Abstract
BackgroundAntirrhinum (snapdragon) species are models for genetic and evolutionary research but recalcitrant to genetic transformation, limiting use of transgenic methods for functional genomics. Transient gene expression from viral vectors and virus-induced gene silencing (VIGS) offer transformation-free alternatives. Here we investigate the utility of Tobacco rattle virus (TRV) for homologous gene expression in Antirrhinum and VIGS in Antirrhinum and its relative Misopates.ResultsA. majus proved highly susceptible to systemic TRV infection. TRV carrying part of the Phytoene Desaturase (PDS) gene triggered efficient PDS silencing, visible as tissue bleaching, providing a reporter for the extent and location of VIGS. VIGS was initiated most frequently in young seedlings, persisted into inflorescences and flowers and was not significantly affected by the orientation of the homologous sequence within the TRV genome. Its utility was further demonstrated by reducing expression of two developmental regulators that act either in the protoderm of young leaf primordia or in developing flowers. The effects of co-silencing PDS and the trichome-suppressing Hairy (H) gene from the same TRV genome showed that tissue bleaching provides a useful marker for VIGS of a second target gene acting in a different cell layer. The ability of TRV-encoded H protein to complement the h mutant phenotype was also tested. TRV carrying the native H coding sequence with PDS to report infection failed to complement h mutations and triggered VIGS of H in wild-type plants. However, a sequence with 43% synonymous substitutions encoding H protein, was able to complement the h mutant phenotype when expressed without a PDS VIGS reporter.ConclusionsWe demonstrate an effective method for VIGS in the model genus Antirrhinum and its relative Misopates that works in vegetative and reproductive tissues. We also show that TRV can be used for complementation of a loss-of-function mutation in Antirrhinum. These methods make rapid tests of gene function possible in these species, which are difficult to transform genetically, and opens up the possibility of using additional cell biological and biochemical techniques that depend on transgene expression.
Highlights
Antirrhinum species are models for genetic and evolutionary research but recalcitrant to genetic transformation, limiting use of transgenic methods for functional genomics
Given the high mortality and low efficiency reported to result from direct Agro-infiltration of A. majus [13], we first produced infectious virus in N. benthamiana by infiltration with a mixture of A. tumefaciens strains carrying pTRV1, which expresses the TRV1 RNA, and either empty or recombinant pTRV2sgP, to express TRV2 RNA (Fig. 1a)
A. majus is susceptible to infection with recombinant Tobacco rattle virus (TRV) Inoculating A. majus seedlings with TRV1 and empty TRV2sgP, led to slightly reduced growth and distortion of leaves compared to mock-inoculated control plants (Fig. 1b,c), suggesting that TRV was able to infect A. majus
Summary
Antirrhinum (snapdragon) species are models for genetic and evolutionary research but recalcitrant to genetic transformation, limiting use of transgenic methods for functional genomics. Though transgenic A. majus plants have been produced by infecting hypocotyl explants with Agrobacterium tumefaciens and regenerating plants from transgenic cells, or by regeneration from root cultures transformed by A. rhizogenes [10,11,12], these methods involve tissue culture and regeneration and are slow, as well as very inefficient [13, 14].
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