An Optimized Protocol to Increase Virus-Induced Gene Silencing Efficiency and Minimize Viral Symptoms in Petunia

Virus-induced gene silencing (VIGS) is used to down-regulate endogenous plant genes. VIGS efficiency depends on viral proliferation and systemic movement throughout the plant. Although tobacco rattle virus (TRV)-based VIGS has been successfully used in petunia (Petunia?×?hybrida), the protocol has not been thoroughly optimized for efficient and uniform gene down-regulation in this species. Therefore, we evaluated six parameters that improved VIGS in petunia. Inoculation of mechanically wounded shoot apical meristems induced the most effective and consistent silencing compared to other methods of inoculation. From an evaluation of ten cultivars, a compact petunia variety, 'Picobella Blue', exhibited a 1.8-fold higher CHS silencing efficiency in corollas. We determined that 20 °C day/18 °C night temperatures induced stronger gene silencing than 23 °C/18 °C or 26 °C/18 °C. The development of silencing was more pronounced in plants that were inoculated at 3–4 versus 5 weeks after sowing. While petunias inoculated with pTRV2-NbPDS or pTRV2-PhCHS showed very minimal viral symptoms, plants inoculated with the pTRV2 empty vector (often used as a control) were stunted and developed severe necrosis, which often led to plant death. Viral symptoms were eliminated by developing a control construct containing a fragment of the green fluorescent protein (pTRV2-sGFP). These optimization steps increased the area of chalcone synthase (CHS) silencing by 69 % and phytoene desaturase (PDS) silencing by 28 %. This improved VIGS protocol, including the use of the pTRV2-sGFP control plants, provides stronger down-regulation for high-throughput analyses of gene function in petunia.     

Heterologous gene silencing induced by tobacco rattle virus (TRV) is efficient for pursuing functional genomics studies in woody plants

Virus-induced gene silencing (VIGS) is an effective tool for studying the functions of plant genes, but only a few VIGS vectors available for woody plants were reported so far. Here we present an effective heterologous VIGS system in woody plants based on tobacco rattle virus (TRV) vectors. We first tested whether the TRV-vector can be directly applied to infect woody plant species, such as Vernicia fordii, Populus tomentosa Carr. and Camellia oleifera. The results revealed that TRV-mediated VIGS could be effectively elicited in V. fordii, weakly in P. tomentosa Carr., but not in C. oleifera. TRV-based VIGS vectors with heterologous phytoene desaturase (PDS) sequences from various woody plant species silenced successfully the endogenous PDS gene in Nicotina benthamiana and V. fordii. The photobleached leaf phenotype of silenced plants significantly correlated with the down-regulation of endogenous PDS as compared with controls. To further confirm the reliability of VIGS in V. fordii, we also isolated the cloroplastos alterados 1 gene from P. tomentosa Carr., and the silencing pheotypes of albino leaves were observed in V. fordii 2 weeks after inoculation using a heterologous TRV-based VIGS system. Taken together, we have successfully developed an Agrobacterium-mediated VIGS assay in V. fordii and demonstrated that V. fordii as a heterologous VIGS system provides a valuable tool for functional genomic analysis in woody plant species.     

Genome editing of polyploid crops: prospects,achievements and bottlenecks

Plant breeding aims to develop improved crop varieties. Many crops have a polyploid and often highly heterozygous genome, which may make breeding of polyploid crops a real challenge. The efficiency of traditional breeding based on crossing and selection has been improved by using marker-assisted selection (MAS), and MAS is also being applied in polyploid crops, which helps e.g. for introgression breeding. However, methods such as random mutation breeding are difficult to apply in polyploid crops because there are multiple homoeologous copies (alleles) of each gene. Genome editing technology has revolutionized mutagenesis as it enables precisely selecting targets. The genome editing tool CRISPR/Cas is especially valuable for targeted mutagenesis in polyploids, as all alleles and/or copies of a gene can be targeted at once. Even multiple genes, each with multiple alleles, may be targeted simultaneously. In addition to targeted mutagenesis, targeted replacement of undesirable alleles by desired ones may become a promising application of genome editing for the improvement of polyploid crops, in the near future. Several examples of the application of genome editing for targeted mutagenesis are described here for a range of polyploid crops, and achievements and bottlenecks are highlighted.

