Update on Small Regulatory RNA Exchanges

Exchange of Small Regulatory RNAs between 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 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. miR166 showed compromised resistance to the fun- A major distinctive feature of p4-siRNAs is that they are gal pathogen, indicating that miR166 contributed to not known to target mRNAs or to function outside of plant defense, possibly through trans-species gene the nucleus. Instead, they function in the nucleus to silencing. target non-protein-coding nascent RNAs. Successful Following the discovery that plant miRNAs could p4-siRNA/AGO targeting is thought to lead to the enter invading V. dahliae cells and induce gene silenc- recruitment of de novo DNA methyltransferases to the ing, studies in Arabidopsis showed that siRNAs were local chromatin, causing de novo DNA methylation. also used to silence target genes in the necrotrophic The outcome of p4-siRNA function is therefore DNA fungal pathogen Botrytis cinerea (Cai et al., 2018) and the modification of sequence-similar loci. Secondary siRNAs hemibiotrophic oomycete pathogen Phytophthora capsici can sometimes be captured by the p4-siRNA-specific (Hou et al., 2019). Two siRNAs derived from the non- AGOs and cause DNA methylation of homologous loci coding, secondary siRNA-spawning loci TAS1 and (Wu et al., 2012; McCue et al., 2015). Thus, all three major TAS2 were found to target genes involved in vesicle typesofplantsRNAsareconnectedtoeachother: trafficking in B. cinerea; as a result, overexpression of miRNAs can stimulate secondary siRNAs, and sec- these siRNAs in Arabidopsis led to reduced virulence ondary siRNAs can act like p4-siRNAs. Although p4- of the fungal pathogen (Cai et al., 2018). During the siRNAs are very abundant in plants, they are not infection of P. capsici, a pool of siRNAs generated from a currently known to be involved in trans-species small few transcripts of pentatricopeptide repeat (PPR) en- RNA interactions and thus will not be discussed fur- coding genes was induced as a defense response (Hou ther here. Interested readers are directed to the excel- et al., 2019). These PPR-derived siRNAs represent a lent reviews by Borges and Martienssen (2015) and large diversity of sequences and presumably silence Matzke and Mosher (2014) for details on p4-siRNAs. pathogen genes using a shotgun approach. Multiple PPR-derived siRNAs were predicted to target several genes in P. capsici, some of which contribute to patho- gen development and colonization. The PPR-derived PATHOGEN GENE SILENCING INDUCED BY siRNAs also have predicted targets in V. dahliae, indi- HOST sRNAS cating that they may contribute to broad-spectrum re- Since the discovery of RNA interference (RNAi) sistance to diverse pathogens. Both the TAS-derived about two decades ago, evidence has accumulated to and PPR-derived siRNAs are secondary siRNAs, whose indicate a role for sRNAs in plant defense. Small RNA- biosynthesis depends on the activity of RDR6. rdr6 mediated immunity is best understood in antiviral mutants of Arabidopsis exhibited hypersusceptibility

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Figure 2. Examples of host plant-to-pathogen (top) and pathogen/parasite-to-host plant (bottom) movement and function of trans- species small RNAs. The rounded rectangles of mRNAs represent open reading frames. Figures are not drawn to scale; miRNA and siRNA duplexes have two-nucleotide 39 overhangs. All experimentally confirmed target RNAs or target RNA families are shown and are listed as they appear from top to bottom and from left to right in each part of the figure. MYB, Myelobastosis (Arabidopsis loci: AT5G06100 and AT3G11440); HD-ZIP III, Homeodomain Leu-zipper class III (Arabidopsis loci: AT2G34710, AT1G30490, AT5G60690, AT4G32880, and AT1G52150); TAS, Trans-acting siRNA (Arabidopsis loci: AT2G27400, AT1G50055, AT2G39675, and AT2G39681); PPR, Pentatricopeptide repeat (Arabidopsis loci: AT1G62590, AT1G62910, AT1G62914, AT1G62930, AT1G63080, AT1G63130, AT1G63150, and AT1G63400); PRXIIF, Peroxiredoxin2-F (Arabidopsis locus: AT3G06050); MPK, Mitogen-activated kinase (Arabidopsis loci: AT1G10210 and AT1G59580); MPKKK, Mitogen-activated kinase kinase kinase; WAK, Wall-associated kinase (Arabidopsis locus: AT5G50290); BIK1, Botrytis-induced kinase1 (Arabidopsis locus: AT2G39660); TIR1, Transport inhibitor related1 (Arabidopsis locus: AT3G62980); AFB, Auxin-related F-box (Arabidopsis loci: AT3G26810 and AT1G12810); SEOR1, Sieve element occlusion related1 (Arabidopsis locus: AT3G01680); SCZ, Schizorhiza (Arabidopsis locus:

