Virus-induced gene silencing as a tool for functional genomics in a legume species

• Published in: The Plant journal : for cell and molecular biology 2004-11, Vol.40 (4), p.622-631
• Main Authors: Constantin, G.D, Krath, B.N, MacFarlane, S.A, Nicolaisen, M, Johansen, I.E, Lund, O.S
• Format: Article
• Language: English
• Subjects: agrobacterium; Arabidopsis thaliana; Biological and medical sciences; carotenoid; cell wall; Diverse techniques; dual silencing; functional genomics; Fundamental and applied biological sciences. Psychology; Gene expression; Gene Expression Regulation, Plant; Gene Silencing; Genes, Plant; Genetic Vectors; Genome, Plant; Genomics; Genomics - methods; korrigan1 gene; leafy gene; Legumes; meristem; Molecular and cellular biology; Molecular Sequence Data; Nematoda; nucleotide sequences; Oxidoreductases - genetics; Oxidoreductases - metabolism; Pea early-browning virus; Phenotype; phytoene desaturase; Pisum sativum; Pisum sativum - genetics; Pisum sativum - metabolism; Pisum sativum - virology; Plant Viruses; Plants, Genetically Modified; reverse genetics; Rhizobium; tobravirus; Transgenes; unifoliata gene; virus-induced gene silencing; Viruses
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/j.1365-313X.2004.02233.x

Virus-induced gene silencing (VIGS) is an attractive reverse-genetics tool for studies of gene function. However, efficient VIGS has only been accomplished in a few plant species. In order to extend the application of VIGS, we examined whether a VIGS vector based on Pea early browning virus (PEBV) would produce recognizable phenotypes in Pisum sativum. A plasmid vector of PEBV was modified to allow agro-inoculation and insertion of heterologous sequences. cDNA fragments of the P. sativum phytoene desaturase (PDS), LEAFY (LFY) and KORRIGAN1 (KOR1) homologues were inserted into the PEBV RNA2 vector, replacing the genes required for nematode transmission. Pisum sativum inoculated with PEBV carrying a fragment of PsPDS developed characteristic photo-bleached leaves and this phenotype was associated with a significant reduction in PsPDS mRNA. The P. sativum homologue of LFY is known as UNIFOLIATA (UNI). Plants inoculated with PEBV carrying a fragment of UNI developed distorted flowers and leaves with modified architecture, which are also observed in UNI-mutants. In Arabidopsis thaliana, the KOR1-mutant is characterized by an extreme dwarf phenotype. Pisum sativum plants inoculated with PEBV carrying a fragment of PsKOR1 displayed a significant reduction in height and inhibition of root growth. The PEBV VIGS vector did not affect the ability of P. sativum to flower, set seeds, and form nodules characteristic of symbiosis with rhizobium. These results suggest that the PEBV vector can be applied to functional genomics in a legume species to study genes involved in a wide range of biological processes.