Bidirectional cross-kingdom RNAi and fungal uptake of external RNAs confer plant protection

Abstract

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: B. cinerea dcl1 dcl2 double mutant, but not the dcl1 or dcl2 single mutants, displays reduced virulence on fruits, vegetables, and flower petals.
Figure 2: Arabidopsis and tomato Bc-DCL1/2–RNAi plants confer enhanced resistance against B. cinerea infection.
Figure 3: Environmental Bc-DCL1/2–sRNAs and –dsRNAs are taken into B. cinerea cells and where they silence fungal DCL genes; Bc-DCL1/2–sRNAs move from plants into fungal cells.
Figure 4: Externally applied Bc-DCL1/2–sRNAs and –dsRNAs inhibited pathogen virulence on fruits, vegetables, and flower petals.
Figure 5: Treatment with N. benthamiana RNA extracts containing Bc-DCL1/2–sRNAs and –dsRNAs reduces grey mould disease symptoms caused by B. cinerea.
Figure 6: Arabidopsis plants expressing hairpin RNAs that simultaneously target DCL genes of B. cinerea and V. dahliae show enhanced disease resistance to both pathogens.

References

  1. 1

    Ghildiyal, M. & Zamore, P. D. Small silencing RNAs an expanding universe. Nat. Rev. Genet. 10, 94–108 (2009).

    CAS  Article  Google Scholar 

  2. 2

    Baulcombe, D. RNA silencing in plants. Nature 431, 356–363 (2004).

    CAS  Article  Google Scholar 

  3. 3

    Vaucheret, H. Plant ARGONAUTES. Trends Plant Sci. 13, 350–358 (2008).

    CAS  Article  Google Scholar 

  4. 4

    Hutvagner, G. & Simard, M. J. Argonaute proteins: key players in RNA silencing. Nat. Rev. Mol. Cell Biol. 9, 22–32 (2008).

    CAS  Article  Google Scholar 

  5. 5

    Weiberg, A. et al. Fungal small RNAs suppress plant immunity by hijacking host RNA interference pathways. Science 342, 118–123 (2013).

    CAS  Article  Google Scholar 

  6. 6

    Weiberg, A. & Jin, H. L. Small RNAs – the secret agents in the plant-pathogen interactions. Curr. Opin. Plant Biol. 26, 87–94 (2015).

    CAS  Article  Google Scholar 

  7. 7

    Weiberg, A., Wang, M., Bellinger, M. & Jin, H. Small RNAs: a new paradigm in plant-microbe interactions. Annu. Rev. Phytopathol. 52, 495–516 (2014).

    CAS  Article  Google Scholar 

  8. 8

    Buck, A. H. et al. Exosomes secreted by nematode parasites transfer small RNAs to mammalian cells and modulate innate immunity. Nat. Commun. 5, 5488 (2014).

    CAS  Article  Google Scholar 

  9. 9

    Cheng, G. F., Luo, R., Hu, C., Cao, J. & Jin, Y. X. Deep sequencing-based identification of pathogen-specific microRNAs in the plasma of rabbits infected with Schistosoma japonicum. Parasitology 140, 1751–1761 (2013).

    CAS  Article  Google Scholar 

  10. 10

    Garcia-Silva, M. R. et al. Extracellular vesicles shed by Trypanosoma cruzi are linked to small RNA pathways, life cycle regulation, and susceptibility to infection of mammalian cells. Parasitol. Res. 113, 285–304 (2014).

    Article  Google Scholar 

  11. 11

    Zamanian, M. et al. Release of small RNA-containing exosome-like vesicles from the human filarial parasite Brugia malayi. PLoS Negl. Trop. Dis. 9, e0004069 (2015).

    Article  Google Scholar 

  12. 12

    Quintana, J. F. et al. Extracellular Onchocerca-derived small RNAs in host nodules and blood. Parasit Vectors 8, 58 (2015).

    Article  Google Scholar 

  13. 13

    Baum, J. A. et al. Control of coleopteran insect pests through RNA interference. Nat. Biotechnol. 25, 1322–1326 (2007).