54 Plant Physiol. Vol. 182, 2020 sRNA Exchanges during Plant-Pest Interactions to B. cinerea, P. capsici,andV. dahliae (Ellendorff et al., infestation of these insects in cotton (Baum et al., 2007) 2009; Cai et al., 2018; Hou et al., 2019). The contribu- and maize (Zea mays; Mao et al., 2007). Following these tion of RDR6 to plant immunity is likely attributed to early studies, HIGS has been demonstrated to be a its essential role in the production of secondary siR- promising tool in controlling parasitic weeds. Differ- NAs that silence pathogen mRNAs. ent from nematodes and insects, parasitic plants exploit External application of both dsRNAs and sRNAs has host plants by connecting to the host’s vasculature been reported to induce gene silencing in pathogens and taking up water and nutrients. Through the con- (Koch et al., 2016; Wang et al., 2016; Rosa et al., 2018), nected vasculature, hosts and pathogens exchange raising the question whether dsRNAs instead of mature RNA molecules including mRNAs and sRNAs (Kim siRNAs are being transferred from plants to pathogens. and Westwood, 2015). Transgenic lettuce (Lactuca sativa) In Arabidopsis, PPR-derived siRNAs were produced expressing hairpin RNAs that target the GUS reporter by DCL4 and its cofactor Double-Stranded-RNA- gene was found to be able to silence GUS expression in Binding Protein4 (DRB4). In addition to the rdr6 mu- the parasitic plant Triphysaria versicolor (Tomilov et al., Downloaded from https://academic.oup.com/plphys/article/182/1/51/6116263 by guest on 01 October 2021 tant, the drb4 mutant of Arabidopsis also showed 2008). A similar approach was successfully used to hypersusceptibility to P. capsici (Hou et al., 2019) and induce gene silencing in Orobanche and spe- V. dahliae (Ellendorff et al., 2009). Because the drb4 cies, thus limiting parasitic growth (Aly et al., 2009; mutant still accumulates the precursor dsRNA, these Alakonya et al., 2012). findings indicate that mature siRNAs or siRNA HIGS has also been successfully employed to confer duplexes, rather than dsRNAs, are likely the antimi- resistance to specific fungi and oomycete pathogens. crobial agents. Taken together, the current data suggest Barley (Hordeum vulgare) and wheat (Triticum spp.) that trans-species RNAi between plant hosts and fun- expressing artificial siRNAs were found to be more gal/oomycete pathogens is an integral component of resistant to the biotrophic fungal pathogen Blumeria plant immunity. In particular, since the endogenous graminis (Nowara et al., 2010) and the hemibiotrophic functions of many plant secondary siRNAs are unclear, fungal pathogen Fusarium graminearum (Koch et al., their main activity may be trans-species gene silencing. 2013). A similar approach was used in tomato (Solanum lycopersicum) and cotton to enhance resistance to wilt- ing disease caused by V. dahliae (Zhang et al., 2016a; Song and Thomma, 2018). Furthermore, successful APPLICATION OF HOST-INDUCED GENE silencing of targeted genes, and hence increased re- SILENCING IN PLANT DISEASE CONTROL sistance, was reported in the hemibiotrophic oomycete Although the natural role of endogenous sRNAs in pathogen Phytophthora infestans using potato (Solanum trans-species gene silencing is a recent discovery in tuberosum) plants expressing siRNA-producing hair- plants, substantial efforts have been invested to engi- pin RNAs (Jahan et al., 2015). These numerous exam- neer plant sRNAs designed to inhibit the infection by ples of HIGS support engineering sRNAs of plants as a cellular pathogens and parasites. This strategy, termed practical tool in agricultural biotechnology. The suc- host-induced gene silencing (HIGS), involves