VIGS for functional genomics

Key words: Virus induced gene silencing (VIGS) – RNA silencing – functional genomics – molecular biology -  plant development – virus resistance

 

Viruses induce a defence response in their hosts known as RNA silencing. This involves recognition by the plant of double-stranded RNA structures formed by the virus in infected cells. The dsRNA is cleaved by plant enzymes into short 21-25 nt RNAs, which subsequently guide degradation by other plant enzymes of homologous single-stranded RNAs, e.g. viral genomic RNA and viral mRNAs. These reactions lead to partial inhibition of viral replication and spread, but only rarely to complete elimination of the virus.

 

The RNA silencing response can be exploited for silencing of selected plant genes by constructing plant viruses carrying fragments of the target plant gene(s). Upon infection, the modified virus induces silencing directed at the inserted plant gene sequences as well as at the viral sequences. The silencing will also lead to degradation of the corresponding plant mRNAs.

 

 

 

We have used the potato virus X VIGS vector from Prof. D. Baulcombe’s lab. to study the specificity and spreading of silencing along target genes known as transitive silencing. We found evidence of transitive silencing along a transgene (beta-glucuronidase, GUS) suggesting unprimed activity of a cellular RdRP enzyme initiating at the 3’ end of target transcripts, but no evidence of transitive silencing along endogenous targets (Braunstein, TH et al., RNA 8: 1034, 2002; Petersen, BO and Albrechtsen, Plant Mol. Biol. 58: 575, 2005).

 

We have also used the PVX vector as well as a Pea early browning virus VIGS vector (see below) to study the function of a plant homologue of the mammalian RNAse L inhibitor gene. The severe developmental effects of silencing the plant homologue RLIh indicated that RLIh plays important role(s) in plant growth and development rather than being involved in virus interactions as suggested for the mammalian gene (Petersen, BO et al. Plant Science 167: 1283, 2004).

 

 
Silencing of the RLIh gene in N. benthamiana using PEBV as vector (left) compared with control plant infected with the PEBV vector carrying a GFP fragment (right).  

 
VIGS in legumes
An infectious clone of Pea early browning virus (PEBV) has been adapted for use as a VIGS vector in pea and some other legumes. Very efficient and durable silencing of several genes has been demonstrated (Constantin, GD et al., Plant J. 40: 622, 2004). We are currently using the PEBV vector for studies of nodulation, plant – mycorrhiza interactions, and genes controlling plant development and flowering.

 

     
Silencing of the phytoene desaturase gene leads to photobleaching in many tissues (left) while silencing of the meristem-expressed UNI gene leads to changes in leaf and flower morphology (right).

 

 

VIGS in monocots
Most monocotyledonous crop species have large genomes and genetic transformation tends to be difficult and/or time consuming. Therefore the VIGS technique can be very useful for functional studies of monocot genes.

We are currently focusing on the use of Barley stripe mosaic virus (BSMV) for VIGS in barley and wheat. An infectious clone of BSMV obtained from Prof. A. Jackson (University of California) has been modified to serve as silencing vector by a strategy similar to that described by Holzberg et al. (Plant J. 30: 315 (2002)). Efficient silencing of the model genes phytoene desaturase in barley and wheat and a subunit of magnesium chelatase in barley has been obtained. A reliable method for quantifying down-regulation of barley genes by real-time RT-PCR has been established. We are currently performing silencing experiments with genes potentially involved in barley – powdery mildew interactions in collaboration with Dr. M.F. Lyngkjær’s group at Risø National Laboratory. Furthermore, experiments to explore the potential of BSMV as a silencing vector in barley and the role of barley RNA-dependent RNA polymerase genes in the silencing process are in progress.

 

 

Silencing af phytoene desaturase (PDS) in barley cv. Black Hulless.

 

VIGS in cassava
Clones of two isolates of East African cassava mosaic virus (EACMV) have been modified to serve as silencing vectors in cassava in collaboration with Dr. S. Winter at the DSMZ, Braunschweig. Very efficient silencing of a cassava magnesium chelatase gene has been demonstrated. Attempts to use this system to interfere with replication of other viral pathogens are in progress.

 

Silencing of the magnesium chelatase gene in cassava.

 

 

VIGS in ornamentals
Plant species grown for ornamental potted plants are very often sprayed with retardants during production in order to produce the compact plants demanded by consumers. Breeding for compactness is desirable but not always feasible. We are collaborating with colleagues at the Institute of Agricultural Sciences at KVL to identify genes involved in stem elongation in the ornamental species Kalanchoë blossfeldiana. Construction of infectious full-length clones of Kalanchoë latent virus for use as VIGS vector in K. blossfeldiana has been initiated. Meanwhile, VIGS experiments with Kalanchoë candidate genes in Nicotiana benthamiana have been used to select a gene apparently involved in stem elongation in Kalanchoë. This gene will now be tested by RNAi in transgenic Kalanchoë by our collaborators at Institute of Agricultural Sciences, KVL.

 

The persons currently working at the project:

 

VIGS in legumes:
Gabriela Constantin, PhD-student,
Birgit S. Olsen, technician,
Elisabeth Johansen, group leader,

 

VIGS in monocots:

Katrin Geisler, PhD student, at JIC until July 2009
Andrzej Pacak, EU Marie Curie postdoc,
Merete Albrechtsen, senior researcher,

 

Insect resistance through RNAi:
Magnus Lundmark, scientist,
Merete Albrechtsen, senior researcher,

 

VIGS in ornamentals:
Malene Dinesen, PhD-student,
Merete Albrechtsen, senior researcher,