    CAS  Article  Google Scholar 

  14. 14

    Mao, Y. B. et al. Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol. Nat. Biotechnol. 25, 1307–1313 (2007).

    CAS  Article  Google Scholar 

  15. 15

    Huang, G. Z., Allen, R., Davis, E. L., Baum, T. J. & Hussey, R. S. Engineering broad root-knot resistance in transgenic plants by RNAi silencing of a conserved and essential root-knot nematode parasitism gene. Proc. Natl. Acad. Sci. USA 103, 14302–14306 (2006).

    CAS  Article  Google Scholar 

  16. 16

    Li, J., Todd, T. C., Oakley, T. R., Lee, J. & Trick, H. N. Host-derived suppression of nematode reproductive and fitness genes decreases fecundity of Heterodera glycines Ichinohe. Planta 232, 775–785 (2010).

    CAS  Article  Google Scholar 

  17. 17

    Nowara, D. et al. HIGS: Host-Induced gene silencing in the obligate biotrophic fungal pathogen Blumeria graminis. Plant Cell 22, 3130–3141 (2010).

    CAS  Article  Google Scholar 

  18. 18

    Koch, A. et al. Host-induced gene silencing of cytochrome P450 lanosterol C14 alpha-demethylase-encoding genes confers strong resistance to Fusarium species. Proc. Natl. Acad. Sci. USA 110, 19324–19329 (2013).

    CAS  Article  Google Scholar 

  19. 19

    Jahan, S. N. et al. Plant-mediated gene silencing restricts growth of the potato late blight pathogen Phytophthora infestans. J. Exp. Bot. 66, 2785–2794 (2015).

    CAS  Article  Google Scholar 

  20. 20

    Vega-Arreguin, J. C., Jalloh, A., Bos, J. I. & Moffett, P. Recognition of an Avr3a homologue plays a major role in mediating nonhost resistance to Phytophthora capsici in Nicotiana species. Mol. Plant Microbe Interact. 27, 770–780 (2014).

    CAS  Article  Google Scholar 

  21. 21

    Nunes, C. C. & Dean, R. A. Host-induced gene silencing: a tool for understanding fungal host interaction and for developing novel disease control strategies. Mol. Plant Pathol. 13, 519–529 (2012).

    CAS  Article  Google Scholar 

  22. 22

    Williamson, B., Tudzynsk, B., Tudzynski, P. & van Kan, J. A. L. Botrytis cinerea: the cause of grey mould disease. Mol. Plant Pathol. 8, 561–580 (2007).

    CAS  Article  Google Scholar 

  23. 23

    van Kan, J. A. L. Licensed to kill: the lifestyle of a necrotrophic plant pathogen. Trends Plant Sci. 11, 247–253 (2006).

    CAS  Article  Google Scholar 

  24. 24

    Fradin, E. F. & Thomma, B. P. Physiology and molecular aspects of Verticillium wilt diseases caused by V. dahliae and V. albo-atrum. Mol. Plant Pathol. 7, 71–86 (2006).

    CAS  Article  Google Scholar 

  25. 25

    Klosterman, S. J., Atallah, Z. K., Vallad, G. E. & Subbarao, K. V. Diversity, pathogenicity, and management of Verticillium species. Annu. Rev. Phytopathol. 47, 39–62 (2009).

    CAS  Article  Google Scholar 

  26. 26

    Pliego, C. et al. Host-Induced gene silencing in barley powdery mildew reveals a class of ribonuclease-like effectors. Mol. Plant Microbe Interact. 26, 633–642 (2013).

    CAS  Article  Google Scholar 

  27. 27

    Whigham, E. et al. Broadly conserved fungal effector BEC1019 suppresses host cell death and enhances pathogen virulence in powdery mildew of barley (Hordeum vulgare L.). Mol. Plant Microbe Interact. 28, 968–983 (2015).

