Update on Small Regulatory RNA Exchanges Exchange of Small Regulatory RNAs between Plants and Their Pests1[OPEN] Collin Hudzik,a,2 Yingnan Hou,b,2 Wenbo Ma,b,3 and Michael J. Axtella,3,4 aDepartment of Biology, Intercollege Ph.D. Program in Plant Biology, and Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, Pennsylvania 16802 bDepartment of Microbiology and Plant Pathology, Center for Plant Cell Biology, University of California, Riverside, California 92521 Downloaded from https://academic.oup.com/plphys/article/182/1/51/6116263 by guest on 01 October 2021 ORCID IDs: 0000-0001-5569-639X (W.M.); 0000-0001-8951-7361 (M.J.A.). Regulatory small RNAs are well known as antiviral agents, regulators of gene expression, and defenders of genome integrity in plants. Several studies over the last decade have also shown that some small RNAs are exchanged between plants and their pathogens and parasites. Naturally occurring trans-species small RNAs are used by host plants to silence mRNAs in pathogens. These gene-silencing events are thought to be detrimental to the pathogen and beneficial to the host. Conversely, trans-species small RNAs from pathogens and parasites are deployed to silence host mRNAs; these events are thought to be beneficial for the pests. The natural ability of plants to exchange small RNAs with invading eukaryotic organisms can be exploited to provide disease resistance. This review gives an overview of the current state of trans-species small RNA research in plants and discusses several outstanding questions for future research. SMALL REGULATORY RNA BACKGROUND of the primary transcript forms an imperfect hairpin structure that is recognized by the DCL1 endonucle- Small regulatory RNAs (sRNAs) are numerous in ase. DCL1, along with several accessory proteins, plants. They usually range in size from 21 to 24 nucle- liberates a miRNA/miRNA* duplex. The duplex is otides and serve as key regulators of gene expression. disassembled, with the mature miRNA becoming sRNAs are involved in myriad processes, including bound to an Argonaute (AGO) protein, most frequently development, cell type designation, responses to abi- AGO1. Once the mature miRNA is bound to an AGO otic stress, and silencing of repetitive elements. sRNAs protein, the miRNA* is typically separated from the are processed from longer precursor RNAs (either the complex and degraded (for a more detailed review of helical stem regions of self-complementary single- plant miRNA biogenesis, see Rogers and Chen, 2013). stranded RNAs or double-stranded RNAs [dsRNAs]) The resulting miRNA/AGO complex directs post- by endonucleases in the Dicer-like (DCL) protein fam- transcriptional regulation of mRNAs and long non- ily. DCL endonucleases produce an initial short duplex coding RNAs. Target selection is primarily based on RNA. One of the two short RNA strands forms a com- complementarity between the miRNA and target RNA plex with a protein in the Argonaute (AGO) family. The AGO-sRNA complex then identifies target RNAs based on complementarity between target and sRNA. sRNAs can be categorized based on differences in their biogen- esis and differences in their modes of targeting (Fig. 1). MicroRNAs (miRNAs) in plants are processed from RNA polymerase II-transcribed primary RNAs. A region 1This work was supported by the U.S. Department of Agriculture- National Institute of Food and Agriculture (award nos. 2018-67013- 28514 and 2018-67014-28488) and by the National Science Foundation (grant nos. IOS-1340001 and IOS-1758889). 2These authors contributed equally to the article. 3Senior authors. 4Author for contact: [email protected]. C.H. and M.J.A. wrote sections on small RNA biology and para- site-to-host transfer of small RNAs; W.M. and Y.H. wrote sections on HIGS and host-to-pathogen transfer of small RNAs; figures were made by M.J.A.; all authors cooperatively merged and polished the final draft. [OPEN]Articles can be viewed without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.19.00931 Plant PhysiologyÒ, January 2020, Vol. 182, pp. 51–62, www.plantphysiol.org Ó 2020 American Society of Plant Biologists. All Rights Reserved. 51 Hudzik et al. Downloaded from https://academic.oup.com/plphys/article/182/1/51/6116263 by guest on 01 October 2021 Figure 1. Schematic overview of endogenous small RNA biogenesis and molecular functions in plants. The rounded rectangle with the mRNA represents the open reading frame. Figures are not drawn to scale; miRNA and siRNA duplexes have two- nucleotide 39 overhangs. DRM, Domains rearranged methyltransferase. For brevity, many details are omitted; see Rogers and Chen (2013), Matzke and Mosher (2014), and Borges and Martienssen (2015) for detailed discussions of plant small RNA bio- genesis. (Mallory et al., 2004; Liu et al., 2014). Plant AGO pro- Two major classes of plant siRNAs have been widely teins are endonucleases that cut target RNAs. (Tang recognized: secondary siRNAs and RNA polymerase et al., 2003; Baumberger and Baulcombe, 2005; Qi