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MicroRNAs from the parasitic plant Cuscuta campestris target host messenger RNAs

Abstract

Dodders (Cuscuta spp.) are obligate parasitic plants that obtain water and nutrients from the stems of host plants via specialized feeding structures called haustoria. Dodder haustoria facilitate bidirectional movement of viruses, proteins and mRNAs between host and parasite1, but the functional effects of these movements are not known. Here we show that Cuscuta campestris haustoria accumulate high levels of many novel microRNAs (miRNAs) while parasitizing Arabidopsis thaliana. Many of these miRNAs are 22 nucleotides in length. Plant miRNAs of this length are uncommon, and are associated with amplification of target silencing through secondary short interfering RNA (siRNA) production2. Several A. thaliana mRNAs are targeted by 22-nucleotide C. campestris miRNAs during parasitism, resulting in mRNA cleavage, secondary siRNA production, and decreased mRNA accumulation. Hosts with mutations in two of the loci that encode target mRNAs supported significantly higher growth of C. campestris. The same miRNAs that are expressed and active when C. campestris parasitizes A. thaliana are also expressed and active when it infects Nicotiana benthamiana. Homologues of target mRNAs from many other plant species also contain the predicted target sites for the induced C. campestris miRNAs. These data show that C. campestris miRNAs act as trans-species regulators of host-gene expression, and suggest that they may act as virulence factors during parasitism.

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Figure 1: C. campestris miRNAs induced at the haustorial interface.
Figure 2: C. campestris miRNAs cause slicing and phased siRNA production from host mRNAs.
Figure 3: Effects of C. campestris miRNAs and their targets.
Figure 4: Conservation of host mRNA targeting by C. campestris.

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Acknowledgements

This research was supported in part by awards from the US National Science Foundation (1238057 to J.H.W. and C.W.D.; 1339207 to M.J.A.) and the US National Institute of Food and Agriculture (135997 to J.H.W.).

Author information

Authors and Affiliations

Authors

Contributions

S.S. and M.J.A. did the bioinformatics analysis. S.S., M.J.A. and N.R.J. prepared figures and tables. G.K., J.H.W., N.R.J., S.S., T.P. and M.J.A. cultivated and harvested plant specimens. E.W., G.K., C.W.D. and J.H.W. performed genome and transcriptome sequencing and assemblies. F.W., S.S. and N.R.J. did RNA blotting. S.S. and M.J.A. performed 5′-RLM-RACE and qRT–PCR. C.C. and T.P. constructed small-RNA-seq libraries. N.R.J. and V.B.-G. performed growth assays. M.J.A. and J.H.W. conceived the project. M.J.A. wrote and revised the manuscript.

Corresponding author

Correspondence to Michael J. Axtell.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks M. Albert, F. Tang and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 PCR of C. campestris miRNA loci.

Genomic DNA isolated from C. campestris seedlings four days after germination was used as template; the seedlings had never attached to nor been near a host plant, ruling out host DNA contamination. trnL-F, positive control plastid locus. Experiment performed once.

Extended Data Figure 2 C. campestris miRNAs cause slicing and phased siRNA production from host mRNAs.

Small-RNA-seq coverage across the indicated A. thaliana transcripts are shown in blue for host stem, interface, and parasite stem samples. For display, the two biological replicates of each type were merged. Red marks and vertical lines show positions of complementary sites to C. campestris miRNAs, with the alignments shown above. Fractions indicate numbers of 5′-RLM-RACE clones with 5′-ends at the indicated positions; the locations in red are the predicted sites for miRNA-directed slicing remnants. Bar charts show the length and polarity distribution of transcript-mapped siRNAs. Radar charts show the fractions of siRNAs in each of the 21 possible phasing registers; the registers highlighted in magenta are those predicted by the miRNA target sites.

Extended Data Figure 3 Possible miRNA target sites within endogenous C. campestris mRNAs.

Note that none of these mRNAs showed evidence of secondary siRNA accumulation, and the complementarity of these sites was generally poor.

Extended Data Figure 4 Growth of C. campestris on A. thaliana sgs2-1 and dcl4-2t mutants with varying methodologies, as indicated.

ad, P values (Wilcoxon rank-sum tests, unpaired, two-tailed) from comparison of mutant to wild-type (Col-0) are shown. Box plots show the median, box edges represent the first and third quartiles, the whiskers extend to 1.5× interquartile range, and all data are shown as dots. a, n = 16 and 9 biologically independent samples for Col-0 and sgs2-1, respectively. 95% confidence interval (Col-0 minus sgs2-1), 0.120 to 0.000. b, n = 10 and 8 biologically independent samples for Col-0 and dcl4-2t, respectively. 95% confidence interval (Col-0 minus dcl4-2t), −0.052 to 0.012. c, n = 11 and 10 biologically independent samples for Col-0 and dcl4-2t, respectively. 95% confidence interval (Col-0 minus dcl4-2t), −0.014 to 0.018. d, n = 8 and 9 biologically independent samples for Col-0 and dcl4-2t, respectively. 95% confidence interval (Col-0 minus dcl4-2t), −0.184 to 0.008. e, n = 14, 14 and 12 biologically independent samples for Col-0, sgs2-1, and dcl4-2t, respectively. 95% confidence interval (Col-0 minus sgs2-1), −0.020 to 0.090. 95% confidence interval (Col-0 minus dcl4-2t), −0.010 to 0.110.

