Dynamic changes in transposable element and gene methylation in mulberry (Morus notabilis) in response to Botrytis cinerea

Abstract

DNA methylation has been proposed to regulate plant stress resistance. However, the dynamic changes in DNA methylation in woody plants and their correlations with pathogenic responses are not fully understood. Here, we present single-base maps of the DNA methylomes of mulberry (Morus notabilis) leaves that were subjected to a mock treatment or inoculation with Botrytis cinerea. Compared with the former, the latter showed decreased mCG and mCHG levels and increased mCHH levels. DNA methylation inhibitors reduced resistance gene methylation levels and enhanced mulberry resistance, suggesting that the hypomethylation of resistance genes affects mulberry resistance to B. cinerea. Virus-induced gene silencing of MnMET1 enhanced the expression of mulberry-resistance genes, thereby increasing the plant’s resistance to B. cinerea. We also found that MITEs play a dominant role in controlling DNA methylation levels. MITEs appear to be the main sources of 24-nt siRNAs that regulate gene expression through the RNA-directed DNA methylation pathway.

References

[1] Dean, R. et al. The Top 10 fungal pathogens in molecular plant pathology. Mol. Plant Pathol. 13, 414-430 (2012).

[2] Legard, D. E., Xiao, C. L., Mertely, J. C. & Chandler, C. K. Effects of plant spacing and cultivar on incidence of Botrytis fruit rot in annual strawberry. Plant Dis. 84, 531-538 (2000).

[3] Angelini, R. M. D. et al. Occurrence of fungicide resistance in populations of Botryotinia fuckeliana (Botrytis cinerea) on table grape and strawberry in southern Italy. Pest Manag. Sci. 70, 1785-1796 (2014).

[4] Lamichhane, J. R., Dachbrodt-Saaydeh, S., Kudsk, P. & Messean, A. Toward a reduced reliance on conventional pesticides in European agriculture. Plant Dis. 100, 10-24 (2016).

[5] Dowen, R. H. et al. Widespread dynamic DNA methylation in response to biotic stress. Proc. Natl Acad. Sci. USA 109, E2183-E2191 (2012).

[6] Hewezi, T., Howe, P., Maier, T. R. & Baum, T. J. Arabidopsis small RNAs and their targets during cyst nematode parasitism. Mol. Plant Microbe 21, 1622-1634 (2008).

[7] Boyko, A. et al. Transgenerational changes in the genome stability and methylation in pathogen-infected plants (virus-induced plant genome instability). Nucleic Acids Res. 35, 1714-1725 (2007).

[8] Yang, S. M., Tang, F., Caixeta, E. T. & Zhu, H. Y. Epigenetic regulation of a powdery mildew resistance gene in medicago truncatula. Mol. Plant 6, 2000-2003 (2013).

[9] Zhu, J. K. Active DNA demethylation mediated by DNA glycosylases. Annu. Rev. Genet. 43, 143-166 (2009).

[10] Law, J. A. & Jacobsen, S. E. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat. Rev. Genet. 11, 204-220 (2010).

[11] Zemach, A. et al. The Arabidopsis nucleosome remodeler DDM1 allows DNA methyltransferases to access H1-containing heterochromatin. Cell 153, 193-205 (2013).

[12] Matzke, M. A., Kanno, T. & Matzke, A. J. RNA-Directed DNA methylation: the evolution of a complex epigenetic pathway in flowering plants. Annu. Rev. Plant Biol. 66, 243-267 (2015).

[13] Matzke, M. A. & Mosher, R. A. RNA-directed DNA methylation: an epigenetic pathway of increasing complexity. Nat. Rev. Genet. 15, 394-408 (2014).

[14] Liu, R. & Lang, Z. B. The mechanism and function of active DNA demethylation in plants. J. Integr. Plant Biol. 62, 148-159 (2020).

[15] Zhang, H. et al. Transposon-derived small RNA is responsible for modified function of WRKY45 locus. Nat. Plants 2, 16016 (2016).

[16] Yoder, J. A., Walsh, C. P. & Bestor, T. H. Cytosine methylation and the ecology of intragenomic parasites. Trends Genet. 13, 335-340 (1997).

[17] Deng, Y. et al. Epigenetic regulation of antagonistic receptors confers rice blast resistance with yield balance. Science 355, 962-965 (2017).

[18] Tirnaz, S. & Batley, J. DNA methylation: toward crop disease resistance improvement. Trends Plant Sci. 24, 1137-1150 (2019).

[19] Jiang, Y. & Nie, W. J. Chemical properties in fruits of mulberry species from the Xinjiang province of China. Food Chem. 174, 460-466 (2015).

[20] Xin, Y. C., Meng, S., Ma, B., He, W. M. & He, N. J. Mulberry genes MnANR and MnLAR confer transgenic plants with resistance to Botrytis cinerea. Plant Sci. 296, 110473 (2020).

[21] He, N. et al. Draft genome sequence of the mulberry tree Morus notabilis. Nat. Commun. 4, 2445 (2013).

[22] Ma, B., Li, T., Xiang, Z. & He, N. MnTEdb, a collective resource for mulberry transposable elements. Database 2015, bav004 (2015).

[23] Xin, Y. C., Ma, B., Xiang, Z. H. & He, N. J. Amplification of miniature inverted-repeat transposable elements and the associated impact on gene regulation and alternative splicing in mulberry (Morus notabilis). Mobile DNA 10, 27 (2019).

[24] Song, Q. X. et al. Genome-wide analysis of DNA methylation in soybean. Mol. Plant 6, 1961-1974 (2013).