    CAS  Article  Google Scholar 

  28. 28

    Panwar, V., McCallum, B. & Bakkeren, G. Host-induced gene silencing of wheat leaf rust fungus Puccinia triticina pathogenicity genes mediated by the Barley stripe mosaic virus. Plant Mol. Biol. 81, 595–608 (2013).

    CAS  Article  Google Scholar 

  29. 29

    Ghag, S. B., Shekhawat, U. K. & Ganapathi, T. R. Host-induced post-transcriptional hairpin RNA-mediated gene silencing of vital fungal genes confers efficient resistance against Fusarium wilt in banana. Plant Biotechnol. J. 12, 541–553 (2014).

    CAS  Article  Google Scholar 

  30. 30

    Cheng, W. et al. Host-induced gene silencing of an essential chitin synthase gene confers durable resistance to Fusarium head blight and seedling blight in wheat. Plant Biotechnol. J. 13, 1335–1345 (2015).

    CAS  Article  Google Scholar 

  31. 31

    Sanju, S. et al. Host-mediated gene silencing of a single effector gene from the potato pathogen Phytophthora infestans imparts partial resistance to late blight disease. Funct. Integr. Genomics 15, 697–706 (2015).

    CAS  Article  Google Scholar 

  32. 32

    Lee, H. C. et al. Diverse pathways generate microRNA-like RNAs and dicer-independent small interfering RNAs in fungi. Mol. Cell 38, 803–814 (2010).

    CAS  Article  Google Scholar 

  33. 33

    Jin, H. L. & Zhu, J. K. How many ways are there to generate small RNAs? Mol. Cell 38, 775–777 (2010).

    CAS  Article  Google Scholar 

  34. 34

    Helliwell, C. A. & Waterhouse, P. M. Constructs and methods for hairpin RNA-mediated gene silencing in plants. Methods Enzymol. 392, 24–35 (2005).

    CAS  Article  Google Scholar 

  35. 35

    Liu, Y. L., Schiff, M. & Dinesh-Kumar, S. P. Virus-induced gene silencing in tomato. Plant J. 31, 777–786 (2002).

    CAS  Article  Google Scholar 

  36. 36

    Song, J. Q. et al. Gene RB cloned from Solanum bulbocastanum confers broad spectrum resistance to potato late blight. Proc. Natl Acad. Sci. USA 100, 9128–9133 (2003).

    CAS  Article  Google Scholar 

  37. 37

    Winston, W. M., Sutherlin, M., Wright, A. J., Feinberg, E. H. & Hunter, C. P. Caenorhabditis elegans SID-2 is required for environmental RNA interference. Proc. Natl Acad. Sci. USA 104, 10565–10570 (2007).

    CAS  Article  Google Scholar 

  38. 38

    Whangbo, J. S. & Hunter, C. P. Environmental RNA interference. Trends Genet. 24, 297–305 (2008).

    CAS  Article  Google Scholar 

  39. 39

    McEwan, D. L., Weisman, A. S. & Huntert, C. P. Uptake of extracellular double-Stranded RNA by SID-2. Mol. Cell 47, 746–754 (2012).

    CAS  Article  Google Scholar 

  40. 40

    Feinberg, E. H. & Hunter, C. P. Transport of dsRNA into cells by the transmembrane protein SID-1. Science 301, 1545–1547 (2003).

    CAS  Article  Google Scholar 

  41. 41

    Ivashuta, S. et al. Environmental RNAi in herbivorous insects. RNA 21, 840–850 (2015).

    CAS  Article  Google Scholar 

  42. 42

    San Miguel, K. & Scott, J. G. The next generation of insecticides: dsRNA is stable as a foliar-applied insecticide. Pest Manage. Sci. 72, 801–809 (2016).

    CAS  Article  Google Scholar 

  43. 43

    Baulcombe, D. C. VIGS, HIGS and FIGS: small RNA silencing in the interactions of viruses or filamentous organisms with their plant hosts. Curr. Opin. Plant Biol. 26, 141–146 (2015).

