An analysis of candidate effector genes in Venturia inaequalis

Abstract

Apple scab (or black spot), caused by the fungal pathogen Venturia inaequalis, is the most important disease of apples (Malus spp.) worldwide and in New Zealand. Fungal plant pathogens cause disease by secreting a variety of effector molecules that interfere with the plant's ability to respond to infection. A subset of effectors can be recognised by plant resistance genes and consequently trigger a resistance response. The durability of plant resistance in the field is dependent on the importance of the recognised effector to the virulence of the pathogen, and whether it can be mutated to evade detection. Thus, understanding the molecular basis of V. inaequalis pathogenicity is paramount to the development of long-term genetic resistance. To this end, the functions of four V. inaequalis candidate effector genes were investigated in this thesis. These are: ViEcp6, an orthologue to a characterised effector in Cladesporium fulvum; AvrRvi4,5, identified from a protein filtrate which elicits a hypersensitive response on apple hosts h(4) and h(5); and Vice12 and Vice16, identified from a comparative analysis between in planta and in vitro EST libraries. Expression profiles of these genes during an infection time-course were measured using real time reverse transcriptase PCR. ViEcp6 transcription was highly and constitutively upregulated in planta versus in vitro; AvrRvi4,5 transcription correlated with cuticle penetration but was not upregulated in planta; Vice12 transcription was constitutively upregulated in planta; Vice16 transcription was upregulated in planta and correlated with initiation of stromata growth and sporulation. Knock-down vectors were constructed and transformants for all genes but AvrRvi4,5, analysed. Silencing of ViEcp6 and Vice16 resulted in reduced virulence in comparison to wildtype controls. Conversely, upregulation of Vice12 transcription resulted in reduced pathogenicity. In vitro phenotypes of all transformants were indistinguishable from the wildtype. Bioinformatic analysis of Vice12 using a gene prediction from the latest genome assembly suggests this protein is involved in ribosomal trafficking, and is therefore unlikely to be an effector. ViEcp6 and Vice16 are considered to be promising effector candidates on which further analysis of protein function should be undertaken.