Efficacy of an injectable toltrazuril - gleptoferron (Forceris®) to control coccidiosis (Cystoisospora suis) in comparison with iron supplemented piglets without anticoccidial treatment.

The efficacy and safety of an injectable combination product containing toltrazuril and gleptoferron (Forceris® - CEVA) for the control of coccidiosis due to Cystoisospora suis was investigated in neonatal piglets. The study was carried out on five European commercial pig farms in France, Germany and Spain and 122 litters consisting of 1508 piglets were selected and randomly allocated to one of two treatment groups. The first group received a single intramuscular injection per piglet of the test product, containing 45 mg toltrazuril and 200 mg iron and this was compared with a control group, which was administered a single intramuscular treatment of iron at 200 mg per piglet only. Body weights, faecal scores and oocysts counts, recorded as oocysts per gram of faeces, (OPG) were observed for 21 days. Only 1138 piglets were actually exposed to coccidiosis and data sets of these animals were selected for statistical analysis. The efficacy of the test product in the control of coccidiosis was shown in higher body weight gains, a lower percentage of animals with diarrhoea, fewer samples with positive oocysts counts as well as a lower excretion peak and a reduced area under the curve of OPG from study day (SD) 4 - SD 21. The combination product of toltrazuril and gleptoferron provided an effective alternative approach to current conventional separate treatment for the prevention of iron deficiency anaemia and coccidiosis in neonatal piglets. It reduced the numbers of potentially stressful interventions and work time.

Efficacy of an injectable toltrazuril - gleptoferron (Forceris®) to control coccidiosis (Cystoisospora suis) in comparison with iron supplemented piglets without anticoccidial treatment.

The efficacy and safety of an injectable combination product containing toltrazuril and gleptoferron (Forceris® - CEVA) for the control of coccidiosis due to Cystoisospora suis was investigated in neonatal piglets. The study was carried out on five European commercial pig farms in France, Germany and Spain and 122 litters consisting of 1508 piglets were selected and randomly allocated to one of two treatment groups. The first group received a single intramuscular injection per piglet of the test product, containing 45 mg toltrazuril and 200 mg iron and this was compared with a control group, which was administered a single intramuscular treatment of iron at 200 mg per piglet only. Body weights, faecal scores and oocysts counts, recorded as oocysts per gram of faeces, (OPG) were observed for 21 days. Only 1138 piglets were actually exposed to coccidiosis and data sets of these animals were selected for statistical analysis. The efficacy of the test product in the control of coccidiosis was shown in higher body weight gains, a lower percentage of animals with diarrhoea, fewer samples with positive oocysts counts as well as a lower excretion peak and a reduced area under the curve of OPG from study day (SD) 4 - SD 21. The combination product of toltrazuril and gleptoferron provided an effective alternative approach to current conventional separate treatment for the prevention of iron deficiency anaemia and coccidiosis in neonatal piglets. It reduced the numbers of potentially stressful interventions and work time.

Cloning of the Arabidopsis rwm1 gene for resistance to Watermelon mosaic virus points to a new function for natural virus resistance genes.

Arabidopsis thaliana represents a valuable and efficient model to understand mechanisms underlying plant susceptibility to viral diseases. Here, we describe the identification and molecular cloning of a new gene responsible for recessive resistance to several isolates of Watermelon mosaic virus (WMV, genus Potyvirus) in the Arabidopsis Cvi-0 accession. rwm1 acts at an early stage of infection by impairing viral accumulation in initially infected leaf tissues. Map-based cloning delimited rwm1 on chromosome 1 in a 114-kb region containing 30 annotated genes. Positional and functional candidate gene analysis suggested that rwm1 encodes cPGK2 (At1g56190), an evolutionary conserved nucleus-encoded chloroplast phosphoglycerate kinase with a key role in cell metabolism. Comparative sequence analysis indicates that a single amino acid substitution (S78G) in the N-terminal domain of cPGK2 is involved in rwm1-mediated resistance. This mutation may have functional consequences because it targets a highly conserved residue, affects a putative phosphorylation site and occurs within a predicted nuclear localization signal. Transgenic complementation in Arabidopsis together with virus-induced gene silencing in Nicotiana benthamiana confirmed that cPGK2 corresponds to rwm1 and that the protein is required for efficient WMV infection. This work uncovers new insight into natural plant resistance mechanisms that may provide interesting opportunities for the genetic control of plant virus diseases.

