Some pathogens and pests deliver small RNAs (sRNAs) into host cells to suppress host immunity. Conversely, hosts also transfer sRNAs into pathogens and pests to inhibit their virulence. Although sRNA ...trafficking has been observed in a wide variety of interactions, how sRNAs are transferred, especially from hosts to pathogens and pests, is still unknown. Here, we show that host
cells secrete exosome-like extracellular vesicles to deliver sRNAs into fungal pathogen
These sRNA-containing vesicles accumulate at the infection sites and are taken up by the fungal cells. Transferred host sRNAs induce silencing of fungal genes critical for pathogenicity. Thus,
has adapted exosome-mediated cross-kingdom RNA interference as part of its immune responses during the evolutionary arms race with the pathogen.
State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, China Affiliation: Department of Microbiology and Plant Pathology, Center for Plant Cell Biology, Institute for ...Integrative Genome Biology, University of California, Riverside, California, United States of America Baoye He Affiliation: Department of Microbiology and Plant Pathology, Center for Plant Cell Biology, Institute for Integrative Genome Biology, University of California, Riverside, California, United States of America ORCID logo http://orcid.org/0000-0003-0401-4955 Arne Weiberg Affiliation: Department of Biology, Ludwig-Maximilians University of Munich (LMU), Munich, Germany ORCID logo http://orcid.org/0000-0003-4300-4864 Amy H. Buck Affiliations Institute of Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom, Centre for Immunity, Infection and Evolution, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom ORCID logo http://orcid.org/0000-0003-2645-7191 Hailing Jin * E-mail: hailingj@ucr.edu Affiliation: Department of Microbiology and Plant Pathology, Center for Plant Cell Biology, Institute for Integrative Genome Biology, University of California, Riverside, California, United States of America ORCID logo http://orcid.org/0000-0001-5778-5193 Citation: Cai Q, He B, Weiberg A, Buck AH, Jin H (2019) Small RNAs and extracellular vesicles: In detail, Botrytis cinerea, the grey mold fungal pathogen that infects over 1,000 plant species, delivers sRNAs into plant cells and hijacks host RNAi machinery by loading its sRNAs into the Arabidopsis AGO1 protein to trigger silencing of host immunity genes, including mitogen-activated protein kinases (MAPKs), cell-wall–associated kinases, and other defense and signaling proteins 2. AGO, Argonaute; EE, early endosome; ER, endoplasmic reticulum; EV, extracellular vesicle; MV, microvesicle; MVB, multivesicular body; RNAi, RNA interference; sRNA, small RNA; TGN, trans-Golgi network. https://doi.org/10.1371/journal.ppat.1008090.g001 Cross-kingdom sRNA trafficking from a fungal pathogen to an animal host was also observed recently. Patients who suffer from sickle cell anemia show abnormal erythrocyte development but exhibit resistance to the malaria parasite Plasmodium falciparum.
Small RNAs (sRNAs) of the fungal pathogen Botrytis cinerea can enter plant cells and hijack host Argonaute protein 1 (AGO1) to silence host immunity genes. However, the mechanism by which these ...fungal sRNAs are secreted and enter host cells remains unclear. Here, we demonstrate that B. cinerea utilizes extracellular vesicles (EVs) to secrete Bc-sRNAs, which are then internalized by plant cells through clathrin-mediated endocytosis (CME). The B. cinerea tetraspanin protein, Punchless 1 (BcPLS1), serves as an EV biomarker and plays an essential role in fungal pathogenicity. We observe numerous Arabidopsis clathrin-coated vesicles (CCVs) around B. cinerea infection sites and the colocalization of B. cinerea EV marker BcPLS1 and Arabidopsis CLATHRIN LIGHT CHAIN 1, one of the core components of CCV. Meanwhile, BcPLS1 and the B. cinerea-secreted sRNAs are detected in purified CCVs after infection. Arabidopsis knockout mutants and inducible dominant-negative mutants of key components of the CME pathway exhibit increased resistance to B. cinerea infection. Furthermore, Bc-sRNA loading into Arabidopsis AGO1 and host target gene suppression are attenuated in those CME mutants. Together, our results demonstrate that fungi secrete sRNAs via EVs, which then enter host plant cells mainly through CME.
