Expression profiling in HcrVf2-transformed apple plants in response to Venturia inaequalis

Abstract

Apple scab resistance is one of the most wellcharacterized plant-pathogen interactions in a woody plant species. While the HcrVf2 gene from the wild apple Malus floribunda 821 has proved capable of conferring scab resistance to the susceptible cv. Gala after genetic transformation, its identification represents only the first step in understanding the molecular mechanisms and, hence, the network of genes underlying the defence response. We used a PCR-based suppression subtractive hybridization to identify apple genes that are differentially expressed after Venturia inaequalis inoculation. Subtractive hybridization was performed between cDNA from challenged leaves of HcrVf2-resistant transgenic Gala and susceptible cv. Gala plants. A library of 523 unigenes was constructed and characterized by assigning a putative function via comparison with public databases. This set of pathogen-modulated apple genes includes many defence-related genes and is therefore an important source of information for understanding the molecular basis of the Malus-V. inaequalis interaction.

dHPLC efficiency for semi-automated cDNA-AFLP analyses and fragment collection in the apple scab-resistance gene model

“…The RNA was extracted from frozen leaf tissues according to Paris et al (2009), with a typical yield of 50-80 lg total RNA (A260/A280 = 1.9-2.2, A260/A230 = 1.99-2.2) from 100 mg tissue. RNA quantification was also done by Nanodrop TM and 40 lg total RNA were treated with 10 U of DNase (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK) at 37°C for 20 min, in 30 ll total volume.…”

“…After the cloning from the cultivar Florina of the resistance gene HcrVf2 (Vinatzer et al 2001), capable of conferring scab resistance to the susceptible cv. Gala (Belfanti et al 2004), the identification and characterization of genes that are differentially expressed after pathogen infection started (Degenhardt et al 2005;Paris et al 2009). …”

Characterization of the defence response to Venturia inaequalis in ‘Honeycrisp’ apple, its ancestors, and progeny

“…Resistant and susceptible apple cultivars have been shown to have differential gene expression when inoculated with V. inaequalis (Holzapfel et al 2012). In infected apple leaves, the increased transcripts related to reactive oxygen species and oxidative burst are often implicated in PCD (Paris et al, 2009).…”

Biphenyl 4-Hydroxylases Involved in Aucuparin Biosynthesis in Rowan and Apple Are Cytochrome P450 736A Proteins

“…Quantitative PCR primer pairs for Md(GD)B4H were designed based on the sequence of unigene MDP0000152900. All samples were normalized using mRNA of the reference gene, MdActin, as an internal control sample for each line (Paris et al, 2009). Scaling of Md(GD)B4H expression was performed in relation to the respective mRNA expression levels in mockinoculated stems, which were set to 1.…”