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A fungal RNA-dependent RNA polymerase is a novel player in plant infection and cross-kingdom RNA interference
by An-Po ChengBernhard LedererLorenz OberkoflerLihong HuangNathan R JohnsonFabian PlattenFlorian DunkerConstance TisserantArne Weiberg

Small RNAs act as fungal pathogen effectors that silence host target genes to promote infection, a virulence mechanism termed cross-kingdom RNA interference (RNAi). The essential pathogen factors of cross-kingdom small RNA production are largely unknown. We here characterized the RNA-dependent RNA polymerase (RDR)1 in the fungal plant pathogen Botrytis cinerea that is required for pathogenicity and cross-kingdom RNAi. B. cinerea bcrdr1 knockout (ko) mutants exhibited reduced pathogenicity and loss of cross-kingdom small RNAs. We developed a "switch-on" GFP reporter to study cross-kingdom RNAi in real-time within the living plant tissue which highlighted that bcrdr1 ko mutants were compromised in cross-kingdom RNAi. Moreover, blocking seven pathogen cross-kingdom small RNAs by expressing a short-tandem target mimic RNA in transgenic Arabidopsis thaliana led to reduced infection levels of the fungal pathogen B. cinerea and the oomycete pathogen Hyaloperonospora arabidopsidis. These results demonstrate that cross-kingdom RNAi is significant to promote host infection and making pathogen small RNAs an effective target for crop protection.

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Spotlight on plant RNA-containing extracellular vesicles

by Alessa Ruf, Lorenz Oberkofler, Silke Robatzek and Arne Weiberg

Extracellular vesicles (EVs) carrying RNA have attracted growing attention in plant cell biology. For a long time, EV release or uptake through the rigid plant cell wall was considered to be impossible and RNA outside cells to be unstable. Identified EV biomarkers have brought new insights into functional roles of EVs to transport their RNA cargo for systemic spread in plants and into plant-invading pathogens. RNA-binding proteins supposedly take over key functions in EV-mediated RNA secretion and transport, but the mechanisms of RNA sorting and EV translocation through the plant cell wall and plasma membrane are not understood. Characterizing the molecular players and the cellular mechanisms of plant RNA-containing EVs will create new knowledge in cell-to-cell and inter-organismal communication.


Retrotransposons as pathogenicity factors of the plant pathogenic fungus Botrytis cinerea.

by Antoine Porquier, Constance Tisserant, Francisco Salinas, Carla Glassl, Lucas Wange, Wolfgang Enard, Andreas Hauser, Matthias Hahn, Arne Weiberg*

Retrotransposons are genetic elements inducing mutations in all domains of life. Despite their detrimental effect, retrotransposons can become temporarily active during epigenetic reprogramming and cellular stress response, which may accelerate host genome evolution. In fungal pathogens, a positive role has been attributed to retrotransposons when shaping genome architecture and expression of genes encoding pathogenicity factors; thus, retrotransposons are known to influence pathogenicity.

We uncover a hitherto unknown role of fungal retrotransposons as being pathogenicity factors, themselves. The aggressive fungal plant pathogen, Botrytis cinerea, is known to deliver some long-terminal repeat (LTR) deriving regulatory trans-species small RNAs (BcsRNAs) into plant cells to suppress host gene expression for infection. We find that naturally occurring, less aggressive B. cinerea strains possess considerably lower copy numbers of LTR retrotransposons and had lost retrotransposon BcsRNA production. Using a transgenic proof-of-concept approach, we reconstitute retrotransposon expression in a BcsRNA-lacking B. cinerea strain, which results in enhanced aggressiveness in a retrotransposon and BcsRNA expression-dependent manner. Moreover, retrotransposon expression in B. cinerea leads to suppression of plant defence-related genes during infection.

We propose that retrotransposons are pathogenicity factors that manipulate host plant gene expression by encoding trans-species BcsRNAs. Taken together, the novelty that retrotransposons are pathogenicity factors will have a broad impact on studies of host-microbe interactions and pathology.

Oomycete small RNAs bind to the plant RNA-induced silencing complex for virulence

by Florian Dunker, Adriana Trutzenberg, Jan S Rothenpieler, Sarah Kuhn, Reinhard Pröls, Tom Schreiber, Alain Tissier, Ariane Kemen, Eric Kemen, Ralph Hückelhoven, Arne Weiberg*


The exchange of small RNAs (sRNAs) between hosts and pathogens can lead to gene silencing in the recipient organism, a mechanism termed cross-kingdom RNAi (ck-RNAi). While fungal sRNAs promoting virulence are established, the significance of ck-RNAi in distinct plant pathogens is not clear. Here, we describe that sRNAs of the pathogen Hyaloperonospora arabidopsidis, which represents the kingdom of oomycetes and is phylogenetically distant from fungi, employ the host plant’s Argonaute (AGO)/RNA-induced silencing complex for virulence. To demonstrate H. arabidopsidis sRNA (HpasRNA) functionality in ck-RNAi, we designed a novel CRISPR endoribonuclease Csy4/GUS reporter that enabled in situ visualization of HpasRNA- induced target suppression in Arabidopsis. The significant role of HpasRNAs together with AtAGO1 in virulence was revealed in plant atago1 mutants and by transgenic Arabidopsis expressing a short-tandem-target-mimic to block HpasRNAs, that both exhibited enhanced resistance. HpasRNA-targeted plant genes contributed to host immunity, as Arabidopsis gene knockout mutants displayed quantitatively enhanced susceptibility.


