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Summary Although it is well known that miRNAs play crucial roles in multiple biological processes, there is currently no evidence indicating that milRNAs from Fusarium oxysporum f. sp. lycopersici (Fol) interfere with tomato resistance during infection.Here, using sRNA‐seq, we demonstrate that Fol‐milR1, a trans‐kingdom small RNA, is exported into tomato cells after infection.The knockout strain ∆Fol‐milR1 displays attenuated pathogenicity to the susceptible tomato cultivar ‘Moneymaker’. On the other hand, Fol‐milR1 overexpression strains exhibit enhanced virulence against the resistant cultivar ‘Motelle’. Several tomato mRNAs are predicted targets of Fol‐milR1. Among these genes, Solyc06g007430 (encoding the CBL‐interacting protein kinase, SlyFRG4) is regulated at the posttranscriptional level by Fol‐milR1. Furthermore, SlyFRG4 loss‐of‐function alleles created using CRISPR/Cas9 in tomato (‘Motelle’) exhibit enhanced disease susceptibility to Fol, further supporting the idea that SlyFRG4 is essential for tomato wilt disease resistance. Notably, our results using immunoprecipitation with specific antiserum suggest that Fol‐milR1 interferes with the host immunity machinery by binding to tomato ARGONAUTE 4a (SlyAGO4a). Furthermore, virus‐induced gene silenced (VIGS) knock‐down SlyAGO4a plants exhibit reduced susceptibility to Fol.Together, our findings support a model in which Fol‐milR1 is an sRNA fungal effector that suppresses host immunity by silencing a disease resistance gene, thus providing a novel virulence strategy to achieve infection., See also the Commentary on this article by Thieron et al., 232: 464–467.

The establishment of host-microbe interactions requires molecular communication between both partners, which involves the mutual transfer of noncoding small RNAs. Previous evidence suggests that this is also true for the barley powdery mildew disease, which is caused by the fungal pathogen Blumeria hordei. However, previous studies lacked spatial resolution regarding the accumulation of small RNAs upon host infection by B. hordei. Here, we analysed site-specific small RNA repertoires in the context of the barley-B. hordei interaction. To this end, we dissected infected leaves into separate fractions representing different sites that are key to the pathogenic process: epiphytic fungal mycelium, infected plant epidermis, isolated haustoria, a vesicle-enriched fraction from infected epidermis, and extracellular vesicles. Unexpectedly, we discovered enrichment of specific 31- to 33-base long 5’-terminal fragments of barley 5.8S ribosomal RNA (rRNA) in extracellular vesicles and infected epidermis, as well as particular B. hordei tRNA fragments in haustoria. We describe canonical small RNAs from both the plant host and the fungal pathogen that may confer cross-kingdom RNA interference activity. Interestingly, we found first evidence of phased small RNAs (phasiRNAs) in B. hordei, a feature usually attributed to plants, which may be associated with the post-transcriptional control of fungal coding genes, pseudogenes, and transposable elements. Our data suggests a key and possibly site-specific role for cross-kingdom RNA interference and noncoding RNA fragments in the host-pathogen communication between B. hordei and its host barley.

One major threat to plant cultivation are fungal pathogens, which can cause substantial yield losses in agriculture. As an example, cereal powdery mildew fungi such as the barley (Hordeum vulgare) pathogen, Blumeria graminis f. sp. hordei (Bgh), are among the ten most relevant fungal plant pathogens in molecular plant pathology and can lead to yield losses of up to 30%. Plant Mildew resistance Locus O (MLO) genes are required for successful colonization of plants by powdery mildew fungi. Accordingly, loss-of-function mlo mutants confer durable resistance against powdery mildew fungi in many plant species. In the case of barley, mlo-based resistance has been used for more than 40 years in agriculture without powdery mildew fungi effectively overcoming this kind of immunity. However, the molecular basis of mlo resistance and function(s) of the transmembrane Mlo protein(s) are still incompletely understood. The generation of transgenic barley plants to study the plant immune response and the involvement of Mlo therein is time-consuming and challenging. Therefore, transient gene expression via gene gun-mediated particle bombardment became a popular, easy, and efficient tool to investigate different aspects of plant defense responses in barley. Since Bgh fails to penetrate leaf epidermal cells of mlo mutants, single-cell complementation upon biolistic transformation resulting in (over-)expression of Mlo can be used to characterize the Mlo protein functionally in vivo. In this chapter, we describe in detail the gene gun-mediated transient expression of Mlo in barley leaf epidermal cells followed by powdery mildew inoculation and the subsequent microscopic evaluation. However, gene gun-mediated transient gene expression may be also used to address other research questions or to transform the epidermal tissues of other plant organs and/or species.

The journal of biological chemistry : JBC 296, 100611 (2021). doi:10.1016/j.jbc.2021.100611, Published by American Society for Biochemistry and Molecular Biology, Bethesda, MD.

International audience; Small RNAs (sRNAs) play a key role in eukaryotic gene regulation, for example by gene silencing via RNA interference (RNAi). The biogenesis of sRNAs depends on proteins that are generally conserved in all eukaryotic lineages, yet some species that lack part or all the components of the mechanism exist. Here we explored the presence of the RNAi machinery and its expression as well as the occurrence of sRNA candidates and their putative endogenous as well as host targets in phytopathogenic powdery mildew fungi. We focused on the species Blumeria graminis, which occurs in various specialized forms (formae speciales) that each have a strictly limited host range. B. graminis f. sp. hordei and B. graminis f. sp. tritici, colonizing barley and wheat, respectively, have genomes that are characterized by extensive gene loss. Nonetheless, we find that the RNAi machinery appears to be largely complete and expressed during infection. sRNA sequencing data enabled the identification of putative sRNAs in both pathogens. While a considerable part of the sRNA candidates have predicted target sites in endogenous genes and transposable elements, a small proportion appears to have targets in planta, suggesting potential cross-kingdom RNA transfer between powdery mildew fungi and their respective plant hosts. (C) 2018 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

PLoS pathogens 15(3), e1007620 - (2019). doi:10.1371/journal.ppat.1007620, Published by PLoS, Lawrence, Kan.

The biotrophic fungal pathogenBlumeria graminiscauses the powdery mildew disease of cereals and grasses. Proteins with a predicted ribonuclease (RNase)-like fold (termed RALPHs) comprise the largest set of secreted effector candidates within theB. graminisf. sp.hordeigenome. Their exceptional abundance suggests they play crucial functions during pathogenesis. We show that transgenic expression of RALPH CSEP0064/BEC1054 increases susceptibility to infection in monocotyledenous and dicotyledonous plants. CSEP0064/BEC1054 interactsin plantawith five host proteins: two translation elongation factors (eEF1α and eEF1γ), two pathogenesis-related proteins (PR5 and PR10) and a glutathione-S-transferase. We present the first crystal structure of a RALPH, CSEP0064/BEC1054, demonstrating it has an RNase-like fold. The protein interacts with total RNA and weakly with DNA. Methyl jasmonate levels modulate susceptibility to aniline-induced host RNA fragmentation.In plantaexpression of CSEP0064/BEC1054 reduces the formation of this RNA fragment. We propose that CSEP0064/BEC1054 is a pseudoenzyme that binds to host ribosomes, thereby inhibiting the action of plant ribosome-inactivating proteins that would otherwise lead to host cell death, an unviable interaction and demise of the fungus.