Fusarium graminearum DICER-like-dependent sRNAs are required for the suppression of host immune genes and full virulence.

In filamentous fungi, gene silencing by RNA interference (RNAi) shapes many biological processes, including pathogenicity. Recently, fungal small RNAs (sRNAs) have been shown to act as effectors that disrupt gene activity in interacting plant hosts, thereby undermining their defence responses. We show here that the devastating mycotoxin-producing ascomycete Fusarium graminearum (Fg) utilizes DICER-like (DCL)-dependent sRNAs to target defence genes in two Poaceae hosts, barley (Hordeum vulgare, Hv) and Brachypodium distachyon (Bd). We identified 104 Fg-sRNAs with sequence homology to host genes that were repressed during interactions of Fg and Hv, while they accumulated in plants infected by the DCL double knock-out (dKO) mutant PH1-dcl1/2. The strength of target gene expression correlated with the abundance of the corresponding Fg-sRNA. Specifically, the abundance of three tRNA-derived fragments (tRFs) targeting immunity-related Ethylene overproducer 1-like 1 (HvEOL1) and three Poaceae orthologues of Arabidopsis thaliana BRI1-associated receptor kinase 1 (HvBAK1, HvSERK2 and BdSERK2) was dependent on fungal DCL. Additionally, RNA-ligase-mediated Rapid Amplification of cDNA Ends (RLM-RACE) identified infection-specific degradation products for the three barley gene transcripts, consistent with the possibility that tRFs contribute to fungal virulence via targeted gene silencing.

Biotic stress-associated microRNA families in plants.

Plants and animals utilize various regulatory mechanisms for control of gene expression during development in different tissues and cell types. About 30 years ago, a new mechanism of gene regulation, termed RNA interference (RNAi), was discovered and proved revolutionary for the mechanistic understanding of gene regulation. Noncoding RNAs, including short, 21-24 nucleotide (nt) long microRNAs (miRNAs), endogenously-generated from MIR genes, are key components of RNAi processes, by post-transcriptionally controlling transcripts with antisense complementarity through either translational repression or mRNA degradation. Since their discovery, important roles in regulation of ontogenetic development, cell differentiation, proliferation, and apoptosis in eukaryotes have been elucidated. In plants, miRNAs are known regulatory elements of basic endogenous functions and responses to the environmental stimuli. While the role of miRNAs in regulation of nutrient uptake, circadian clock and general response to abiotic stress is already well understood, a comprehensive understanding of their immune-regulatory roles in response to various biotic stress factors has not yet been achieved. This review summarizes the current understanding of the function of miRNAs and their targets in plants during interaction with microbial pathogens and symbionts. Additionally, we provide a consensus conclusion regarding the typical induction or repression response of conserved miRNA families to pathogenic and beneficial fungi, bacteria, and oomycetes, as well as an outlook of agronomic application of miRNAs in plants. Further investigation of plant miRNAs responsive to microbes, aided with novel sequencing and bioinformatics approaches for discovery and prediction in non-model organisms holds great potential for development of new forms of plant protection.

Requirements for fungal uptake of dsRNA and gene silencing in RNAi-based crop protection strategies.

Growing evidence indicates that RNAi is an effective control strategy for agronomically important fungi. To implement RNAi-based crop protection strategies, dsRNA molecules are either sprayed on foliage or generated by genetically engineered plants. Here, we summarize current knowledge of the mechanisms governing dsRNA uptake and RNAi-mediated gene silencing in fungi, as well as the factors that influence these phenomena. Of primary importance is dsRNA design, as identifying an appropriate gene for silencing and determining which region of the gene to target are critical for maximizing efficiency. Strategies for enhancing dsRNA uptake, potentially by using formulations and/or carriers that prevent dsRNA degradation by (a)biotic factors and possibly facilitate translocation, also are a key consideration. Finally, determining whether the fungal pathogen of interest contains a functional RNAi machinery is a major consideration. Integrated experimental confirmation of these important factors is necessary for the successful development of crop protection strategies against fungal pathogens.

Comparative Analysis of Transcriptome and sRNAs Expression Patterns in the Brachypodium distachyon- Magnaporthe oryzae Pathosystems.

