RNA silencing is a key regulatory mechanism in the biocontrol fungus Clonostachys rosea-wheat interactions

Abstract

Background: Small RNA (sRNAs)- mediated RNA silencing is emerging as a key player in host-microbe interactions. However, its role in fungus-plant interactions relevant to biocontrol of plant diseases is yet to be explored. This study aimed to investigate Dicer (DCL)-mediated endogenous and cross-kingdom gene expression regulation in the biocontrol fungus Clonostachys rosea and wheat roots during interactions.

Results: C. rosea Δdcl2 strain exhibited significantly higher root colonization than the WT, whereas no significant differences were observed for Δdcl1 strains. Dual RNA-seq revealed the upregulation of CAZymes, membrane transporters, and effector coding genes in C. rosea, whereas wheat roots responded with the upregulation of stress-related genes and the downregulation of growth-related genes. The expression of many of these genes was downregulated in wheat during the interaction with DCL deletion strains, underscoring the influence of fungal DCL genes on wheat defense response. sRNA sequencing identified 18 wheat miRNAs responsive to C. rosea, and three were predicted to target the C. rosea polyketide synthase gene pks29. Two of these miRNAs (mir_17532_x1 and mir_12061_x13) were observed to enter C. rosea from wheat roots with fluorescence analyses and to downregulate the expression of pks29, showing plausible cross-kingdom RNA silencing of the C. rosea gene by wheat miRNAs.

Conclusions: We provide insights into the mechanisms underlying the interaction between biocontrol fungi and plant roots. Moreover, the study sheds light on the role of sRNA-mediated gene expression regulation in C. rosea-wheat interactions and provides preliminary evidence of cross-kingdom RNA silencing between plants and biocontrol fungi.

Keywords: Triticum aestivum; Beneficial fungi; Cross-kingdom RNA silencing; DCL; Defense induction; Gene silencing; Growth promotion; RNA interference; miRNA; sRNAs.

Fig. 1

Determination of C. rosea root colonization in wheat roots. Wheat roots were harvested five days post-inoculation of C. rosea spores, and the fungal biomass was quantified using RT-qPCR. C. rosea colonization is expressed as the ratio between C. rosea DNA and wheat DNA. Actin and Hor1 were used as target genes for DNA quantification for C. rosea and wheat, respectively. Error bars represent standard deviation based on five biological replicates. Different letters indicate statistically significant differences (p < 0.05) based on Fisher’s exact test

Fig. 2

The transcriptomic response of wheat to interaction with C. rosea. A Pie chart showing the proportion of the stress-related genes in wheat upregulated during Cr-Wr. B Pie chart showing the proportion of stress-related genes, specialized metabolism, and cell wall-related genes downregulated during Cr-Wr

Fig. 3

The number of differentially expressed wheat genes during the interactions with C. rosea WT or DCL gene deletion strains. The Venn diagram was generated with https://bioinformatics.psb.ugent.be/webtools/Venn/

Fig. 4

The heatmap shows the expression (Log2FC) of selected wheat genes of interest. All genes were differentially expressed during Cr-Wr (log2(FC) > 1.5 or < −1.5 and FDR < 0.05) but not during both or either Δdcl1-Wr or Δdcl2-Wr. Wr indicates wheat roots; Cr indicates C. rosea wildtype

Fig. 5

Percentage of genes annotated with gene ontology terms referring to biological processes enriched in wheat genes upregulated (A) or downregulated (B) during the interaction between wheat and the WT but not when the plant interacted with the Δdcl1 or Δdcl2 mutant. For each of these GO terms, the percentage of genes having the term in the whole wheat genome is compared with the percentage of genes with the term in the situation of enrichment (FDR < 0.05)

Fig. 6

Number of differentially expressed C. rosea genes during the interaction with wheat roots. Venn diagram generated with https://bioinformatics.psb.ugent.be/webtools/Venn/

Fig. 7

A Read length distribution of sRNA reads mapped to wheat. B 5′ base distribution of sRNA reads mapped to wheat. C Average transcriptome read counts of wheat genes, depending on their percentile rank of antisense sRNA counts. Percentile ranks were assigned to each gene based on its antisense sRNA counts. Therefore, genes in the group “1–20” are the 20% of genes with the lowest amount of antisense sRNAs mapping to them, while genes in the group “81–100” are the ones with the highest number of antisense sRNAs. Genes with an antisense sRNA count of zero were not considered. Lowercase, uppercase, Greek letters and numbers indicate groups not significantly different according to separate Tukey tests with a maximum p-value of 0.05

