Specific detection and quantification of the phytopathogenic agent 'Candidatus Phytoplasma prunorum'.

'Candidatus Phytoplasma prunorum' is a wall-less bacterium associated with European stone fruit yellows (ESFY), a severe disease of Prunus spp. (mainly apricot and Japanese plum trees). It can be spread by one insect vector, Cacopsylla pruni, and by the trade of infected material. The availability of PCR-based methods allowing a sensitive and specific detection of 'Ca. P. prunorum' is crucial for this phytoplasma because, at present, it is uncultured and cannot be detected serologically. We developed a PCR test which, in contrast to the existing detection tools, provides a fast, specific and sensitive detection of 'Ca. P. prunorum' in plants and insects. For studies requiring an absolute quantification of the phytoplasma titer, the same primers were used to develop a real-time PCR assay, including a standard for C. pruni. The sensitivity of these molecular tools was compared by serial dilutions and their specificity was assessed both in silico and experimentally for reference strains and field samples of the closely related phytoplasma 'Ca. P. prunorum', 'Ca. P. pyri' (pear decline agent) and 'Ca. P. mali' (apple proliferation agent), as well as for representative strains of the 'Ca. Phytoplasma' genus.

Efficient transmission of 'Candidatus phytoplasma prunorum' Is delayed by eight months due to a long latency in its host-alternating vector.

Understanding at which spatiotemporal scale a disease causes significant secondary spread has both theoretical and practical implications. We investigated this issue in the case of European stone fruit yellows (ESFY), a quarantine vector-borne phytoplasma disease of Prunus trees. Our work was focused on the processes underlying disease spread: the interplay between the life cycles of the pathogen ('Candidatus Phytoplasma prunorum') and of the vector (Cacopsylla pruni). We demonstrated experimentally that C. pruni has only one generation per year and we showed that, at least in southeastern France, C. pruni migrates between conifers in mountainous regions (where it overwinters) and Prunus spp. at lower altitude (where it breeds). In acquisition-inoculation experiments performed with C. pruni over its period of presence on Prunus spp., both immature and mature C. pruni were hardly infectious (0.6%) despite effective phytoplasma acquisition and multiplication. We demonstrated that most immature vectors born on infected plants reach their maximum phytoplasma load (10(7) genomes per insect) only after migrating to conifers and that, after a life-long retention of the phytoplasma, their transmission efficiency was very high (60%) at the end of winter (when they migrate back to their Prunus host). Thus, most transmissions occur only after an effective latency of 8 months, following vector migrations and overwintering on conifers in mountainous regions. From this transmission cycle, we can infer that local secondary spread of ESFY in apricot orchards is marginal, and recommend that disease management strategies take more into account the processes occurring at a regional scale, including the role of wild Prunus spp. in ESFY epidemics.

Two differentially regulated phosphate transporters from the symbiotic fungus Hebeloma cylindrosporum and phosphorus acquisition by ectomycorrhizal Pinus pinaster.

Ectomycorrhizal symbiosis markedly improves plant phosphate uptake, but the molecular mechanisms underlying this benefit are still poorly understood. We identified two ESTs in a cDNA library prepared from the ectomycorrhizal basidiomycete Hebeloma cylindrosporum with significant similarities to phosphate transporters from the endomycorrhizal fungus Glomus versiforme and from non-mycorrhizal fungi. The full-length cDNAs corresponding to these two ESTs complemented a yeast phosphate transport mutant (Deltapho84). Measurements of (33)P-phosphate influx into yeast expressing either cDNA demonstrated that the encoded proteins, named HcPT1 and HcPT2, were able to mediate Pi:H(+) symport with different affinities for Pi (K(m) values of 55 and 4 mum, respectively). Real-time RT-PCR showed that Pi starvation increased the levels of HcPT1 transcripts in H. cylindrosporum hyphae grown in pure culture. Transcript levels of HcPT2 were less dependent on Pi availability. The two transporters were expressed in H. cylindrosporum associated with its natural host plant, Pinus pinaster, grown under low or high P conditions. The presence of ectomycorrhizae increased net Pi uptake rates into intact Pinus pinaster roots at low or high soil P levels. The expression patterns of HcPT1 and HcPT2 indicate that the two fungal phosphate transporters may be involved in uptake of phosphate from the soil solution under the two soil P availability conditions used.

