Fungal Genomics & Biology
Open Access

Small RNAs at the Interface of the Plant-Fungal Interactions

Cole R. Sawyer^1,2 and Jessy L. Labbé^{1,2* *}Correspondence: Dr. Jessy L. Labbé, Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, USA, Email:

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Description

Small RNAs are noncoding RNA molecules ranging from 20 to 30 nucleotides (i.e., not translated into protein), found across all eukaryotic organisms [1]. There are several distinctive groups of small RNAs, based on their biogenesis and precursor structure, of which the most characterized are: small interfering RNAs (siRNAs), microRNAs (miRNAs), Piwi-associated RNAs (piRNAs) and long noncoding RNAs (lncRNAs) [2,3]. The molecular mechanisms and structural features of these different types of noncoding RNAs (ncRNAs) have been well characterized over the last decade [4-7], and the list of their regulatory roles continue expanding [1,4,8-10]. Indeed, ncRNAs have been implicated broadly in epigenetic regulatory mechanisms, targeting degradation and/or translational silencing of mRNAs at the post-transcriptional level [11,12] influencing almost every biological process in animal, plant and fungi with significant impacts on a wide range of developmental, metabolic and stress responses [13-15].

The life cycle of many plants relies heavily on the use of small RNAs. Developmental processes including seed germination [16,17], root elongation [18] and leaf development [19,20] are finely tuned by the use of various small RNAs. Unsurprisingly, plants respond to colonization by fungal organisms using similar means, employing sRNAs to regulate the immune response [21,22]. For instance, Oryza sativa responds to effectors from the rice blast fungus, Magnaporthe oryzae, by increasing transcription of multiple miRNAs. Analysis of this RNA pool discovered osamiR7695, a miRNA that downregulates OsNramp6 (Natural resistance-associated macrophage protein 6) which plays a role in defense against pathogens [22]. When overexpressed in-planta, one can observe an enhanced resistance to the M. oryzae, showing the positive role of this miRNA in combating the antagonistic fungus. A more recent example in wheat (Triticum aestivum) revealed the presence of many previously unknown miRNAs when challenged with its foliar pathogen Puccinia striiformis. Of these, 998 species-specific miRNAs were identified, increasing the list of previously known wheat miRNAs [23]. One of these miRNAs, PC-3P-7484, was found to be highly upregulated during the infection period and was selected for further study. The targeted transcript encoded ubiquilin, which assists in protein degradation in the cell [24]. The exact role of this interaction and its consequences remains to be determined, but it is hypothesized that ubiquilin may increase pathogenicity. Reducing ubiquilin expression may reduce pathogen infectivity, making PC-3P-7484 a good target for overexpression studies in wheat to increase disease resistance.

Similarly, several studies have now confirmed that sRNAs are necessary in mutualistic plant-fungal associations [25-29]. For example, colonization of Medicago truncatula roots by Rhizophagus irregularis is shaped by the actions of miRNAs [27,30]. miR171h was found to cleave the transcript encoding Nodulation signalling pathway 2 (NSP2), a transcription factor up-regulated during symbiosis with nodule forming bacteria [30]. Overexpression of miR171h led to decreased levels of fungal occupation indicating its role in preventing overgrowth of the fungus [27]. Likewise, in our recent study, we characterized the sRNAs generated by Populus spp. in response to two different mutualistic symbionts the arbuscular mycorrhizal Rhizophagus irregularis and the Ectomycorrhizal Laccaria bicolor. Using this dataset, we curated a list of miRNAs and putative gene targets potentially involved in formation and maintenance of these beneficial associations [31]. Both species of Populus tested (P. trichocarpa and P. deltoides) generated different sRNA responses, with P. trichocarpa accumulating 4 times more unique miRNAs than P. deltoides. Additionally, miR393, which negatively regulates mycorrhization by arbuscular fungi [28], was found to be downregulated in P. trichocarpa, but not in P. deltoides during treatment with R. irregularis [31]. These two lines of evidence point towards a species-specific response and may help in understanding why P. trichocarpa is more readily colonized by mycorrhizal fungi than P. deltoides. Examination of the data between treatments indicates that Populus spp. may respond to both fungi with the same toolset [32]. In P. trichocarpa, 39 of the 44 miRNAs shared between fungal treatments had comparable expression trends. The same was found in the P. deltoides dataset where all shared miRNAs (n=4) showed similar values over time.

