Michela Guidarelli

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.

First report of Colletotrichum acutatum causing bitter rot on apple in Italy.

Italy could be considered the main apple-producing country in the European Union. Italian apple (Malus domestica L. Borkh.) production is estimated at approximately 2.1 million tons and encompasses a wide range of cultivars, harvested from August to November. Colletotrichum acutatum, which causes severe losses to strawberry production, was a regulated organism for all European countries until 2008, when it was removed from the EPPO quarantine pathogen list because of its wide distribution in strawberry-production areas. During the growing season of 2010, fungi were isolated from apple fruits exhibiting bitter rot symptoms after 4 months of storage in several packinghouses in the Emilia Romagna Region. The apples belonged to the Golden Delicious, Granny Smith, Pink Lady and Crisp Pink cultivars. Lesions on the fruit surface were circular and 1 to 3 cm in diameter. When lesions enlarged, they became sunken with relatively firm rotten tissues. The fungal fruiting structures, acervuli, were distributed sparsely or densely on old lesions, and under humid conditions, they discharged an orange conidial mass. Conidia observed with a light microscope appeared hyaline and fusiform, 8 to 16 × 2.5 to 4 μm, with two pointed ends or one rounded end. The fungal isolates were grown on potato dextrose agar (PDA) plates incubated at 25°C. After 7 days, colonies were white, becoming gray to pale orange, and when viewed from the reverse side, the color ranged from pink to reddish orange. Both cultural and morphological characteristics of the pathogen were similar to those described for C. acutatum J.H. Simmonds (3), which is responsible for bitter rot of apple (2). Kochs postulates were performed with one representative isolate from each host by artificial inoculation of 30 healthy apples from the cultivars listed above. Fruit surfaces were disinfected with 70% ethanol, wounded with a sterile needle, and then inoculated with 20 μl of a spore suspension (105 conidia ml-1) prepared from a 15-day-old culture on PDA. Inoculated fruits were sealed in a plastic bag and incubated at 25°C for 10 days. In 92% of fruits, symptoms appeared 10 days later, forming lesions with cream-to-salmon pink fruiting structures. The fungus was reisolated onto PDA from the lesions on the inoculated apples. After 7 days of incubation, the colonies and the morphology of conidia were the same as those of the original isolates. The tests were performed on all four cultivars with similar results. The PCR analysis, carried out using universal primers ITS1 and ITS4 (4) directly from single-spore-derived mycelium (1), resulted in an amplification product with 100% sequence homology with C. acutatum isolate AB626881 from GenBank database. Considering the results obtained, to our knowledge, this is the first report of C. acutatum in Italy causing bitter rot on apple. The disease is common in practically all countries where apples are commercially grown and since the losses could be severe under prolonged warm and wet weather conditions, C. acutatum could represent a serious issue for the Italian apple industry. References: (1) M. Iotti and A. Zambonelli. Mycol. Res. 110:60, 2006. (2) A. L. Jones et al. Plant Dis. 80:1294, 1996. (3) B. C. Sutton. Page 1 in: Colletotrichum: Biology, Pathology and Control. Brit. Soc. Plant Pathol. Oxon. UK 1992. (4) T. J. White et al. Page 315 in: PCR Protocols. A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

The mannose‐binding lectin gene FaMBL1 is involved in the resistance of unripe strawberry fruits to Colletotrichum acutatum

The fungal pathogen Colletotrichum acutatum is the causal agent of strawberry (Fragaria × ananassa) anthracnose. Although the fungus can infect strawberry fruits at both unripe and ripe stages, the symptoms appear only on red ripe fruits. On white unripe fruits, the pathogen becomes quiescent as melanized appressoria after 24 h of interaction. Previous transcriptome analysis has indicated that a mannose-binding lectin (MBL) gene is the most up-regulated gene in 24-h-infected white strawberries, suggesting a role for this gene in the low susceptibility of unripe stages. A time course analysis of the expression of this MBL gene, named FaMBL1 (Fragaria × ananassa MBL 1a), was undertaken to monitor its expression profile in white and red fruits at early interaction times: FaMBL1 was expressed exclusively in white fruit after 24 h, when the pathogen was quiescent. Agrobacterium-mediated transient transformation was used to silence and overexpress the FaMBL1 gene in 24-h-infected white and red strawberries, respectively. FaMBL1-silenced unripe fruits showed an increase in susceptibility to C. acutatum. These 24-h-infected tissues contained subcuticular hyphae, indicating pathogen penetration and active growth. In contrast, overexpression of FaMBL1 in ripe fruits decreased susceptibility; here, 24-h-infected tissues showed a high percentage of ungerminated appressoria, suggesting that the growth of the pathogen had slowed. These data suggest that FaMBL1 plays a crucial role in the resistance of unripe strawberry fruits to C. acutatum.

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.