•Small RNAs regulate plant immune responses and pathogen virulence.•Small RNAs can move between interacting organisms and induce cross-kingdom RNAi.•Advanced plant pathogens use cross-kingdom RNAi to ...suppress host immunity genes.•Host induced gene silencing provides a mechanism whereby crops produce small RNAs to silence pathogen genes
Eukaryotic regulatory small RNAs (sRNAs) that induce RNA interference (RNAi) are involved in a plethora of biological processes, including host immunity and pathogen virulence. In plants, diverse classes of sRNAs contribute to the regulation of host innate immunity. These immune-regulatory sRNAs operate through distinct RNAi pathways that trigger transcriptional or post-transcriptional gene silencing. Similarly, many pathogen-derived sRNAs also regulate pathogen virulence. Remarkably, the influence of regulatory sRNAs is not limited to the individual organism in which they are generated. It can sometimes extend to interacting species from even different kingdoms. There they trigger gene silencing in the interacting organism, a phenomenon called cross-kingdom RNAi. This is exhibited in advanced pathogens and parasites that produce sRNAs to suppress host immunity. Conversely, in host-induced gene silencing (HIGS), diverse plants are engineered to trigger RNAi against pathogens and pests to confer host resistance. Cross-kingdom RNAi opens up a vastly unexplored area of research on mobile sRNAs in the battlefield between hosts and pathogens.
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A never-ending arms race drives coevolution between pathogens and hosts. In plants, pathogen attacks invoke multiple layers of host immune responses. Many pathogens deliver effector proteins into ...host cells to suppress host immunity, and many plants have evolved resistance proteins to recognize effectors and trigger robust resistance. Here, we discuss findings on noncoding small RNAs (sRNAs) from plants and pathogens, which regulate host immunity and pathogen virulence. Recent discoveries have unveiled the role of noncoding sRNAs from eukaryotic pathogens and bacteria in pathogenicity in both plant and animal hosts. The discovery of fungal sRNAs that are delivered into host cells to suppress plant immunity added sRNAs to the list of pathogen effectors. Similar to protein effector genes, many of these sRNAs are generated from transposable element (TE) regions, which are likely to contribute to rapidly evolving virulence and host adaptation. We also discuss RNA silencing that occurs between organisms.
Botrytis cinerea, the causative agent of gray mold disease, is an aggressive fungal pathogen that infects more than 200 plant species. Here, we show that some B. cinerea small RNAs (Bc-sRNAs) can ...silence Arabidopsis and tomato genes involved in immunity. These Bc-sRNAs hijack the host RNA interference (RNAi) machinery by binding to Arabidopsis Argonaute 1 (AGO1) and selectively silencing host immunity genes. The Arabidopsis ago1 mutant exhibits reduced susceptibility to B. cinerea, and the B. cinerea dcl1 dcl2 double mutant that can no longer produce these Bc-sRNAs displays reduced pathogenicity on Arabidopsis and tomato. Thus, this fungal pathogen transfers "virulent" sRNA effectors into host plant cells to suppress host immunity and achieve infection, which demonstrates a naturally occurring cross-kingdom RNAi as an advanced virulence mechanism.
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
, 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
sRNA (
sRNA) functionality in ck-RNAi, we designed a novel CRISPR endoribonuclease Csy4/GUS reporter that enabled in situ visualization of
sRNA-induced target suppression in Arabidopsis. The significant role of
sRNAs together with
AGO1 in virulence was revealed in plant
mutants and by transgenic Arabidopsis expressing a short-tandem-target-mimic to block
sRNAs, that both exhibited enhanced resistance.
sRNA-targeted plant genes contributed to host immunity, as Arabidopsis gene knockout mutants displayed quantitatively enhanced susceptibility.
Pathogens secrete effector proteins to suppress host immune responses. Recently, we showed that an aggressive plant fungal pathogen Botrytis cinerea can also deliver small RNA effectors into host ...cells to suppress host immunity. B. cinerea sRNAs (Bc-sRNAs) translocate into host plants and hijack the plant RNAi machinery to induce cross-kingdom RNAi of host immune responsive genes. Here, we functionally characterized another Bc-sRNA effector Bc-siR37 that is predicted to target at least 15 Arabidopsis genes, including WRKY transcription factors, receptor-like kinases, and cell wall-modifying enzymes. Upon B. cinerea infection, the expression level of Bc-siR37 was induced, and at least eight predicted Arabidopsis target genes were downregulated. These target genes were also suppressed in the transgenic Arabidopsis plants overexpressing Bc-siR37, which exhibited enhanced disease susceptibility to B. cinerea. Furthermore, the knockout mutants of the Bc-siR37 targets, At-WRKY7, At-PMR6, and At-FEI2, also exhibited enhanced disease susceptibility to B. cinerea, giving further support that these genes indeed play a positive role in plant defense against B. cinerea. Our study demonstrates that analysis of pathogen sRNA effectors can be a useful tool to help identify host immunity genes against the corresponding pathogen.
Jasmonates are vital plant hormones that not only act in the stress response to biotic and abiotic influences, such as wounding, pathogen attack, and cold acclimation, but also drive developmental ...processes in cooperation with other plant hormones. The biogenesis of jasmonates starts in the chloroplast, where several enzymatic steps produce the jasmonate precursor 12-oxophytodienoic acid (OPDA) from α-linolenic acid. OPDA in turn is exported into the cytosol for further conversion into active jasmonates, which subsequently induces the expression of multiple genes in the nucleus. Despite its obvious importance, the export of OPDA across the chloroplast membranes has remained elusive. In this study, we characterized a protein residing in the chloroplast outer membrane, JASSY, which has proven indispensable for the export of OPDA from the chloroplast. We provide evidence that JASSY has channel-like properties and propose that it thereby facilitates OPDA transport. Consequently, a lack of JASSY in Arabidopsis leads to a deficiency in accumulation of jasmonic acids, which results in impaired expression of jasmonate target genes on exposure to various stresses. This results in plants that are more susceptible to pathogen attack and also exhibit defects in cold acclimation.
Aggressive fungal pathogens such as Botrytis and Verticillium spp. cause severe crop losses worldwide. We recently discovered that Botrytis cinerea delivers small RNAs (Bc-sRNAs) into plant cells to ...silence host immunity genes. Such sRNA effectors are mostly produced by Botrytis cinerea Dicer-like protein 1 (Bc-DCL1) and Bc-DCL2. Here we show that expressing sRNAs that target Bc-DCL1 and Bc-DCL2 in Arabidopsis and tomato silences Bc-DCL genes and attenuates fungal pathogenicity and growth, exemplifying bidirectional cross-kingdom RNAi and sRNA trafficking between plants and fungi. This strategy can be adapted to simultaneously control multiple fungal diseases. We also show that Botrytis can take up external sRNAs and double-stranded RNAs (dsRNAs). Applying sRNAs or dsRNAs that target Botrytis DCL1 and DCL2 genes on the surface of fruits, vegetables and flowers significantly inhibits grey mould disease. Such pathogen gene-targeting RNAs represent a new generation of environmentally friendly fungicides.
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.
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
during infection of its host plant
. 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.
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