trans- cess of HIGS in such diverse systems also indicates genic plants developed to produce dsRNA precursors that sRNAs may frequently move from host to path- with homology to target mRNAs in invading pests. It is ogen/parasite. Thus, we expect the number of known presumed that the dsRNA precursors produce artificial natural examples of host-to-pathogen/parasite sRNA sRNAs, which are then taken up by the invaders and movement to continue to grow. induce gene silencing using the pathogen’s RNA si- Nearly all of the reports of successful HIGS have used lencing machinery. Some of these plants were indeed a strategy of producing siRNAs from long hairpin RNA found to exhibit enhanced resistance to the targeted precursors. Long hairpin-induced RNAi primarily en- organism. gages the DCL4/DCL2-dependent secondary siRNA The first successful example of HIGS for nonviral pathway, and to a lesser extent the DCL3-dependent pests was reported in Arabidopsis, where the expres- p4-siRNA pathway, to produce a potent mixture of sion of specific hairpin RNAs induced the silencing of a 21-, 22-, and 24-nucleotide-long siRNAs (Fusaro et al., gene encoding a secretory peptide in root-knot nema- 2006). However, empirical evidence showing that the todes (Meloidogyne spp.) and resulted in disease resis- plant-produced 21-, 22-, and 24-nucleotide siRNAs are tance (Huang et al., 2006). dsRNAs designed to target a directly responsible for HIGS is lacking. Further ex- cytochrome P450 gene in cotton bollworm (Helicoverpa periments using dcl and drb mutants are required to armigera) and a vacuolar ATPase gene in coleopteran demonstrate whether intact dsRNAs can enter the insect pests were also found to significantly reduce the pathogen/parasite and be subsequently processed by

Figure 2. (Continued.) AT1G46264); HiC-51, Isotrichodermin C-15 hydroxylase (V. dahliae locus: VDAG_09950); Clp-1, Calpain1 (V. dahliae locus: VDAG_09736); Bc-VPS51, B. cinerea vacuolar protein sorting51 (B. cinerea locus: BC1G_10728); Bc-DCTN, B. cinerea dynactin complex large subunit (B. cinerea locus: BC1G_10508); Bc-SAC1, B. cinerea suppressor of actin-like (B. cinerea locus: BC1G_08464); LTR, Long-terminal repeat retrotransposon.

Plant Physiol. Vol. 182, 2020 55 Hudzik et al. the endogenous machinery of the recipient organism mutation in host dcl4 or rdr6) fails to noticeably affect for gene silencing. C. campestris growth (Shahid et al., 2018). Thus, the importance of the secondary siRNAs in this interaction remains unknown. PATHOGEN/PARASITE-TO-HOST sRNA MOVEMENT Small RNA movement has also been shown to occur MECHANISM(S) OF sRNA MOVEMENT BETWEEN from pathogens and parasites (Fig. 2), which employ PATHOGENS AND PLANT CELLS sRNAs as one of their numerous molecular strategies to subvert host defenses and remodel host physiology to Small RNAs are known to be highly mobile mole- their advantage. In at least two known cases, these cules that traffic intercellularly and systemically in strategies include the delivery of sRNAs into host tis- plants (Liu and Chen, 2018). In plants, intercellular sues to silence host mRNAs. The pathogenic fun- movement involves sRNA transfer through plasmo- gus B. cinerea accumulates several sRNAs during desmata, and systemic movement is believed to occur Downloaded from https://academic.oup.com/plphys/article/182/1/51/6116263 by guest on 01 October 2021 plant infection (Weiberg et al., 2013). Some of these in the phloem. The phenomenon of HIGS as well as the sRNAs have complementarity to host immune signal- natural examples of trans-species sRNA movement ing mRNAs that are down-regulated during B. cinerea raise the question: How are sRNAs transferred between infection. The incoming B. cinerea sRNAs interact with different cellular organisms? Recent studies suggest a the host’s AGO1 protein, and Arabidopsis ago1 hypo- potential role of extracellular vesicles (EVs) in the morphic mutants are resistant to B. cinerea infection translocation of sRNAs from Arabidopsis to P. capsici (Weiberg et al., 2013). A dcl1/dcl2 double mutant of (Hou et al., 2019) and B. cinerea (Cai et al., 2018). B. cinerea lost accumulation