Engineering of alfalfa mosaic virus RNA 3 into an expression vector.

Abstract:

:RNA 3 of alfalfa mosaic virus (AMV) encodes the 5'-proximal movement protein (MP) gene and the 3'-proximal coat protein (CP) gene which is expressed from a subgenomic RNA. Several strategies were explored to use this RNA as a vector for expression of the green fluorescent protein (GFP) in Nicotiana tabaccum plants expressing the viral polymerase proteins P1 and P2 (P12 plants). Insertion of a subgenomic promoter (sgp)-GFP cassette between the CP gene and the 3'-untranslated region (UTR) interfered with RNA accumulation in protoplasts, indicating that cis-acting sequences required for replication were disrupted. When GFP was fused to the N-terminus of MP or CP, the chimeric RNAs accumulated in protoplasts but cell-to-cell movement in plants was blocked. Insertion of a GFP-sgp cassette immediately upstream of the CP gene caused a hypersensitive host response. However, insertion of a GFP-sgp cassette upstream of the MP gene did not affect symptom formation and yielded a vector that expressed GFP in inoculated but not in the systemic leaves of both P12 tobacco and non-transgenic N. benthamina plants. When the size of the GFP gene was reduced from 700 to 300 nucleotides, virus infection was observed in the non-inoculated leaves. Analysis of the progeny of some chimera revealed novel data on replication, encapsidation and recombination of AMV RNA 3.

journal_name

Arch Virol

journal_title

Archives of virology

authors

Sanchez-Navarro J,Miglino R,Ragozzino A,Bol JF

doi

10.1007/s007050170125

keywords:

subject

Has Abstract

pub_date

2001-01-01 00:00:00

pages

923-39

issue

5

eissn

0304-8608

issn

1432-8798

journal_volume

146

pub_type

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