There is also evidence that RNAs generated by fungal colonizers can move through the plant cell wall and interact with argonaute proteins in the cytoplasm. This process, termed “Cross-Kingdom RNAi” has been observed in multiple pathogenic fungal species. For instance, the grey mold pathogen, Botrytis cinerea sends multiple sRNAs to cleave plant messenger RNAs [33]. Targets of these RNAs include mitogen activated protein kinase, peroxiredoxin and cell-wall associated kinase, all of which were down-regulated during infection by B. cinerea. Cross-Kingdom RNAi has been observed in other pathogenic fungi including Verticillium dahliae and Puccinia striiformis as well as other eukaryotic pathogens like the oomycete Phytphthora capsici and the parasitic plant Cuscuta campestris [34-37]. Movement of RNAs also appears to be bi-directional, as studies have shown that plants are capable of sending sRNAs to their associated fungi using extracellular vesicles [38,39]. Vesicles extracted from Arabidopsis thaliana infected with B. cinerea were found to contain trans-acting silencing RNAs (ta-siRNAs). Fungal protoplasts isolated from infected plant tissue via enzymatic digestion were found to contain RNAs from the plant. Two sRNA species, TAS1c-siR483 and TAS2-siR453 were found at high levels in fungal material. The RNAs targeted multiple fungal transcripts, all of which played a role in vesicle trafficking. These mRNAs were also downregulated, indicating that plant RNAs were able to lower expression of fungal transcripts.

Whether Cross-Kingdom RNAi plays a role in mutualistic symbiosis is unclear. In-silico analysis of the transcriptome of Rhizophagus irregularis revealed a substantial number of small RNAs [40]. These RNAs were then compared against the degradome of Medicago truncatula. Upwards of 237 plant transcripts were found to be potential targets for cross-kingdom silencing events [40]. Our analysis found no evidence for Populus RNAs that could interact with R. irregularis. We did, however, find multiple miRNAs that could interact with L. bicolor [31]. Comparing these miRNAs against the host genome determined that they could potentially target multiple messenger RNAs. These mRNAs encode for proteins involved in vesicular transport, transcriptional regulation and several with unknown function. More experimentation is required to determine whether or not Cross-Kingdom RNAi occurs between plants and their mycorrhizal fungi and, if it does, the ramifications of these events.

Until recently, investigations of small RNAs have been largely limited by a lack of high-throughput experimental studies, and because of their small sizes in the sequencing samples, were often misidentified as background noise [1,41,42]. Many of the studies cited here were made possible by the increase in sequencing capabilities over the last decade. Additionally, the aggregation of miRNA sequence data into large repositories like the plant microRNA database [43] and plant microRNA encyclopedia [44] have allowed researchers to compare their datasets against those collected in previous publications. Bioinformatic tools like sPARTA [45] and miTRATA [46] which allow for analysis of 3’ modifications and mRNA targets, respectively, give researchers the ability to ask specific questions of their miRNA libraries. Future research will focus on miRNA interactions and networks, capturing the web of regulatory RNAs as they affect one another. The topic of plant extracellular vesicles is also of great interest, but remains in its infancy, and many valid criticisms have been levied toward previous works in the field [47]. Differences in isolation methods, lack of proper controls and inconsistent characterization techniques has left several studies in question. Further optimization of protocols to identify extracellular vesicles and the contents they contain will be required for forthcoming studies.