An Arabidopsis downy mildew non-RxLR effector suppresses induced plant cell death to promote biotroph infection

by Florian Dunker, Lorenz Oberkofler, Bernhard Lederer, Adriana Trutzenberg and Arne Weiberg*

Our understanding of obligate biotrophic pathogens is limited by lack of knowledge concerning the molecular func- tion of virulence factors. We established Arabidopsis host-induced gene silencing (HIGS) to explore gene functions of Hyaloperonospora arabidopsidis, including CYSTEINE-RICH PROTEIN (HaCR)1, a potential secreted effector gene of this obligate biotrophic pathogen. HaCR1 HIGS resulted in H. arabidopsidis-induced local plant cell death and reduced pathogen reproduction. We functionally characterized HaCR1 by ectopic expression in Nicotiana benthamiana. HaCR1 was capable of inhibiting effector-triggered plant cell death. Consistent with this, HaCR1 ex- pression in N. benthamiana led to stronger disease symptoms caused by the hemibiotrophic oomycete pathogen Phytophthora capsici, but reduced disease symptoms caused by the necrotrophic fungal pathogen Botrytis cinerea. Expressing HaCR1 in transgenic Arabidopsis confirmed higher susceptibility to H. arabidopsidis and to the bac- terial hemibiotrophic pathogen Pseudomonas syringae. Increased H. arabidopsidis infection was in accordance with reduced PATHOGENESIS RELATED (PR)1 induction. Expression of full-length HaCR1 was required for its function, which was lost if the signal peptide was deleted, suggesting its site of action in the plant apoplast. This study pro- vides phytopathological and molecular evidence for the importance of this widespread, but largely unexplored class of non-RxLR effectors in biotrophic oomycetes.

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Plant ARGONAUTE Protein Immunopurification for Pathogen Cross Kingdom Small RNA Analysis

Florian Dunker, Bernhard Lederer and Arne Weiberg*  

Over the last decade, it has been noticed that microbial pathogens and pests deliver small RNA (sRNA) effectors into their host plants to manipulate plant physiology and immunity for infection, known as cross kingdom RNA interference. In this process, fungal and oomycete parasite sRNAs hijack the plant ARGONAUTE (AGO)/RNA-induced silencing complex to post-transcriptionally silence host target genes. We hereby describe the methodological details of how we recovered cross kingdom sRNA effectors of the oomycete pathogen Hyaloperonospora arabidopsidis during infection of its host plant Arabidopsis thaliana. This Bio-protocol contains two parts: first, a detailed description on the procedure of plant AGO/sRNA co-immunopurification and sRNA recovery for Illumina high throughput sequencing analysis. Second, we explain how to perform bioinformatics analysis of sRNA sequence reads using a Galaxy server. In principle, this protocol is suitable to investigate AGO-bound sRNAs from diverse host plants and plant-interacting (micro)organisms.


Extracellular vesicles in plant host-microbe interaction

by Constance Tisserant and Arne Weiberg*  

Extracellular vesicles (EVs) are secreted lipid bilayer membrane particles that are increasingly drawing attention due to their potential role in intercellular communication. EVs have been mainly reported in mammalian systems, but are also found in non-mammalian classes, such as Archeae, bacteria, fungi, oomycetes, protozoa, invertebrates and plants. Over the last decade, EV research on mammalian systems has been massively advanced driven by the interests and applications of the biomedical field, while research on non-mammalian EVs that aims to understand the biological origins and functions of EVs remains rather descriptive and premature. Nevertheless, recent pioneering works resulted in novel concepts that place EVs carrying regulatory small RNAs as central players in inter-species and cross-kingdom communication with emphasis on host-pathogen, host-parasite and host-microbiome interactions. EVs transport small RNAs from microbe/pathogen/parasite to animal or plant hosts, and vice versa, which results in the manipulation of host immunity or microbial virulence, respectively. This article highlights some of the latest discoveries regarding EV-mediated communication across species and kingdoms with a special focus on plants and their interacting microbes.

Full list of publications of our team members

Cheng A.P., Lederer B., Oberkofler L., Huang J., Johnson, N.R., Platten, F., Dunker, F., Tisserant, C., Weiberg A.. (2023). A fungal RNA-dependent RNA polymerase is a novel player in plant infection and cross-kingdom RNA interference. PLoS Pathogens, e1011885.

An-Po ChengSeomun KwonTrusha AdesharaVera GöhreMichael FeldbrüggeArne Weiberg. (2023). Extracellular RNAs released by plant-associated fungi: from fundamental mechanisms to biotechnological applications. Appl. Microbiol Biotechnol, 107: 5935-5945.

