US 8,637,653 B2 · Infectious plant viral vector and an artificial bipartite plant viral vector an infectious plant viral vector and an artificial bipartite plant viral vector

Abstract

We have developed a versatile plant viral vector system based on Alternanthera mosaic virus (AltMV), suitable for infection by agroinfiltration or in vivo T7 transcripts from the same clone; agroinfection is enhanced by coinfiltration of a T7 RNA polymerase construct. Variants adapted for efficient protein expression, or for virus-induced gene silencing (VIGS), are based on a specific amino acid substitution (L88P) in the triple gene block 1 (TGB1) protein affecting RNA silencing suppression. A bipartite delivery system developed for AltMV delivers replicase (RdRp) functions separately from movement and encapsidation (TGB and coat protein, CP) functions by agroinfiltration, resulting in precise recombination of RdRp and TGB-CP constructs in planta. The bipartite delivery system has potential for high throughput protein expression or VIGS with the appropriate TGB1 variant, for hosts including Nicotiana benthamiana and Arabidopsis thaliana. Equivalent TGB1 substitutions in other potexviruses also reduced RNA silencing suppression, demonstrated with Potato virus X.

Claims16

Dependent claims

An Alternanthera mosaic virus (AItMV) bipartite launch system comprising:

(a) a first construct comprising an AItMV RNA-dependent RNA polymerase (RdRp), operably linked to a CaMV 35S promoter or other suitable plant promoter and a T7 promoter or other suitable bacteriophage promoter;
(b) a second construct comprising in order an AItMV partial RdRp, shown schematically in FIG. 19D, an AItMV Triple Gene Block 1 (TGB1), an AItMV Triple Gene Block 2 (TGB2), an AItMV Triple Gene Block 3 (TGB3), a subgenomic promoter operably linked to a multiple cloning site (MCS), and a subgenomic promoter operably linked to an AItMV Coat Protein (CP);
and
(c) a third construct encoding a T7 RNA polymerase (T7RNAP);
wherein the second construct is operably linked to a CaMV 35S promoter or other suitable plant promoter and a T7 promoter or other suitable bacteriophage promoter,
the TGB1 amino acid sequence is the sequence set forth in SEQ ID NO:
84 or SEQ ID NO:
88,
the first and second construct undergo recombination in planta within a short overlap of a common sequence of the partial RdRp present in both constructs,
and the AItMV bipartite launch system is capable of high level protein expression in a plant.

7 more elements

Dependencies are inferred from the claim text.

Infectious plant viral vector and an artificial bipartite plant viral vector an infectious plant viral vector and an artificial bipartite plant viral vector

Life of this patent

Infectious plant viral vector and an artificial bipartite plant viral vector an infectious plant viral vector and an artificial bipartite plant viral vector

Life of this patent

Abstract

Claims16