Ine Pertry

pFiD188, the Linear Virulence Plasmid of Rhodococcus fascians D188

Rhodococcus fascians is currently the only phytopathogen of which the virulence genes occur on a linear plasmid. To get insight into the origin of this replicon and into the virulence strategy of this broad-spectrum phytopathogen, the sequence of the linear plasmid of strain D188, pFiD188, was determined. Analysis of the 198,917 bp revealed four syntenic regions with linear plasmids of R. erythropolis, R. jostii, and R. opacus, suggesting a common origin of these replicons. Mutational analysis of pFi_086 and pFi_102, similar to cutinases and type IV peptidases, respectively, showed that conserved region R2 was involved in plasmid dispersal and pointed toward a novel function for actinobacterial cutinases in conjugation. Additionally, pFiD188 had three regions that were unique for R. fascians. Functional analysis of the stk and nrp loci of regions U2 and U3, respectively, indicated that their role in symptom development was limited compared with that of the previously identified fas, att, and hyp virulence loci situated in region U1. Thus, pFiD188 is a typical rhodococcal linear plasmid with a composite structure that encodes core functions involved in plasmid maintenance and accessory functions, some possibly acquired through horizontal gene transfer, implicated in virulence and the interaction with the host.

Transcriptome Analysis and Systemic RNAi Response in the African Sweetpotato Weevil (Cylas puncticollis, Coleoptera, Brentidae)

The African sweetpotato weevil (SPW) Cylas puncticollis 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 laccase2, 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.

RNAi-based gene silencing through dsRNA injection or ingestion against the African sweet potato weevil Cylas puncticollis (Coleoptera: Brentidae)

BACKGROUND RNA interference (RNAi) technology can potentially serve as a suitable strategy to control the African sweet potato weevil Cylas puncticollis (SPW), which is a critical pest in sub-Saharan Africa. Important prerequisites are required to use RNAi in pest control, such as the presence of an efficient RNAi response and the identification of suitable target genes. RESULTS Here we evaluated the toxicity of dsRNAs targeting essential genes by injection and oral feeding in SPW. In injection assays, 12 of 24 dsRNAs were as toxic as the one targeting Snf7, a gene used commercially against Diabrotica virgifera virgifera. Three dsRNAs with high insecticidal activity were then chosen for oral feeding experiments. The data confirmed that oral delivery can elicit a significant toxicity, albeit lower compared with injection. Subsequently, ex vivo assays revealed that dsRNA is affected by degradation in the SPW digestive system, possibly explaining the lower RNAi effect by oral ingestion. CONCLUSION We conclude that the full potential of RNAi in SPW is affected by the presence of nucleases. Therefore, for future application in crop protection, it is necessary constantly to provide new dsRNA and/or protect it against possible degradation in order to obtain a higher RNAi efficacy.

Biosafety capacity building: experiences and challenges from a distance learning approach

Biotechnology is revolutionizing industrial and agricultural practice as the number of commercial biotechnology products is increasing each year. Simultaneously, several regulatory approaches are put into place to allow technological advancement while preserving public health and the environment. Developing and/or emerging countries often face major barriers to access biotechnologies and biotechnology derived products as they frequently lack the institutional capacities and professional competence in exercising regulatory oversight. To address this need, intensive biosafety capacity building is required. Different training approaches can be used to train individuals in biosafety ranging from long-term leading to a postgraduate certificate or a Masters degree, to short term courses. In this paper, we discuss the applicability of a different approach to biosafety capacity building based on a distance e-learning system, the UNIDO e-Biosafety program that has been annually organized at the Marche Polytechnic University (MPU) in Italy and Ghent University (UGent) in Belgium since 2006. Even though there are some challenges, we can conclude based on our experience that distance learning in combination with on-campus tuition is amendable for biosafety capacity building.

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 prosα2, 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 μg 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.

DTREEv2, a computer-based support system for the risk assessment of genetically modified plants

Risk assessment of genetically modified organisms (GMOs) remains a contentious area and a major factor influencing the adoption of agricultural biotech. Methodologically, in many countries, risk assessment is conducted by expert committees with little or no recourse to databases and expert systems that can facilitate the risk assessment process. In this paper we describe DTREEv2, a computer-based decision support system for the identification of hazards related to the introduction of GM-crops into the environment. DTREEv2 structures hazard identification and evaluation by means of an Event-Tree type of analysis. The system produces an output flagging identified hazards and potential risks. It is intended to be used for the preparation and evaluation of biosafety dossiers and, as such, its usefulness extends to researchers, risk assessors and regulators in government and industry.

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.