of the relevant trans-species EVs are membrane-bound particles containing sRNAs and became avirulent (note that B. cinerea DCL1 transmembrane proteins and soluble cargoes. They are and DCL2 are not functionally homologous to plant released by donor cells into their surrounding envi- DCL1 and DCL2). Altogether, these data show that ronment. EVs have been extensively characterized in B. cinerea hijacks host sRNA machinery with its own animals and humans, where they are classified based sRNAs. An analysis of mRNA 59 ends has also indicated on their size and origins: exosomes, which are 30 to 100 that the oomycete Plasmopara viticola has a great number nm in diameter; shedding microvesicles, which are 100 of sRNAs that may target host (grapevine, Vitis spp.) nm to 1 mm in diameter; and apoptotic bodies, which mRNAs for cleavage during infection (Brilli et al., 2018). are 50 nm to 5 mm in diameter (Théry et al., 2009; Ressel Another example of parasite-to-host movement of et al., 2019). Microvesicles are shed from the plasma sRNAs comes from the parasitic plant Cuscuta cam- membrane; apoptotic bodies contain parts of dying pestris. Cuscuta spp. is a widespread genus of obligate cells formed during programmed cell death; and exo- parasitic plants that attach to host plant stems using a somes are formed in the cytosol by inward budding of specialized organ called haustorium. Cuscuta haustoria the limiting membrane of endocytic compartments, are permissive and known to permit bidirectional leading to vesicle-containing endosomes called multi- movement of mRNAs, proteins, and secondary me- vesicular bodies (MVBs). The vesicles are released into tabolites (Kim and Westwood, 2015). the environment when MVBs fuse with the plasma produces many miRNAs that specifically accumulate membrane (Raposo and Stoorvogel, 2013; Ressel et al., in haustorial tissues (Shahid et al., 2018). Some of these 2019). EVs have been shown to be key players in in- miRNAs specifically target host mRNAs involved in tercellular communication in animal systems. In par- pathogen defense, vascular system function, and ticular, EVs of animal cells participate in the transport hormone signaling. Examples include Sieve Element of proteins, lipids, mRNAs, miRNAs, and other non- Occlusion Related1 (SEOR1), which functions to reduce coding RNAs (Valadi et al., 2007; Yáñez-Mó et al., sap loss from wounded phloem (Ernst et al., 2012), and 2015). These discoveries led to the interesting possibil- Auxin F-Box Related3 (AFB3), which couples auxin ity that EVs may also shuttle sRNAs between plant sensing to transcriptional responses (Parry and Estelle, hosts and their invading pathogens or parasites. 2006). C. campestris growth is increased on Arabidopsis Plant EVs were first successfully isolated by Rutter seor1 or afb3 mutants (Shahid et al., 2018). These data and Innes (2017) from apoplastic wash fluid recovered suggest that trans-species miRNAs from C. campestris from Arabidopsis leaves. Proteomic analysis of the function to silence host genes in order to increase para- purified EVs revealed enrichment of defense-related site growth and fitness. Interestingly, many of the proteins. Examples of these proteins include the syn- C. campestris trans-speciesmiRNAsare22nucleotides taxin AtSYP121/PENETRATION1 (PEN1), which is long and induce secondary siRNA accumulation from related to papillae formation during fungal infection their targets. Secondary siRNA accumulation requires (Assaad et al., 2004), the ATP-binding cassette trans- host DCL4 and RDR6, which provides evidence that porter PEN3, which accumulates around haustoria of the C. campestris miRNAsareactinginsidehostcells. powdery mildew (Underwood and Somerville, 2013), An attractive hypothesis is that secondary siRNA in- and RPM1-INTERACTING PROTEIN4 (RIN4), which duction serves to strengthen the silencing caused by participates in immune responses triggered by bacterial the initial miRNA from the parasitic plant. However, infection (Mackey et al., 2003). These results suggest that reduction or removal of the secondary siRNAs (by plant EVs are associated with biotic stress responses.

