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Mycoreovirus genome rearrangements associated with RNA silencing deficiency.

Eusebio-Cope A, Suzuki N - Nucleic Acids Res. (2015)

Bottom Line: Here, we explored a possible link between MyRV1 genome rearrangements and the host RNA silencing pathway using wild-type (WT) and mutant strains of both MyRV1 and the host fungus.Consequently, intragenic rearrangements with nearly complete duplication of the three largest segments, i.e. S1, S2 and S3, were observed even more frequently in the RNA silencing-deficient strains Δdcl2 and Δagl2 infected with MyRV1/S4ss, but not with any other viral/host strain combinations.An interesting difference was noted between genome rearrangement events in the two host strains, i.e. generation of the rearrangement required prolonged culture for Δagl2 in comparison with Δdcl2.

View Article: PubMed Central - PubMed

Affiliation: Agrivirology Laboratory, Institute of Plant Science and Resources, Okayama University, Kurashiki, Okayama 710-0046, Japan.

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RT-PCR for specific detection of DLSs in Δdcl2 and Δagl2. A primer set was designed for back-to-back targeting of the jointed 5′- and 3′-terminal sequence of each DLS rearrangement (see Figure 2). When two-week-old culture materials were used, an approximately 1.2-kb PCR fragment of S1Da was detected in five Δdcl2 subcultures and one Δagl2 subculture. Results obtained with S2D and S3D-specific primers are shown in the middle and bottom panels, respectively. No PCR fragments were obtained in any Δagl2 subculture using the S2Db- or S3Da-specific primer. Three samples per replicate (Rep) were assayed. The MyRV1/S4ss+S1Da, MyRV1/S4ss+S2Db and MyRV1/S4ss+S3Da dsRNAs were employed as internal controls for quantitative analysis (+). DLS-free WT MyRV1 was included as a negative control (−). M represents the DNA size marker.
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Figure 4: RT-PCR for specific detection of DLSs in Δdcl2 and Δagl2. A primer set was designed for back-to-back targeting of the jointed 5′- and 3′-terminal sequence of each DLS rearrangement (see Figure 2). When two-week-old culture materials were used, an approximately 1.2-kb PCR fragment of S1Da was detected in five Δdcl2 subcultures and one Δagl2 subculture. Results obtained with S2D and S3D-specific primers are shown in the middle and bottom panels, respectively. No PCR fragments were obtained in any Δagl2 subculture using the S2Db- or S3Da-specific primer. Three samples per replicate (Rep) were assayed. The MyRV1/S4ss+S1Da, MyRV1/S4ss+S2Db and MyRV1/S4ss+S3Da dsRNAs were employed as internal controls for quantitative analysis (+). DLS-free WT MyRV1 was included as a negative control (−). M represents the DNA size marker.

Mentions: To further confirm this difference in the rate of the appearance of DLSs in different host fungal strains, we utilized a highly sensitive method originally developed by (9) for detecting rotavirus rearrangements. This method allows for the detection of the junction of rearranged segments with duplications even at a ≥10 000:1 ratio of normal to rearranged molecules (the sensitivity threshold of RT-PCR). MyRV1/S4ss was horizontally transferred into Δdcl2 and Δagl2 via hyphal anastomosis, and a total of nine samples (three samples each from three independent co-cultures) per host were assayed by RT-PCR at six different time points (3, 5, 7, 10, 14 and 25 days a.c.; RT-PCR data are shown in Table 3 and in Figure 4). No PCR products were detected in either host strain until 7 days a.c. (Table 3). At 10 days a.c., only one sample from Δdcl2 was S1Da-positive, whereas no other samples provided PCR fragments. RT-PCR data for Δagl2 and Δdcl-2 infected with MyRV1/S4ss at 14 days a.c. are presented in Figure 4. Of nine Δdcl2 samples, five, two and one contained S1D, S2D and S3D, respectively, while only S1D was detected in a single Δagl2 sub-culture (Figure 4). Longer culture (25 days a.c.) resulted in an increased frequency of DLS generation in both strains (Table 3). As shown in Table 3, all types of DLSs (S1D–S3D) were formed in Δdcl2. However, no S2D was found in Δagl2. The dominance of S1D in Δdcl2 and the failure of S2D in Δagl2 were also repeatedly observed in a similar independent assay (Supplementary Table S2). It is also noteworthy that only one of the DLSs (S1D, S2D and S3D) was observed in certain single fungal subcultures. Consistent with the results of agarose gel electrophoresis from previous experiments (data not shown), no DLS-carrying isolate was detected in WT EP155 even by this sensitive method (Supplementary Table S2).


