Unique Terminal Regions and Specific Deletions of the Segmented Double-Stranded RNA Genome of Alternaria Alternata Virus 1, in the Proposed Family Alternaviridae.

Alternaria alternata virus 1 (AaV1) has been identified in the saprophytic fungus Alternaria alternata strain EGS 35-193. AaV1 has four genomic double-stranded (ds)RNA segments (dsRNA1-4) packaged in isometric particles. The 3' end of each coding strand is polyadenylated (36-50nt), but the presence of a cap structure at each 5' end has not previously been investigated. Here, we have characterized the AaV1 genome and found that it has unique features among the mycoviruses. We confirmed the existence of cap structures on the 5' ends of the AaV1 genomic dsRNAs using RNA dot blots with anti-cap antibodies and the oligo-capping method. Polyclonal antibodies against purified AaV1 particles specifically bound to an 82kDa protein, suggesting that this protein is the major capsid component. Subsequent Edman degradation indicated that the AaV1 dsRNA3 segment encodes the major coat protein. Two kinds of defective AaV1 dsRNA2, which is 2,794bp (844 aa) in length when intact, appeared in EGS 35-193 during subculturing, as confirmed by RT-PCR and northern hybridization. Sequence analysis revealed that one of the two defective dsRNA2s contained a 231bp deletion, while the other carried both the 231bp deletion and an additional 465bp deletion in the open reading frame. Both deletions occurred in-frame, resulting in predicted proteins of 767 aa and 612 aa. The fungal isolates carrying virions with the defective dsRNA2s showed impaired growth and abnormal pigmentation. To our best knowledge, AaV1 is the first dsRNA virus to be identified with both 5' cap and 3'poly(A) structures on its genomic segments, as well as the specific deletions of dsRNA2.

Molecular characterization of two evolutionarily distinct endornaviruses co-infecting common bean (Phaseolus vulgaris).

Two high-molecular-mass dsRNAs of approximately 14 and 15 kbp were isolated from the common bean (Phaseolus vulgaris) cultivar Black Turtle Soup. These dsRNAs did not appear to cause obvious disease symptoms, and were transmitted through seeds at nearly 100% efficiency. Sequence information indicates that they are the genomes of distinct endornavirus species, for which the names Phaseolus vulgaris endornavirus 1 (PvEV-1) and Phaseolus vulgaris endornavirus 2 (PvEV-2) are proposed. The PvEV-1 genome consists of 13,908 bp and potentially encodes a single polyprotein of 4496 aa, while that of PvEV-2 consists of 14 820 bp and potentially encodes a single ORF of 4851 aa. PvEV-1 is more similar to Oryza sativa endornavirus, while PvEV-2 is more similar to bell pepper endornavirus. Both viruses have a site-specific nick near the 5' region of the coding strand, which is a common property of the endornaviruses. Their polyproteins contain domains of RNA helicase, UDP-glycosyltransferase and RNA-dependent RNA polymerase, which are conserved in other endornaviruses. However, a viral methyltransferase domain was found in the N-terminal region of PvEV-2, but was absent in PvEV-1. Results of cell-fractionation studies suggested that their subcellular localizations were different. Most endornavirus-infected bean cultivars tested were co-infected with both viruses.

Mycoviruses related to chrysovirus affect vegetative growth in the rice blast fungus Magnaporthe oryzae.

Mycoviruses causing impaired growth and abnormal pigmentation of the host were found in the rice blast fungus, Magnaporthe oryzae. Four dsRNAs, dsRNA 1 (3554 bp), dsRNA 2 (3250 bp), dsRNA 3 (307 bp) and dsRNA 4 (3043 bp), were detected in isolate S-0412-II 1a of M. oryzae. By picking up single conidia of S-0412-II 1a, cured strains of the fungus were isolated that had completely lost the mycovirus. The cured strains had normal mycelial growth and pigmentation, suggesting that this mycovirus modulates host traits. The buoyant densities of isometric virus particles (∼35 nm diameter) containing these dsRNAs in CsCl ranged from 1.37 to 1.40 g cm⁻³. The single ORF (3384 nt) of dsRNA 1 encoded a gene product highly homologous to the viral RNA-dependent RNA polymerase of members of the family Chrysoviridae. It is noteworthy that mycovirus S-0412-II 1a was detected not only in host cells but also in culture supernatant. Furthermore, abnormal aggregation of mycelia was observed after adding the mycovirus-containing culture supernatant to an uninfected strain of M. oryzae and mycoviral dsRNAs were detectable from the aggregated mycelia. This novel dsRNA mycovirus was named Magnaporthe oryzae chrysovirus 1.

Knock-down of OsDCL2 in rice negatively affects maintenance of the endogenous dsRNA virus, Oryza sativa endornavirus.

An endogenous double-stranded RNA (dsRNA), which has recently been recognized as the dsRNA virus Oryza sativa endornavirus (OsEV), is found in many strains of cultivated rice (Oryza sativa). Small RNAs derived from OsEV dsRNA were detected, indicating that the RNA silencing machinery recognizes OsEV dsRNA. The existence of OsEV in knock-down (KD) lines of five genes of RNA-dependent RNA polymerase (OsRDR1-OsRDR5) or two genes of Dicer-like protein (OsDCL2 or OsDCL3a) was examined to characterize the relationship between the host RNA silencing system and the propagation of this dsRNA virus. OsEV was not detected in OsRDR4-KD or OsDCL2-KD T(1) lines. We attempted to introduce OsEV into these KD lines by crossing them with OsEV-carrying plants because of the efficient transmission of OsEV to F(1) plants via pollen or ova. All OsRDR4-KD but only some OsDCL2-KD F(1) plants contained OsEV. Some OsDCL2-KD F(1) plants consisted of OsEV-carrying and OsEV-free cells. These results suggest that the maintenance of OsEV is unstable in OsDCL2-KD plants. Furthermore, the amount of OsEV-derived small interfering RNA (vsiRNA) in the OsDCL2-KD plants increased relative to the wild type. This increased level of vsiRNA may cause OsEV instability during cell division.

A novel mycovirus associated with four double-stranded RNAs affects host fungal growth in Alternaria alternata.

Four double-stranded RNAs (dsRNAs), referred to as dsRNA 1 (3617 bp), dsRNA 2 (2794 bp), dsRNA 3 (2576 bp) and dsRNA 4 (1420 bp), were detected in the EGS 35-193 strain of Alternaria alternata at high concentration ( approximately 3 microg/g dried mycelium). This strain had an impaired growth phenotype. By exposing the strain to cycloheximide during hyphal tip isolation, we isolated strains which had normal mycelial growth and pigmentation, in which decreased levels of the dsRNAs were observed ( approximately 0.3 microg/g dried mycelium). These results indicate that this dsRNA mycovirus might be involved in modulating traits of its fungal host, A. alternata. The buoyant density of isometric virus particles (about 33 nm in diameter) containing these dsRNAs in CsCl was 1.35-1.40 g/cm(3) depending on the size of the packaged dsRNAs. The dsRNA 1 encodes a single open reading frame (3447 nt) containing the conserved motifs of viral RNA-dependent RNA polymerase (RdRp), which is related to the ORF encoded by dsRNA 1 of Aspergillus mycovirus 341. It is noteworthy that all of the coding strands of the four dsRNA genomes have 3'-poly (A) tails ranging from 33 to 50 nt in length. We named this novel dsRNA mycovirus in the EGS 35-193 strain A. alternata virus-1 (AaV-1).