Virus-induced gene complementation reveals a transcription factor network in modulation of tomato fruit ripening

Plant virus technology, in particular virus-induced gene silencing, is a widely used reverse- and forward-genetics tool in plant functional genomics. However the potential of virus technology to express genes to induce phenotypes or to complement mutants in order to understand the function of plant genes is not well documented. Here we exploit Potato virus X as a tool for virus-induced gene complementation (VIGC). Using VIGC in tomato, we demonstrated that ectopic viral expression of LeMADS-RIN, which encodes a MADS-box transcription factor (TF), resulted in functional complementation of the non-ripening rin mutant phenotype and caused fruits to ripen. Comparative gene expression analysis indicated that LeMADS-RIN up-regulated expression of the SBP-box (SQUAMOSA promoter binding protein-like) gene LeSPL-CNR, but down-regulated the expression of LeHB-1, an HD-Zip homeobox TF gene. Our data support the hypothesis that a transcriptional network may exist among key TFs in the modulation of fruit ripening in tomato

Foxtail Mosaic Virus-induced Flowering Assays in Monocot Crops

Virus-induced flowering (VIF) exploits RNA or DNA viruses to express flowering time genes to induce flowering in plants. Such plant virus-based tools have recently attracted widespread attention for their fundamental and applied uses in flowering physiology and in accelerating breeding in dicotyledonous crops and woody fruit-trees. We now extend this technology to a monocot grass and a cereal crop. Using the Foxtail mosaic virus-based VIF system, dubbed FoMViF, we showed that expression of florigenic Flowering Locus T (FT) genes can promote early flowering and spikelet development in proso millet, a C4 grass species with potential for nutritional food and biofuel resources, and in non-vermalized C3 wheat, a major food crop worldwide. Floral and spikelet/grain induction in the two monocot plants was caused by the virally expressed untagged or FLAG-tagged FT orthologues, and the florigenic activity of rice Hd3a was more pronounced than its dicotyledonous counterparts in proso millet. The FoMViF system is easy to perform and its efficacy to induce flowering and early spikelet/grain production is high. In addition to proso millet and wheat, we envisage that FoMViF will be also applicable to many economically important monocotyledonous food and biofuel crops

Influence of retinoblastoma-related gene silencing on the initiation of DNA replication by African cassava mosaic virus Rep in cells of mature leaves in Nicotiana benthamiana plants

Background Geminiviruses mainly infect terminally differentiated tissues and cells in plants. They need to reprogramme host cellular machinery for DNA replication. This process is thought to be mediated by inactivation of cell-cycle repressor proteins and by induction of host DNA synthesis protein expression through actions of the geminviral replication initiator protein (Rep). Findings Exploiting a Nicotiana benthamiana pOri2 line, which is transformed with a transgene consisting of a direct repeat of the African cassava mosaic virus (ACMV)-replication origin (Ori) flanking a non-viral DNA region, and virus-induced RNA silencing (VIGS), the impact of host gene expression on replication of the ACMV-derived replicon was investigated. The ACMV Rep trans-replicated the viral episomal replicon in leaves of young but not older pOri2 plants. Upon VIGS-mediated down-regulation of N. benthamiana NbRBR1, the retinoblastoma-related protein gene coding for a negative cell-cycle suppressor, recovered the ability of ACMV Rep for trans DNA replication, whereas the silencing of NbPCNA coding for the sliding clamp of DNA polymerase had no effect. Conclusions These results suggest that the cellular machinery for DNA replication in differentiated tissues of older leaves cannot be reprogrammed by Rep alone but may need other uncharacterised viral and plant factors

Mobile FT mRNA contributes to the systemic florigen signalling in floral induction

In inducing photoperiodic conditions, plants produce a signal dubbed "florigen" in leaves. Florigen moves through the phloem to the shoot apical meristem (SAM) where it induces flowering. In Arabidopsis, the FLOWERING LOCUS T (FT) protein acts as a component of this phloem-mobile signal. However whether the transportable FT mRNA also contributes to systemic florigen signaling remains to be elucidated. Using non-conventional approaches that exploit virus-induced RNA silencing and meristem exclusion of virus infection, we demonstrated that the Arabidopsis FT mRNA, independent of the FT protein, can move into the SAM. Viral ectopic expression of a non-translatable FT mRNA promoted earlier flowering in the short-day (SD) Nicotiana tabacum Maryland Mammoth tobacco in SD. These data suggest a possible role for FT mRNA in systemic floral signalling, and also demonstrate that cis-transportation of cellular mRNA into SAM and meristem exclusion of pathogenic RNAs are two mechanistically distinct processes

Tuning LeSPL-CNR expression by SlymiR157 affects tomato fruit ripening

In plants, microRNAs (miRNAs) play essential roles in growth, development, yield, stress response and interactions with pathogens. However no miRNA has been experimentally documented to be functionally involved in fruit ripening although many miRNAs have been profiled in fruits. Here we show that SlymiR157 and SlymiR156 differentially modulate ripening and softening in tomato (Solanum lycopersicum). SlymiR157 is expressed and developmentally regulated in normal tomato fruits and in those of the Colourless non-ripening (Cnr) epimutant. It regulates expression of the key ripening gene LeSPL-CNR in a likely dose-dependent manner through miRNA-induced mRNA degradation and translation repression. Viral delivery of either pre-SlymiR157 or mature SlymiR157 results in delayed ripening. Furthermore, qRT-PCR profiling of key ripening regulatory genes indicates that the SlymiR157-target LeSPL-CNR may affect expression of LeMADS-RIN, LeHB1, SlAP2a and SlTAGL1. However SlymiR156 does not affect the onset of ripening, but it impacts fruit softening after the red ripe stage. Our findings reveal that working together with a ripening network of transcription factors, SlymiR157 and SlymiR156 form a critical additional layer of regulatory control over the fruit ripening process in tomato