RNA binding properties of the US11 protein from four primate simplexviruses

<p>Abstract</p> <p>Background</p> <p>The protein encoded by the Us11 gene of herpes simplex viruses is a dsRNA binding protein which inhibits protein kinase R activity, thereby preventing the interferon-induced shut down of protein synthesis following viral infection. Us11 protein is not essential for infectivity <it>in vitro </it>and in mice in herpes simplex virus type 1 (HSV1), however this virus has a second, and apparently more important, inhibitor of PKR activity, the γ₁34.5 protein. Recently sequenced simian simplexviruses SA8, HVP2 and B virus do not have an ORF corresponding to the γ₁34.5 protein, yet they have similar, or greater, infectivity as HSV1 and HSV2.</p> <p>Methods</p> <p>We have expressed the US11 proteins of the simplexviruses HSV1, HSV2, HVP2 and B virus and measured their abilities to bind dsRNA, in order to investigate possible differences that could complement the absence of the γ₁34.5 protein. We employed a filter binding technique that allows binding of the Us11 protein under condition of excess dsRNA substrate and therefore a measurement of the true Kd value of Us11-dsRNA binding.</p> <p>Results and Conclusions</p> <p>The results show a Kd of binding in the range of 0.89 nM to 1.82 nM, with no significant difference among the four Us11 proteins.</p

A new genome allows the identification of genes associated with natural variation in aluminium tolerance in Brachiaria grasses

Toxic concentrations of aluminium cations and low phosphorus availability are the main yield-limiting factors in acidic soils, which represent half of the potentially available arable land. Brachiaria grasses, which are commonly sown as forage in the tropics because of their resilience and low demand for nutrients, show greater tolerance to high concentrations of aluminium cations (Al3+) than most other grass crops. In this work, we explored the natural variation in tolerance to Al3+ between high and low tolerant Brachiaria species and characterized their transcriptional differences during stress. We identified three QTLs (quantitative trait loci) associated with root vigour during Al3+ stress in their hybrid progeny. By integrating these results with a new Brachiaria reference genome, we identified 30 genes putatively responsible for Al3+ tolerance in Brachiaria. We observed differential expression during stress of genes involved in RNA translation, response signalling, cell wall composition, and vesicle location homologous to aluminiuminduced proteins involved in limiting uptake or localizing the toxin. However, there was limited regulation of malate transporters in Brachiaria, which suggests that exudation of organic acids and other external tolerance mechanisms, common in other grasses, might not be relevant in Brachiaria. The contrasting regulation of RNA translation and response signalling suggests that response timing is critical in high Al3+-tolerant Brachiaria

Overexpression of Arabidopsis FLOWERING LOCUS T (FT) gene improves floral development in cassava (Manihot esculenta, Crantz)

Cassava is a tropical storage-root crop that serves as a worldwide source of staple food for over 800 million people. Flowering is one of the most important breeding challenges in cassava because in most lines flowering is late and non-synchronized, and flower production is sparse. The FLOWERING LOCUS T (FT) gene is pivotal for floral induction in all examined angiosperms. The objective of the current work was to determine the potential roles of the FT signaling system in cassava. The Arabidopsis thaliana FT gene (atFT) was transformed into the cassava cultivar 60444 through Agrobacterium-mediated transformation and was found to be overexpressed constitutively. FT overexpression hastened flower initiation and associated fork-type branching, indicating that cassava has the necessary signaling factors to interact with and respond to the atFT gene product. In addition, overexpression stimulated lateral branching, increased the prolificacy of flower production and extended the longevity of flower development. While FT homologs in some plant species stimulate development of vegetative storage organs, atFT inhibited storage-root development and decreased root harvest index in cassava. These findings collectively contribute to our understanding of flower development in cassava and have the potential for applications in breeding

Identification and characterization of the Cassava core-clock gene Early Flowering 4

