Genetic variability of the stolbur phytoplasma vmp1 gene in grapevines,bindweeds and vegetables

Aim: Evaluation of the genetic variability of stolbur phytoplasma infecting grapevines, bindweeds and vegetables, collected in different central and southern Italian regions. Materials and Results: Phytoplasma isolates belonging to stolbur subgroup 16SrXII‐A were subjected to molecular characterization by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP), to investigate two different nonribosomal genes: tuf and vmp1. In grapevines, 32% of samples were infected by tuf‐a type and 68% by tuf‐b type, with different relative incidences in the regions surveyed. All herbaceous samples (bindweeds, tomato, tobacco, pepper, celery) were infected by tuf‐b. The gene vmp1 showed higher polymorphism in grapevines (nine profiles) than herbaceous plants (six) by RFLP analysis, in agreement with nucleotide sequences’ analysis and virtual digestions. Conclusions: The phylogenetic analysis of vmp1 gene sequences supports the RFLP data and demonstrates the accuracy of RFLP for preliminary assessments of genetic diversity of stolbur phytoplasmas and for screening different vmp types. Significance and Impact of the Study: Stolbur represents a serious phytosanitary problem in the areas under investigation, owing to heavy economic losses in infected grapevines and vegetables. Molecular information about the complex genotyping of the vmp1 gene provides useful data towards a better understanding of stolbur epidemiology. Moreover, this study clarifies some different vmp1 genotype classifications of stolbur, providing molecular data in comparison with previous investigations.     

Response of the Vitis vinifera L. cv. 'Nebbiolo' proteome to Flavescence dorée phytoplasma infection

Flavescence dorée is a serious phytoplasma disease affecting grapevine in several European countries. We studied the interaction of Flavescence dorée phytoplasma with its natural plant host by monitoring the effects of infection on the protein expression profile. Among the 576 analyzed spots, 33 proteins were differentially regulated in infected grapevines. Grouping into MIPS functional categories showed proteins involved in metabolism (21%), energy processes (9%), protein synthesis (3%), protein fate (18%), cellular transport and transport routes (6%), cell defense and virulence (42%). Among the differentially regulated proteins, we selected six targets (thaumatin I, thaumatin II, osmotin-like protein, plant basic secretory protein, AAA(+) Rubisco activase and proteasome α5 subunit) and we analyzed their expression by quantitative RT-PCR on samples collected in 2008 and 2009 in several vineyards in Piedmont region, Italy. There was a positive correlation between mRNA and protein expression for most of the genes in both the years. We discuss the involvement of these proteins in the specific response to phytoplasma infection. To our knowledge, this work is the first to investigate the response of the grapevine proteome to Flavescence dorée phytoplasma infection, and provides reference protein profiles for future comparative proteomic and genomic studies.     

Markov chain Monte Carlo fitting of single-channel data from inositol trisphosphate receptors

In many cell types, the inositol trisphosphate receptor (IPR) is one of the important components that control intracellular calcium dynamics, and an understanding of this receptor (which is also a calcium channel) is necessary for an understanding of calcium oscillations and waves. Recent advances in experimental techniques now allow for the measurement of single-channel activity of the IPR in conditions similar to its native environment, and these data can be used to determine the rate constants in Markov models of the IPR. We illustrate a parameter estimation method based on Markov chain Monte Carlo, which can be used to fit directly to single-channel data, and determining, as an intrinsic part of the fit, the times at which the IPR is opening and closing. We show, using simulated data, the most complex Markov model that can be unambiguously determined from steady-state data and show that non-steady-state data is required to determine more complex models.     

Identification of effector genes from the phytopathogenic Oomycete Plasmopara viticola through the analysis of gene expression in germinated zoospores

Grapevine downy mildew caused by the Oomycete Plasmopara viticola is one of the most important diseases affecting Vitis spp. The current strategy of control relies on chemical fungicides. An alternative to the use of fungicides is using downy mildew resistant varieties, which is cost-effective and environmentally friendly. Knowledge about the genetic basis of the resistance to P. viticola has progressed in the recent years, but little data are available about P. viticola genetics, in particular concerning the nature of its avirulence genes. Identifying pathogen effectors as putative avirulence genes is a necessary step in order to understand the biology of the interaction. It is also important in order to select the most efficient combination of resistance genes in a strategy of pyramiding. On the basis of knowledge from other Oomycetes, P. viticola effectors can be identified by using a candidate gene strategy based on data mining of genomic resources. In this paper we describe the development of Expressed Sequence Tags (ESTs) from P. viticola by creating a cDNA library from in vitro germinated zoospores and the sequencing of 1543 clones. We present 563 putative nuclear P. viticola unigenes. Sequence analysis reveals 54 ESTs from putative secreted hydrolytic enzymes and effectors, showing the suitability of this material for the analysis of the P. viticola secretome and identification of effector genes. Next generation sequencing of cDNA from in vitro germinated zoospores should result in the identification of numerous candidate avirulence genes in the grapevine/downy mildew interaction.     

Computerized matching of relevés and association tables,with an application to the British National Vegetation Classification

When a new relevé is to be assigned to a pre-existing type, its composition is compared with an association table. Bayesian inference may seem a good way to make the comparison, but presents difficulties. In an alternative approach, three indices of goodness-of-fit are proposed. Compositional satisfaction is a measure of how well the species composition of the relevé fits the constancy classes in the table; it is a minor modification of the Czekanowski coefficient of similarity between observed and expected numbers of species in each constancy class. Dominance satisfaction is a modification of the Czekanowski similarity between the relevé and cover values that might be expected from the association table. Dominance constancy is a weighted mean of the constancy class of the four most abundant species in the relevé. A computer program, TABLEFIT, combines them into a single index. It has been tested on British mire vegetation.     

Recent duplications dominate NBS-encoding gene expansion in two woody species

Most disease resistance genes in plants encode NBS-LRR proteins. However, in woody species, little is known about the evolutionary history of these genes. Here, we identified 459 and 330 respective NBS-LRRs in grapevine and poplar genomes. We subsequently investigated protein motif composition, phylogenetic relationships and physical locations. We found significant excesses of recent duplications in perennial species, compared with those of annuals, represented by rice and Arabidopsis. Consequently, we observed higher nucleotide identity among paralogs and a higher percentage of NBS-encoding genes positioned in numerous clusters in the grapevine and poplar. These results suggested that recent tandem duplication played a major role in NBS-encoding gene expansion in perennial species. These duplication events, together with a higher probability of recombination revealed in this study, could compensate for the longer generation time in woody perennial species e.g. duplication and recombination could serve to generate novel resistance specificities. In addition, we observed extensive species-specific expansion in TIR-NBS-encoding genes. Non-TIR-NBS-encoding genes were poly- or paraphyletic, i.e. genes from three or more plant species were nested in different clades, suggesting different evolutionary patterns between these two gene types.