A large number of small RNAs have been discovered, and smaller RNAs are expected to be further characterized along with the rapidly expanding sequencing power, despite the multiple challenges in distinguishing functional sRNAs and non-functional background. sRNAs seem to serve as hubs of gene networks that are rich in information flow. The recent development of machine learning approach will improve reliability of sRNAs identification and determine the interacting features and models. Better understanding the ways these sRNAs affect symbiosis and the broader plant microbiome will pave the way for novel and precise manipulation of natural or managed ecosystems to improve ecosystem resilience and productivity.

Acknowledgements

This research was sponsored by the Genomic Science Program, U.S. Department of Energy, Office of Science, Biological and Environmental Research as part of the Secure Ecosystem Engineering and Design Scientific Focus Area and the Plant Microbe Interfaces Scientific Focus Area, at the Oak Ridge National Laboratory. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. Department of Energy under contract DE-AC05-00OR22725.

Sourcing of Funding

This work has been funded by the Genomic Science Program of the U.S. Department of Energy, Office of Science, Biological and Environmental Research as part of the Secure Ecosystem Engineering and Design Scientific Focus Area and the Plant Microbe Interfaces Scientific Focus Area, at the Oak Ridge National Laboratory. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. Department of Energy under contract DE-AC05-00OR22725.

Conflicts of Interest

None declared.