56 Plant Physiol. Vol. 182, 2020 sRNA Exchanges during Plant-Pest Interactions

In addition to defense-related proteins, EVs in adjacent to the EHM (Takemoto et al., 2003; Lu et al., ArabidopsisalsocarrysRNAs(Caietal.,2018; 2012). Similarly, the accumulation of secretory vesicles Baldrich et al., 2019; Hou et al., 2019). A comparison of and MVBs in the proximity of the EHM was also ob- sRNA profilesinapoplastandEVsrevealedthat served in Arabidopsis during the infection of Golovino- sRNAs are differentially secreted and enriched in EVs myces orontii (Micali et al., 2011). These findings indicate (Baldrich et al., 2019). Interestingly, the majority of sRNA that plant sRNAs, together with other antimicrobial cargos in EVs are only 10 to 17 nucleotides in length proteins and metabolites, could be targeted to the EHM (Baldrich et al., 2019). These so-called tiny RNAs (tyR- and released into the EHMx. It is noteworthy that NAs) appear to be processing by-products of miRNA fungal/oomycete haustoriaareencasedbythepath- precursors and mRNAs. Whether they regulate gene ogen cell wall; it remains to be determined how the expression is currently unknown. sRNA profiling of pathogen cell wall may function as a barrier for the AGO-associated sRNAs has not found any evidence endocytosis of host sRNAs, especially those shuffled that AGO proteins bind 10- to 17-nucleotide RNAs as cargos in EVs. Downloaded from https://academic.oup.com/plphys/article/182/1/51/6116263 by guest on 01 October 2021 in vivo (Mi et al., 2008; Wang et al., 2011), so it seems Although they share the same name, the haustoria of likely that tyRNAs function differently than miRNAs parasitic plants are very distinct from those of patho- and siRNAs. Although miRNAs and siRNAs were also genic fungi and oomycetes. One fundamental differ- detected in EVs, their abundance was relatively low ence is that parasitic plant haustoria function to bridge compared with tyRNAs (Baldrich et al., 2019). Fur- the vascular systems of the host and parasite, thus en- thermore, specific miRNAs and siRNAs were found to abling large-scale flow of water, minerals, carbohy- be enriched in EVs, indicating a possible sorting drates, and other compounds into the parasitic plant mechanism. (Yoshida et al., 2016). Another fundamental difference Two PPR-derived siRNAs that silence target genes in is that parasitic plant haustoria are multicellular, mac- P. capsici were found to be EV cargos in Arabidopsis roscopic organs that contain several different special- (Hou et al., 2019). Their abundance increased in EVs ized cell types. The trans-species miRNAs that are isolated from P. capsici-infected leaves, consistent with delivered from C. campestris to hosts thus move in the the observation that PPR-derived siRNAs were in- opposite direction relative to the major flow of material duced during infection. The tasiRNAs that target across the haustorium; therefore, their delivery is not by mRNAs in the fungal pathogen B. cinerea were also simple bulk flow. The mature Cuscuta spp. haustorium present in EVs (Cai et al., 2018). However, secondary contains several distinct tissues in close contact with siRNAs, including multiple PPR-derived siRNAs and host cells (Dawson et al., 1994). The main endophyte tasiRNAs, were highly enriched in apoplast but not in body is a large papillar organ that penetrates the host EVs (Baldrich et al., 2019), indicating that these siRNAs epidermis and is lodged within host cortical tissue. were actively secreted by plant cells but possibly Long filamentous searching hyphae cells are projected through an EV-independent pathway. On the other from this main body, which thread through the middle hand, EV cargos may change in infected tissues and lamellae of host tissues and can penetrate into host cells. EV-dependent sRNA secretion could still be involved Searching hyphae that contact host xylem differentiate in defense responses. into xylem, while searching hyphae that contact host Following the release from plant cells, sRNAs need to phloem branch form finger-like projections that sur- enter pathogen cells, or vice versa in cases where patho- round the host phloem (Dawson et al., 1994). Each of gen sRNAs enter plant hosts. Biotrophic/hemibiotrophic these distinct Cuscuta spp. tissues is in direct contact filamentous pathogens and parasitic plants form spe- with host cells; which ones actually deliver trans- cialized infection structures called haustoria (Catanzariti species miRNAs is not clear. The tips of searching et al., 2006; Yoshida et al., 2016), which extend into hyphae that penetrate host cells have numerous plas- host cells and may act as the gateway for