IV-dependent siRNAs (p4-siRNAs; Fig. 1). et al., 2005). This target slicing destabilizes the RNA. Secondary siRNA biogenesis depends on an initial The association of AGO/miRNA complexes with mRNAs AGO-miRNA or AGO-siRNA interaction with a target can also cause translational repression and in certain RNA. This interaction stimulates the activity of a spe- cases trigger the biogenesis of secondary short inter- cific RNA-dependent RNA polymerase (RDR6) on the fering RNAs (siRNAs; Fig. 1). Most plant miRNAs are target,creatingdsRNA.Typically,thedsRNAispro- 21 nucleotides long. miRNAs of 22 nucleotides also cessed into siRNA duplexes by both DCL4 (which sometimes occur, but sizes other than 21 or 22 nucle- makes 21-nucleotide-long duplexes) and DCL2 (which otides are much less common. makes 22-nucleotide-long duplexes). The resulting Besides miRNAs, many other sRNAs are produced population of 21- to 22-nucleotide secondary siRNAs and used by the plant DCL/AGO system. These are can be bound to AGO proteins and target additional collectively termed siRNAs. Plant siRNAs are typically copies of the original transcript as well as other tran- generated from dsRNA and can be processed by mul- scripts with sufficient complementarity. The result is a tiple DCLs. They are distinguished from miRNAs by positive feedback loop where an initial miRNA or the absence of a precisely processed stem-loop precur- siRNA trigger can amplify its effects and cause potent sor. Also, unlike miRNAs, which target RNAs distinct gene silencing. Not all AGO/miRNA or AGO/siRNA from their own precursors, plant siRNAs typically tar- targets spawn secondary siRNAs. For reasons that get transcripts from the same loci where they originate. remain murky, targeting by a 22-nucleotide miRNA or 52 Plant Physiol. Vol. 182, 2020 sRNA Exchanges during Plant-Pest Interactions siRNA (Chen et al., 2010; Cuperus et al., 2010) and/or defense (Baulcombe, 2004; Ding, 2010). Plants infected multiple target sites on the same RNA (Axtell et al., with viruses acquire immunity by producing DCL- 2006) promote secondary siRNA biogenesis; in con- dependent and virus-derived siRNAs, which guide trast, targeting at single sites by typical 21-nucleotide AGO proteins to viral RNAs and thus help to arrest the miRNAs does not promote secondary siRNA accumu- infection (Guo et al., 2019). However, it was not until lation. Secondary siRNA biogenesis does not strictly recently that a role of sRNAs was established in plant require AGO-catalyzed slicing of the precursor (Axtell defense during infections by cellular pathogens, especially et al., 2006; Arribas-Hernández et al., 2016), but slicing eukaryotic pathogens including fungi and oomycetes. often occurs. When it does occur at a single predomi- An early example of pathogen gene silencing in- nant site, the resulting dsRNA production will all be- duced by host sRNAs was from the observation that gin at the same position. Because the relevant DCLs native miRNAs produced from human erythrocytes liberate secondary siRNA duplexes sequentially from translocate into the malaria-causing parasite Plasmo- the dsRNA terminus, the resulting siRNA population has dium falciparum and inhibit pathogen gene expression Downloaded from https://academic.oup.com/plphys/article/182/1/51/6116263 by guest on 01 October 2021 59 and 39 ends at regularly defined 21- to 22-nucleotide (LaMonte et al., 2012). Whether endogenous sRNAs in intervals. This property is known as phasing, and the plants could mediate trans-species RNAi remained resulting secondary siRNAs are thus known as phased unknown until the report from Zhang et al. (2016b), siRNAs (Fei et al., 2013). In some cases, secondary which described two cotton (Gossypium hirsutum) siRNAs can also target mRNAs that are distinct from miRNAs, miR159 and miR166, that conferred resistance their precursor RNA; these siRNAs have been called to the fungal pathogen Verticillium dahliae (Fig. 2). trans-acting siRNAs (tasiRNAs; Vazquez et al., 2004). miR159 and miR166 are induced upon the fungal in- We regard tasiRNAs and phased siRNAs as subsets fection. Importantly, they were detected in fungal within the more general class of secondary siRNAs. hyphae isolated from the infected cotton tissues and The third major type of plant sRNAs are the predicted to target specific transcripts encoding p4-siRNAs. Both the biogenesis and function of p4- virulence-related proteins in the fungus. As a result, siRNAs are distinct from miRNAs and secondary siR- these miRNAs promoted resistance to V. dahliae.This NAs. RDR2 is attached to RNA polymerase IV and defense mechanism seems to be conserved in Arabi- generates a short (;40 nucleotides) dsRNA. This dsRNA dopsis (Arabidopsis thaliana), in which miR159 and is in turn processed into a 24-nucleotide-long siRNA miR166 were also induced by V. dahliae infection (Zhang duplex by DCL3. A mature 24-nucleotide p4-siRNA is et al., 2016b). Furthermore, knockdown mutants of then loaded onto a specialized AGO in the AGO4 clade.