Chrysoviruses in Magnaporthe oryzae

Abstract

Magnaporthe oryzae, the fungus that causes rice blast, is the most destructive pathogen of rice worldwide. A number of M. oryzae mycoviruses have been identified. These include Magnaporthe oryzae. viruses 1, 2, and 3 (MoV1, MoV2, and MoV3) belonging to the genus, Victorivirus, in the family, Totiviridae; Magnaporthe oryzae. partitivirus 1 (MoPV1) in the family, Partitiviridae; Magnaporthe oryzae. chrysovirus 1 strains A and B (MoCV1-A and MoCV1-B) belonging to cluster II of the family, Chrysoviridae; a mycovirus related to plant viruses of the family, Tombusviridae (Magnaporthe oryzae. virus A); and a (+)ssRNA mycovirus closely related to the ourmia-like viruses (Magnaporthe oryzae. ourmia-like virus 1). Among these, MoCV1-A and MoCV1-B were the first reported mycoviruses that cause hypovirulence traits in their host fungus, such as impaired growth, altered colony morphology, and reduced pigmentation. Recently we reported that, although MoCV1-A infection generally confers hypovirulence to fungi, it is also a driving force behind the development of physiological diversity, including pathogenic races. Another example of modulated pathogenicity caused by mycovirus infection is that of Alternaria alternata chrysovirus 1 (AaCV1), which is closely related to MoCV1-A. AaCV1 exhibits two contrasting effects: Impaired growth of the host fungus while rendering the host hypervirulent to the plant, through increased production of the host-specific AK-toxin. It is inferred that these mycoviruses might be epigenetic factors that cause changes in the pathogenicity of phytopathogenic fungi.

Keywords: Magnaporthe oryzae. chrysovirus 1; Mycovirus; double-stranded RNA virus; hypovirulence; rice blast fungus.

Figure 1

Distribution map of rice blast fungus infected with mycoviruses. Mycoviruses with dsRNA genomes were found in 11 M. oryzae isolates from three provinces in the Mekong Delta region of Vietnam. The plant diagrams show the sites where disease lesions were sampled. The agarose gel shows viral dsRNA segments present in the infected M. oryzae isolates. Two mycoviruses were identified: MoCV1 had five dsRNA genomic segments of 2.6 kbp to 3.6 kbp, and a partitivirus had three segments of 1.8 kbp to 2.4 kbp. The M. oryzae isolates, S-0412-II 1a, S-0412-II 1c, and S-0412-II 2a, were infected with the MoCV1 strains, MOCV1-A, MOCV1-A-a, and MOCV1-B, respectively. Isolate S-0412-II 1c was also infected with the partitivirus.

Figure 2

Analysis of mycoviruses isolated from M. oryzae in Vietnam. Top left, agarose gel electrophoresis of dsRNAs derived from mycoviruses purified from rice blast isolates. Lower left, northern hybridisation with a cDNA probe derived from MoCV1-A dsRNA3, showing a weak signal for MoCV1-B dsRNA3. Right, fungal flora of the M. oryzae isolates infected with MoCV1-A (top), MoCV1-A-a (middle), and MoCV1-B (bottom). These isolates were cultured on PDA medium at 26 °C for 10 days.

Figure 3

Influence of MoCV1-B on cell wall formation in M. oryzae hyphae. Infected and non-infected hyphae were stained with calcofluor-white (Sigma Chemical, St. Louis, MO, USA) and examined at 1000× magnification under a light microscope (Olympus IX71, Tokyo, Japan) with differential interference contrast (DIC) optics. Calcofluor-white (CW) binds strongly to structures containing cellulose and chitin.

Figure 4

Isolation and analysis of mycovirus MoCV1-A from the rice blast fungus, M. oryzae. Upper panels, process of isolation and purification of the mycovirus. Lower left, dsRNA genomic segments extracted from purified MoCV1-A virus particles were subjected to 5% (w/v) native PAGE. Lower middle, open reading frames (ORFs) within each of the five genomic segments. Lower right, MoCV1-A viral proteins were separated by denaturing PAGE.

Figure 5

Determination of the C-terminal residue of the MoCV1-A 60 kDa protein (labeled P58 in the figure). The C-terminus of P58 is a truncated version of the ORF3 protein missing 200 amino acids. Mass spectrometry of tryptic peptides derived from P58 identified peptides specific to the deduced amino acid sequence of ORF3, but no peptide sequence mapped to the C terminus of the deduced amino acid sequence of ORF3 (M565 to L799).

Figure 6

Model for partial degradation of the ORF3 and ORF4 proteins. In the early stage of culture, the MoCV1-A viral particles contained full-length ORF3 and ORF4 proteins. After long-term culture and nutrient depletion, the viral particles were composed of partially degraded ORF3 and ORF4 proteins.

Figure 7

Release of mycoviruses from the mycelium of mycovirus-infected M. oryzae into the culture supernatant. The mycoviruses gradually appeared and accumulated in the liquid medium during the long-term suspension culture. After five weeks of liquid culture, 250 μL samples of the culture supernatant were subjected to stepwise centrifugation treatments. Total nucleic acids were extracted from each supernatant and then subjected to agarose gel electrophoresis.

Figure 8

Expression of the MoCV1-A ORF4 protein induced cytological damage in yeast (S. cerevisiae) cells. The MoCV1-A ORF4 gene was inserted in a high-expression vector (2 μ ori, TDH3 promoter) and the vector was used to transform the yeast strain, W303-1A. The morphology and growth of the yeast cells were observed. The MoCV1-A ORF4-GFP fusion protein caused aggregation in the yeast cells.

Figure 9

Influence of MoCV1-A on pathogenicity of M. oryzae. Upper panel: Spray inoculation assays revealed that MoCV1-A infection altered M. oryzae pathogenicity from virulence to avirulence (sensitive to resistance) in IRBL 5-M rice, and from avirulence to virulence (resistance to sensitive) in IRBL 9-W rice. Lower panel: The changes in pathogenicity were confirmed by changes in the growth indices of infectious hyphae in leaf sheath inoculation assays (lower panel). Details about the pathogenicity assays and statistical analyses are given in Aihara et al. [39].