Detection of phloem restricted bacteria responsible for strawberry marginal chlorosis (SMC) by real-time PCR in a single assay

Two uncultured phloem restricted plant pathogens, the γ3 proteobacterium «Candidatus Phlomobacter fragariae » and the stolbur phytoplasma (group 16SrXII-A) are associated with strawberry marginal chlorosis (SMC) in France. As “Ca. P. fragariae” and stolbur phytoplasma induce identical symptoms, the only way to identify the pathogen infecting a given diseased plant is to perform conventional PCR assays. Because using two PCR techniques for detecting separately each of the two bacteria is time consuming and because specificity and sensitivity of the detection test needed to be improved, a new approach using triplex real time PCR was developed for the routine detection of “Ca. P. fragariae “ and stolbur phytoplasma. The real time PCR has the advantage of being faster reduces the risks of producing false positives. Furthermore, real-time PCR techniques provide the possibility of multiplexing by using probes with different compatible fluorescent dyes. Here, we present a new sensitive Taqman® method which permits the simultaneous amplification of three DNA targets in one test: the map gene of stolbur phytoplasma, the spoT gene of “Ca. P. fragariae” and the cox gene of strawberry chloroplast taken as an internal control. The specificity and the efficiency of this method were determined.Keywords: Strawberry Marginal Chlorosis, Triplex taqman® PCR ,Candidatus Phlomobacter fragariae, stolbur phytoplasma

Triplex real-time PCR assay for sensitive and simultaneous detection of grapevine phytoplasmas of the 16SrV and 16SrXII-A groups with an endogenous analytical control

Flavescence dorée (FD) and Bois noir (BN) are the two main yellows of grapevine in Europe and are caused by phytoplasmas of the 16SrV and 16SrXII-A groups respectively. A new triplex real-time PCR assay was developed in order to detect simultaneously the FD and BN phytoplasmas as well as grapevine chloroplastic DNA with TaqMan minor groove binder probes. Each set of designed primers and probes specifically detected the map gene of the FD and BN phytoplasmas, respectively and did not detect phytoplasmas from other phylogenetic groups. PCR efficiencies varied from 90 to 110 %. The PCR assay showed good intra-test and inter-test reproducibility. Triplex real-time PCR was compared to the conventional biplex nested-PCR method. The sensitivity of the real-time PCR, tested on several infected periwinkle and grapevine samples, was up to 5 and 100 times higher for the BN-P and the FD-P targets, respectively. Out of 109 grapevine samples analysed 10, which were negative with the nested PCR, turned to be FD-P positive with the real-time PCR. A decision scheme was set up according to the Ct values of the FD-P, BN-P and grapevine targets in order to assess the routine detection results

Using ESTs to improve the accuracy of de novo gene prediction

BACKGROUND: ESTs are a tremendous resource for determining the exon-intron structures of genes, but even extensive EST sequencing tends to leave many exons and genes untouched. Gene prediction systems based exclusively on EST alignments miss these exons and genes, leading to poor sensitivity. De novo gene prediction systems, which ignore ESTs in favor of genomic sequence, can predict such "untouched" exons, but they are less accurate when predicting exons to which ESTs align. TWINSCAN is the most accurate de novo gene finder available for nematodes and N-SCAN is the most accurate for mammals, as measured by exact CDS gene prediction and exact exon prediction. RESULTS: TWINSCAN_EST is a new system that successfully combines EST alignments with TWINSCAN. On the whole C. elegans genome TWINSCAN_EST shows 14% improvement in sensitivity and 13% in specificity in predicting exact gene structures compared to TWINSCAN without EST alignments. Not only are the structures revealed by EST alignments predicted correctly, but these also constrain the predictions without alignments, improving their accuracy. For the human genome, we used the same approach with N-SCAN, creating N-SCAN_EST. On the whole genome, N-SCAN_EST produced a 6% improvement in sensitivity and 1% in specificity of exact gene structure predictions compared to N-SCAN. CONCLUSION: TWINSCAN_EST and N-SCAN_EST are more accurate than TWINSCAN and N-SCAN, while retaining their ability to discover novel genes to which no ESTs align. Thus, we recommend using the EST versions of these programs to annotate any genome for which EST information is available. TWINSCAN_EST and N-SCAN_EST are part of the TWINSCAN open source software package

Effector diversification within compartments of the Leptosphaeria maculans genome affected by Repeat-Induced Point mutations

Fungi are of primary ecological, biotechnological and economic importance. Many fundamental biological processes that are shared by animals and fungi are studied in fungi due to their experimental tractability. Many fungi are pathogens or mutualists and are model systems to analyse effector genes and their mechanisms of diversification. In this study, we report the genome sequence of the phytopathogenic ascomycete Leptosphaeria maculans and characterize its repertoire of protein effectors. The L. maculans genome has an unusual bipartite structure with alternating distinct guanine and cytosine-equilibrated and adenine and thymine (AT)-rich blocks of homogenous nucleotide composition. The AT-rich blocks comprise one-third of the genome and contain effector genes and families of transposable elements, both of which are affected by repeat-induced point mutation, a fungal-specific genome defence mechanism. This genomic environment for effectors promotes rapid sequence diversification and underpins the evolutionary potential of the fungus to adapt rapidly to novel host-derived constraints

The Major Antigenic Membrane Protein of “Candidatus Phytoplasma asteris” Selectively Interacts with ATP Synthase and Actin of Leafhopper Vectors

Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp) and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of “Candidatus Phytoplasma asteris”, the chrysanthemum yellows phytoplasmas (CYP) strain, and three others as non-vectors. Interactions between a labelled (recombinant) CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the α and β subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity