Plant pathogenic fungi represent the largest group of disease-causing agents on crop plants, and are a constant and major threat to agriculture worldwide. Recent studies have shown that engineered production of RNA interference (RNAi)-inducing dsRNA in host plants can trigger specific fungal gene silencing and confer resistance to fungal pathogens1–7. Although these findings illustrate efficient uptake of host RNAi triggers by pathogenic fungi, it is unknown whether or not such an uptake mechanism has been evolved for a natural biological function in fungus–host interactions. Here, we show that in response to infection with Verticillium dahliae (a vascular fungal pathogen responsible for devastating wilt diseases in many crops) cotton plants increase production of microRNA 166 (miR166) and miR159 and export both to the fungal hyphae for specific silencing. We found that two V. dahliae genes encoding a Ca2+-dependent cysteine protease (Clp-1) and an isotrichodermin C-15 hydroxylase (HiC-15), and targeted by miR166 and miR159, respectively, are both essential for fungal virulence. Notably, V. dahliae strains expressing either Clp-1 or HiC-15 rendered resistant to the respective miRNA exhibited drastically enhanced virulence in cotton plants. Together, our findings identify a novel defence strategy of host plants by exporting specific miRNAs to induce cross-kingdom gene silencing in pathogenic fungi and confer disease resistance.
Subscribe to Journal
Get full journal access for 1 year
only $5.42 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Nowara, D. et al. HIGS: host-induced gene silencing in the obligate biotrophic fungal pathogen Blumeria graminis. Plant Cell 22, 3130–3141 (2010).
Tinoco, M. L., Dias, B. B., Dall'Astta, R. C., Pamphile, J. A. & Aragao, F. J. In vivo trans-specific gene silencing in fungal cells by in planta expression of a double-stranded RNA. BMC Biol. 8, 27 (2010).
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).
Panwar, V., McCallum, B. & Bakkeren, G. Endogenous silencing of Puccinia triticina pathogenicity genes through in planta-expressed sequences leads to the suppression of rust diseases on wheat. Plant J. 73, 521–532 (2013).
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).
Baulcombe, D. Small RNA—the secret of noble rot. Science 342, 45–46 (2013).
Weiberg, A. & Jin, H. Small RNAs—the secret agents in the plant-pathogen interactions. Curr. Opin. Plant Biol. 26, 87–94 (2015).
Zhengjun, X., Achar, P. N. & Benkang, G. Vegetative compatibility groupings of Verticillium dahliae from cotton in mainland China. Eur. J. Plant Pathol. 104, 871–876 (1998).
Bowman, D. Public cotton breeders—do we need them? J. Cotton Sci. 3, 139–152 (1999).
Koroleva, O. V., Stepanova, E. V., Gavrilova, V. P., Biniukov, V. I. & Pronin, A. M. Comparative characterization of methods for removal of Cu(II) from the active sites of fungal laccases. Biochemistry (Moscow) 66, 960–966 (2001).
Gerik, J. & Huisman, O. Study of field-grown cotton roots infected with Verticillium dahliae using an immunoenzymatic staining technique. Phytopathology 78, 1174–1178 (1988).
Bell, A. A. Verticillium wilt. Cotton Dis. 87–126 (1992).
Klosterman, S. J. et al. Comparative genomics yields insights into niche adaptation of plant vascular wilt pathogens. PLoS Pathog. 7, e1002137 (2011).
de Jonge, R. et al. Extensive chromosomal reshuffling drives evolution of virulence in an asexual pathogen. Genome Res. 23, 1271–1282 (2013).
Gong, L., Kakrana, A., Arikit, S., Meyers, B. C. & Wendel, J. F. Composition and expression of conserved microRNA genes in diploid cotton (Gossypium) species. Genome Biol. Evol. 5, 2449–2459 (2013).
Liu, N. et al. Small RNA and degradome profiling reveals a role for miRNAs and their targets in the developing fibers of Gossypium barbadense. Plant J. 80, 331–344 (2014).
Sorimachi, H., Hata, S., Ono, Y. Calpain chronicle—an enzyme family under multidisciplinary characterization. Proc. Jpn Acad. Ser. B Phys. Biol. Sci. 87, 287 (2011).
Zamir, L. O., Devor, K. A., Nikolakakis, A. & Sauriol, F. Biosynthesis of Fusarium culmorum trichothecenes. The roles of isotrichodermin and 12, 13-epoxytrichothec-9-ene. J. Biol. Chem. 265, 6713–6725 (1990).
Yan, J. et al. Effective small RNA destruction by the expression of a short tandem target mimic in Arabidopsis. Plant Cell 24, 415–427 (2012).
Campo, S. et al. Identification of a novel microRNA (miRNA) from rice that targets an alternatively spliced transcript of the Nramp6 (Natural resistance-associated macrophage protein 6) gene involved in pathogen resistance. New Phytol. 199, 212–227 (2013).
Wong, J. et al. Roles of small RNAs in soybean defense against Phytophthora sojae infection. Plant J. 79, 928–940 (2014).
Jung, J. H. & Park, C. M. MIR166/165 genes exhibit dynamic expression patterns in regulating shoot apical meristem and floral development in Arabidopsis. Planta 225, 1327–1338 (2007).
Zhao, P., Zhao, Y.-L., Jin, Y., Zhang, T. & Guo, H.-S. Colonization process of Arabidopsis thaliana roots by a green fluorescent protein-tagged isolate of Verticillium dahliae. Protein Cell 5, 94–98 (2014).
Kalantidis, K. Grafting the way to the systemic silencing signal in plants. PLoS Biol. 2, E224 (2004).
Molnar, A. et al. Small silencing RNAs in plants are mobile and direct epigenetic modification in recipient cells. Science 328, 872–875 (2010).
Zhou, B. J., Jia, P. S., Gao, F. & Guo, H. S. Molecular characterization and functional analysis of a necrosis- and ethylene-inducing, protein-encoding gene family from Verticillium dahliae. Mol. Plant Microbe Interact. 25, 964–975 (2012).
Xu, L. et al. Functional characterization of cotton genes responsive to Verticillium dahliae through bioinformatics and reverse genetics strategies. J. Exp. Bot. 65, 6679–6692 (2014).
Frandsen, R., Frandsen, M. & Giese, H. Targeted gene replacement in fungal pathogens via Agrobacterium tumefaciens- mediated transformation. Methods Mol. Biol. 835 (2012).
Gao, F. et al. A glutamic acid-rich protein identified in Verticillium dahliae from an insertional mutagenesis affects microsclerotial formation and pathogenicity. PloS ONE 5, e15319 (2010).
Takemoto, D., Tanaka, A. & Scott, B. A p67Phox-like regulator is recruited to control hyphal branching in a fungal-grass mutualistic symbiosis. Plant Cell 18, 2807–2821 (2006).
We thank B. Scott for plasmid pPN94, Y.-L. Peng for plasmids pKOV21, G.-L. Tang for STTM166 Arabidopsis seeds, and B. Thomma for the VdLs17 strain. This work was supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB11040500) and the China Transgenic Research and Commercialization Key Special Project (2014ZX00800908B).
The authors declare no competing financial interests.
About this article
Cite this article
Zhang, T., Zhao, Y., Zhao, J. et al. Cotton plants export microRNAs to inhibit virulence gene expression in a fungal pathogen. Nature Plants 2, 16153 (2016). https://doi.org/10.1038/nplants.2016.153
Trends in Microbiology (2020)
Host-induced gene silencing of the acetolactate synthases VdILV2 and VdILV6 confers resistance to Verticillium wilt in cotton (Gossypium hirsutum L.)
Biochemical and Biophysical Research Communications (2020)
Molecular Plant Pathology (2020)
Molecular Plant-Microbe Interactions® (2020)
Frontiers in Plant Science (2020)