovense and P. taurinense. Chestnuts proved to be an optimal substrate for species of Penicillium with different mycotoxigenic potential, opening the door to risks related to the occurrence of multiple mycotoxins in the same food matrix.

Functional analysis of the apple fruit microbiome based on shotgun metagenomic sequencing of conventional and organic orchard samples.

Fruits harbour abundant and diverse microbial communities that protect them from post-harvest pathogens. Identification of functional traits associated with a given microbiota can provide a better understanding of their potential influence. Here, we focused on the epiphytic microbiome of apple fruit. We suggest that shotgun metagenomic data can indicate specific functions carried out by different groups and provide information on their potential impact. Samples were collected from the surface of 'Golden Delicious' apples from four orchards that differ in their geographic location and management practice. Approximately 1 million metagenes were predicted based on a high-quality assembly. Functional profiling of the microbiome of fruits from orchards differing in their management practice revealed a functional shift in the microbiota. The organic orchard microbiome was enriched in pathways involved in plant defence activities; the conventional orchard microbiome was enriched in pathways related to the synthesis of antibiotics. The functional significance of the variations was explored using microbial network modelling algorithms to reveal the metabolic role of specific phylogenetic groups. The analysis identified several associations supported by other published studies. For example, the analysis revealed the nutritional dependencies of the Capnodiales group, including the Alternaria pathogen, on aromatic compounds.

Comparative Small RNA and Degradome Sequencing Provides Insights into Antagonistic Interactions in the Biocontrol Fungus Clonostachys rosea.

Necrotrophic mycoparasitism is an intricate process involving recognition, physical mycelial contact, and killing of host fungi (mycohosts). During such interactions, mycoparasites undergo a complex developmental process involving massive regulatory changes of gene expression to produce a range of chemical compounds and proteins that contribute to the parasitism of the mycohosts. Small RNAs (sRNAs) are vital components of posttranscriptional gene regulation, although their role in gene expression regulation during mycoparasitisms remain understudied. Here, we investigated the role of sRNA-mediated gene regulation in mycoparasitism by performing sRNA and degradome tag sequencing of the mycoparasitic fungus Clonostachys rosea interacting with the plant-pathogenic mycohosts Botrytis cinerea and Fusarium graminearum at two time points. The majority of differentially expressed sRNAs were downregulated during the interactions with the mycohosts compared to a C. rosea self-interaction control, thus allowing desuppression (upregulation) of mycohost-responsive genes. Degradome analysis showed a positive correlation between high degradome counts and antisense sRNA mapping and led to the identification of 201 sRNA-mediated potential gene targets for 282 differentially expressed sRNAs. Analysis of sRNA potential gene targets revealed that the regulation of genes coding for membrane proteins was a common response against both mycohosts. The regulation of genes involved in oxidative stress tolerance and cellular metabolic and biosynthetic processes was exclusive against F. graminearum, highlighting common and mycohost-specific gene regulation of C. rosea. By combining these results with transcriptome data collected during a previous study, we expand the understanding of the role of sRNA in regulating interspecific fungal interactions and mycoparasitism. IMPORTANCE Small RNAs (sRNAs) are emerging as key players in pathogenic and mutualistic fungus-plant interactions; however, their role in fungus-fungus interactions remains elusive. In this study, we employed the necrotrophic mycoparasite Clonostachys rosea and the plant-pathogenic mycohosts Botrytis cinerea and Fusarium graminearum and investigated the sRNA-mediated gene regulation in mycoparasitic interactions. The combined approach of sRNA and degradome tag sequencing identified 201 sRNA-mediated putative gene targets for 282 differentially expressed sRNAs, highlighting the role of sRNA-mediated regulation of mycoparasitism in C. rosea. We also identified 36 known and 13 novel microRNAs (miRNAs) and their potential gene targets at the endogenous level and at a cross-species level in B. cinerea and F. graminearum, indicating a role of cross-species RNA interference (RNAi) in mycoparasitism, representing a novel mechanism in biocontrol interactions. Furthermore, we showed that C. rosea adapts its transcriptional response, and thereby its interaction mechanisms, based on the interaction stages and identity of the mycohost.

