Virus-mediated suppression of host non-self recognition facilitates horizontal transmission of heterologous viruses

Abstract

Non-self recognition is a common phenomenon among organisms; it often leads to innate immunity to prevent the invasion of parasites and maintain the genetic polymorphism of organisms. Fungal vegetative incompatibility is a type of non-self recognition which often induces programmed cell death (PCD) and restricts the spread of molecular parasites. It is not clearly known whether virus infection could attenuate non-self recognition among host individuals to facilitate its spread. Here, we report that a hypovirulence-associated mycoreovirus, named Sclerotinia sclerotiorum mycoreovirus 4 (SsMYRV4), could suppress host non-self recognition and facilitate horizontal transmission of heterologous viruses. We found that cell death in intermingled colony regions between SsMYRV4-infected Sclerotinia sclerotiorum strain and other tested vegetatively incompatible strains was markedly reduced and inhibition barrage lines were not clearly observed. Vegetative incompatibility, which involves Heterotrimeric guanine nucleotide-binding proteins (G proteins) signaling pathway, is controlled by specific loci termed het (heterokaryon incompatibility) loci. Reactive oxygen species (ROS) plays a key role in vegetative incompatibility-mediated PCD. The expression of G protein subunit genes, het genes, and ROS-related genes were significantly down-regulated, and cellular production of ROS was suppressed in the presence of SsMYRV4. Furthermore, SsMYRV4-infected strain could easily accept other viruses through hyphal contact and these viruses could be efficiently transmitted from SsMYRV4-infected strain to other vegetatively incompatible individuals. Thus, we concluded that SsMYRV4 is capable of suppressing host non-self recognition and facilitating heterologous viruses transmission among host individuals. These findings may enhance our understanding of virus ecology, and provide a potential strategy to utilize hypovirulence-associated mycoviruses to control fungal diseases.

Fig 1. SsMYRV4 confers hypovirulence on S. sclerotiorum.

(A) Hypovirulent strain SX10 was dual-cultured with a virulent strain (such as Ep-1PNA367R). When the hyphae of the two strains contacted each other for 2 days, a mycelial plug potentially containing a newly transmitted mycovirus was picked up from the colony margin (marked with a blue star) of virulent strain (a site farthest from the hypovirulent strain). (B) Biological characteristics of strains Ep-1PNA367, SX10 and Ep-1PNA367T1 (derived from a dual culture of strains Ep-1PNA367 and SX10; as shown in A). All strains were cultured on PDA plates for 10 days prior to photography. Pathogenicity assay of S. sclerotiorum strains were carried out on detached leaves (left panels) and whole rapeseed plants (right panels). Photos and data were taken at 72 hour post inoculation. (C) Ultrastructure of fungal cells of strains Ep-1PNA367 and Ep-1PNA367T1 as observed under TEM. The two strains were cultured for 4 days and then examined by TEM. The right panel is an enlargement of nuclear area that is indicated by boxes in left panels. VLPs in the cell (right panel) were marked with blue arrows. N = Nucleus, W = cell wall. (D) TEM images of negatively stained SsMYRV4 particles. (E) PAGE analysis on 5% polyacrylamide gel of dsRNA directly extracted from mycelia of strains SX10, Ep-1PNA367T1 (A367T1), Ep-1PNA367 (A367), or extracted from purified virus particles from SX10, Ep-1PNA367T1. Lane M, λ Hind III-digested DNA used as size markers. All dsRNA samples were treated with DNase I and S1 nucleases prior to electrophoresis. (F) SDS-PAGE analysis of purified SsMYRV4 particles. Samples collected from sucrose gradient fractions were subjected to SDS-PAGE analysis on a 12% polyacrylamide gel. The sizes of the Coomassie blue-stained proteins were estimated by comparison with protein size markers (lane M). (G) Phylogenetic analysis of SsMYRV4. A neighbor-joining phylogenetic tree was constructed based on the complete amino acid sequences of viral RdRp. The virus name (Sclerotinia sclerotiorum mycoreovirus 4) was printed in blue color and pertinent information on other reoviruses selected for phylogenetic analysis is shown in S3 Table. Bootstrap values (%) obtained with 2000 replicates are indicated on the branches, and branch lengths correspond to genetic distance; scale bar at lower left corresponds to genetic distance.

Fig 2. SsMYRV4-mediated suppression of vegetative incompatibility reaction and PCD response.

(A) Strains Ep-1PNA367 (SsMYRV4-free) and Ep-1PNA367T1 (SsMYRV4-infected) were each dual-cultured with strains Ep-1PNA367, 1980, RL19, RL26 and DT47-39 on PDA amended with 75 μl/L of McCormick’s red food coloring. A narrow intense-black-pigmentation zone was easily observed in the interface region of Ep-1PNA367 and other referenced strains, but such a black-pigmented zone was not detected when Ep-1PNA367 was replaced with Ep-1PNA367T1. The two strains were individually dual-cultured for 10 days. (B) Evans blue staining to check the PCD response. PCD response was detected at the interface regions after the hyphae of individuals S. sclerotiorum strains contacted each other for 3 hours.

Fig 3. SsMYRV4-mediated suppression of G protein subunit genes expression in S. sclerotiorum.

