How the fas locus contributes to Rhodococcus fascians cytokinin production: an in-depth molecular and biochemical analysis

The fine-tuned balance of plant regulators plays a key role in growth and development of plants. Many plant-associated bacteria can influence their hosts either by modulating phytohormone production or by producing phytohormones themselves. The Actinomycete Rhodococcus fascians provokes the formation of differentiated leafy galls consisting of numerous shoot primordia that are inhibited in further outgrowth. Based on the shooty phenotype and the presence of an ipt gene on the linear virulence plasmid of R. fascians D188, the role for cytokinins in the pathology had been anticipated for a long time. Subsequent studies identified and characterized the fas operon as a key genetic determinant of virulence and likely cytokinin biosynthesis. Nevertheless, many aspects concerning regulation of fas gene expression, Fas protein function, and, importantly, the encoded cytokinin biosynthetic pathway and the identity of the produced morphogens remained to be uncovered. Therefore, the main objectives of this research were to identify the bacterial cytokinins responsible for the R. fascians pathology, to unravel how they exerted their function, and to elucidate the role of the fas locus and its expression in their production. In conclusion, our data have largely uncovered the role of cytokinins and the fas locus in the R. fascians pathology: the continuous challenge with defined ratios of synergistically acting cytokinins eventually defeats nearly all plants and transforms them into shooty niches. Many intriguing questions derived from the novel insights obtained during this work remain to be answered. Nevertheless, we feel that the results presented here have shed some light on the remaining secrets of this fascinating pathogen

RNA interference : a promising biopesticide strategy against the African sweetpotato weevil Cylas brunneus

The African sweetpotato weevil Cylas brunneus is one of the most devastating pests affecting the production of sweetpotatoes, an important staple food in Sub-Saharan Africa. Current available control methods against this coleopteran pest are limited. In this study, we analyzed the potential of RNA interference as a novel crop protection strategy against this insect pest. First, the C. brunneus transcriptome was sequenced and RNAi functionality was confirmed by successfully silencing the laccase2 gene. Next, 24 potential target genes were chosen, based on their critical role in vital biological processes. A first screening via injection of gene-specific dsRNAs showed that the dsRNAs were highly toxic for C. brunneus. Injected doses of 200ng/mg body weight led to mortality rates of 90% or higher for 14 of the 24 tested genes after 14 days. The three best performing dsRNAs, targeting pros alpha 2, rps13 and the homolog of Diabrotica virgifera snf7, were then used in further feeding trials to investigate RNAi by oral delivery. Different concentrations of dsRNAs mixed with artificial diet were tested and concentrations as low as 1 mu g dsRNA/mL diet led to significant mortality rates higher than 50%. These results proved that dsRNAs targeting essential genes show great potential to control C. brunneus

Pathogenicity strategies in the gall-forming bacterium Rhodococcus fascians

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Analog electronic teaching – a new approach for bipolar transistors

a) This work was based in a single individual sample; b) Learning Support Means (LSM) received poor acceptance; c) Other Alternative Support Means are under development; d) For these, a broader test sample was used; e) Preliminary results from this test are encouraging to further pursue this workN/

Transcriptome analysis and systemic RNAi response in the African sweetpotato weevil (Cylas puncticollis, Coleoptera, Brentidae

<div><p>The African sweetpotato weevil (SPW) <i>Cylas puncticollis</i> Boheman is one of the most important constraints of sweetpotato production in Sub-Saharan Africa and yet is largely an uncharacterized insect pest. Here, we report on the transcriptome analysis of SPW generated using an Illumina platform. More than 213 million sequencing reads were obtained and assembled into 89,599 contigs. This assembly was followed by a gene ontology annotation. Subsequently, a transcriptome search showed that the necessary RNAi components relevant to the three major RNAi pathways, were found to be expressed in SPW. To address the functionality of the RNAi mechanism in this species, dsRNA was injected into second instar larvae targeting <i>laccase2</i>, a gene which encodes an enzyme involved in the sclerotization of insect exoskeleton. The body of treated insects showed inhibition of sclerotization, leading eventually to death. Quantitative Real Time PCR (qPCR) confirmed this phenotype to be the result of gene silencing. Together, our results provide valuable sequence data on this important insect pest and demonstrate that a functional RNAi pathway with a strong and systemic effect is present in SPW and can further be explored as a new strategy for controlling this important pest.</p></div