Studio molecolare dei meccanismi dell'autoincompatibilità gametofitica in pero europeo (Pyrus communis)

Self-incompatibility (SI) systems have evolved in many flowering plants to prevent self-fertilization and thus promote outbreeding. Pear and apple, as many of the species belonging to the Rosaceae, exhibit RNase-mediated gametophytic self-incompatibility, a widespread system carried also by the Solanaceae and Plantaginaceae. Pear orchards must for this reason contain at least two different cultivars that pollenize each other; to guarantee an efficient cross-pollination, they should have overlapping flowering periods and must be genetically compatible. This compatibility is determined by the S-locus, containing at least two genes encoding for a female (pistil) and a male (pollen) determinant. The female determinant in the Rosaceae, Solanaceae and Plantaginaceae system is a stylar glycoprotein with ribonuclease activity (S-RNase), that acts as a specific cytotoxin in incompatible pollen tubes degrading cellular RNAs. Since its identification, the S-RNase gene has been intensively studied and the sequences of a large number of alleles are available in online databases. On the contrary, the male determinant has been only recently identified as a pollen-expressed protein containing a F-box motif, called S-Locus F-box (abbreviated SLF or SFB). Since F-box proteins are best known for their participation to the SCF (Skp1 - Cullin - F-box) E3 ubiquitine ligase enzymatic complex, that is involved in protein degradation through the 26S proteasome pathway, the male determinant is supposed to act mediating the ubiquitination of the S-RNases, targeting them for the degradation in compatible pollen tubes. Attempts to clone SLF/SFB genes in the Pyrinae produced no results until very recently; in apple, the use of genomic libraries allowed the detection of two F-box genes linked to each S haplotype, called SFBB (S-locus F-Box Brothers). In Japanese pear, three SFBB genes linked to each haplotype were cloned from pollen cDNA. The SFBB genes exhibit S haplotype-specific sequence divergence and pollen-specific expression; their multiplicity is a feature whose interpretation is unclear: it has been hypothesized that all of them participate in the S-specific interaction with the RNase, but it is also possible that only one of them is involved in this function. Moreover, even if the S locus male and female determinants are the only responsible for the specificity of the pollen-pistil recognition, many other factors are supposed to play a role in GSI; these are not linked to the S locus and act in a S-haplotype independent manner. They can have a function in regulating the expression of S determinants (group 1 factors), modulating their activity (group 2) or acting downstream, in the accomplishment of the reaction of acceptance or rejection of the pollen tube (group 3). This study was aimed to the elucidation of the molecular mechanism of GSI in European pear (Pyrus communis) as well as in the other Pyrinae; it was divided in two parts, the first focusing on the characterization of male determinants, and the second on factors external to the S locus. The research of S locus F-box genes was primarily aimed to the identification of such genes in European pear, for which sequence data are still not available; moreover, it allowed also to investigate about the S locus structure in the Pyrinae. The analysis was carried out on a pool of varieties of the three species Pyrus communis (European pear), Pyrus pyrifolia (Japanese pear), and Malus × domestica (apple); varieties carrying S haplotypes whose RNases are highly similar were chosen, in order to check whether or not the same level of similarity is maintained also between the male determinants. A total of 82 sequences was obtained, 47 of which represent the first S-locus F-box genes sequenced from European pear. The sequence data strongly support the hypothesis that the S locus structure is conserved among the three species, and presumably among all the Pyrinae; at least five genes have homologs in the analysed S haplotypes, but the number of F-box genes surrounding the S-RNase could be even greater. The high level of sequence divergence and the similarity between alleles linked to highly conserved RNases, suggest a shared ancestral polymorphism also for the F-box genes. The F-box genes identified in European pear were mapped on a segregating population of 91 individuals from the cross 'Abbé Fétel' × 'Max Red Bartlett'. All the genes were placed on the linkage group 17, where the S locus has been placed both in pear and apple maps, and resulted strongly associated to the S-RNase gene. The linkage with the RNase was perfect for some of the F-box genes, while for others very rare single recombination events were identified. The second part of this study was focused on the research of other genes involved in the SI response in pear; it was aimed on one side to the identification of genes differentially expressed in compatible and incompatible crosses, and on the other to the cloning and characterization of the transglutaminase (TGase) gene, whose role may be crucial in pollen rejection. For the identification of differentially expressed genes, controlled pollinations were carried out in four combinations (self pollination, incompatible, half-compatible and fully compatible cross-pollination); expression profiles were compared through cDNA-AFLP. 28 fragments displaying an expression pattern related to compatibility or incompatibility were identified, cloned and sequenced; the sequence analysis allowed to assign a putative annotation to a part of them. The identified genes are involved in very different cellular processes or in defense mechanisms, suggesting a very complex change in gene expression following the pollen/pistil recognition. The pool of genes identified with this technique offers a good basis for further study toward a better understanding of how the SI response is carried out. Among the factors involved in SI response, moreover, an important role may be played by transglutaminase (TGase), an enzyme involved both in post-translational protein modification and in protein cross-linking. The TGase activity detected in pear styles was significantly higher when pollinated in incompatible combinations than in compatible ones, suggesting a role of this enzyme in the abnormal cytoskeletal reorganization observed during pollen rejection reaction. The aim of this part of the work was thus to identify and clone the pear TGase gene; the PCR amplification of fragments of this gene was achieved using primers realized on the alignment between the Arabidopsis TGase gene sequence and several apple EST fragments; the full-length coding sequence of the pear TGase gene was then cloned from cDNA, and provided a precious tool for further study of the in vitro and in vivo action of this enzyme.

