Roles of Dicer-Like Proteins 2 and 4 in Intra- and Intercellular Antiviral Silencing

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

The Roles of the Cation Transporters CHX21 and CHX23 in the Development of Arabidopsis Thaliana

The Arabidopsis thaliana genome encodes a family of 28 proteins whose members have been associated with the transport of monovalent cations across membranes. Experiments have been performed to elucidate the biochemical function and the role in plant development of two closely related members of this CHX family. A genotype carrying a knockout of the AtCHX23 gene (At1g05580) showed no phenotype when grown in glasshouse conditions. In particular, it did not exhibit the reduced root growth phenotype observed for a knockout of its homologue AtCHX21 when exposed to elevated sodium concentration. However, it was not possible to produce plants that were homozygous knockout for both AtCHX21 and AtCHX23. Reverse transcription-PCR (RT-PCR) experiments revealed that both genes are highly expressed in flower buds, flowers, and pollen. However, examination of pollen grain viability and pollen tube growth through excised styles did not reveal a phenotypic difference between the chx21–chx23– condition and other haplotypes. Crosses between selected mutants and wild-type plants in which the chx21–chx23– haplotype was produced by either the male or female parent demonstrated unequivocally that the chx21–chx23– haplotype could not pass through the female line. This suggests that the genes share a critical function in the development and/or function of the female gametophyte and that this function cannot be provided by other members of the AtCHX gene family. Experiments were carried out using the heterologous expression of AtCHX23 in Saccharomyces cerevisiae genotypes carrying combinations of deletions of genes involved in the transport of sodium or potassium across membranes. The results show that CHX23 would only complement the poor colony growth phenotype associated with the deletion of the yeast gene kha1. The conclusion is that both AtCHX21 and AtCHX23 act in potassium homeostasis within the female gametophyte and this is discussed in terms of the diversification of gene sequence and function within the CHX gene family

Wildflower strips enhance pest regulation services in citrus orchards

Contemporary approaches to agriculture must be reimaged to include ecological techniques that maximise ecosystem services, so that food can be produced sustainably whilst simultaneously meeting yield demands. Pest regulation services, harnessed through the conservation of natural enemies in the agri-environment are an economically important service degraded by conventional citrus production practices. For the first time, a sown wildflower strip composed of native forbs and tussock-forming grasses has been investigated for its influence on natural enemies and their pest regulation services in citrus orchards. A novel management strategy was applied, using the predicted generation times of Aonidiella aurantii Maskell (Hemiptera: Diaspididae), a key pest in citrus, to determine whether cutting the wildflower strips could force spill-over of natural enemies onto the adjacent crop, enhancing pest regulation services. Three treatments applied to orange orchard alleyways were compared: i) a control treatment, the standard orchard practice of regular cutting to 5 cm throughout the year, ii) a sown wildflower treatment managed with cutting once a year in February to a height of 10 cm (standard management wildflower treatment, SMWT), and iii) the same sown wildflower treatment but managed with two additional cuts in May and June (active management wildflower treatment, AMWT). Orange tree canopies were sampled for natural enemies, and pest regulation services were quantified using sentinel prey cards baited with Ephestia kuehniella eggs. Natural enemy richness was greatest in canopies with SMWT, supporting a greater relative abundance of primary parasitoids and lower relative abundances of antagonists (ants) compared to the control. This was associated with enhanced pest regulation services (depletion of sentinel prey from baited cards), especially during the early summer months, which coincides with a critical period to control A. aurantii and other key citrus pests. In contrast, AMWT did not enhance natural enemy richness, and pest regulation services were diminished. This study suggests that leaving wildflower strips uncut throughout the season, as in SMWT, may help to mitigate pest incidence through enhanced pest regulation services. Further studies are now required to determine how this would influence populations of target pests