Aberrant Topologies of Bacterial Membrane Proteins Revealed by High Sensitivity Fluorescence Labelling.

The cytoplasmic membrane compartmentalises the bacterial cell into cytoplasm and periplasm. Proteins located in this membrane have a defined topology that is established during their biogenesis. However, the accuracy of this fundamental biosynthetic process is unknown. We developed compartment-specific fluorescence labelling methods with up to single-molecule sensitivity. Application of these methods to the single and multi-spanning membrane proteins of the Tat protein transport system revealed rare topogenesis errors. This methodology also detected low level soluble protein mislocalization from the cytoplasm to the periplasm. This study shows that it is possible to uncover rare errors in protein localization by leveraging the high sensitivity of fluorescence methods.

Rapid, high efficiency virus-mediated mutant complementation and gene silencing in Antirrhinum.

BACKGROUND: Antirrhinum (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. RESULTS: A. 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. CONCLUSIONS: We 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.

Shared Mutations in a Novel Glutaredoxin Repressor of Multicellular Trichome Fate Underlie Parallel Evolution of Antirrhinum Species.

Most angiosperms produce trichomes-epidermal hairs that have protective or more specialized roles. Trichomes are multicellular in almost all species and, in the majority, secretory. Despite the importance of multicellular trichomes for plant protection and as a source of high-value products, the mechanisms that control their development are only poorly understood. Here, we investigate the control of multicellular trichome patterns using natural variation within the genus Antirrhinum (snapdragons), which has evolved hairy alpine-adapted species or lowland species with a restricted trichome pattern multiple times in parallel. We find that a single gene, Hairy (H), which is needed to repress trichome fate, underlies variation in trichome patterns between all Antirrhinum species except one. We show that H encodes a novel epidermis-specific glutaredoxin and that the pattern of trichome distribution within individuals reflects the location of H expression. Phylogenetic and functional tests suggest that H gained its trichome-repressing role late in the history of eudicots and that the ancestral Antirrhinum had an active H gene and restricted trichome distribution. Loss of H function was involved in an early divergence of alpine and lowland Antirrhinum lineages, and the alleles underlying this split were later reused in parallel evolution of alpines from lowland ancestors, and vice versa. We also find evidence for an evolutionary reversal from a widespread to restricted trichome distribution involving a suppressor mutation and for a pleiotropic effect of H on plant growth that might constrain the evolution of trichome pattern.