Identification of differentially expressed genes in a resistant versus a susceptible blueberry cultivar after infection by Colletotrichum acutatum

Abstract

Anthracnose fruit rot, caused by the fungus Colletotrichum acutatum, is an important disease of blueberry worldwide. The cultivar Elliott is resistant, severely restricting fungal growth and sporulation relative to the susceptible cultivar Jersey. The objectives of this research were as follows: (i) to analyse pre-penetration events in 'Elliott' and 'Jersey' at different stages of fruit development; (ii) to identify putative defence genes in 'Elliott' fruit; and (iii) to monitor the timing of the oxidative burst in both cultivars. Light microscopy revealed no differences in the pre-penetration activities of C. acutatum on the immature fruit of both cultivars. However, at fruit ripening, conidia germinated and formed appressoria more rapidly on 'Jersey' than on 'Elliott' fruit. Using suppression subtractive hybridization, 37 differentially expressed sequence tags (ESTs) were detected in 'Elliott' versus 'Jersey' upon infection. Several of the ESTs had homology to known plant defence genes, such as a class II chitinase, pathogenesis-related protein 10 (PR10) and β-1,3-glucanase. Two putative genes involved in oxidative stress were identified: a metallothionein-like protein and monodehydroascorbate reductase. ESTs of fungal origin were also detected. Many ESTs had no homology to known genes. Using semi-quantitative and quantitative reverse transcription-polymerase chain reaction (RT-PCR), the expression of most of the candidate genes was detected earlier in 'Elliott' than in 'Jersey', some within 24 h post inoculation (hpi). Monitoring of the oxidative burst showed that the overall H(2) O(2) concentration was two to three times higher in 'Elliott' than in 'Jersey' at 24 hpi. The elucidation of the basis of resistance to C. acutatum in blueberry will facilitate the development of new anthracnose fruit rot-resistant cultivars.

Figure 1

Symptoms and signs of Colletotrichum acutatum infection of highbush blueberry fruit. Fruit appearance 8 days after inoculation in the susceptible cultivar Jersey (A) and the resistant cultivar Elliott (B). Scanning electron micrograph of acervuli on the fruit surface of ‘Jersey’ (C) and ‘Elliott’ (D). Bar, 0.5 mm.

Figure 2

(A) Average diameter of acervuli of Colletotrichum acutatum on ripe fruit of the susceptible blueberry cultivar Jersey (open bars) and resistant cultivar Elliott (filled bars) (n= 50). (B) Quantity of C. acutatum conidia produced on ‘Jersey’ (open bars) and ‘Elliott’ (filled bars) after 8 days of incubation (n= 5). Error bars denote the standard error of the mean. Means with the same letter are not significantly different from each other according to Student's paired t‐test (P≤ 0.05).

Figure 3

Percentage of Colletotrichum acutatum conidia that had formed melanized appressoria at 24 h post inoculation on blueberry fruit at different stages of development in the susceptible cultivar Jersey (open bars) and resistant cultivar Elliott (filled bars). Error bars denote the standard error of the mean (n= 5). Within each development stage, means with the same letter are not significantly different from each other according to Student's paired t‐test (P≤ 0.05).

Figure 4

Suppression subtractive hybridization to detect differential expression of genes in ‘Elliott’ (resistant) compared with ‘Jersey’ (susceptible) blueberry fruit after inoculation with Colletotrichum acutatum. (A) Colony polymerase chain reaction (PCR) products obtained from the forward‐subtracted ‘Elliott’ cDNA library with T7 and Sp6 primers used for dot blot analysis and visualized through gel electrophoresis. Lanes 1 and 24, 1‐kb+ DNA ladder; lane 2, negative control; lane 3, EV = empty vector; lanes 4–23, PCR products from the forward‐subtracted ‘Elliott’ cDNA library. (B) PCR products from the forward‐subtracted ‘Elliott’ library hybridized with the reverse‐subtracted ‘Jersey’ library DIG probe. (C) PCR products from the forward‐subtracted ‘Elliott’ library hybridized with the forward‐subtracted ‘Elliott’ DIG probe. Broken lines surround the actin PCR product which served as an internal standard.

Figure 5

Temporal pattern of transcript accumulation and origin determination of putative genes in blueberry fruit of the resistant cultivar Elliott and susceptible cultivar Jersey after inoculation with Colletotrichum acutatum. (A) Transcript accumulation of expressed sequence tags (ESTs) (Table 1) at 0, 24, 48, 96 and 144 h post‐inoculation in semi‐quantitative reverse transcription‐polymerase chain reaction (RT‐PCR). (B) The origin of ESTs as determined by PCR using genomic DNA from ‘Elliott’, ‘Jersey’ and C. acutatum. In (A) and (B), the experiments were performed with specific primers constructed for each individual EST of interest (Table 2). Controls were performed with primers specific for actin (Table 2) and the fungal internal transcribed spacer (ITS) region (ITS1F–ITS4).

Figure 6

Ratio of transcript accumulation of expressed sequence tags (ESTs) relative to actin in quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) at 0, 24, 48, 96 and 144 h post‐inoculation of ripe blueberry fruit of the susceptible cultivar Jersey and resistant cultivar Elliott with Colletotrichum acutatum or water (control). Mean expression levels were normalized with mean endogenous actin levels, and an induced sample was used to generate the standard curve plots for each individual EST. ΔR_n thresholds varied between 0.15 and 0.75. Error bars denote the standard error of the means of the replicates within a representative experiment (n= 3).

Figure 7

H₂O₂ accumulation in epidermal peels of green (A) and ripe (B) fruit of the susceptible blueberry cultivar Jersey and resistant cultivar Elliott inoculated with Colletotrichum acutatum or water (control) at 0, 12, 18, 24 and 48 h post‐inoculation (n= 5). H₂O₂ was detected by the Amplex® Red Hydrogen Peroxide/Peroxidase Kit. Results from a representative experiment of three independent experiments are displayed.