Plants use extracellular vesicles (EVs) to transport small RNAs (sRNAs) into their fungal pathogens and silence fungal virulence-related genes through a phenomenon called 'cross-kingdom RNAi'. It ...remains unknown, however, how sRNAs are selectively loaded into EVs. Here, we identified several RNA-binding proteins in Arabidopsis, including Argonaute 1 (AGO1), RNA helicases (RHs) and annexins (ANNs), which are secreted by exosome-like EVs. AGO1, RH11 and RH37 selectively bind to EV-enriched sRNAs but not to non-EV-associated sRNAs, suggesting that they contribute to the selective loading of sRNAs into EVs. Conversely, ANN1 and ANN2 bind to sRNAs non-specifically. The ago1, rh11 rh37 and ann1 ann2 mutants showed reduced secretion of sRNAs in EVs, demonstrating that these RNA-binding proteins play an important role in sRNA loading and/or stabilization in EVs. Furthermore, rh11 rh37 and ann1 ann2 showed increased susceptibility to Botrytis cinerea, suggesting that RH11, RH37, ANN1 and ANN2 positively regulate plant immunity against B. cinerea.
Plants communicate with their interacting microorganisms through the exchange of functional molecules. This communication is critical for plant immunity, for pathogen virulence, and for establishing ...and maintaining symbioses. Extracellular vesicles (EVs) are lipid bilayer‐enclosed spheres that are released by both the host and the microbe into the extracellular environment. Emerging evidence has shown that EVs play a prominent role in plant–microbe interactions by safely transporting functional molecules, such as proteins and RNAs to interacting organisms. Recent studies revealed that plant EVs deliver fungal gene‐targeting small RNAs into fungal pathogens to suppress infection via cross‐kingdom RNA interference (RNAi). In this review, we focus on the recent advances in our understanding of plant EVs and their role in plant–microbe interactions.
A key characteristic of chloroplast gene expression is the predominance of posttranscriptional control via numerous nucleus-encoded RNA binding factors. Here, we explored the essential roles of the ...S1-domain-containing protein
(
)/
Stabilizing Factor (BSF) in the stabilization and translation of chloroplast mRNAs. BSF binds to the intergenic region of
-
, thereby stabilizing 3' processed
transcripts and stimulating
translation. BSF also binds to the 5' untranslated region of
and activates its translation. BSF displayed nucleic-acid-melting activity in vitro, and its absence induces structural changes to target RNAs in vivo, suggesting that BSF functions as an RNA chaperone to remodel RNA structure. BSF physically interacts with the pentatricopeptide repeat protein Chloroplast RNA Processing 1 (AtCRP1) and the ribosomal release factor-like protein Peptide chain Release Factor 3 (PrfB3), whose established RNA ligands overlap with those of BSF. In addition, PrfB3 stimulated the RNA binding ability of BSF in vitro. We propose that BSF and PrfB3 cooperatively reduce the formation of secondary RNA structures within target mRNAs and facilitate AtCRP1 binding. The translation activation function of BSF for
is conserved in Arabidopsis (
) and maize (
), but that for
operates specifically in Arabidopsis. Our study sheds light on the mechanisms by which RNA binding proteins cooperatively regulate mRNA stability and translation in chloroplasts.
Significance The assembly of chloroplast ATP synthase, a multiprotein complex made of subunits of mixed genetic origin, is essential for organelle biogenesis and organism survival. Assembly of ...multiprotein complexes relies not only on the proper action of universal folding chaperones but also on that of specific assembly chaperones that orchestrate the joining of individual components into higher-order complexes. In this work, we have identified a specific assembly chaperone for chloroplast ATP synthase and shown that it functions downstream of the Cpn60 chaperone to promote assembly of the catalytically active core of the chloroplast ATP synthase. Such collaboration between folding and assembly chaperones appears to be a widely used cellular strategy for the folding and assembly of nucleus-encoded proteins into multiprotein photosynthetic complexes.