Source data

Extended Data Figure 5 Highly reproducible induction of C. campestris miRNAs in different hosts.

a, Mean abundance plot from original experiment on A. thaliana hosts of C. campestris small-RNA loci comparing interface to parasite stem samples. Significantly upregulated loci are highlighted (alternative hypothesis: true difference >2-fold, FDR ≤ 0.05 after correction for multiple testing with the Benjamini–Hochberg procedure). Reproduced from Fig. 1a. b, c, As a, except for a new set of A. thaliana hosts (b) or from an experiment using N. benthamiana as hosts (c). Significantly upregulated loci are highlighted (alternative hypothesis: true difference > 2-fold, FDR ≤ 0.05 after correction for multiple testing with the Benjamini–Hochberg procedure). d, Area-proportional Euler diagram showing overlaps of upregulated C. campestris 21–22-nucleotide miRNA loci among the three small-RNA-seq experiments. The locations of the six miRNA loci of special interest are highlighted in green.

Extended Data Figure 6 C. campestris miRNAs cause slicing and phased siRNA production from N.benthamiana mRNAs.

Small-RNA-seq coverage across the indicated N. benthamiana transcripts are shown in blue for host stem, interface, and parasite stem samples. For display, the two biological replicates of each type were merged. Red marks and vertical lines show position of complementary sites to C. campestris miRNAs, with the alignments shown above. Fraction indicates numbers of 5′-RLM-RACE clones with 5′-ends at the indicated positions; the locations in red are the predicted sites for miRNA-directed slicing remnants. Bar charts show the length and polarity distribution of transcript-mapped siRNAs. Radar charts show the fractions of siRNAs in each of the 21 possible phasing registers; the registers highlighted in magenta are those predicted by the miRNA target sites.

Extended Data Figure 7 C. campestris pre-haustoria.

a, C. campestris seedling wound around a bamboo stake. b, The same seedling, removed from the stake to show the prominent pre-haustorial bumps. Seedling was scarified, germinated on moist paper towels for three days at ~28 °C, and then placed next to a bamboo stake for four days with far-red LED lighting. Approximately 30 such seedlings were used for the pre-haustoria RNA in Fig. 4b. Scale bars, 1 mm.

Extended Data Table 1 Predicted miRNA targets multiple plant species

Supplementary information

Life Sciences Reporting Summary (PDF 72 kb)

Supplementary Data 1

This file contains A. thaliana and C. campestris small RNA loci. Output from ShortStack 3.8.3 (https://github.com/MikeAxtell/ShortStack) showing all small RNA loci identified in this study. (.xlsx format) (XLSX 21429 kb)

Supplementary Data 2

This file contains mature miRNAs and miRNA*s fromC. campestris Interface-induced MIRNA loci. (.xlsx format) (XLSX 51 kb)

Supplementary Data 3

This file contains details ofC. campestris MIRNA loci: Text-based sequences, predicted secondary structures, and aligned small RNA reads (all six libraries). Lower-case letters indicate small RNA bases that are mismatched to the genomic sequence. Plain-text (ASCII) format. (TXT 798 kb)

Supplementary Data 4

This file contains images showing C. campestris hairpins overlaid with color-codes representing total read-depth (all six 'original' C. campestris x A. thaliana small RNA libraries). (pdf, 43 pages). (PDF 1722 kb)

Supplementary Data 5

This file contains alignments of mature miRNAs and miRNA*s (from Supplementary Data 2 -- the induced C. campestris miRNAs) against the A. thaliana genome. Note that most have three or more mismatches. (SAM format). (TXT 10 kb)

Supplementary Data 6

This file contains oligonucleotide sequences. Excel (.xlsx) format. (XLSX 33 kb)

Supplementary Fig. 1

This file contains uncropped gel figures. (PDF 4403 kb)

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Shahid, S., Kim, G., Johnson, N. et al. MicroRNAs from the parasitic plant Cuscuta campestris target host messenger RNAs. Nature 553, 82–85 (2018). https://doi.org/10.1038/nature25027

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