Family-based linkage and association mapping reveals novel genes affecting Plum pox virus infection in Arabidopsis thaliana.

Sharka is a devastating viral disease caused by the Plum pox virus (PPV) in stone fruit trees and few sources of resistance are known in its natural hosts. Since any knowledge gained from Arabidopsis on plant virus susceptibility factors is likely to be transferable to crop species, Arabidopsis's natural variation was searched for host factors essential for PPV infection. To locate regions of the genome associated with susceptibility to PPV, linkage analysis was performed on six biparental populations as well as on multiparental lines. To refine quantitative trait locus (QTL) mapping, a genome-wide association analysis was carried out using 147 Arabidopsis accessions. Evidence was found for linkage on chromosomes 1, 3 and 5 with restriction of PPV long-distance movement. The most relevant signals occurred within a region at the bottom of chromosome 3, which comprises seven RTM3-like TRAF domain-containing genes. Since the resistance mechanism analyzed here is recessive and the rtm3 knockout mutant is susceptible to PPV infection, it suggests that other gene(s) present in the small identified region encompassing RTM3 are necessary for PPV long-distance movement. In consequence, we report here the occurrence of host factor(s) that are indispensable for virus long-distance movement.

The 20S proteasome α5 subunit of Arabidopsis thaliana carries an RNase activity and interacts in planta with the lettuce mosaic potyvirus HcPro protein.

In plants, the ubiquitin/26S proteasome system (UPS) plays a central role in protein degradation and is involved in many steps of defence mechanisms, regardless of the types of pathogen targeted. In addition to its proteolytic activities, the UPS ribonuclease (RNase) activity, previously detected in 20S proteasome preparations from cauliflower and sunflower (Helianthus annuus), has been shown to specifically target plant viral RNAs in vitro. In this study, we show that recombinant Arabidopsis thaliana proteasomal α(5) subunit expressed in Escherichia coli harbours an RNase activity that degrades Tobacco mosaic virus (TMV, Tobamovirus)- and Lettuce mosaic virus (LMV, Potyvirus)-derived RNAs in vitro. The analysis of mutated forms of the α(5) subunit demonstrated that mutation of a glutamic acid at position 110 affects RNase activity. Furthermore, it was demonstrated, using a bimolecular fluorescence complement assay, that the multifunctional helper component proteinase (HcPro) of LMV, already known to interfere with the 20S proteasome RNase activity in vitro, can interact in vivo with the recombinant α(5) subunit. Further experiments demonstrated that, in LMV-infected lettuce cells, α(5) is partially relocalized to HcPro-containing infection-specific inclusions. Susceptibility analyses of Arabidopsis mutants, knocked out for each At-PAE gene encoding α(5) , showed that one (KO-pae1) of the two mutants exhibited a significantly increased susceptibility to LMV infection. Taken together, these results extend to A. thaliana α(5) the range of HcPro-interacting proteasomal subunits, and suggest that HcPro may modulate its associated RNase activity which may contribute to an antiviral response.

Single amino acid changes in the turnip mosaic virus viral genome-linked protein (VPg) confer virulence towards Arabidopsis thaliana mutants knocked out for eukaryotic initiation factors eIF(iso)4E and eIF(iso)4G.

Previous resistance analyses of Arabidopsis thaliana mutants knocked out for eukaryotic translation initiation factors showed that disruption of the At-eIF(iso)4E or both the At-eIF(iso)4G1 and At-eIF(iso)4G2 genes resulted in resistance against turnip mosaic virus (TuMV). This study selected TuMV virulent variants that overcame this resistance and showed that two independent mutations in the region coding for the viral genome-linked protein (VPg) were sufficient to restore TuMV virulence in At-eIF(iso)4E and At-eIF(iso)4G1xAt-eIF(iso)4G2 knockout plants. As a VPg-eIF(iso)4E interaction has been shown previously to be critical for TuMV infection, a systematic analysis of the interactions between A. thaliana eIF4Es and VPgs of virulent and avirulent TuMVs was performed. The results suggest that virulent TuMV variants may use an eIF4F-independent pathway.

Optimizing TILLING populations for reverse genetics in Medicago truncatula.

