PR genes of apple: identification and expression in response to elicitors and inoculation with Erwinia amylovora

Abstract

Background: In the past decade, much work has been done to dissect the molecular basis of the defence signalling pathway in plants known as Systemic Acquired Resistance (SAR). Most of the work has been carried out in model species such as Arabidopsis, with little attention paid to woody plants. However within the range of species examined, components of the pathway seem to be highly conserved. In this study, we attempted to identify downstream components of the SAR pathway in apple to serve as markers for its activation.

Results: We identified three pathogenesis related (PR) genes from apple, PR-2, PR-5 and PR-8, which are induced in response to inoculation with the apple pathogen, Erwinia amylovora, but they are not induced in young apple shoots by treatment with known elicitors of SAR in herbaceous plants. We also identified three PR-1-like genes from apple, PR-1a, PR-1b and PR-1c, based solely on sequence similarity to known PR-1 genes of model (intensively researched) herbaceous plants. The PR-1-like genes were not induced in response to inoculation with E. amylovora or by treatment with elicitors; however, each showed a distinct pattern of expression.

Conclusion: Four PR genes from apple were partially characterized. PR-1a, PR-2, PR-5 and PR-8 from apple are not markers for SAR in young apple shoots. Two additional PR-1-like genes were identified through in-silico analysis of apple ESTs deposited in GenBank. PR-1a, PR-1b and PR-1c are not involved in defence response or SAR in young apple shoots; this conclusion differs from that reported previously for young apple seedlings.

Figure 1

Alignment of the deduced amino acid sequences of three apple PR-1 genes and the type member, PR-1a from tobacco (GeneBank:CAA29392). Residues shown in red are a predicted or known signal sequence. Boxed residues are the six conserved cysteine residues requisite in PR-1 type proteins. Residues shown in green were used in a tblastn query to generate data for table 2.

Figure 2

Expression patterns of three different PR-1 genes from apple during flower development, and in several cultivars. Two micrograms of total RNA was reverse-transcribed in a 20 μl reaction volume. Two μl of the resulting cDNA template from blossoms of cultivars Gala and Red Delicious at stages; tight-cluster (TC), pink (P), full-bloom (F) and 6 days post full-bloom (+6) or from shoots of the cultivars Jonagold (J), Gala (G), Mutsu (M), Rogers Mac (RM), Red Delicious (RD) and Liberty (L) were used in PCR with primers for PR-1a, PR-1b or PR-1c for 45 cycles. Ten μl of 25 μl reaction mixtures were loaded for each sample. For the EF1α control, 2 μl of the same cDNA template were amplified for 30 cycles with primers for EF1α. Ten μl of 25 μl reaction mixtures were loaded for each sample. Genomic DNA from cultivar Gala was used as the positive PCR control (+). The negative control (-) did not contain template. Note that the EF1α primers span an intron.

Figure 3

Expression of PR-2 and PR-5 and PR-8 following inoculation of apple shoots with Erwinia amylovora by three different methods. Northern hybridization of RNA preparations from young apple shoots following inoculation with E. amylovora Ea273 by piercing shoot tips with a contaminated florist's frog (Frog), slicing the two youngest unfolded leaves on either side of the mid-vein with contaminated scissors (Slice) or by snipping off the distal 1/3 of the two youngest unfolded leaves with contaminated scissors (Snip). Shoots or leaves were sampled at 6, 12, 22, 32 and 45 hours following inoculation.

Figure 4

Expression of apple PR genes in response to inoculation with plant pathogenic bacteria. Northern hybridization of RNA preparations from young apple shoots just prior to (Pre-treatment), and following inoculation with E. amylovora Ea273 (E), P. syringae DC3000 (P), or mock inoculation with 5 mM potassium phosphate buffer pH 6.5 (B).

Figure 5

Expression of apple PR genes in response to treatment with SAR elicitors. Northern hybridization of RNA preparations from young apple shoots following spray application of water (W), Actigard^® (A), INA (I) or ProAct^® (P). 15 μg of total RNA was loaded in each lane.

Figure 6

Phenotype of apple shoots 96 hours following inoculation. Apple shoots were either mock-inoculated (B), or inoculated with E. amylovora Ea273 (E) or P. syringae DC3000 (P). Note the wounds made by the inoculating pins. Wounds are not evident in E because the inoculated leaves were totally necrotic when photographed.