sRNA deliv- modesmatal connections between parasite and host ery. Fungal/oomycete haustoria are enveloped by the (Dawson et al., 1994); thus, one possibility is that extrahaustorial membrane (EHM), a modified plant miRNA delivery in this system occurs through plas- plasma membrane, and are separated from plant cells by modesmata. Another possibility is delivery by EVs. the extrahaustorial matrix (EHMx), in which active ma- Further studies are required to understand the cell and terial exchange is believed to occur (Koh et al., 2005). For molecular biology of trans-species sRNA movement in example, haustoria provide portals for nutrient uptake fungal, oomycete, and parasitic plant systems. from the host to the pathogen and the delivery of viru- lence effectors from the pathogen to the host (Catanzariti et al., 2006). On the other hand, antimicrobial agents EVOLUTION OF TRANS-SPECIES sRNAS produced by the host are also targeted to accumulate in the EHMx in order to arrest infection. Systematic studies Few studies have explicitly studied the evolution of of fluorescence-tagged cell components during oomy- trans-species small RNAs. Endogenous host secondary cete infection, including Phytophthora sojae, Peronospora siRNAs induced upon infection of P. capsici have the parasitica,andPhytophthora parasitica, showed the accu- capability to alter pathogen gene expression mediated mulation of Golgi stacks, endoplasmic reticulum, secre- by the induction of miR161 triggering siRNA accumu- tory vesicles, and MVBs in the infected plant cells lation from PPR transcripts (Hou et al., 2019). PPR

Plant Physiol. Vol. 182, 2020 57 Hudzik et al. proteins are primarily known as sequence-specific siRNAs (Xia et al., 2013). This is further evidence that RNA-binding proteins that are critical for mRNA mat- the Arabidopsis-specificmiR173/TAS1/TAS2 cascade uration in plastids (Miranda et al., 2018). However, is really a component or offshoot of the more wide- some PPR proteins also play roles in abiotic and biotic spread PPR secondary siRNA system. It is also im- defense (Kobayashi et al., 2007; Tang et al., 2010). The portant to consider that because secondary siRNA pool of secondary siRNAs made from PPR transcripts is accumulation seems to be a general defense response, extremely diverse, which might increase the chances of there may be subsets of secondary siRNAs that have successfully targeting of pathogen mRNAs (Hou et al., been specifically selected to target mRNAs from cer- 2019). The PPR gene family is large, but only members tain individual pathogens. This would result in host of a small clade spawn secondary siRNAs. This clade production of a massive number of secondary siRNAs evolves at a faster rate compared with other clades of with only a few being actually effective for any par- PPR genes (Dahan and Mireau, 2013), further suggest- ticular infection. An interesting hypothesis on how a ing that PPR-derived secondary siRNA accumulation host responds to pathogen infection is that the accu- Downloaded from https://academic.oup.com/plphys/article/182/1/51/6116263 by guest on 01 October 2021 could be under selective pressure to maximize sequence mulation of these antimicrobial sRNAs may not be diversity rather than to maintain pairing between any under any strong selective pressure but that the host particular siRNA and pathogen mRNA. Indeed, PPR- responds with a shotgun approach and by chance is derived secondary siRNAs have complementarity to able to target many different kinds of transcripts. Al- mRNAs from diverse pathogens. The induction of ternatively, this may also suggest that because path- miR161 requires an intact pattern recognition receptor ogenic transcripts are under such high selective complex on the plant cell surface (Hou et al., 2019). pressure to alter their target sites, the hosts have Furthermore, the miR161 level was increased in Ara- evolved a way to produce a wide variety of sequence bidopsis treated with the bacterial elicitor flg22 (Li et al., variations in an attempt to always remain one step 2010). These findings suggest that miR161 induction, ahead of their pathogens. and hence elevated accumulation of PPR-derived siRNAs, A counterpoint to the shotgun approach of using is a basal immune response that confers broad-spectrum secondarysiRNAsistheuseofmiRNAsintrans- resistance (Hou et al., 2019). PPR-based secondary siRNA species small RNA silencing. MicroRNAs by defini- production is commonly seen from diverse plants (Xia tion are loci where just one or two functional small et al., 2013), which suggests that this could be an an- RNAsaremadefromasingleprecursor(Axtelland cient evolutionary response