Biology subjects

Piombo, E.

Publications and source records attributed to Piombo, E..

3 recordsLinked to original sources

bioRxiv2023

Dicer-mediated RNA silencing is the key regulatory mechanism in the biocontrol fungus Clonostachys rosea-wheat interactions

The intricate molecular interplay between beneficial fungi and plants is vital to plant growth promotion and induced defense response. This study explored the role of DCL-mediated RNA silencing in the interaction between the biocontrol fungus Clonostachys rosea and wheat roots. We investigated the impact of DCL (Dicer-like) gene deletions in C. rosea on its root colonization ability. Our results revealed that the deletion of dcl2 significantly enhanced C. rosea biomass on wheat roots, indicating a pivotal role of DCL2 in root colonization. Transcriptome sequencing of C. rosea and wheat during their interactions unveiled extensive gene expression changes. In wheat, genes related to stress responses were upregulated during C. rosea interactions, while genes associated with plant cell wall modification and metabolic processes were downregulated, suggesting complex regulatory responses and a trade-off between defense mechanisms and growth promotion. Deletion of C. rosea dcl1 and dcl2 altered the transcriptomic responses of wheat roots during interactions. Wheat genes associated with stress responses were downregulated during interactions with DCL deletion strains. In contrast, genes involved in metabolic processes and growth were upregulated, emphasizing the cross-kingdom regulatory role of C. rosea small RNAs (sRNAs). We identified 18 wheat miRNAs responsive to C. rosea interactions. Furthermore, we identified 24 endogenous and six cross-kingdom potential gene targets for seven and five differentially expressed miRNAs, supported by their inverse gene expression pattern. In C. rosea, we found a large transcriptional reprogramming of genes during interaction with wheat roots. The upregulated genes were associated with carbohydrate and polysaccharide catabolic processes, membrane transporters and effectors. Conversely, downregulated genes were mainly associated with transition metal ion transport and homeostasis processes. The deletion of dcl1 and dcl2 had significant effects on gene expression. A higher number of genes upregulated in WT during the interaction were restored in DCL deletion mutants, suggesting DCL-mediated gene expression regulation. Furthermore, we identified 21 differentially expressed micro-RNA-like RNAs (milRNAs) in C. rosea; nine were DCL-dependent. They had putative gene targets in C. rosea, including transcription factors, effectors, transporters, and enzymes involved in specialized metabolite production. Cross-kingdom RNA silencing was also observed, with seven DCL-dependent C. rosea milRNAs potentially targeting 29 genes in wheat. These findings provide valuable insights into the molecular mechanisms underlying the beneficial interaction between fungi and plant roots. In addition, the study shed light on the role of sRNA-mediated gene regulation in the C. rosea-wheat interaction, with potential implications for sustainable agriculture and biocontrol strategies.

genomics↗

bioRxiv2022

Comparative small RNA and degradome sequencing provide new 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 post-transcriptional gene regulation, although their role in gene expression regulation during mycoparasitism remain understudied. Here, we investigated the role of sRNA-mediated gene regulation in mycoparasitism by performing sRNA and degradome tags 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 down-regulated during the interactions with the mycohosts compared to a C. rosea self-interaction control, thus allowing de-suppression (up-regulation) 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 gene targets for 282 differentially expressed sRNAs. Analysis of sRNA gene targets revealed that the regulation of genes coding for membrane proteins was a common response against both mycohosts. While the regulation of genes involved in oxidative stress tolerance and cellular metabolic and biosynthetic processes was exclusive against F. graminearum highlighting common and mycohosts-specific gene regulation of C. rosea. By combining these results with transcriptome data collected in similar experimental conditions during a previous study, we expand the understanding of the role of sRNA in regulating interspecific fungal interactions and mycoparasitism. ImportanceSmall-RNAs (sRNAs) are emerging as key players in pathogenic and symbiotic fungus-plant interactions, however, their role in fungal-fungal interactions remains elusive. In this study, we employed the necrotrophic mycoparasite Clonostachys rosea and plant pathogenic mycohots 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 miRNAs and their potential gene targets at endogenous level, and at a cross-species level in B. cinerea and F. graminearum indicating a role of cross-species 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.

microbiology↗

bioRxiv2021

The role of Dicer-dependent RNA interference in regulating cross-species communication during fungus-fungus interactions

Dicer-like (DCL) proteins play a vital role in transcriptional and post-transcriptional gene silencing, also known as RNA interference (RNAi), by cleaving double-stranded RNAs or single-stranded RNAs with stem-loop structures into small RNAs. Although DCL-mediated RNAi can regulate interspecific communication between pathogenic/mutualistic organisms and their hosts, its role in parasitic fungus-fungus interactions is yet to be investigated. In this study, we deleted dcl genes in the mycoparasitic fungus Clonostachys rosea and analyzed the transcriptome and secondary metabolome to characterize the regulatory functions of DCL-dependent RNAi in mycoparasitism. Deletion of dcl2 resulted in a mutant with reduced growth rate, pigment production and antagonism towards the plant pathogenic fungus Botrytis cinerea. Moreover, the {Delta}dcl2 mutant displayed a reduced ability to control fusarium foot rot disease on wheat, caused by Fusarium graminearum, and reduced production of 62 secondary metabolites (SM) including yellow-coloured sorbicillinoids. Transcriptome sequencing of the in vitro interaction between the C. rosea {Delta}dcl2 strain and B. cinerea or F. graminearum identified downregulation of genes coding for transcription factors, membrane transporters, hydrolytic enzymes and SM biosynthesis enzymes putatively involved in antagonistic interactions, in comparison with the C. rosea wild type interaction. Sixty-one putative novel microRNA-like RNAs (milRNAs) were identified in C. rosea, and 11 was upregulated in the {Delta}dcl2 mutant. In addition to putative endogenous gene targets, these DCL2-dependent milRNAs were predicted to target B. cinerea and F. graminearum virulence factor genes, which showed an increased expression during interaction with the {Delta}dcl2 mutant incapable of producing the targeting milRNAs. This paper constitutes the first step in elucidating the role of RNAi in mycoparasitism, with important implications for biological control of plant diseases. This study further indicates a possible cross-species regulatory activity of fungal milRNAs, emphasizing a novel role of RNAi in fungal interactions and ecology. Author summaryRNA interference (RNAi) is a conserved cellular mechanism mediated by small RNAs (sRNAs) regulating biological processes through the targeted destruction or modulation of RNA filaments necessary for protein synthesis. Dicer-like endoribonucleases (DCL) play a vital role in the RNAi pathway by generating sRNAs. In this study, we identified two DCL-encoding genes in the mycoparasitic fungus Clonostachys rosea and investigated a role of DCL-mediated RNAi in interference interactions between Clonostachys rosea and the two important fungal pathogens Botrytis cinerea and Fusarium graminearum (here called mycohost). Using transcriptome (sRNA and mRNA) sequencing and secondary metabolome analysis approach, we found that the dcl mutants were not able to produce 11 sRNAs predicted to finetune the regulatory network of genes known to be involved in production of hydrolytic enzymes, antifungal compounds, and membrane transporters needed for antagonistic action of C. rosea. We also found C. rosea sRNAs putatively targeting known virulence factors in the mycohost, indicating RNAi-mediated cross-species communication. Our study expanded the understanding of underlying mechanisms of cross-species communication during interference interactions and showed that DCL-mediated RNAi is an important regulator of parasitic fungus-fungus interactions. The results pose the base for future works studying the role of DCL-based cross-species RNAi in fungal interactions.

genomics↗