Medicago truncatula has been widely adopted as a model plant for crop legume species of the Vicieae. Despite the availability of transformation and regeneration protocols, there are currently limited tools available in this species for the systematic investigation of gene function. Within the framework of the European Grain Legumes Integrated Project (http://www.eugrainlegumes.org), chemical mutagenesis was applied to M. truncatula to create two mutant populations that were used to establish a TILLING (targeting induced local lesions in genomes) platform and a phenotypic database, allowing both reverse and forward genetics screens. Both populations had the same M2 line number, but differed in their M1 population size: population 1 was derived from a small M1 population (one-tenth the size of the M2 generation), whereas population 2 was generated by single seed descent and therefore has M1 and M2 generations of equal size. Fifty-six targets were screened, 10 on both populations, and 546 point mutations were identified. Population 2 had a mutation frequency of 1/485 kb, twice that of population 1. The strategy used to generate population 2 is more efficient than that used to generate population 1, with regard to mutagenesis density and mutation recovery. However, the design of population 1 allowed us to estimate the genetically effective cell number to be three in M. truncatula. Phenotyping data to help forward screenings are publicly available, as well as a web tool for ordering seeds at http://www.inra.fr/legumbase.

An investigation of carotenoid biosynthesis in Coffea canephora and Coffea arabica.

Carotenoids are essential components of the photosynthetic apparatus in a wide range of organisms. They participate in the adaptation of plastids to changing environmental light conditions and prevent photo-oxidative damage of the photosynthetic apparatus by detoxifying reactive oxygen species. We identified eight cDNAs from the carotenoid biosynthetic pathway (PSY, PDS, ZDS, PTOX, LCY-E, CRTR-B, ZEP and VDE) and two cDNA encoding carotenoid cleavage dioxygenase family members (NCED3 and CCD1) in Coffea canephora. We also obtained cDNA encoding several different fibrillin proteins involved in carotenoid sequestration (FIB). Expression of the coffee carotenoid genes was determined in leaf, branch and flower tissues using quantitative RT-PCR. Expression analysis of these genes in leaf tissue from osmotically stressed plants was also carried out. These experiments showed that the transcript levels of PTOX, CRTR-B, NCED3, CCD1 and FIB1 increased under these stress conditions, while LCY-E decreased, indicating that the metabolic flux towards the xanthophyll cycle branch of the carotenoid biosynthetic pathway may be favoured in leaves under drought conditions. Functional analysis of CcCRTR-B using an in vivo method employing Escherichia coli strains engineered to make carotenoids confirmed that the beta-carotene hydroxylase activity of CcCRTR-B generates beta-cryptoxanthin and zeaxanthin from beta-carotene. A similar approach was also used to show that CcCCD1 encoded a functional 9,10(9'10') carotenoid cleavage dioxygenase, and thus that this enzyme is capable of forming one or more apocarotenoids in vivo. Finally, high-performance liquid chromatography analysis of coffee leaves revealed the presence of alpha-carotene and suggests that Coffea arabica may have higher levels of alpha-carotene than C. canephora.

Characterization of genetically modified maize in weakly contaminated seed batches and identification of the origin of the adventitious contamination.

So far, relatively few genetically modified plants (GMPs) have been planted in the European Union (EU). However, in France, seed batches weakly contaminated by unidentified GM materials have recently been detected among commercial maize seeds (14 seed batches positive out of 447 analyzed). We have developed a 3-step approach to precisely identify the genetic modifications detected in such maize seed batches. First, to isolate GMPs derived from the contaminated seed batches, 10 000 maize seeds of each batch were planted and screened by polymerase chain reaction (PCR) on 100-plant batches, then on 10-plant subbatches, and finally, plant by plant. In a second step, specific identification of the individual GMPs was performed. Finally, to determine the origin of the contamination, each individual GMP was analyzed by simple sequence repeat (SSR) markers. The results showed that all batches were contaminated by few GM seeds, having a GM content < 0.1%. Finally, 12 individual GMPs have been isolated from 17 plant pools that were tested positive either for P35-S and/or T-Nos. MON810 and T25 transformation events approved for cultivation in the EU were detected in 7 individual GMPs. The other seed batches were contaminated by genetically modified organisms (GMOs) that are not approved in the EU, including GA21 or the stacking MON810/T25. Presumable identification of T14 was also achieved following sequencing of 1 individual GMP. The data also showed that most of the seed batches were contaminated by several transformation events. Finally, analysis of SSR markers indicated that the contaminations were essentially due to cross-pollination in the seed production process.