that continues to confer Meyers, 2018). This contrasts with the large number effective resistance. Interestingly, although the exis- of siRNAs spawned from a single secondary siRNA tence of PPR-derived secondary siRNAs is quite con- precursor. The case described by Zhang et al. (2016b), served, the trigger miRNA, miR161, is restricted to the wherecottonmiR159andmiR166areactiveagainst Brassicaceae (Chávez Montes et al., 2014; Kozomara V. dahliae mRNAs, is especially curious from an evo- and Griffiths-Jones, 2014). The conservation of PPR- lutionary point of view. Both miR159 and miR166 are derived secondary siRNAs implies that some other ancient miRNAs with complete sequence conservation miRNA(s) serves as the trigger in non-Brassicaceae between ferns, gymnosperms, and angiosperms (Cuperus plants. One possibility is that the descendants of a et al., 2011; Chávez Montes et al., 2014). miR159 targets single ancestral miR161 have diversified substantially endogenous Myeloblastosis transcription factor mRNAs, in sequence in different plant lineages, confounding while miR166 targets endogenous Homeodomain leucine- the normal rules for miRNA family assignment but zipper class III mRNAs; these target relationships are retaining the ability to target PPR mRNAs. The iden- also universally conserved among land plants (Floyd tification of expressed small RNAs from some basal and Bowman, 2004; Jones-Rhoades and Bartel, 2004). lineages of plants (ferns and lycophytes) that have These conserved regulatory interactions are critical for some sequence homology to miR161 (You et al., 2017) plant development (Mallory et al., 2004; Allen et al., is consistent with this hypothesis. 2007). It seems clear that these two miRNAs have Secondary siRNAs from Arabidopsis TAS1 and TAS2 been selected for their roles in endogenous gene regu- noncoding RNAs can target mRNAs from B. cinerea lation. Moreover, any pathogen that has attacked any (Cai et al., 2018). The trigger for TAS1/TAS2 secondary land plant in the last ;250 million years would have siRNA accumulation is miR173. Some TAS1/TAS2- been exposed to both miR159 and miR166 as well as a derived secondary siRNAs are also able to target PPR handful of other ultraconserved plant miRNAs. It is transcripts (Allen et al., 2005; Howell et al., 2007); thus, possible that miR159 and miR166 function in both en- the two pools of secondary siRNAs are linked (Fig. 2). dogenous gene regulation and pathogen defense. Similar to PPR-derived secondary siRNAs, there is no However, the possibility that miR159 and miR166 tar- evidence that the sequence of any particular TAS1/ geting of V. dahliae mRNAs is accidental should not be TAS2-derived secondary siRNA is under strong puri- dismissed. fying selection. Both miR173 and the TAS1 and TAS2 The parasitic plant C. campestris also uses miRNAs as loci are only found in Arabidopsis. However, miR173 trans-species effectors. Unlike miR159 and miR166, the is a member of a larger superfamily of miRNAs, miRNAs that C. campestris uses as effectors are not members of which are found in many other plants, and present in other plants (Shahid et al., 2018); instead, most of which are triggers for PPR-derived secondary they seem to be specialized for use in trans-species

58 Plant Physiol. Vol. 182, 2020 sRNA Exchanges during Plant-Pest Interactions silencing. But how could single parasite-derived miR- 2010; Dou and Zhou, 2012). Effectors with RNAi sup- NAs persist over evolutionary time? Presumably, the pression activities have been reported in numerous vi- targeting event is not beneficial for the host, so one ruses (Burgyán and Havelda, 2011), several species of might expect that host target sites would have been Phytophthora (Qiao et al., 2013; Xiong et al., 2014; Zhang selected over time to diminish complementarity to the et al., 2019), and the fungus Puccinia graminis (Yin et al., incoming parasite miRNAs. Another possibility is that 2019). The identification of these viral, Phytophthora,or the trans-species C. campestris miRNAs target regions of fungal suppressors of RNA silencing supports plant host mRNAs that code for highly conserved or critical sRNAs as antimicrobial agents in the corresponding amino acids. The fact that some C. campestris miRNAs pathosystems. The fact that P. syringae produces effec- silence homologous target mRNAs in both Arabidopsis tors that can target the sRNA pathway in Arabidopsis and Nicotiana benthamiana (Shahid et al., 2018) supports indicates that sRNAs may also contribute to plant the hypothesis of conserved target sites. If that were the defense against P. syringae. It is important to note that case, non-synonymous changes to