Differential regulation of the expression of two high-affinity sulfate transporters, SULTR1.1 and SULTR1.2, in Arabidopsis.

The molecular mechanisms regulating the initial uptake of inorganic sulfate in plants are still largely unknown. The current model for the regulation of sulfate uptake and assimilation attributes positive and negative regulatory roles to O-acetyl-serine (O-acetyl-Ser) and glutathione, respectively. This model seems to suffer from exceptions and it has not yet been clearly validated whether intracellular O-acetyl-Ser and glutathione levels have impacts on regulation. The transcript level of the two high-affinity sulfate transporters SULTR1.1 and SULTR1.2 responsible for sulfate uptake from the soil solution was compared to the intracellular contents of O-acetyl-Ser, glutathione, and sulfate in roots of plants submitted to a wide diversity of experimental conditions. SULTR1.1 and SULTR1.2 were differentially expressed and neither of the genes was regulated in accordance with the current model. The SULTR1.1 transcript level was mainly altered in response to the sulfur-related treatments. Split-root experiments show that the expression of SULTR1.1 is locally regulated in response to sulfate starvation. In contrast, accumulation of SULTR1.2 transcripts appeared to be mainly related to metabolic demand and is controlled by photoperiod. On the basis of the new molecular insights provided in this study, we suggest that the expression of the two transporters depends on different regulatory networks. We hypothesize that interplay between SULTR1.1 and SULTR1.2 transporters could be an important mechanism to regulate sulfate content in the roots.

Wheat non-specific lipid transfer protein genes display a complex pattern of expression in developing seeds.

Nine cDNA clones encoding non-specific lipid transfer proteins (nsLTPs) were isolated from Triticum aestivum and Triticum durum cDNA libraries and characterized. One cDNA is predicted to encode a type 2 nsLTP (7 kDa) while others encode type 1 nsLTPs (9 kDa). All encoded proteins contain an N-terminal signal sequence and possess the characteristic features of nsLTPs. The genomic structures of the wheat nsLtp genes show that type 2 TaLtp7.1a, TaLtp7.2a and type 1 TaLtp9.2b genes lack introns while the other type 1 genes consist of one intron. Construction of a phylogenic tree of Poaceae nsLTPs shows that wheat nsLTPs can be divided into eleven distinct groups and are closely related to barley sequences. Using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, the expression patterns of nine nsLtp genes were studied during wheat seed development and germination. We identified three different profiles of nsLtp gene transcript accumulation. Whereas TdLtp7.1a, TdLtp9.4a and TdLtp9.7a transcripts were detected during all maturation stages, TdLtp7.2a, TdLtp9.2a, TdLtp9.3a, TdLtp9.5a and TdLtp9.6a transcripts were only present in the first and TdLtp9.1a in the last stages of seed development. Moreover, these nine wheat nsLtp genes are not seed-specific and are also expressed in the coleoptile of young seedlings. The present study revealed the complexity of the wheat nsLtp gene family and showed that the expression of nsLtp genes is developmentally regulated in the seeds, suggesting a specific function for each of the corresponding proteins.

Comparative expression of five Lea Genes during wheat seed development and in response to abiotic stresses by real-time quantitative RT-PCR.

Gene expression profiles of group 2 (dehydrins) and group 4 Late embryogenesis abundant (Lea) genes in developing seeds of Triticum durum and T. aestivum and in coleoptiles and coleorhizae of T. durum seedlings were monitored by real-time quantitative RT-PCR. The five genes exhibited clear differences in their accumulation pattern in wheat seed and in response to dehydration, low temperature, salinity and ABA. Td29b, Td16 and Td27e gene transcripts accumulate late in embryogenesis as expected for Lea genes, Td11 gene transcripts were present throughout seed development whereas no Td25a gene transcripts were detected in seeds. Drastic changes in the relative levels of Td29b, Td16, Td27e and Td11 transcripts occurred at the shift between the cell expansion and desiccation phases. All genes except the Td11 gene are more highly induced by dehydration in coleorhizae than in coleoptiles. In contrast, response to low temperature, salinity or ABA is higher in coleoptiles than in coleorhizae. Depending on both the gene and on the type of stress, a wide range of induction levels (8- to 100,000-fold) was observed.