The hemibiotrophic fungus Magnaporthe oryzae (Mo) is the causative agent of rice blast and can infect aerial and root tissues of a variety of Poaceae, including the model Brachypodium distachyon (Bd). To gain insight in gene regulation processes occurring at early disease stages, we comparatively analyzed fungal and plant mRNA and sRNA expression in leaves and roots. A total of 310 Mo genes were detected consistently and differentially expressed in both leaves and roots. Contrary to Mo, only minor overlaps were observed in plant differentially expressed genes (DEGs), with 233 Bd-DEGs in infected leaves at 2 days post inoculation (DPI), compared to 4978 at 4 DPI, and 138 in infected roots. sRNA sequencing revealed a broad spectrum of Mo-sRNAs that accumulated in infected tissues, including candidates predicted to target Bd mRNAs. Conversely, we identified a subset of potential Bd-sRNAs directed against fungal cell wall components, virulence genes and transcription factors. We also show a requirement of operable RNAi genes from the DICER-like (DCL) and ARGONAUTE (AGO) families for fungal virulence. Overall, our work elucidates the extensive reprogramming of transcriptomes and sRNAs in both plant host (Bd) and fungal pathogen (Mo), further corroborating the critical role played by sRNA species in the establishment of the interaction and its outcome.

Further Elucidation of the Argonaute and Dicer Protein Families in the Model Grass Species Brachypodium distachyon.

RNA interference (RNAi) is a biological process in which small RNAs regulate gene silencing at the transcriptional or posttranscriptional level. The trigger for gene silencing is double-stranded RNA generated from an endogenous genomic locus or a foreign source, such as a transgene or virus. In addition to regulating endogenous gene expression, RNAi provides the mechanistic basis for small RNA-mediated communication between plant hosts and interacting pathogenic microbes, known as cross-kingdom RNAi. Two core protein components, Argonaute (AGO) and Dicer (DCL), are central to the RNAi machinery of eukaryotes. Plants encode for several copies of AGO and DCL genes; in Arabidopsis thaliana, the AGO protein family contains 10 members, and the DCL family contains four. Little is known about the conservation and specific roles of these proteins in monocotyledonous plants, which account for the most important food staples. Here, we utilized in silico tools to investigate the structure and related functions of AGO and DCL proteins from the model grass Brachypodium distachyon. Based on the presence of characteristic domains, 16 BdAGO- and 6 BdDCL-predicted proteins were identified. Phylogenetic analysis showed that both protein families were expanded in Brachypodium as compared with Arabidopsis. For BdDCL proteins, both plant species contain a single copy of DCL1 and DCL4; however, Brachypodium contains two copies each of DCL2 and DCL3. Members of the BdAGO family were placed in all three functional clades of AGO proteins previously described in Arabidopsis. The greatest expansion occurred in the AtAGO1/5/10 clade, which contains nine BdAGOs (BdAGO5/6/7/9/10/11/12/15/16). The catalytic tetrad of the AGO P-element-induced wimpy testis domain (PIWI), which is required for endonuclease activity, is conserved in most BdAGOs, with the exception of BdAGO1, which lacks the last D/H residue. Three-dimensional modeling of BdAGO proteins using tertiary structure prediction software supported the phylogenetic classification. We also predicted a provisional interactome network for BdAGOs, their localization within the cell, and organ/tissue-specific expression. Exploring the specifics of RNAi machinery proteins in a model grass species can serve as a proxy for agronomically important cereals such as barley and wheat, where the development of RNAi-based plant protection strategies is of great interest.

A Bioinformatics Pipeline for the Analysis and Target Prediction of RNA Effectors in Bidirectional Communication During Plant-Microbe Interactions.

Small RNA (sRNA) molecules are key factors in the communication between hosts and their interacting pathogens, where they function as effectors that can modulate both host defense and microbial virulence/pathogenicity through a mechanism termed cross-kingdom RNA interference (ck-RNAi). Consistent with this recent knowledge, sRNAs and their double-stranded RNA precursor have been adopted to control diseases in crop plants, demonstrating a straight forward application of the new findings to approach agricultural problems. Despite the great interest in natural ck-RNAi, it is astonishing to find just a few additional examples in the literature since the first report was published in 2013. One reason might be that the identification of sRNA effectors is hampered both by technical challenges and lack of routine bioinformatics application strategies. Here, we suggest a practical procedure to find, characterize, and validate sRNA effectors in plant-microbe interaction. The aim of this review is not to present and discuss all possible tools, but to give guidelines toward the best established software available for the analysis.