References

1. Grosshans H, Filipowicz W. Molecular biology: The expanding world of small RNAs. Nature. 2008;451(7177):414-416.
2. Borges F, Martienssen RA. The expanding world of small RNAs in plants. Nat Rev Mol Cell Biol. 2015;16(12):727-741.
3. Katiyar-Agarwal S, Jin H. Role of small RNAs in host-microbe interactions. Annu Rev Phytopathol. 2010;48:225-246.
4. Ruiz-Ferrer V, Voinnet O. Roles of plant small RNAs in biotic stress responses. Ann Rev Plant Biol. 2009;60:485-510.
5. Chekanova JA. Long non-coding RNAs and their functions in plants. Curr Opin Plant Biol. 2015;27:207-216.
6. Mohanta TK, Bae H. The diversity of fungal genome. Biol Proced Online. 2015;17(1):8.
7. Huang J, Yang M, Lu L, Zhang X. Diverse functions of small RNAs in different plant–pathogen communications. Front Microbiol. 2016;7:1552.
8. Mallory AC, Vaucheret H. Functions of microRNAs and related small RNAs in plants. Nat Gen. 2006;38(6):S31-6.
9. Chen X. Small RNAs in development: Insights from plants. Curr Opin Genet Dev. 2012;22(4):361-367.
10. Zhang YC, Chen YQ. Long noncoding RNAs: New regulators in plant development. Biochem Biophys Res Commun. 2013;436(2):111-114.
11. Phillips T. Small non-coding RNA and gene expression. Nature Educ. 2008;1(1):115.
12. Pattanayak D, Solanke AU, Kumar PA. Plant RNA interference pathways: Diversity in function, similarity in action. Plant Mol Biol Rep. 2013;31(3):493-506.
13. Watanabe T, Totoki Y, Toyoda A, Kaneda M, Kuramochi-Miyagawa S, Obata Y, et al. Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes. Nature. 2008;453(7194):539-43.
14. Verdel A, Moazed D. RNAi-directed assembly of heterochromatin in fission yeast. FEBS Lett. 2005;579(26):5872-5878.
15. Budak H, Akpinar BA. Plant miRNAs: biogenesis, organization and origins. Funct Integr Genomics. 2015;15(5):523-531.
16. Liu PP, Montgomery TA, Fahlgren N, Kasschau KD, Nonogaki H, Carrington JC. Repression of AUXIN RESPONSE FACTOR10 by microRNA160 is critical for seed germination and post‐germination stages. Plant J. 2007;52(1):133-146.
17. Das SS, Yadav S, Singh A, Gautam V, Sarkar AK, Nandi AK, et al. Expression dynamics of miRNAs and their targets in seed germination conditions reveals miRNA-ta-siRNA crosstalk as regulator of seed germination. Sci Rep. 2018;8(1):1-3.
18. Parry G, Villalobos CLI, Prigge M, Peret B, Dharmasiri S, Itoh H, et al. Complex regulation of the TIR1/AFB family of auxin receptors. Proc Natl Acad Sci. 2009;106(52):22540-22545.
19. Liu D, Song Y, Chen Z, Yu D. Ectopic expression of miR396 suppresses GRF target gene expression and alters leaf growth in Arabidopsis. Physiol Plant. 2009;136(2):223-236.
20. Ori N, Cohen AR, Etzioni A, Brand A, Yanai O, Shleizer S, et al. Regulation of LANCEOLATE by miR319 is required for compound-leaf development in tomato. Nat Gen. 2007;39(6):787-791.
21. Katiyar-Agarwal S, Morgan R, Dahlbeck D, Borsani O, Villegas A, Zhu JK, et al. A pathogen-inducible endogenous siRNA in plant immunity. Proc Natl Acad Sci. 2006;103(47):18002-18007.
22. Campo S, Peris‐Peris C, Siré C, Moreno AB, Donaire L, Zytnicki M, et al. Identification of a novel micro-RNA (mi RNA) from rice that targets an alternatively spliced transcript of the N ramp6 (N atural resistance‐associated macrophage protein 6) gene involved in pathogen resistance. New Phytol. 2013;199(1):212-227.
23. Feng H, Wang T, Feng C, Zhang Q, Zhang X, Huang L, et al. Identification of microRNAs and their corresponding targets involved in the susceptibility interaction of wheat response to Puccinia striiformis f. sp. tritici. Physiol Plant. 2016;157(1):95-107.
24. Marín I. The ubiquilin gene family: evolutionary patterns and functional insights. BMC Evol Biol. 2014;14(1):63.
25. Branscheid A, Sieh D, Pant BD, May P, Devers EA, Elkrog A, et al. Expression pattern suggests a role of MiR399 in the regulation of the cellular response to local Pi increase during arbuscular mycorrhizal symbiosis. Mol Plant Microbe Interact. 2010;23(7):915-926.
26. Devers EA, Branscheid A, May P, Krajinski F. Stars and symbiosis: microRNA-and microRNA*-mediated transcript cleavage involved in arbuscular mycorrhizal symbiosis. Plant Physiol. 2011;156(4)RNA-and%20microRNA*-mediated%20transcript%20cleavage%20involved%20in%20arbuscular%20mycorrhizal%20symbiosis.%20Plant%20physiology.%202011%20Aug%201;156(4):1990-2010' target='_blank'>:1990-2010.
