Candidate effector gene identification in the ascomycete fungal phytopathogen Venturia inaequalis by expressed sequence tag analysis

Abstract

The hemi-biotrophic fungus Venturia inaequalis infects members of the Maloideae, causing the economically important apple disease, scab. The plant-pathogen interaction of Malus and V. inaequalis follows the gene-for-gene model. cDNA libraries were constructed, and bioinformatic analysis of the resulting expressed sequence tags (ESTs) was used to characterize potential effector genes. Effectors are small proteins, secreted in planta, that are assumed to facilitate infection. Therefore, a cDNA library was constructed from a compatible interaction. To distinguish pathogen from plant sequences, the library was probed with genomic DNA from V. inaequalis to enrich for pathogen genes, and cDNA libraries were constructed from in vitro-grown material. A suppression subtractive hybridization library enriched for cellophane-induced genes was included, as growth on cellophane may mimic that in planta, with the differentiation of structures resembling those formed during plant colonization. Clustering of ESTs from the in planta and in vitro libraries indicated a fungal origin of the resulting non-redundant sequence. A total of 937 ESTs was classified as putatively fungal, which could be assembled into 633 non-redundant sequences. Sixteen new candidate effector genes were identified from V. inaequalis based on features common to characterized effector genes from filamentous fungi, i.e. they encode a small, novel, cysteine-rich protein, with a putative signal peptide. Three of the 16 candidates, in particular, conformed to most of the protein structural characteristics expected of fungal effectors and showed significant levels of transcriptional up-regulation during in planta growth. In addition to candidate effector genes, this collection of ESTs represents a valuable genomic resource for V. inaequalis.

Figure 1

The distribution of putatively fungal expressed sequence tags (ESTs) and non‐redundant sequences (nr) between the in planta libraries (ABEA and ABEB), the potato dextrose broth (PDB) in vitro libraries (IAAA and MAAB) and the suppression subtractive hybridization (SSH) cellophane in vitro library (MAAD). Where there are nr with representative ESTs from more than one library (overlapping sectors), the numbers given are totals from all contributing libraries.

Figure 2

Strategy used for the identification of candidate effectors from Venturia inaequalis expressed sequence tags (ESTs). ORF, open reading frame.

Figure 3

Infection by Venturia inaequalis during a detached leaf assay at (a) 5 days post‐inoculation (dpi), with stromata beginning to form, and (b) 10 dpi, with sporulating stromata. Scale bar, 50 µm.

Figure 4

The average expression of four Venturia inaequalis candidate effector (Vice) genes at two time points during detached leaf infection compared with that during growth in potato dextrose broth (PDB). A gene expression normalization factor was calculated for each sample based on the geometric mean of two stably expressed reference genes (β‐tubulin; EU853839) and 60S ribosomal protein L12 (EU853840) using geNorm software v3.4 (Vandesompele et al., 2002). Averages were calculated from six expression measurements from each time point of infection or growth stage, gathered from two independent infection/growth assays; each expression measurement was the average of three biological samples (each sample comprising three infected leaves or material gathered from three flasks of PDB), and the polymerase chain reaction (PCR) for each sample was conducted in triplicate. The error bars represent the standard deviation of the six expression measurements. The expression of each of these genes during infection was significantly different (P = 0.01) from that during growth in PDB, with the exception of Vice5 (light grey) at 10 dpi, which only showed a 1.39‐fold increase. Vice4, diagonal lines; Vice5, light grey; Vice12, black; Vice16, horizontal lines.