A protocol for the rapid isolation of full geminivirus genomes from dried plant tissue

Shepherd, Dionne N.; Martin, Darren P.; Lefeuvre, Pierre; Monjane, Adérito Luis; Owor, Betty E.; Rybicki, Edward P.; Varsani, Arvind

doi:10.1016/j.jviromet.2007.12.014

Cited by 114 publications

(57 citation statements)

References 21 publications

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“…Viral DNA was isolated from symptomatic leaf material at between 20 and 127 days (median, 47 days) postinoculation (p.i.) by using the Extract-n-Amp kit (SigmaAldrich, USA), followed by rolling-circle amplification as previously described (37,38). Amplified DNA was digested with the restriction enzyme BamHI (Fermentas, USA) to generate ϳ2.7-kb monomeric MSV genomes, which were gel purified (GFX; GE Healthcare), ligated into BamHI-digested pGEM-3Zf(ϩ) (Promega, USA) using T4 DNA ligase (Fermentas, USA), and used to transform competent E. cloni cells (Lucigen Corporation, USA).…”

Section: Methodsmentioning

confidence: 99%

Extensive Recombination-Induced Disruption of Genetic Interactions Is Highly Deleterious but Can Be Partially Reversed by Small Numbers of Secondary Recombination Events

Monjane

Martin

Lakay

et al. 2014

J Virol

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Although homologous recombination can potentially provide viruses with vastly more evolutionary options than are available through mutation alone, there are considerable limits on the adaptive potential of this important evolutionary process. Primary among these is the disruption of favorable coevolved genetic interactions that can occur following the transfer of foreign genetic material into a genome. Although the fitness costs of such disruptions can be severe, in some cases they can be rapidly recouped by either compensatory mutations or secondary recombination events. Here, we used a maize streak virus (MSV) experimental model to explore both the extremes of recombination-induced genetic disruption and the capacity of secondary recombination to adaptively reverse almost lethal recombination events. Starting with two naturally occurring parental viruses, we synthesized two of the most extreme conceivable MSV chimeras, each effectively carrying 182 recombination breakpoints and containing thorough reciprocal mixtures of parental polymorphisms. Although both chimeras were severely defective and apparently noninfectious, neither had individual movement-, encapsidation-, or replication-associated genome regions that were on their own "lethally recombinant." Surprisingly, mixed inoculations of the chimeras yielded symptomatic infections with viruses with secondary recombination events. These recombinants had only 2 to 6 breakpoints, had predominantly inherited the least defective of the chimeric parental genome fragments, and were obviously far more fit than their synthetic parents. It is clearly evident, therefore, that even when recombinationally disrupted virus genomes have extremely low fitness and there are no easily accessible routes to full recovery, small numbers of secondary recombination events can still yield tremendous fitness gains. IMPORTANCERecombination between viruses can generate strains with enhanced pathological properties but also runs the risk of producing hybrid genomes with decreased fitness due to the disruption of favorable genetic interactions. Using two synthetic maize streak virus genome chimeras containing alternating genome segments derived from two natural viral strains, we examined both the fitness costs of extreme degrees of recombination (both chimeras had 182 recombination breakpoints) and the capacity of secondary recombination events to recoup these costs. After the severely defective chimeras were introduced together into a suitable host, viruses with between 1 and 3 secondary recombination events arose, which had greatly increased replication and infective capacities. This indicates that even in extreme cases where recombination-induced genetic disruptions are almost lethal, and 91 consecutive secondary recombination events would be required to reconstitute either one of the parental viruses, moderate degrees of fitness recovery can be achieved through relatively small numbers of secondary recombination events.

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Section: Methodsmentioning

confidence: 99%

Extensive Recombination-Induced Disruption of Genetic Interactions Is Highly Deleterious but Can Be Partially Reversed by Small Numbers of Secondary Recombination Events

Monjane

Martin

Lakay

et al. 2014

J Virol

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“…Circular viral DNA molecules were amplified from a crude total DNA extract from the two maize samples using Phi29 DNA polymerase (TempliPhi TM , GE Healthcare, USA) as described previously [15,16,18] and cut with KpnI (PR50) and BamHI (PR52) to yield *2.7-kb DNA fragments, which were subsequently ligated to the KpnI and BamHI sites of pGEMZf? (Promega Biotech, USA).…”

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confidence: 99%

A novel maize-infecting mastrevirus from La Réunion Island

et al. 2012

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Despite extensive sampling, only one virus belonging to the genus Mastrevirus of the family Geminiviridae, maize streak virus (MSV), has until now been detected in maize with maize streak disease (MSD) symptoms. Here, we report for the first time a second, highly divergent, mastrevirus isolated from two maize plants displaying characteristic MSD-like symptoms, sampled on the South-west Indian Ocean Island, La Réunion. The two isolates shared \57 % genome-wide identity with all other known mastreviruses. We propose calling the new species Maize streak Réunion virus.

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“…To determine the suspected begomoviruses associated with CMD symptoms, leaf samples were collected from five symptomatic cassava plants in Cameroon, and from three symptomatic plants in Togo in August 2014 (Table 1). Total DNA was isolated as described by Shepherd et al (2008). Circular, single-stranded DNA was enriched by rolling circle amplification (RCA).…”

Section: Figurementioning

confidence: 99%

First report of Soybean chlorotic blotch virus and West African Asystasia virus 1 infecting cassava and a wild cassava relative in Cameroon and Togo

Leke¹,

Mignouna²,

Brown³

et al. 2016

New Disease Reports

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A protocol for the rapid isolation of full geminivirus genomes from dried plant tissue

Cited by 114 publications

References 21 publications

Extensive Recombination-Induced Disruption of Genetic Interactions Is Highly Deleterious but Can Be Partially Reversed by Small Numbers of Secondary Recombination Events

Extensive Recombination-Induced Disruption of Genetic Interactions Is Highly Deleterious but Can Be Partially Reversed by Small Numbers of Secondary Recombination Events

A novel maize-infecting mastrevirus from La Réunion Island

First report of Soybean chlorotic blotch virus and West African Asystasia virus 1 infecting cassava and a wild cassava relative in Cameroon and Togo

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