27. Lauressergues D, Delaux PM, Formey D, Lelandais‐Brière C, Fort S, Cottaz S, et al. The microRNA miR171h modulates arbuscular mycorrhizal colonization of Medicago truncatula by targeting NSP2. Plant J. 2012;72(3):512-522.
28. Etemadi M, Gutjahr C, Couzigou JM, Zouine M, Lauressergues D, Timmers A, et al. Auxin perception is required for arbuscule development in arbuscular mycorrhizal symbiosis. Plant Physiol. 2014;166(1):281-292.
29. Wu P, Wu Y, Liu CC, Liu LW, Ma FF, Wu XY, et al. Identification of arbuscular mycorrhiza (AM)-responsive microRNAs in tomato. Front Plant Sci. 2016;7:429.
30. Hofferek V, Mendrinna A, Gaude N, Krajinski F, Devers EA. MiR171h restricts root symbioses and shows like its target NSP2 a complex transcriptional regulation in Medicago truncatula. BMC Plant Biol. 2014;14(1):1-6.
31. Mewalal R, Yin H, Hu R, Jawdy S, Vion P, Tuskan GA, et al. Identification of populus small RNAs responsive to mutualistic interactions with mycorrhizal fungi, Laccaria bicolor and Rhizophagus irregularis. Front Microbiol. 2019;10:515.
32. Labbé J, Jorge V, Kohler A, Vion P, Marçais B, Bastien C, et al. Identification of quantitative trait loci affecting ectomycorrhizal symbiosis in an interspecific F 1 poplar cross and differential expression of genes in ectomycorrhizas of the two parents: Populus deltoides and Populus trichocarpa. Tree Genet Genomes. 2011;7(3):617-627.
33. Weiberg A, Wang M, Lin FM, Zhao H, Zhang Z, Kaloshian I, et al. Fungal small RNAs suppress plant immunity by hijacking host RNA interference pathways. Science. 2013;342(6154):118-123.
34. Wang M, Weiberg A, Lin FM, Thomma BP, Huang HD, Jin H. Bidirectional cross-kingdom RNAi and fungal uptake of external RNAs confer plant protection. Nat Plants. 2016;2(10):1-0.
35. Wang B, Sun Y, Song N, Zhao M, Liu R, Feng H, et al. Puccinia striiformis f. sp. tritici mi croRNA‐like RNA 1 (Pst‐milR1), an important pathogenicity factor of Pst, impairs wheat resistance to Pst by suppressing the wheat pathogenesis‐related 2 gene. New Phytol. 2017;215(1):338-350.
36. Hou Y, Zhai Y, Feng L, Karimi HZ, Rutter BD, Zeng L, et al. A Phytophthora effector suppresses trans-kingdom RNAi to promote disease susceptibility. Cell Host Microbe. 2019;25(1):153-165.
37. Shahid S, Kim G, Johnson NR, Wafula E, Wang F, Coruh C, et al. MicroRNAs from the parasitic plant Cuscuta campestris target host messenger RNAs. Nature. 2018;553(7686):82-85.
38. Cai Q, Qiao L, Wang M, He B, Lin FM, Palmquist J, et al. Plants send small RNAs in extracellular vesicles to fungal pathogen to silence virulence genes. Science. 2018;360(6393):1126-1129.
39. Baldrich P, Rutter BD, Karimi HZ, Podicheti R, Meyers BC, Innes RW. Plant extracellular vesicles contain diverse small RNA species and are enriched in 10-to 17-nucleotide “tiny” RNAs. Plant Cell. 2019;31(2):315-324.
40. Silvestri A, Fiorilli V, Miozzi L, Accotto GP, Turina M, Lanfranco L. In silico analysis of fungal small RNA accumulation reveals putative plant mRNA targets in the symbiosis between an arbuscular mycorrhizal fungus and its host plant. BMC Genomics. 2019;20(1):1-8.
41. Kawaji H, Hayashizaki Y. Exploration of small RNAs. PLoS Genet. 2008;4(1):e22.
42. Budak H, Zhang B. MicroRNAs in model and complex organisms. Funct Integr Genomics. 2017; 17(2-3):121-124.
43. Zhang Z, Yu J, Li D, Zhang Z, Liu F, Zhou X, et al. PMRD: plant microRNA database. Nucleic acids research. 2010;38(suppl_1):D806-D813.
44. Guo Z, Kuang Z, Wang Y, Zhao Y, Tao Y, Cheng C, et al. PmiREN: A comprehensive encyclopedia of plant miRNAs. Nucleic Acids Res. 2020;48(D1):D1114-D1121.
45. Kakrana A, Hammond R, Patel P, Nakano M, Meyers BC. sPARTA: A parallelized pipeline for integrated analysis of plant miRNA and cleaved mRNA data sets, including new miRNA target-identification software. Nucleic Acids Res. 2014;42(18):e139.
46. Patel P, Ramachandruni SD, Kakrana A, Nakano M, Meyers BC. miTRATA: A web-based tool for microRNA Truncation and T ailing Analysis. Bioinformatics. 2016;32(3):450-452.
47. Rutter BD, Innes RW. Growing pains: Addressing the pitfalls of plant extracellular vesicle research. New Phytol. 2020;228(5):1505-1510.