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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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Related in: MedlinePlus

Differences in DLS-generation profiles between the two RNA-silencing deficient hosts. Agarose gel electrophoresis of MyRV1/S4ss dsRNAs from Δdcl2 and Δagl2 at 44 and 66 days a.c. Progression of DLSs between Δdcl2 and Δagl2 were compared. All virus-infected isolates of Δdcl2 developed DLSs even at 44 days a.c. While some virus-infected Δagl2 subcultures showed very faint DLS bands (shown by white asterisks), DLSs were observed in all MyRV1/S4ss-infected Δagl2 isolates at 66 days a.c. with even lower intensity than those in Δdcl2. Red bars in the Δdcl2 panel indicate the loss of cognate normal segments in agarose gel, while yellow arrowheads in the Δagl2 panel represent remnants of standard segments. Red asterisks show migration positions of S4.
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Figure 3: Differences in DLS-generation profiles between the two RNA-silencing deficient hosts. Agarose gel electrophoresis of MyRV1/S4ss dsRNAs from Δdcl2 and Δagl2 at 44 and 66 days a.c. Progression of DLSs between Δdcl2 and Δagl2 were compared. All virus-infected isolates of Δdcl2 developed DLSs even at 44 days a.c. While some virus-infected Δagl2 subcultures showed very faint DLS bands (shown by white asterisks), DLSs were observed in all MyRV1/S4ss-infected Δagl2 isolates at 66 days a.c. with even lower intensity than those in Δdcl2. Red bars in the Δdcl2 panel indicate the loss of cognate normal segments in agarose gel, while yellow arrowheads in the Δagl2 panel represent remnants of standard segments. Red asterisks show migration positions of S4.

Mentions: While DLSs were detected in both MyRV1/S4ss-infected Δdcl2 and Δagl2, a lower frequency of DLSs occurred in Δagl2 than in Δdcl2 at a certain time point after receiving MyRV1/S4ss (Figure 1). That is, most Δdcl2 derived isolates harbored DLSs 40 days a.c., but only some of the Δagl2 derived subcultures contained DLSs. To monitor the appearance of DLS-carrying isolates in these two hosts, a set of ten isolates per host was taken from the recipient side of the co-cultures used in the previous experiment shown in Figure 1. These were cultured sequentially in solid medium until specific time points: 44 days a.c., 66 days a.c. and 120 days a.c. (Figure 3 and data not shown). At 44 days a.c., all ten sub-isolates of Δdcl2 were confirmed to carry DLSs, although some subcultures contained DLSs showing weak band intensity, as expected from Figure 1. An increase in the number of DLS-carrying isolates in Δagl2 was observed only beyond 44 days a.c. After prolonged incubation (66 days a.c.), all subcultures carried DLSs in Δagl2. At this time point, there was a difference between Δagl2 and Δdcl2; while intact corresponding segments were completely replaced by DLSs in Δdcl2-infected MyRV1/S4ss (see the red bars in lanes 4–9 for S1 and in lane 10 for S3 in Figure 3; Δdcl2), they were still visible in Δagl2 with lower band intensity (see yellow arrowheads in Figure 3; Δagl2). It was also noteworthy that two DLSs found in a single isolate at day 44 (Figure 3; Δdcl2 lane 8) converged into one at 66 days a.c. Even at 120 days a.c. the gel profiles remained the same in the two fungal strains (data not shown). It should be noted that S10ss occasionally appeared below the S4ss band in some DLS-carrying strains after prolonged culture (data not shown). These results suggest that Δagl2 and Δdcl2 differ at least in the rate at which DLSs develop and in the robustness of replacement of normal unaltered segments.


Mycoreovirus genome rearrangements associated with RNA silencing deficiency.

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

Differences in DLS-generation profiles between the two RNA-silencing deficient hosts. Agarose gel electrophoresis of MyRV1/S4ss dsRNAs from Δdcl2 and Δagl2 at 44 and 66 days a.c. Progression of DLSs between Δdcl2 and Δagl2 were compared. All virus-infected isolates of Δdcl2 developed DLSs even at 44 days a.c. While some virus-infected Δagl2 subcultures showed very faint DLS bands (shown by white asterisks), DLSs were observed in all MyRV1/S4ss-infected Δagl2 isolates at 66 days a.c. with even lower intensity than those in Δdcl2. Red bars in the Δdcl2 panel indicate the loss of cognate normal segments in agarose gel, while yellow arrowheads in the Δagl2 panel represent remnants of standard segments. Red asterisks show migration positions of S4.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4402544&req=5

Figure 3: Differences in DLS-generation profiles between the two RNA-silencing deficient hosts. Agarose gel electrophoresis of MyRV1/S4ss dsRNAs from Δdcl2 and Δagl2 at 44 and 66 days a.c. Progression of DLSs between Δdcl2 and Δagl2 were compared. All virus-infected isolates of Δdcl2 developed DLSs even at 44 days a.c. While some virus-infected Δagl2 subcultures showed very faint DLS bands (shown by white asterisks), DLSs were observed in all MyRV1/S4ss-infected Δagl2 isolates at 66 days a.c. with even lower intensity than those in Δdcl2. Red bars in the Δdcl2 panel indicate the loss of cognate normal segments in agarose gel, while yellow arrowheads in the Δagl2 panel represent remnants of standard segments. Red asterisks show migration positions of S4.
Mentions: While DLSs were detected in both MyRV1/S4ss-infected Δdcl2 and Δagl2, a lower frequency of DLSs occurred in Δagl2 than in Δdcl2 at a certain time point after receiving MyRV1/S4ss (Figure 1). That is, most Δdcl2 derived isolates harbored DLSs 40 days a.c., but only some of the Δagl2 derived subcultures contained DLSs. To monitor the appearance of DLS-carrying isolates in these two hosts, a set of ten isolates per host was taken from the recipient side of the co-cultures used in the previous experiment shown in Figure 1. These were cultured sequentially in solid medium until specific time points: 44 days a.c., 66 days a.c. and 120 days a.c. (Figure 3 and data not shown). At 44 days a.c., all ten sub-isolates of Δdcl2 were confirmed to carry DLSs, although some subcultures contained DLSs showing weak band intensity, as expected from Figure 1. An increase in the number of DLS-carrying isolates in Δagl2 was observed only beyond 44 days a.c. After prolonged incubation (66 days a.c.), all subcultures carried DLSs in Δagl2. At this time point, there was a difference between Δagl2 and Δdcl2; while intact corresponding segments were completely replaced by DLSs in Δdcl2-infected MyRV1/S4ss (see the red bars in lanes 4–9 for S1 and in lane 10 for S3 in Figure 3; Δdcl2), they were still visible in Δagl2 with lower band intensity (see yellow arrowheads in Figure 3; Δagl2). It was also noteworthy that two DLSs found in a single isolate at day 44 (Figure 3; Δdcl2 lane 8) converged into one at 66 days a.c. Even at 120 days a.c. the gel profiles remained the same in the two fungal strains (data not shown). It should be noted that S10ss occasionally appeared below the S4ss band in some DLS-carrying strains after prolonged culture (data not shown). These results suggest that Δagl2 and Δdcl2 differ at least in the rate at which DLSs develop and in the robustness of replacement of normal unaltered segments.

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