Abstract
A versatile green fluorescent protein (GFP) expression cassette containing the replication origins of the monopartite begomovirus Tomato yellow leaf curl Sardinia virus (TYLCSV) is described. Transgenic Nicotiana benthamiana plants containing one copy of the cassette stably integrated into their genome were superinfected with TYLCSV, which mobilized and replicated the cassette as an episomal replicon. The expression of the reporter gene (the GFP gene) was thereby modified. Whereas GFP fluorescence was dimmed in the intercostal areas, an increase of green fluorescence in veins of all leaves placed above the inoculation site, as well as in transport tissues of roots and stems, was observed. The release of episomal trans replicons from the transgene and the increase in GFP expression were dependent on the cognate geminiviral replication-associated protein (Rep) and required interaction between Rep and the intergenic region of TYLCSV. This expression system is able to monitor the replication status of TYLCSV in plants, as induction of GFP expression is only produced in those tissues where Rep is present. To further confirm this notion, the expression of a host factor required for geminivirus replication, the proliferating cellular nuclear antigen (PCNA) was transiently silenced. Inhibition of PCNA prevented GFP induction in veins and reduced viral DNA. We propose that these plants could be widely used to easily identify host factors required for geminivirus replication by virus-induced gene silencing.
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Claims 7
Claim · model-written summary
Superinfection with TYLCSV increased green fluorescence in veins of leaves above the inoculation site, while GFP fluorescence was dimmed in intercostal areas.
- Taxon
- Nicotiana benthamiana
Nicotiana benthamianae1 - Organ / stage
- veinse2
- Treatment
- superinfected with TYLCSVe1
- Interactor
- TYLCSVTomato yellow leaf curl Sardinia viruse1pathogen
- Gene
- GFPe0
- Outcome
- green fluorescence in veinse2
- Direction
- increasee2
- Comparator
- intercostal arease2
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“A versatile green fluorescent protein (GFP) expression cassette containing the replication origins of the monopartite begomovirus Tomato yellow leaf curl Sardinia virus (TYLCSV) is described.”
e1“Transgenic Nicotiana benthamiana plants containing one copy of the cassette stably integrated into their genome were superinfected with TYLCSV, which mobilized and replicated the cassette as an episomal replicon.”
e2“Whereas GFP fluorescence was dimmed in the intercostal areas, an increase of green fluorescence in veins of all leaves placed above the inoculation site, as well as in transport tissues of roots and stems, was observed.”
Claim · model-written summary
The release of episomal replicons and the increase in GFP expression depended on the replication-associated protein Rep.
- Taxon
- Nicotiana benthamianaNicotiana benthamianae1
- Gene
- replication-associated protein (Rep)e3
- Outcome
- increase in GFP expressione3
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e1“Transgenic Nicotiana benthamiana plants containing one copy of the cassette stably integrated into their genome were superinfected with TYLCSV, which mobilized and replicated the cassette as an episomal replicon.”
e3“The release of episomal trans replicons from the transgene and the increase in GFP expression were dependent on the cognate geminiviral replication-associated protein (Rep) and required interaction between Rep and the intergenic region of TYLCSV.”
Claim · model-written summary
Replication of TYLCSV required an interaction between Rep and the intergenic region of the virus.
- Taxon
- Nicotiana benthamianaNicotiana benthamianae1
- Interactor
- TYLCSVTomato yellow leaf curl Sardinia viruse3pathogen
- Gene
- Repe3
- Outcome
- interaction between Rep and the intergenic regione3
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e1“Transgenic Nicotiana benthamiana plants containing one copy of the cassette stably integrated into their genome were superinfected with TYLCSV, which mobilized and replicated the cassette as an episomal replicon.”
e3“The release of episomal trans replicons from the transgene and the increase in GFP expression were dependent on the cognate geminiviral replication-associated protein (Rep) and required interaction between Rep and the intergenic region of TYLCSV.”
Claim · model-written summary
The expression system is able to monitor the replication status of TYLCSV in plants, with GFP induced only in tissues where Rep is present.
- Taxon
- Nicotiana benthamianaNicotiana benthamianae1
- Interactor
- TYLCSVTomato yellow leaf curl Sardinia viruse4pathogen
- Outcome
- replication status of TYLCSVe4
- Comparator
- tissues where Rep is presente4
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e1“Transgenic Nicotiana benthamiana plants containing one copy of the cassette stably integrated into their genome were superinfected with TYLCSV, which mobilized and replicated the cassette as an episomal replicon.”
e4“This expression system is able to monitor the replication status of TYLCSV in plants, as induction of GFP expression is only produced in those tissues where Rep is present.”
Claim · model-written summary
Inhibition of PCNA prevented GFP induction in veins.
- Taxon
- Nicotiana benthamianaNicotiana benthamianae1
- Treatment
- Inhibition of PCNAe5
- Gene
- proliferating cellular nuclear antigen (PCNA)e5
- Outcome
- GFP induction in veinse5
- Direction
- preventede5
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e1“Transgenic Nicotiana benthamiana plants containing one copy of the cassette stably integrated into their genome were superinfected with TYLCSV, which mobilized and replicated the cassette as an episomal replicon.”
e5“the expression of a host factor required for geminivirus replication, the proliferating cellular nuclear antigen (PCNA) was transiently silenced. Inhibition of PCNA prevented GFP induction in veins and reduced viral DNA.”
Claim · model-written summary
Inhibition of PCNA reduced viral DNA.
- Taxon
- Nicotiana benthamianaNicotiana benthamianae1
- Treatment
- Inhibition of PCNAe5
- Interactor
- TYLCSVTomato yellow leaf curl Sardinia viruse1pathogen
- Gene
- PCNAe5
- Outcome
- viral DNAe5
- Direction
- reducede5
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e1“Transgenic Nicotiana benthamiana plants containing one copy of the cassette stably integrated into their genome were superinfected with TYLCSV, which mobilized and replicated the cassette as an episomal replicon.”
e5“the expression of a host factor required for geminivirus replication, the proliferating cellular nuclear antigen (PCNA) was transiently silenced. Inhibition of PCNA prevented GFP induction in veins and reduced viral DNA.”
Claim · model-written summary
The authors propose that these plants could identify host factors required for geminivirus replication by virus-induced gene silencing.
- Taxon
- Nicotiana benthamianaNicotiana benthamianae1
- Outcome
- identify host factors required for geminivirus replicatione6
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e1“Transgenic Nicotiana benthamiana plants containing one copy of the cassette stably integrated into their genome were superinfected with TYLCSV, which mobilized and replicated the cassette as an episomal replicon.”
e6“We propose that these plants could be widely used to easily identify host factors required for geminivirus replication by virus-induced gene silencing.”