Simultaneous analysis of the bidirectional African cassava mosaic virus promoter activity using two different luciferase genes.

Abstract:

:The expression of geminivirus genes is controlled by bidirectional promoters which are located in the large intergenic region of the circular DNA genomes and specifically regulated by virus encoded proteins. In order to study the simultaneous regulation of both orientations of the DNA A and DNA B promoters of African cassava mosaic virus (ACMV), they were cloned between two different luciferase genes with the firefly luciferase gene in complementary-sense and the Renilla luciferase gene in virion-sense orientation. The regulation of the ACMV promoters by proteins encoded by the complete DNA A, as well as by the individually expressed transactivator (TrAP) or replication-associated (Rep) proteins was assessed in tobacco and cassava protoplasts using dual luciferase assays. In addition, the regulation of the DNA A promoter integrated into tobacco genome was also assessed. The results show that TrAP activates virion-sense expression strongly both in cassava and tobacco protoplasts, but not in transgenic tobacco plants. In contrast to this, DNA A encoded proteins activate virion-sense expression both in protoplasts and in transgenic plants. At the same time they reduce the expression of the complementary-sense Rep gene on DNA A but activate the expression of the complementary-sense movement protein (MPB) gene on DNA B. The degree of MBP activation is higher in cassava than in tobacco protoplasts, indicating that the plant host also influences the promoter strength. Transient transformation experiments using linearized DNA indicate that the different regulation of the ACMV DNA A promoter in protoplasts and transgenic plants could be due to different DNA curvature in free plasmids and in genes integrated in plant genomic DNA.

journal_name

Virus Genes

journal_title

Virus genes

authors

Frey PM,Schärer-Hernández NG,Fütterer J,Potrykus I,Puonti-Kaerlas J

doi

10.1023/a:1008183827072

subject

Has Abstract

pub_date

2001-03-01 00:00:00

pages

231-42

issue

2

eissn

0920-8569

issn

1572-994X

journal_volume

22

pub_type

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