Sequencing of non-virulent strains of Fusarium fujikuroi reveals genes putatively involved in bakanae disease of rice.

Bakanae, one of the most important diseases of rice, is caused by the fungal pathogen Fusarium fujikuroi. The elongation of internodes is the most common symptom induced by the pathogen, and it is related to the production of gibberellins. Despite this, the pathogenicity mechanism of F. fujikuroi is still not completely clear, and there are some strains inducing stunting instead of elongation. Even if there are relatively many genomes of F. fujikuroi strains available in online databases, none of them belongs to an isolate of proven non-virulence, and therefore there has been no comparative genomics study conducted between virulent and non-virulent strains. In the present work, the genomes of non-virulent strain SG4 and scarcely virulent strain C2S were compared to the ones of 12 available virulent isolates. Genes present in the majority of available virulent strains, but not in the non-virulent one, underwent functional annotation with multiple tools, and their expression level during rice infection was checked using pre-existing data. Nine genes putatively related to pathogenicity in F. fujikuroi were identified throughout comparative and functional analyses. Among these, many are involved in the degradation of plant cell wall, which is poorly studied in F. fujikuroi-rice interactions. Three of them were validated through qPCR, showing higher expression in the virulent strain and low to no expression in the low virulent and non virulent strains during rice infection. This work helps to clarify the mechanisms of pathogenicity of F. fujikuroi on rice.

Different Phenotypes, Similar Genomes: Three Newly Sequenced Fusarium fujikuroi Strains Induce Different Symptoms in Rice Depending on Temperature.

Bakanae, caused by the hemibiotrophic fungus Fusarium fujikuroi, is one of the most important diseases of rice and is attributed to up to 75% of losses, depending on the strain and environmental conditions. Some strains cause elongation and thin leaves, whereas others induce stunting and chlorotic seedlings. Differences in symptoms are attributed to genetic differences in the strains. F. fujikuroi strains Augusto2, CSV1, and I1.3 were sequenced with Illumina MiSeq, and pathogenicity trials were conducted on rice cultivar Galileo, which is susceptible to bakanae. By performing gene prediction, single nucleotide polymorphism (SNP) calling, and structural variant analysis with a reference genome, we show how an extremely limited number of polymorphisms in genes not commonly associated with bakanae disease can cause strong differences in phenotype. CSV1 and Augusto2 were particularly close, with only 21,887 SNPs between them, but they differed in virulence, reaction to temperature, induced symptoms, colony morphology and color, growth speed, fumonisin, and gibberellin production. Genes potentially involved in the shift in phenotype were identified. Furthermore, we show how temperature variation may result in different symptoms even in rice plants inoculated with the same F. fujikuroi strain. Moreover, all of the F. fujikuroi strains became more virulent at higher temperatures. Significant differences were likewise observed in gibberellic acid production and in the expression of both fungal and plant gibberellin biosynthetic genes.

Transcriptomic and functional analyses on a Botrytis cinerea multidrug-resistant (MDR) strain provides new insights into the potential molecular mechanisms of MDR and fitness.

Botrytis cinerea is a notorious pathogen causing pre- and post-harvest spoilage in many economically important crops. Excessive application of site-specific fungicides to control the pathogen has led to the selection of strains possessing target site alterations associated with resistance to these fungicides and/or strains overexpressing efflux transporters associated with multidrug resistance (MDR). MDR in B. cinerea has been correlated with the overexpression of atrB and mfsM2, encoding an ATP-binding cassette (ABC) and a major facilitator superfamily (MFS) transporter, respectively. However, it remains unknown whether other transporters may also contribute to the MDR phenotype. In the current study, the transcriptome of a B. cinerea multidrug-resistant (MDR) field strain was analysed upon exposure to the fungicide fludioxonil, and compared to the B05.10 reference strain. The transcriptome of this field strain displayed significant differences as compared to B05.10, including genes involved in sugar membrane transport, toxin production and virulence. Among the induced genes in the field strain, even before exposure to fludioxonil, were several putatively encoding ABC and MFS transmembrane transporters. Overexpression of a highly induced MFS transporter gene in the B05.10 strain led to an increased tolerance to the fungicides fluopyram and boscalid, indicating an involvement in efflux transport of these compounds. Overall, the data from this study give insights towards better understanding the molecular mechanisms involved in MDR and fitness cost, contributing to the development of more efficient control strategies against this pathogen.