the target site might although bacteria do not have eukaryote-like RNA- Downloaded from https://academic.oup.com/plphys/article/182/1/51/6116263 by guest on 01 October 2021 affect host fitness due to disruption of the relevant silencing machineries, some of them encode AGO or proteins. Thus, purifying selection would act to main- AGO-like enzymes (Swarts et al., 2014) that may guide tain the non-synonymous nucleotides within target gene silencing triggered by plant sRNAs, should they sites, and sequence changes in target sites might be be transferred into bacterial cells during infection. In- constrained to only synonymous site variation. deed, bacterial gene silencing triggered by animal Alternatively, it may also be possible that C. cam- sRNAs has been reported in gutmicrobiota.Extracel- pestris does not produce these trans-species miRNAs in lular miRNAs of mouse and human feces were found order to affect host physiology but instead to protect to be functional to regulate bacterial gene expression itself from the translation of certain incoming host in the gut (Liu et al., 2016). In addition, miRNAs mRNAs. Many host-derived mRNAs are present within enclosed in plant-derived exosome-like nanoparticles Cuscuta spp., and there does not seem to be selectivity could enter specificbacterialspeciesinthegut with respect to which specific mRNAs are imported microbiota for gene silencing, thus altering the com- (Kim et al., 2014). Whether these incoming host mRNAs position of the microbial community (Teng et al., can be translated once they enter Cuscuta spp. is not 2018). It would be interesting to determine whether known. If translation of host mRNAs did occur, the ap- plant bacterial pathogens have AGO-like proteins and pearance of foreign proteins could be detrimental. In that how plant sRNAs may affect bacterial gene expression case, it would be advantageous for the parasite to have during infection. mechanisms in place to rapidly degrade particularly Although accumulating evidence supports a role of dangerous host mRNAs. In this scenario, the trans- trans-species RNAi in host-pathogen interactions, it species miRNAs would defend the parasite rather than may not be involved in all pathosystems. For example, attack the host. Testing this alternative hypothesis will be dcl and ago mutants of the fungal pathogen Zymoseptoria an interesting goal for the future. tritici were still able to cause disease on wheat with uncompromised virulence activities (Kettles et al., 2019). Moreover, dsRNAs externally applied in vitro or generated from transgenic wheat plants did not in- THE PREVALENCE OF TRANS-SPECIES RNAi duce target gene silencing in the fungal pathogen IN PLANTS (Kettles et al., 2019). The lack of trans-species RNAi Current research, although mainly centered on the between some fungal pathogens and their host is not model plant Arabidopsis and several examples of eu- surprising because many fungal species have lost the karyotic invaders (including fungi, oomycetes, and canonical RNA-silencing machinery. In a study that parasitic weeds), has established a role of trans-species analyzed 54 fungal genomes (Nunes et al., 2011), 13 RNAi during the host-pathogen arms race. However, were found to have lost all the RNA-silencing genes whether trans-species RNAi also contributes to plant such as the DCL, AGO,andRDR families. Among them defense against prokaryotic pathogens remains unclear. is the fungal pathogen Ustilago maydis that infects The intriguing observation that dsRNAs expressed in maize. It is intriguing to hypothesize that the lack of Escherichia coli could induce gene silencing in nematode an RNAi mechanism could be a consequence of a co- larvae that feed on them raised the possibility that evolutionary arms race with the hosts. In any case, dsRNAs may be transferred from prokaryotic pathogens these findings indicate that trans-species RNAi or to their hosts (Timmons and Fire, 1998). Plant mutants HIGS may not be effective against some specificplant deficient in sRNA biogenesis, including dcls and hen1, pathogens. were also hypersusceptible to infection by the bacterial pathogen Pseudomonas syringae (Navarro et al., 2008). Interestingly, three effector proteins produced by CONCLUDING REMARKS P. syringae were shown to possess RNAi suppression activity in Arabidopsis (Navarro et al., 2008). Effectors Multiple plant pathosystems have been shown to are essential virulence proteins produced by microbial involve the exchange of small RNAs between cellular pathogens to defeat host immunity (Dodds and Rathjen, organisms. Exploiting this small RNA transfer may

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