The angiosperm circadian clock has been well established from molecular-genetic studies in a temperate plant model. Conservation of clock function is less explored in plants from the tropics. Cassava (Manihot esculenta) is a staple crop grown in the tropics that has been of limited research interest, and more generally, research on photoperiod and clock genes has been sparse. EARLY FLOWERING 4 (AtELF4) of the temperate plant Arabidopsis thaliana (Arabidopsis) has been reported to be required for photoperiod perception and circadian function. Here, we describe our start to identify circadian and photoperiod genes in cassava with an account on the characterization of its ELF4 gene (MeELF4). After isolating MeELF4, a phylogenetic study was conducted and it was found to cluster within the ELF4 subclade of the ELF4/EFL super-family. Similar to studies in temperate plants, MeELF4 was shown to be an evening-expressed gene in cassava. This collectively suggested to us that MeELF4 could be a functional ortholog of AtELF4. To test this, complementation studies of MeELF4 were performed in the Arabidopsis elf4 mutant. Hypocotyl-length measurements and flowering-time analysis were performed. MeELF4-complementation transgenics in the elf4 background were restored to the wild-type growth habit, suggesting a total rescue of photoperiodic perception. To expand on the molecular role of MeELF4 in the resulting transgenic-complementation lines, the CCA1 and CCR2 promoter-luciferase markers where respectively introduced and bioluminescence-imaging experiments revealed a restoration of circadian-regulated gene expression. The collective results showed that the cassava gene MeELF4 is a functional clock ortholog of AtELF4

Identification of Cassava MicroRNAs under Abiotic Stress

The study of microRNAs (miRNAs) in plants has gained significant attention in recent years due to their regulatory role during development and in response to biotic and abiotic stresses. Although cassava (Manihot esculenta Crantz) is tolerant to drought and other adverse conditions, most cassava miRNAs have been predicted using bioinformatics alone or through sequencing of plants challenged by biotic stress. Here, we use high-throughput sequencing and different bioinformatics methods to identify potential cassava miRNAs expressed in different tissues subject to heat and drought conditions. We identified 60 miRNAs conserved in other plant species and 821 potential cassava-specific miRNAs. We also predicted 134 and 1002 potential target genes for these two sets of sequences. Using real time PCR, we verified the condition-specific expression of 5 cassava small RNAs relative to a non-stress control. We also found, using publicly available expression data, a significantly lower expression of the predicted target genes of conserved and nonconserved miRNAs under drought stress compared to other cassava genes. Gene Ontology enrichment analysis along with condition specific expression of predicted miRNA targets, allowed us to identify several interesting miRNAs which may play a role in stress-induced posttranscriptional regulation in cassava and other plants

Flowering traits in non‐transformed wildtype line (60444) and in the four independent transformants.

<p>(a) Flowering time in days from establishment in soil to flowering at the 1st, 2nd, and 3rd tier of flowering, as defined by fork-type branching at the apical meristems. (b) Number of shoot nodes to forking events where inflorescences develop. The number of nodes between the soil surface and the first fork, between the first-tier and second-tier forks, and between the second- and third-tier forks. (c) Number of flowers per tier, per plant. (d) Time to start of floral and/or inflorescence senescence. Floral traits were recorded weekly to determine the date of inflorescence appearance, and initial date of floral senescence. The total number of days from flower appearance to start of inflorescence and/or flower senescence was calculated from these weekly records. Shown are the means ± SEM.</p

Schematic representation of the transformation vector.

<p><i>Arabidopsis FT</i> cDNA was inserted into the construct through Gateway cloning. pAnos, nopaline synthase polyadenylation signal; pat, phosphinothricin acetyltransferase; Tnos, terminator of nopaline synthase; pAlcA, promoter of alcohol dehydrogenase I (Adh-I) encoded by the <i>alc</i>A gene; <i>FT</i> cDNA, cDNA of Flowering Locus (FT) gene; pA35S, polyadenylation sequence of Cauliflower mosaic virus 35S gene; nos, nopaline synthase terminator; ALCR, transcriptional factor which binds to <i>AlcA promoter</i>; p35S, Cauliflower Mosaic Virus 35S promoter; LB, left border; RB, right border.</p