     

Diversity of sequences and expression patterns among alleles of a sugarcane loading stem gene

Modern sugarcane cultivars are highly polyploid and aneuploid hybrids, which are propagated as clones. Their complex genome structure comprises 100–130 chromosomes and 10–13 hom(e)ologous copies of most loci. There is preliminary evidence of very high heterozygosity, with implications for genetic improvement approaches ranging from marker-assisted selection to transgenics. Here, we report that sugarcane cultivar Q200 has at least nine alleles at the Loading Stem Gene (ScLSG) locus. Exon–intron structure is identical and the predicted protein products show at least 92 % identity, across sugarcane alleles and the Sorghum homologue Sb07g027880. There is substantial variation in the 5′ UTR and promoter regions including numerous allele-specific nucleotide polymorphisms, insertions and deletions. We developed an allele-specific qRT-PCR method to undertake the first compelling test of allele-specific expression in polyploid sugarcane. Seven alleles distinguished by this method all showed peak expression in the sucrose-loading zone of the stem, but there was apparent variability in expression patterns across other tissues. The ScLSG2 and ScLSG5 alleles appear promising for specificity of expression in stems, relative to leaf, meristem, emerging shoot and root tissues. Within the stem, there was activity in parenchyma, vascular and rind tissues. This expression pattern is of interest in basic research and biotechnology aimed at enhanced sucrose content, engineering value-added products, and manipulation of stem biomass composition.     

Virus-Induced Gene Silencing as a Tool for Comparative Functional Studies in Thalictrum

Perennial woodland herbs in the genus Thalictrum exhibit high diversity of floral morphology, including four breeding and two pollination systems. Their phylogenetic position, in the early-diverging eudicots, makes them especially suitable for exploring the evolution of floral traits and the fate of gene paralogs that may have shaped the radiation of the eudicots. A current limitation in evolution of plant development studies is the lack of genetic tools for conducting functional assays in key taxa spanning the angiosperm phylogeny. We first show that virus-induced gene silencing (VIGS) of a PHYTOENE DESATURASE ortholog (TdPDS) can be achieved in Thalictrum dioicum with an efficiency of 42% and a survival rate of 97%, using tobacco rattle virus (TRV) vectors. The photobleached leaf phenotype of silenced plants significantly correlates with the down-regulation of endogenous TdPDS (P<0.05), as compared to controls. Floral silencing of PDS was achieved in the faster flowering spring ephemeral T. thalictroides. In its close relative, T. clavatum, silencing of the floral MADS box gene AGAMOUS (AG) resulted in strong homeotic conversions of floral organs. In conclusion, we set forth our optimized protocol for VIGS by vacuum-infiltration of Thalictrum seedlings or dormant tubers as a reference for the research community. The three species reported here span the range of floral morphologies and pollination syndromes present in Thalictrum. The evidence presented on floral silencing of orthologs of the marker gene PDS and the floral homeotic gene AG will enable a comparative approach to the study of the evolution of flower development in this group.     

Gene silencing in potato: allelic differences and effect of ploidy

Silencing of genes is mostly studied in diploid, homozygous, self-fertile and sexually propagated species. However, conclusions drawn for these species are not always applicable to crops like potato, which is an autotetraploid, highly heterozygous, vegetatively propagated species. Factors influencing the level of silencing in potato are discussed, with emphasis on inhibition of the granule-bound starch synthase I (GBSSI) gene. Type of construct, number of integrated T-DNA copies, structural arrangement of the T-DNA locus, integration site, target tissue and genetic background are important factors for all plant species. Ploidy level and multiple allelism are factors deserving special attention when the efficiency of silencing of endogenous genes is studied in polyploid, heterozygous species such as potato.