Studio genetico ed epidemiologico su Erysiphe necator agente eziologico dell'Oidio della vite

The objective was to analyse population structure and to determine genetic diversity of Erysiphe necator (syn. Uncinula necator) populations obtained from some vineyards located in the South-East Po valley (Italy). Powdery mildew is one of the most important fungal diseases of grapes (Vitis vinifera L.) throughout the world. The causal agent is the haploid, heterothallic ascomycete E. necator. It is an obligate biotrophic fungus and it can be found only on green organs of plants belonging to the family Vitaceae. For this pathogen, two sympatric populations (groups A and B) have been described in Europe and Australia. The two genetic groups differ at multiple genetic loci and previous studies reported a lack of interfertility among isolates of the two groups. There are now several well documented examples of plant pathogen species, such as Leptosphaeria maculans, Gaeumannomyces graminis var. tritici, Botrytis cinerea and Erysiphe syringae, which are indeed composed of genetically differentiated clades, that have led to the description of new groups or even new species. Several studies have suggested that genetic E. necator group A and B correlated with ecological features of the pathogen; some researchers proposed that group A isolates over-winter as resting mycelium within dormant buds, and in spring originate infected shoots, known as Flag shoots, while group B isolates would survive as ascospores in overwintering cleistothecia. However, the association between genetic groups and mode of over-wintering has been challenged by recent studies reporting that flag-shoot may be originated indifferently by group A or group B isolate. Previous studies observed a strong association between the levels of disease severity at the end of the growing season and the initial compositions of E. necator populations in commercial vineyards. The frequencies of E. necator genetic groups vary considerably among vineyards, and the two groups may coexist in the same vineyard. This finding suggests that we need more information on the genetics and epidemiology of E. necator for optimize the crop management In this study we monitored E. necator populations in different vineyards in Emilia – Romagna region (Italy), where the pathogen overwinters both as flagshoots and as cleistothecia. During the grape growing season, symptomatic leaves were sampled early in the growing season and both leaves and berries later during the epidemic growth of the disease. From each sample, single-conidial isolate was obtained. Each isolates was grown on V. vinifera leaf cv. Primitivo and after harvesting the mycelium, the DNA was purified and used as template for PCR amplification with SCAR primers (Sequences Characterised Amplified Region ), -tubulin, IGS sequences and Microsatellite markers (SSR). Amplified DNA from b-tubulin and IGS loci was digested with AciI and XhoI restriction enzymes, respectively, to show single-nucleotide polymorphisms specific for the two genetic groups. The results obtained indicated that SCAR primers are not useful to study the epidemiology. of E. necator conversely the b-tubulin IGS sequences and SSR. Summarize the results obtained with b-tubulin, IGS sequences, in treated vineyards we have found individuals of group B along all grape growing season, whereas in the untreated vineyard individuals of the two genetic groups A and B coexisted throughout the season, with no significant change of their frequency. DNA amplified from ascospores of single cleistothecia showed the presence of markers diagnostic for either groups A and B and were seldom observed also the coexistence of both groups within a claistothecium. These results indicate that individuals of the two groups mated in nature and were able to produced ascospores. With SSR we showed the possibility of recombination between A and B groups in field isolates. During winter, cleistothecia were collected repeatedly in the same vineyards sampling leaves fallen on ground, exfoliating bark from trunks, and from soil. From each substrate, was assess the percentage of cleistothecia containing viable ascospores. Our results confirmed that cleisthotecia contained viable ascospores, therefore they have the potential to be an additional and important source of primary inoculum in Emilia-Romagna vineyards.