Transient transformation meets gene function discovery: The strawberry fruit case

Beside the well known nutritional and health benefits, strawberry (Fragaria X ananassa) crop draws increasing attention as plant model system for the Rosaceae family, due to the short generation time, the rapid in vitro regeneration, and to the availability of the genome sequence of F. X ananassa and F. vesca species. In the last years, the use of high-throughput sequence technologies provided large amounts of molecular information on the genes possibly related to several biological processes of this crop. Nevertheless, the function of most genes or gene products is still poorly understood and needs investigation. Transient transformation technology provides a powerful tool to study gene function in vivo, avoiding difficult drawbacks that typically affect the stable transformation protocols, such as transformation efficiency, transformants selection, and regeneration. In this review we provide an overview of the use of transient expression in the investigation of the function of genes important for strawberry fruit development, defense and nutritional properties. The technical aspects related to an efficient use of this technique are described, and the possible impact and application in strawberry crop improvement are discussed

Induced expression of the Fragaria × ananassa Rapid alkalinization factor-33-like gene decreases anthracnose ontogenic resistance of unripe strawberry fruit stages

Rapid alkalinization factor (RALF) genes encode for ubiquitous small peptides that stimulate apoplastic alkalinization through interaction with malectin-like receptor kinase. RALF peptides may act as negative regulators of plant immune response, inhibiting the formation of the signal receptor complex for immune activation. Recently RALF homologues were identified in different fungal pathogen genomes contributing to host infection ability. Here, FaRALF-33-like gene expression was evaluated in strawberry fruits inoculated with Colletotrichum acutatum, Botrytis cinerea, or Penicillium expansum after 24 and 48 h post-infection. To investigate the role of FaRALF-33-like in strawberry susceptibility, transient transformation was used to overexpress it in white unripe fruits and silence it in red ripe fruits. Agroinfiltrated fruits were inoculated with C. acutatum and expression, and histological analysis of infection were performed. Silencing of FaRALF-33-like expression in C. acutatum-inoculated red fruits led to a delay in fruit colonization by the fungal pathogen, and infected tissues showed less penetrated infective hyphae than in wild-type fruits. In contrast, C. acutatum-inoculated white unripe fruits overexpressing the FaRALF-33-like gene decreased the ontogenic resistance of these fruits, leading to the appearance of disease symptoms and penetrated subcuticular hyphae, normally absent in white unripe fruits. The different response of transfected strawberry fruits to C. acutatum supports the hypothesis that the FaRALF-33-like gene plays an important role in the susceptibility of fruits to the fungal pathogen C. acutatum.Fil: Merino, Maria Cecilia. Universidad de Bologna; Italia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Guidarelli, Michela. Universidad de Bologna; ItaliaFil: Negrini, Francesca. Universidad de Bologna; ItaliaFil: De Biase, Dario. Universidad de Bologna; ItaliaFil: Pession, Annalisa. Universidad de Bologna; ItaliaFil: Baraldi, Elena. Universidad de Bologna; Itali

Preliminary studies on partial reduction of Colletotrichum acutatum infection by proteinase inhibitors extarcted from apple skin

Colletotrichum acutatum, a fungal pathogen that causes soft rot in fruit, produced protease when grown on casein or apple cell wall, as revealed by a clear zone around the well filled with C. acutatum medium in a radial diffusion assay. A protease-inhibiting protein (PI) was also extracted from healthy stored apple, cultivar Cripps Pink, and its activity was tested in vitro and in vivo against a protease produced by C. acutatum. In in vitro trials the inhibition rate determined by radial diffusion assay was over 41% after 24 h, while in inoculated fruit the inhibition ranged from 23.5% to 45% after 5 days at 20 C. The protease inhibitor extracted from healthy apple skin was a heat-denaturable protein since the halo produced by protein extracted from C. acutatum and added to boiled protein extracted from healthy apple skin tissue was 338.7 mm2, significantly higher than the halo produced by protein extracted from C. acutatum diluted with fresh protein extracted from healthy tissue (220.7 mm2). Protein secreted by C. acutatum grown in induced buffer media was tested by polyacrylamide gel electrophoresis. SDS-PAGE of crude enzyme extract revealed the presence of various protein bands that could be ascribed by their molecular mass as putative aspartic proteinase, extracellular alkaline proteinase and serine protease. Genomic analyses are, however, in progress to identify the proteins involved in Colletotrichum patogenicity. More investigations are required to identify the nature of the substance responsible for C. acutatum inhibition in apple and to evaluate the possibility of manipulating the protease inhibitor levels in fruit to reduce soft rot caused by C. acutatum

Layers of Venice. Architecture, Arts and Antiquities at Rialto

The book offers a greater understanding of the multiple layers of meaning that have been superimposed in the course of the Medieval and Modern Ages in the Rialto area. The authors follow the Leitmotiv of the memorial component that each operation of architectural reuse has carried in the history of the church of San Giacomo di Rialto, a building which stands, emblematically, as a sort of architectural relic. Adopting this principle, the book offers an in-depth analysis of the spatial model, the reuse of individual architectural and decorative elements but also, on a larger scale, the different configurations of the urban context (the Rialto market) through the centuries, after repeated destruction, reconstruction and transformation. By adopting this multi-scale approach, the book reveals the key role played by the church in the narrative strategy adopted to perpetually renew the myth of Venice, taking on a conceptual and polysemantic dimension where each component (object, context, meaning, function, image) constitutes an element cultural memory, with each leaving a tangible trace of its own

Layers of Venice. Architecture, Art and Antiquities at Rialto.

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First Report of Asiatic Brown Rot Caused by Monilinia polystroma on Peach in Italy

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Gene expression analysis of peach fruit at different growth stages and with different susceptibility to Monilinia laxa

Monilinia brown rot is one of the most important diseases of peach (Prunus persica L. Batsch). In this study, the susceptibility to Monilinia rot of peach fruit during ripening was analysed weekly by assessing infected fruits upon artificial inoculation. Fruit drastically reduced their susceptibility to Monilinia rot along with ripening, becoming resistant in correspondence to pit hardening (a two-week period). Susceptibility increases again thereafter. With the aim to identify genes possibly correlated with the variation of brown rot susceptibility, a microarray based-transcriptome analysis was undertaken to compare the expression of genes between susceptible fruit (two weeks before the pit hardening stage) and resistant fruit (at the pit hardening stage). This approach pointed out that genes involved in defence and primary and secondary metabolism, in particular some phenylpropanoid and flavonoid related genes, are differentially expressed in susceptible and resistant fruit. Considering that several aromatic compounds with antifungal properties are known to accumulate during endocarp lignification, the expression levels of genes encoding key enzymes of the phenylpropanoid and jasmonate pathways was quantified by real time RT-PCR in the peel of both susceptible and resistant fruit. Results show that during the two-week time between the susceptible and resistant fruit stages the expression of several genes involved in the synthesis of phenylpropanoid and jasmonate compounds drastically changes, supporting a role for these metabolites in the fruit response to Monilinia