Göhre V., Weiberg A. (2022). RNA dialogs in fungal-plant relationships. The Mycota – Vol.5 Plant relationships, 31-51.

Ruf A., Oberkofler L., Robatzek S., Weiberg A. (2022). Spotlight on plant RNA-containing extracellular vesicles. Curr Opin Plant Biol, 69: 102272.

Porquier A., Tisserant C., Salinas F., Glassl C., Wange L., Enard W., Hauser A., Hahn M., Weiberg A. (2021). Retrotransposons as pathogenicity factors of the plant pathogenic fungus Botrytis cinerea. Genome Biol, 22:225.

Dunker F., Lederer B., Weiberg A. (2021). Plant ARGONAUTE Protein Immunopurification for Pathogen Cross Kingdom Small RNA Analysis. Bio-Protocol 11 (3): e3911. DOI: 10.21769/BioProtoc.3911.

Dunker F., Oberkofler L., Lederer B., Trutzenberg A., Weiberg A. (2021). An Arabidopsis downy mildew non-RxLR effector suppresses induced plant cell death to promote biotroph infection. J Exp Bot,

Dunker F., Trutzenberg A., Rothenpieler J.S., Kuhn S., Pröls R., Schreiber T., Tissier A., Kemen A., Kemen E., Hückelhoven R., Weiberg A. (2020). Oomycete small RNAs bind to the plant RNA-induced silencing complex for virulence. eLife, doi: 10.7554/eLife.56096.

Kwon S., Tisserant C., Tulinski M., Weiberg A., Feldbrügge M. (2020). Inside-out: from endosomes to extracellular vesicles in fungal RNA transport. Fungal Biol Rev, 34: 89-99.

Cai Q., He B., Weiberg A., Buck A.H., Jin H. (2019). Small RNAs and extracellular vesicles – new mechanisms of cross-species communication and innovative tools for disease control. PLoS Pathogens, doi: 10.1371/journal.ppat.1008090.

Tisserant C., Weiberg A. (2019). Extracellular vesicles in plant host-microbe interaction. Trillium Extracell Vesicles, 1.

Guan L., Denkert N., Eisa A., Lehmann M., Sjuts I., Weiberg A., Soll J., Meinecke M., Schwenkert S. (2019). JASSY, a chloroplast outer membrane protein required for jasmonate biosynthesis. PNAS, 116: 10568-10575.

Bielska E., Birch P.R.J., Buck A.H., Abreu-Goodger C., Inner R.W., Jin H., Paffl M.W., Robatzek S., Regev-Rudzki N., Tisserant C., Wang D., Weiberg A. (2019). Highlights of the mini-symposium on extracellular vesicles in inter-organismal communication, held in Munich, Germany, August 2018. J Extracell Vesicles, doi: 10.1080/20013078.2019

Fiedler I.C., Weiberg A., van der Linde K. (2019). Using Ustilago maydis as a Trojan Horse for in situ delivery of maize proteins. J Vis Exp, doi: 10.3791/58746

Wang, M., Weiberg, A., Dellota, E. Jr., Yamane D., Jin, H. (2017). Botrytis small RNA Bc-siR37 suppresses plant defense genes by cross-kingdom RNAi. RNA Biol, doi:10. 1080/15476286.2017.1291112

Wang, M., Weiberg, A., Lin, F-M., Thomma, B.P.H.J., Huang, H-D., Jin, H. (2016). Bidirectional cross-kingdom RNAi and fungal uptake of external RNAs confer plant protection. Nature Plants, doi:10.1038/nplants.2016.151

Weiberg, A. & Jin, H. (2015). Small RNAs-the secret agents in the plant-pathogen interactions. Curr Opin Plant Biol 26, 87-94

Weiberg, A., Bellinger, M. & Jin, H. (2015). Conversations between kingdoms: small RNAs. Curr Opin Biotechnol 32C, 207-215

Wang, M., Weiberg, A. & Jin, H. (2015). Pathogen small RNAs: a new class of effectors for pathogen attacks. Mol Plant Pathol 16, 219-223

Weiberg, A., Wang, M., Bellinger, M. & Jin, H. (2014). Small RNAs: a new paradigm in plant-microbe interactions. Annu Rev Phytopathol 52, 495-516

Weiberg, A.#, Wang, M.#, Lin F-M., Zhao, H-W., Zhang Z-H., Kaloshian I., Huang H-D., Jin, H. (2013). Fungal small RNAs suppress plant immunity by hijacking host RNA inference pathways. Science 342, 118-123

Weiberg, A. & Karlovsky, P. (2009). Components of variance in transcriptomics based on electrophoretic separation of cDNA fragments (cDNA-AFLP). Electrophoresis 30, 2549-2557

Weiberg, A., Pohler, D., Morgenstern, B. & Karlovsky, P. (2008). Improved coverage of cDNA-AFLP by sequential digestion of immobilized cDNA. BMC Genomics 9, 480

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