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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Off-Patent Transgenic Events: Challenges and Opportunities for New Actors and Markets in Agriculture

More than 20 years ago, the first genetically modified (GM) plants entered the seed market. The patents covering the first GM plants have begun to expire and these can now be considered as Off-Patent Events. Here we describe the challenges that will be faced by a Secondary Party by further use and development of these Off-Patent Events. Indeed, the conditions for Off-Patent Events are not available yet to form the basis for a new viable industry similar to the generic manufacturers of agrochemicals or pharmaceutical products, primarily because of (i) unharmonized global regulatory requirements for GM organisms, (ii) inaccessibility of regulatory submissions and data, and (iii) potential difficulties to obtain seeds and genetic material of the unique genotypes used to generate regulatory data. We propose certain adaptations by comparing what has been done in the agrochemical and pharmaceutical markets to facilitate the development of generics. Finally, we present opportunities that still exist for further development of Off-Patent Events in collaboration with Proprietary Regulatory Property Holders in emerging markets, provided (i) various countries approve these events without additional regulatory burdens (i.e., acceptance of the concept of data transportability), and (ii) local breeders agree to meet product stewardship requirements

The leafy gall syndrome induced by Rhodococcus fascians

The Actinomycete Rhodococcus fascians causes the leafy gall syndrome, an infectious plant disease that affects a wide range of plants, primarily dicotyledonous herbs. The syndrome is associated with delayed senescence, loss of apical dominance, activation of dormant axillary meristems, and formation of multiple inflorescences, leading to a stunted and bushy plant appearance. A major breakthrough in the elucidation of the virulence strategy of this pathogen was the discovery of a linear virulence plasmid, pFiD188 for R.fascians strain D188. Upon perception of a compatible host plant, an autoregulatory mechanism mediated by the att operon directs a switch in the bacterial life style from a harmless epiphyte into a pathogenic endophyte and, concomitantly, activates gene expression of the fas operon that encodes a cytokinin biosynthesis pathway. A mixture of five cytokinins determines the cytokinin activity of R.fascians that directly affects plant responses and development. Moreover, the bacterial cytokinins stimulate the host to produce auxins and polyamines, that function as accessory signals to aid in symptom development. The plant reacts against the developmental hijacking by R.fascians by activating a set of counteracting measures that ultimately results in a delicate balance, allowing a long-lasting biotrophic interaction