Fig. 8

A Read length distribution of sRNA reads mapped to C. rosea. B 5′ base distribution of sRNA reads mapped to C. rosea. C Average transcriptome read counts of C. rosea genes, depending on their percentile rank of antisense sRNA counts. Percentile ranks were assigned to each gene based on its antisense sRNA counts. Therefore, genes in the group “1–20” are the 20% of genes with the lowest amount of antisense sRNAs mapping to them, while genes in the group “81–100” are the ones with the highest number of antisense sRNAs. Genes with an antisense sRNA count of zero were not considered. Lowercase, uppercase, Greek letters and numbers indicate groups not significantly different according to separate Tukey tests with a maximum p-value of 0.05

Fig. 9

Uptake of dsRNA by C. rosea and miRNA mimics by wheat roots. A GFP-tagged C. rosea conidia (C. rosea-GFP) was incubated with Cyanine 3-UTP labeled dsRNA (Cy3-dsRNACt) and examined under a confocal microscope at 24 h post incubation (hpi). Representative confocal microscopy images showing uptake of Cy3-dsRNA by C. rosea hyphae and conidia (left panel, magenta), C. rosea-GFP control (middle panel), and colocalization of GFP (Green) with Cy3 (Right panel) in Cy3-dsRNACt-treated C. rosea-GFP (right panel). B Representative confocal microscopy images show the uptake of Cy3-labeled miRNA mimics by wheat roots. The left panel shows a control treatment with no Cy3 fluorescence signal (Magenta). The right panel shows Cy3 miRNA 12061 mimic (Magenta) internalization into wheat roots (confocal microscopy images of root cross sections). For the experiment, artificially synthesized mir_17532_x1 miRNA mimic miR17532 was applied on wheat roots, and a Confocal microscopic analysis of the root surface and horizontal cross-section was performed 24 hpi

Fig. 10

Trafficking of Cy3 labeled wheat mir_17532_x1 mimics (miR17532) from wheat roots to C. rosea-GFP conidia and hyphae. A Representative confocal images showing the co-localization of Cy3-miR17532 (Magenta), C. rosea-GFP (Green), and merge (right panel). White arrows indicate germinated C. rosea conidia. B A representative confocal image (enlarged) shows the internalization of Cy3-miR17532 mimic (Magenta) by C. rosea conidia (Green) and submerge (right panel). After 24 h of incubation with miR17532, wheat roots were washed with 0.1M KCl and 0.01 M Triton X100 to remove surface-bound miRNA oligos. Conidia from C. rosea-GFP were applied to the roots, and Cy3 fluorescence was determined t72 hpi. C–E Detection and quantification of wheat miRNAs mimic inside wheat roots using stem-loop-RT-qPCR (CP. infestans miR8788 mimic miR8788; D wheat mir_17532_x1 mimic miR17532; E wheat mir_12061_x13 mimic miR12061). F RT-qPCR showing the expression of the pks29 gene in C. rosea cells after import of miR8788, miR1532, and miR1203 during interaction with wheat roots treated with these miRNA mimics

Fig. 11

Illustration of molecular dialogue and cross-kingdom RNA silencing between C. rosea and wheat. A The Clonostachys rosea-wheat interaction triggers the upregulation of wheat genes associated with biotic and abiotic stress tolerance and the downregulation of genes involved in cell wall loosening and expansion, leading to controlled colonization of wheat roots by C. rosea. Concurrently, genes related to carbohydrate catabolism, transport, and effector production are upregulated in C. rosea. B During the interaction between Δdcl2 and wheat roots, the expression patterns of many of these genes in wheat and C. rosea are altered, resulting in enhanced root colonization by C. rosea. The regulation of these gene expression patterns in wheat and C. rosea is potentially mediated by small RNAs at both endogenous and cross-kingdom levels. ↑ Indicates gene upregulation, ↓ indicates gene downregulation, ? Indicate lack of experimental validation for sRNA movement from C. rosea to wheat roots