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 signalling 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.

Naturally-occurring epimutants are rare and have mainly been described in plants. However how these mutants maintain their epigenetic marks and how they are inherited remain unknown. Here we report that CHROMOMETHYLASE3 (SlCMT3) and other methyltransferases are required for maintenance of a spontaneous epimutation and its cognate Colourless non-ripening (Cnr) phenotype in tomato. We screened a series of DNA methylation-related genes that could rescue the hypermethylated Cnr mutant. Silencing of the developmentally-regulated SlCMT3 gene results in increased expression of LeSPL-CNR, the gene encodes the SBP-box transcription factor residing at the Cnr locus and triggers Cnr fruits to ripen normally. Expression of other key ripening-genes was also up-regulated. Targeted and whole-genome bisulfite sequencing showed that the induced ripening of Cnr fruits is associated with reduction of methylation at CHG sites in a 286-bp region of the LeSPL-CNR promoter, and a decrease of DNA methylation in differentially-methylated regions associated with the LeMADS-RIN binding sites. Our results indicate that there is likely a concerted effect of different methyltransferases at the Cnr locus and the plant-specific SlCMT3 is essential for sustaining Cnr epi-allele. Maintenance of DNA methylation dynamics is critical for the somatic stability of Cnr epimutation and for the inheritance of tomato non-ripening phenotype.

No abstract

RNA silencing is an innate antiviral mechanism conserved in organisms across kingdoms. Such a cellular defense involves DICER or DICER-LIKEs (DCLs) that process plant virus RNAs into viral small interfering RNAs (vsiRNAs). Plants encode four DCLs that play diverse roles in cell-autonomous intracellular virus-induced RNA silencing (known as VIGS) against viral invasion. VIGS can spread between cells. However, the genetic basis and involvement of vsiRNAs in non-cell-autonomous intercellular VIGS remains poorly understood. Using GFP as a reporter gene together with a suite of DCL RNAi transgenic lines, here we show that despite the well-established activities of DCLs in intracellular VIGS and vsiRNA biogenesis, DCL4 acts to inhibit intercellular VIGS whereas DCL2 is required (likely along with DCL2-processed/dependent vsiRNAs and their precursor RNAs) for efficient intercellular VIGS trafficking from epidermal to adjacent cells. DCL4 imposed an epistatic effect on DCL2 to impede cell-to-cell spread of VIGS. Our results reveal previously unknown functions for DCL2 and DCL4 that may form a dual defensive frontline for intra-and intercellular silencing to double-protect cells from virus infection in Nicotiana benthamiana.

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.

No abstract

The genome difference(s) that enable the European pathotype 2 isolates of Barley yellow mosaic virus (BaYMV-2) to infect barley genotypes with the rym4 resistance gene were investigated. Stable deletions of different sizes occurred in RNA2 of laboratory isolates of the common pathotype (BaYMV-1) and BaYMV-2. After mechanical inoculation of susceptible or rym4 genotypes with a mixture of both isolates, immunocapture-RT-PCR with RNA2-specific primers flanking stable deletion regions was used to detect and distinguish the two pathotypes. Individual leaves contained RNA2 of either or both isolates, showing that RNA2 of BaYMV-1 can replicate and move systemically in rym4 plants when co-inoculated with BaYMV-2. In contrast, sequences of RNA1-specific RT-PCR fragments showed that in resistant plants these were always BaYMV-2, suggesting that the pathogenicity determinant was on RNA1. The complete ORFs of RNA1 of three BaYMV-1 and four BaYMV-2 isolates from the UK and Germany were sequenced, and the RNA2 sequences of one BaYMV-1 and two BaYMV-2 isolates from the UK were also determined. All sequences were very similar to one another and to the published German BaYMV-1 isolate. The only consistent amino acid difference between the BaYMV-1 and BaYMV-2 isolates was in the RNA1-encoded polyproteins and this was confirmed by sequencing the relevant region of eight further German isolates. All BaYMV-1 isolates had lysine at aa 1307, whereas BaYMV-2 isolates had asparagine (or, in one isolate, histidine). The polymorphism occurred in the central region of VPg, which has been shown to be required for pathogenicity on genotypes carrying recessive resistance genes in several potyvirus/dicotyledonous plant pathosystems.

Spring orchid (Cymbidium goeringii) is a popular flowering plant species. There have been few molecular studies of the genetic diversity and conservation genetics on this species. An assessment of the level of genetic diversity in cultivated spring orchid would facilitate development of the future germplasm conservation for cultivar improvement. In the present study, DNA markers of intersimple sequence repeats (ISSR) were identified and the ISSR fingerprinting technique was used to evaluate genetic diversity in C. goeringii cultivars. Twenty-five ISSR primers were selected to produce a total of 224 ISSR loci for evaluation of the genetic diversity. A wide genetic variation was found in the 50 tested cultivars with Nei's gene diversity (H = 0.2241) and 93.75% of polymorphic loci. Fifty cultivars were unequivocally distinguished based on ISSR fingerprinting. Cultivar-specific ISSR markers were identified in seven of 50 tested cultivars. Unweighted pair-group mean analysis (UPGMA) and principal coordinates analysis (PCA) grouped them into two clusters: one composed the cultivars mainly from Japan, and the other contained three major subclusters mainly from China. Two Chinese subclusters were generally consistent with horticultural classification, and the third Chinese subcluster contained cultivars from various horticultural groups. Our results suggest that the ISSR technique provides a powerful tool for cultivar identification and establishment of genetic relationships of cultivars in C. goeringii.