[25] Zakrzewski, F., Schmidt, M., Van Lijsebettens, M. & Schmidt, T. DNA methylation of retrotransposons, DNA transposons and genes in sugar beet (Beta vulgaris L.). Plant J. 90, 1156-1175 (2017).

[26] Huang, H. et al. Global increase in DNA methylation during orange fruit development and ripening. Proc. Natl Acad. Sci. USA 116, 1430-1436 (2019).

[27] Cheng, J. et al. Downregulation of RdDM during strawberry fruit ripening. Genome Biol. 19, 212 (2018).

[28] Cokus, S. J. et al. Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452, 215-219 (2008).

[29] Zhang, J. et al. Autotetraploid rice methylome analysis reveals methylation variation of transposable elements and their effects on gene expression. Proc. Natl Acad. Sci. USA 112, E7022-E7029 (2015).

[30] Zhong, S. et al. Single-base resolution methylomes of tomato fruit development reveal epigenome modifications associated with ripening. Nat. Biotechnol. 31, 154-159 (2013).

[31] Xu, J. et al. Single-base methylome analysis reveals dynamic epigenomic differences associated with water deficit in apple. Plant Biotechnol. J. 16, 672-687 (2018).

[32] Liang, D. et al. Single-base-resolution methylomes of Populus trichocarpa reveal the association between DNA methylation and drought stress. BMC Genet. 15, S9 (2014).

[33] Ausin, I. et al. DNA methylome of the 20-gigabase Norway spruce genome. Proc. Natl Acad. Sci. USA 113, E8106-E8113 (2016).

[34] Niederhuth, C. E. et al. Widespread natural variation of DNA methylation within angiosperms. Genome Biol. 17, 194 (2016).

[35] Hsieh, T. F. et al. Genome-wide demethylation of Arabidopsis endosperm. Science 324, 1451-1454 (2009).

[36] Zemach, A. et al. Local DNA hypomethylation activates genes in rice endosperm. Proc. Natl Acad. Sci. USA 107, 18729-18734 (2010).

[37] Singer, T., Yordan, C. & Martienssen, R. A. Robertson’s mutator transposons in A. thaliana are regulated by the chromatin-remodeling gene decrease in DNA methylation (DDM1). Genes Dev. 15, 591-602 (2001).

[38] Biemont, C. & Vieira, C. Genetics: junk DNA as an evolutionary force. Nature 443, 521-524 (2006).

[39] Wang, C. et al. A transposon-directed epigenetic change in ZmCCT underlies quantitative resistance to Gibberella stalk rot in maize. New Phytol. 215, 1503-1515 (2017).

[40] Schumann, U. et al. DEMETER plays a role in DNA demethylation and disease response in somatic tissues of Arabidopsis. Epigenetics 14, 1074-1087 (2019).

[41] Schumann, U., Lee, J., Kazan, K., Ayliffe, M. & Wang, M. B. DNA-demethylase regulated genes show methylation-independent spatiotemporal expression patterns. Front. Plant Sci. 8, 1449 (2017).

[42] Le, T. N. et al. DNA demethylases target promoter transposable elements to positively regulate stress responsive genes in Arabidopsis. Genome Biol. 15, 458 (2014).

[43] Cao, X. & Jacobsen, S. E. Role of the arabidopsis DRM methyltransferases in de novo DNA methylation and gene silencing. Curr. Biol. 12, 1138-1144 (2002).

[44] Mosher, R. A. & Melnyk, C. W. siRNAs and DNA methylation: seedy epigenetics. Trends Plant Sci. 15, 204-210 (2010).

[45] Wang, H. et al. CG gene body DNA methylation changes and evolution of duplicated genes in cassava. Proc. Natl Acad. Sci. USA 112, 13729-13734 (2015).

[46] Zhang, X. et al. Genome-wide high-resolution mapping and functional analysis of DNA methylation in arabidopsis. Cell 126, 1189-1201 (2006).

[47] Jones, P. A. Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat. Rev. Genet. 13, 484-492 (2012).

[48] Akimoto, K. et al. Epigenetic inheritance in rice plants. Ann. Bot. 100, 205-217 (2007).

[49] Agorio, A. & Vera, P. ARGONAUTE4 is required for resistance to Pseudomonas syringae in Arabidopsis. Plant Cell 19, 3778-3790 (2007).

[50] Lu, X. et al. Single-base resolution methylomes of upland cotton (Gossypium hirsutum L.) reveal epigenome modifications in response to drought stress. BMC Genomics 18, 297 (2017).

[51] Xi, Y. & Li, W. BSMAP: whole genome bisulfite sequence MAPping program. BMC Bioinform. 10, 232 (2009).

[52] Lister, R. et al. Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 462, 315-322 (2009).

[53] Xiang, H. et al. Single base-resolution methylome of the silkworm reveals a sparse epigenomic map. Nat. Biotechnol. 28, 516-520 (2010).

[54] Juhling, F. et al. metilene: fast and sensitive calling of differentially methylated regions from bisulfite sequencing data. Genome Res. 26, 256-262 (2016).

[55] Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725-2729 (2013).

[56] Huang, C., Xie, Y. & Zhou, X. Efficient virus-induced gene silencing in plants using a modified geminivirus DNA1 component. Plant Biotechnol. J. 7, 254-265 (2009).

[57] Ma, Y. et al. Identification and molecular characterization of a novel monopartite geminivirus associated with mulberry mosaic dwarf disease. J. Gen. Virol. 96, 2421-2434 (2015).