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


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.

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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.


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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




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.

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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)

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Wang, M., Weiberg, A., Lin, FM. et al. Bidirectional cross-kingdom RNAi and fungal uptake of external RNAs confer plant protection. Nature Plants 2, 16151 (2016).

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