Mycoreovirus genome rearrangements associated with RNA silencing deficiency.

Eusebio-Cope A, Suzuki N - Nucleic Acids Res. (2015)

RT-PCR for specific detection of DLSs in Δdcl2 and Δagl2. A primer set was designed for back-to-back targeting of the jointed 5′- and 3′-terminal sequence of each DLS rearrangement (see Figure 2). When two-week-old culture materials were used, an approximately 1.2-kb PCR fragment of S1Da was detected in five Δdcl2 subcultures and one Δagl2 subculture. Results obtained with S2D and S3D-specific primers are shown in the middle and bottom panels, respectively. No PCR fragments were obtained in any Δagl2 subculture using the S2Db- or S3Da-specific primer. Three samples per replicate (Rep) were assayed. The MyRV1/S4ss+S1Da, MyRV1/S4ss+S2Db and MyRV1/S4ss+S3Da dsRNAs were employed as internal controls for quantitative analysis (+). DLS-free WT MyRV1 was included as a negative control (−). M represents the DNA size marker.
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Figure 4: RT-PCR for specific detection of DLSs in Δdcl2 and Δagl2. A primer set was designed for back-to-back targeting of the jointed 5′- and 3′-terminal sequence of each DLS rearrangement (see Figure 2). When two-week-old culture materials were used, an approximately 1.2-kb PCR fragment of S1Da was detected in five Δdcl2 subcultures and one Δagl2 subculture. Results obtained with S2D and S3D-specific primers are shown in the middle and bottom panels, respectively. No PCR fragments were obtained in any Δagl2 subculture using the S2Db- or S3Da-specific primer. Three samples per replicate (Rep) were assayed. The MyRV1/S4ss+S1Da, MyRV1/S4ss+S2Db and MyRV1/S4ss+S3Da dsRNAs were employed as internal controls for quantitative analysis (+). DLS-free WT MyRV1 was included as a negative control (−). M represents the DNA size marker.
Mentions: To further confirm this difference in the rate of the appearance of DLSs in different host fungal strains, we utilized a highly sensitive method originally developed by (9) for detecting rotavirus rearrangements. This method allows for the detection of the junction of rearranged segments with duplications even at a ≥10 000:1 ratio of normal to rearranged molecules (the sensitivity threshold of RT-PCR). MyRV1/S4ss was horizontally transferred into Δdcl2 and Δagl2 via hyphal anastomosis, and a total of nine samples (three samples each from three independent co-cultures) per host were assayed by RT-PCR at six different time points (3, 5, 7, 10, 14 and 25 days a.c.; RT-PCR data are shown in Table 3 and in Figure 4). No PCR products were detected in either host strain until 7 days a.c. (Table 3). At 10 days a.c., only one sample from Δdcl2 was S1Da-positive, whereas no other samples provided PCR fragments. RT-PCR data for Δagl2 and Δdcl-2 infected with MyRV1/S4ss at 14 days a.c. are presented in Figure 4. Of nine Δdcl2 samples, five, two and one contained S1D, S2D and S3D, respectively, while only S1D was detected in a single Δagl2 sub-culture (Figure 4). Longer culture (25 days a.c.) resulted in an increased frequency of DLS generation in both strains (Table 3). As shown in Table 3, all types of DLSs (S1D–S3D) were formed in Δdcl2. However, no S2D was found in Δagl2. The dominance of S1D in Δdcl2 and the failure of S2D in Δagl2 were also repeatedly observed in a similar independent assay (Supplementary Table S2). It is also noteworthy that only one of the DLSs (S1D, S2D and S3D) was observed in certain single fungal subcultures. Consistent with the results of agarose gel electrophoresis from previous experiments (data not shown), no DLS-carrying isolate was detected in WT EP155 even by this sensitive method (Supplementary Table S2).

Bottom Line: Here, we explored a possible link between MyRV1 genome rearrangements and the host RNA silencing pathway using wild-type (WT) and mutant strains of both MyRV1 and the host fungus.Consequently, intragenic rearrangements with nearly complete duplication of the three largest segments, i.e. S1, S2 and S3, were observed even more frequently in the RNA silencing-deficient strains Δdcl2 and Δagl2 infected with MyRV1/S4ss, but not with any other viral/host strain combinations.An interesting difference was noted between genome rearrangement events in the two host strains, i.e. generation of the rearrangement required prolonged culture for Δagl2 in comparison with Δdcl2.

View Article: PubMed Central - PubMed

Affiliation: Agrivirology Laboratory, Institute of Plant Science and Resources, Okayama University, Kurashiki, Okayama 710-0046, Japan.

Show MeSH
Related in: MedlinePlus