(A) qRT-PCR analysis of expression level of G protein subunit genes in mycelium from strains Ep-1PNA367 (Ep-1PNA367 VS Ep-1PNA367), Ep-1PNA367T1 (Ep-1PNA367T1 VS Ep-1PNA367T1), and mycelium from Ep-1PNA367 dual-cultured with strain 1980 (Ep-1PNA367 VS 1980), Ep-1PNA367T1 dual-cultured with 1980 (Ep-1PNA367T1 VS 1980). (B) qRT-PCR analysis of expression level of G protein subunit genes in mycelium from strain 1980 (1980 VS 1980), strain 1980 dual-cultured with Ep-1PNA367T (1980 VS Ep-1PNA367) or Ep-1PNA367T1 (1980 VS Ep-1PNA367T1). (C) qRT-PCR analysis of expression level of G protein subunit genes in mycelium from strain RL19 (RL19 VS RL19), strain RL19 dual-cultured with Ep-1PNA367T (RL19 VS Ep-1PNA367) or Ep-1PNA367T1 (RL19 VS Ep-1PNA367T1). The mycelium of S. sclerotiorum strains was collected at 5 hours post-contact with vegetatively incompatible or compatible strains.

Fig 4. Transcriptional analysis and organization of the candidate het genes that encoded proteins containing HET conserved domains in S. sclerotiorum.

(A). Expression cluster analysis of het genes in strains Ep-1PNA367 and Ep-1PNA367T1. The relative expression of het genes was analyzed based on the threshold of RPKM value. Red means high expression level and green indicates low expression level. The expression profile was analyzed based on RNA-seq data (GEO accession number GSE94575) (B) Organization of het genes in S. sclerotiorum. The HET conserved domains were shown with blue frame. The information (conserved domain, e-value and gene length) of het genes was obtained from NCBI database. The genes selected for qRT-PCR analysis were marked with a black star. (C) qRT-PCR analysis of expression level of twenty het genes during hyphal fusion progress of two vegetatively incompatible or compatible strains. Total RNA was extracted from mycelium of strains Ep-1PNA367 (Ep-1PNA367 VS Ep-1PNA367), Ep-1PNA367T1 (Ep-1PNA367T1 VS Ep-1PNA367T1), and mycelium from Ep-1PNA367 dual-cultured with strain 1980 (Ep-1PNA367 VS 1980), Ep-1PNA367T1 dual-cultured with 1980 (Ep-1PNA367T1 VS 1980)

Fig 5. SsMYRV4-mediated inhibition of ROS production in hyphae of S. sclerotiorum.

(A) NBT staining of hyphae of strains Ep-1PNA367 and Ep-1PNA367T1. Strains were cultured on CM-PDA plates for 2 days and stained with 0.5% NBT in phosphate buffer solution (1 mol/L) for 1 hour under dark conditions, and then the reaction was terminated with anhydrous ethanol. (B) Quantitative detection of H₂O₂ level in hyphae of strains Ep-1PNA367 and Ep-1PNA367T1. Samples were harvested and H₂O₂ level was measured at 3 and 4 dpi. (C) Relative expression of two ROS-associated genes, Ssnox1 and Ssonx2, in Ep-1PNA367 and Ep-1PNA367T1 via Real-time PCR. Total RNA was extracted from mycelium cultured for 3 days and used for qRT-PCR analysis. Comparison of hyphal growth inhibition (D) and colony morphology (E) of strains Ep-1PNA367 and Ep-1PNA367T1 grown on PDA containing 7.5 mM, 15 mM or 20 mM H₂O₂.

Fig 6. SsMYRV4-mediated enhancement of horizontal transmission of unrelated mycoviruses among S. sclerotiorum individuals belonging to different VCGs.

(A) Horizontal transmission of three mycoviruses SsDRV, SsRVL and SsMV1 using a dual-culture method on PDA plates (15-cm in diameter). SsDRV and SsRVL co-infect hypovirulent strain Ep-1PN, whereas SsMV1 naturally infects hypovirulent strain HC025. SsMV1 was horizontally transmitted into strain Ep-1PNA367 under laboratory conditions and the newly obtained SsMV1-infected strain was named HC-A367. Ep-A367T1 harbors three mycoviruses (SsDRV, SsRVL, and SsMYRV4). HC-A367T1 carries two mycoviruses (SsMV1 and SsMYRV4). After the mycelia of two different strains (a hypovirulent strain and a virulent strain) contact each other for 7 days, the new isolates (indicated by blue stars) were picked up from colony margin of the virulent strain, and the designations of newly obtained isolates are shown at the bottom of each plate. (B) Mycovirus content was assessed by dsRNA extraction and RT-PCR amplification. Primers SsMYRV4F and SsMYRV4R, SsDRV-F and SsDRV-R, SsRVLF and SsRVLR, and SsMV1-F and SsMV1-R (S5 Table) were used for detection of SsMYRV4, SsDRV, SsRVL, and SsMV1, respectively. The actin gene served as an internal control; sizes of molecular mass standards (M) are indicated to the left of each panel.

Fig 7. SsMYRV4-mediated enhancement of horizontal transmission between different VCGs effectively prevents and controls Sclerotinia diseases.

(A) SsMYRV4-mediated horizontal transmission of SsDRV controls Sclerotinia diseases on plants of B. napus. Plant leaveas were pre-treated with mycelial suspension (2 g/40ml) of Ep-1PN (infected with SsDRV), Ep-1PNA367T1 (SsMYRV4) and Ep-A367T1 (co-infected with SsDRV and SsMYRV4) for 3 days, and then treated with mycelial suspension of Ep-1PNA367R and 1980R. Photographs were taken at 5 days posted inoculation of Ep-1PNA367R and 1980R. (B) A model for SsMYRV4-mediated horizontal transmission between different VCGs. Strains 1980, Ep-1PN and the bridge donor strain belong to different VCGs. Vegetative incompatibility results in death of the interacting hyphae via PCD (red dotted line area), and in blocking virus horizontal transmission.