The chloroplast ATP synthase, a multisubunit complex in the thylakoid membrane, catalyzes the light-driven synthesis of ATP, thereby supplying the energy for carbon fixation during photosynthesis. The chloroplast ATP synthase is composed of both nucleus- and chloroplast-encoded proteins that have required the evolution of novel mechanisms to coordinate the biosynthesis and assembly of chloroplast ATP synthase subunits temporally and spatially. Here we have elucidated the assembly mechanism of the α ₃β ₃γ core complex of the chloroplast ATP synthase by identification and functional characterization of a key assembly factor, PAB (PROTEIN IN CHLOROPLAST ATPASE BIOGENESIS). PAB directly interacts with the nucleus-encoded γ subunit and functions downstream of chaperonin 60 (Cpn60)-mediated CF ₁γ subunit folding to promote its assembly into the catalytic core. PAB does not have any recognizable motifs or domains but is conserved in photosynthetic eukaryotes. It is likely that PAB evolved together with the transfer of chloroplast genes into the nucleus to assist nucleus-encoded CF ₁γ assembly into the CF ₁ core. Such coordination might represent an evolutionarily conserved mechanism for folding and assembly of nucleus-encoded proteins to ensure proper assembly of multiprotein photosynthetic complexes.
The chloroplast ribosome is a large and dynamic ribonucleoprotein machine that is composed of the 30S and 50S subunits. Although the components of the chloroplast ribosome have been identified in the ...last decade, the molecular mechanisms driving chloroplast ribosome biogenesis remain largely elusive. Here, we show that RNA helicase 22 (RH22), a putative DEAD RNA helicase, is involved in chloroplast ribosome assembly in Arabidopsis (Arabidopsis thaliana). A loss of RH22 was lethal, whereas a knockdown of RH22 expression resulted in virescent seedlings with clear defects in chloroplast ribosomal RNA (rRNA) accumulation. The precursors of 23S and 4.5S, but not 16S, rRNA accumulated in rh22 mutants. Further analysis showed that RH22 was associated with the precursors of 50S ribosomal subunits. These results suggest that RH22 may function in the assembly of 50S ribosomal subunits in chloroplasts. In addition, RH22 interacted with the 50S ribosomal protein RPL24 through yeast two-hybrid and pull-down assays, and it was also bound to a small 23S rRNA fragment encompassing RPL24-binding sites. This action of RH22 may be similar to, but distinct from, that of SrmB, a DEAD RNA helicase that is involved in the ribosomal assembly in Escherichia coli, which suggests that DEAD RNA helicases and rRNA structures may have coevolved with respect to ribosomal assembly and function.
•Plant send small RNAs in extracellular vesicles to fungal pathogens.•Cross-kingdom RNAi is an important regulatory mechanism between plant and eukaryotic pathogens.•Extracellular vesicles and ...membrane trafficking are essential for plant – pathogen communication.
Communication between plants and pathogens requires the transport of regulatory molecules across cellular boundaries, which is essential for host defense and pathogen virulence. Previous research has largely focused on protein transport, but, which other molecules function in communication, and how they are transported remains under explored. Recent studies discovered that small RNAs (sRNAs) are transported between plants and pathogens, which can silence target genes in the interacting organisms and regulate host immunity and pathogen infection, a mechanism called ‘cross-kingdom RNA interference (RNAi)’. Further studies indicate that plant extracellular vesicles (EVs) are essential for sRNA trafficking and host-pathogen communication. This review will focus on the latest advances in our understanding of plant EVs and their roles in transporting regulatory molecules, especially sRNAs, between hosts and pathogens.
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