A fungal milRNA mediates epigenetic repression of a virulence gene in Verticillium dahliae

Abstract

MiRNAs in animals and plants play crucial roles in diverse developmental processes under both normal and stress conditions. miRNA-like small RNAs (milRNAs) identified in some fungi remain functionally uncharacterized. Here, we identified a number of milRNAs in Verticillium dahliae, a soil-borne fungal pathogen responsible for devastating wilt diseases in many crops. Accumulation of a V. dahliae milRNA1, named VdmilR1, was detected by RNA gel blotting. We show that the precursor gene VdMILR1 is transcribed by RNA polymerase II and is able to produce the mature VdmilR1, in a process independent of V. dahliae DCL (Dicer-like) and AGO (Argonaute) proteins. We found that an RNaseIII domain-containing protein, VdR3, is essential for V. dahliae and participates in VdmilR1 biogenesis. VdmilR1 targets a hypothetical protein-coding gene, VdHy1, at the 3'UTR for transcriptional repression through increased histone H3K9 methylation of VdHy1. Pathogenicity analysis reveals that VdHy1 is essential for fungal virulence. Together with the time difference in the expression patterns of VdmilR1 and VdHy1 during fungal infection in cotton plants, our findings identify a novel milRNA, VdmilR1, in V. dahliae synthesized by a noncanonical pathway that plays a regulatory role in pathogenicity and uncover an epigenetic mechanism for VdmilR1 in regulating a virulence target gene. This article is part of the theme issue 'Biotic signalling sheds light on smart pest management'.

Keywords: Verticillium dahliae; histone modification; milRNA; pathogenicity.

Figure 1.

RNA-silencing components and the profiles of small RNAs in V. dahliae. (a) Conserved domains of RNA-silencing components RdRP, DCL and AGO in V. dahliae. (b) The length distribution of total Vd-sRNAs and Vd-sRNAs after removing 3′ or 5′ truncated homologues in V. dahliae. (c) Ratio of the first nucleotide preference of Vd-sRNAs in V592. (d) Predicted stem-loop structures of seven highly expressed (RPM > 10) VdmilRNA precursors in V592, including VdMILR1. The mature VdmilRNA sequences are labelled in red. (e) Small RNA gel blotting of VdmilR1 in V592 detected by using the LNA™ (Locked Nucleotide) enhanced probe.

Figure 2.

VdmilR1, produced by an RNaseIII protein, VdR3, epigenetically represses its target virulence gene VdHy1. (a) Colony morphology of wild-type V592, overexpression strains Tef-VdMILR1 and Tef-VdR3, and knockout mutant Δvdhy1 and its complement strain Δvdhy1/VdHy1 on PDA plates after two weeks of incubation. We found no obvious developmental defect but the slightly dense growth of hyphae was observed in Tef-VdMILR1 and Tef-VdR3. Δvdhy1 reduced hyphal growth and melanin production, and the complemented strain Δvdhy1/VdHy1 restored the colony morphology. (b) Detection of VdmilR1 in Tef-VdMILR1 by small RNA gel blotting. Higher accumulation of VdmilR1 was detected in Tef-VdMILR1 compared with wild-type V592. (c) Pol II is required for the production of VdmilR1. Schematic of the overlapping locus of VdMILR1 and Asp-tRNA in the genome (left). The arrows indicate the transcription direction. Small RNA gel blotting shows the level of VdmilR1 in the indicated strains (right) treated with α-amanitin (Pol II inhibitor) or H₂O (as solvent control), or with ML-60218 (Pol III-specific inhibitor) or DMSO (solvent control). The rRNA in the bottom panel shows equal loading of RNA samples. (d) Small RNA hybridization shows that mature VdmilR1 was markedly increased in Tef-VdR3 strains. (e) Quantitative RT-PCR (qRT-PCR) of VdHy1 in wild-type, Tef-VdMILR1 and Δvdmilr1. The V. dahliae tubulin beta chain gene was used as the internal control. Error bars show ±s.d. from three replicates. The level of VdHy1 mRNA in V592 was set to 1. (f) Chromatin immunoprecipitation (ChIP) assay at VdHy1 chromatin using H3K9me3-specific antibody. The top schematic shows the genomic structure of VdHy1 and the relative positions of the primers (P1–P3) used for ChIP assays and the VdmilR1 target site. Black box: coding sequence. White boxes: 5′UTR and 3′UTR sequences. ChIP-qPCR data were normalized to a sample of input DNA. The V. dahliae tubulin beta chain gene was used as the internal control. Error bars show ±s.d. from three replicates. The values in V592 were arbitrarily designated 1. (g) Disease symptoms of cotton plants infected with V592, Tef-VdMILR1, Δvdhy1 or complemented strain Δvdhy1/VdHy1 at 25 days postinoculation (dpi). (h) The expression patterns of VdmilR1/VdHy1 during the infection process. The V. dahliae tubulin gene and V. dahliae U6 were used as internal controls for VdHy1 and VdmilR1, respectively. Error bars show ±s.d. from three replicates. Data obtained from the time point of 0.5 (first lanes) were set to 1.