Insights into the ecological generalist lifestyle of Clonostachys fungi through analysis of their predicted secretomes.

Introduction: The fungal secretome comprise diverse proteins that are involved in various aspects of fungal lifestyles, including adaptation to ecological niches and environmental interactions. The aim of this study was to investigate the composition and activity of fungal secretomes in mycoparasitic and beneficial fungal-plant interactions. Methods: We used six Clonostachys spp. that exhibit saprotrophic, mycotrophic and plant endophytic lifestyles. Genome-wide analyses was performed to investigate the composition, diversity, evolution and gene expression of Clonostachys secretomes in relation to their potential role in mycoparasitic and endophytic lifestyles. Results and discussion: Our analyses showed that the predicted secretomes of the analyzed species comprised between 7 and 8% of the respective proteomes. Mining of transcriptome data collected during previous studies showed that 18% of the genes encoding predicted secreted proteins were upregulated during the interactions with the mycohosts Fusarium graminearum and Helminthosporium solani. Functional annotation of the predicted secretomes revealed that the most represented protease family was subclass S8A (11-14% of the total), which include members that are shown to be involved in the response to nematodes and mycohosts. Conversely, the most numerous lipases and carbohydrate-active enzyme (CAZyme) groups appeared to be potentially involved in eliciting defense responses in the plants. For example, analysis of gene family evolution identified nine CAZyme orthogroups evolving for gene gains (p ≤ 0.05), predicted to be involved in hemicellulose degradation, potentially producing plant defense-inducing oligomers. Moreover, 8-10% of the secretomes was composed of cysteine-enriched proteins, including hydrophobins, important for root colonization. Effectors were more numerous, comprising 35-37% of the secretomes, where certain members belonged to seven orthogroups evolving for gene gains and were induced during the C. rosea response to F. graminearum or H. solani. Furthermore, the considered Clonostachys spp. possessed high numbers of proteins containing Common in Fungal Extracellular Membranes (CFEM) modules, known for their role in fungal virulence. Overall, this study improves our understanding of Clonostachys spp. adaptation to diverse ecological niches and establishes a basis for future investigation aiming at sustainable biocontrol of plant diseases.

Verticillium longisporum phospholipase VlsPLA2 is a virulence factor that targets host nuclei and modulates plant immunity.

Phospholipase A2 (PLA2 ) is a lipolytic enzyme that hydrolyses phospholipids in the cell membrane. In the present study, we investigated the role of secreted PLA2 (VlsPLA2 ) in Verticillium longisporum, a fungal phytopathogen that mostly infects plants belonging to the Brassicaceae family, causing severe annual yield loss worldwide. Expression of the VlsPLA2 gene, which encodes active PLA2 , is highly induced during the interaction of the fungus with the host plant Brassica napus. Heterologous expression of VlsPLA2 in Nicotiana benthamiana resulted in increased synthesis of certain phospholipids compared to plants in which enzymatically inactive PLA2 was expressed (VlsPLA2 ΔCD ). Moreover, VlsPLA2 suppresses the hypersensitive response triggered by the Cf4/Avr4 complex, thereby suppressing the chitin-induced reactive oxygen species burst. VlsPLA2 -overexpressing V. longisporum strains showed increased virulence in Arabidopsis plants, and transcriptomic analysis of this fungal strain revealed that the induction of the gene contributed to increased virulence. VlsPLA2 was initially localized to the host nucleus and then translocated to the chloroplasts at later time points. In addition, VlsPLA2 bound to the vesicle-associated membrane protein A (VAMPA) and was transported to the nuclear membrane. In the nucleus, VlsPLA2 caused major alterations in the expression levels of genes encoding transcription factors and subtilisin-like proteases, which play a role in plant immunity. In conclusion, our study showed that VlsPLA2 acts as a virulence factor, possibly by hydrolysing host nuclear envelope phospholipids, which, through a signal transduction cascade, may suppress basal plant immune responses.