    CAS  Article  Google Scholar 

  44. 44

    Govindarajulu, M., Epstein, L., Wroblewski, T. & Michelmore, R. W. Host-induced gene silencing inhibits the biotrophic pathogen causing downy mildew of lettuce. Plant Biotechnol. J. 13, 875–883 (2015).

    CAS  Article  Google Scholar 

  45. 45

    Saleh, M. C. et al. The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing. Nat. Cell Biol. 8, 793–802 (2006).

    CAS  Article  Google Scholar 

  46. 46

    Bolognesi, R. et al. Characterizing the mechanism of action of double-stranded RNA activity against western corn rootworm (Diabrotica virgifera virgifera LeConte). PLoS ONE 7, e47534 (2012).

    CAS  Article  Google Scholar 

  47. 47

    Ellendorff, U., Fradin, E. F., de Jonge, R. & Thomma, B. P. H. J. RNA silencing is required for Arabidopsis defence against Verticillium wilt disease. J. Exp. Bot. 60, 591–602 (2009).

    CAS  Article  Google Scholar 

  48. 48

    Zhang, X. et al. Arabidopsis argonaute 2 regulates innate immunity via miRNA393(*)-mediated silencing of a Golgi-localized SNARE gene, MEMB12. Mol. Cell 42, 356–366 (2011).

    CAS  Article  Google Scholar 

  49. 49

    Hinas, A., Wright, A. J. & Hunter, C. P. SID-5 Is an endosome-associated protein required for efficient systemic RNAi in C. elegans. Curr. Biol. 22, 1938–1943 (2012).

    CAS  Article  Google Scholar 

  50. 50

    Wang, M., Weiberg, A. & Jin, H. Pathogen small RNAs: a new class of effectors for pathogen attacks. Mol. Plant Pathol. 16, 219–223 (2015).

    Article  Google Scholar 

  51. 51

    Helliwell, C. & Waterhouse, P. Constructs and methods for high-throughput gene silencing in plants. Methods 30, 289–295 (2003).

    CAS  Article  Google Scholar 

  52. 52

    Yoo, S. D., Cho, Y. H. & Sheen, J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat. Protoc. 2, 1565–1572 (2007).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank H. Vaucheret for the ago1-27 seeds, M. Coffey for the V. dahliae JR2 strain, I. Kaloshian for providing the growth room space for VIGS experiments, and Y. Lii for editing the paper. This work was supported by grants from National Institute of Health (R01 GM093008), National Science Foundation (IOS-1257576, IOS-1557812) and an AES-CE Award (PPA-7517H) awarded to H.J.

Author information

Affiliations

Authors

Contributions

H.J. conceived the idea. M.W. and H.J. designed the experiments. M.W. performed most of the experiments and analysed data. A.W. profiled the sRNAs from dcl1 dcl2 and WT strains and analysed the data. F.M.L. and H.D.H. conducted bioinformatics analysis on sRNA libraries. B.T. provided Vd genome sequence for JR2 strain. M.W., A.W. and H.J. wrote the manuscript.

Corresponding author

Correspondence to Hailing Jin.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Methods, Supplementary Figures 1–9, Supplementary References (PDF 3132 kb)

Supplementary Table 1

The normalized read counts of previously predicted Bc-sRNA effector candidates in B. cinerea WT and dcl1 dcl2 strains (XLSX 76 kb)

Supplementary Table 2

At-AGO1-associated Vd-sRNA effector candidates and their targets. (XLSX 36 kb)

Supplementary Table 3

At-AGO2-associated Vd-sRNAs and their targets. (XLSX 13 kb)

Supplementary Table 4

The list of primers and oligoes used in the manuscript. (XLSX 51 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Wang, M., Weiberg, A., Lin, F. et al. Bidirectional cross-kingdom RNAi and fungal uptake of external RNAs confer plant protection. Nature Plants 2, 16151 (2016). https://doi.org/10.1038/nplants.2016.151

Download citation

Further reading

Search

Quick links

Sign up for the Nature Briefing newsletter for a daily update on COVID-19 science.
Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing