Identification and characterization of Dothistroma septosporum effectors : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics

Abstract

Dothistroma septosporum is the main causal agent of Dothistroma needle blight of pines. However little is known about mechanisms of pine resistance against D. septosporum, or whether there is any classical gene-for-gene resistance involved. The molecular basis of how fungal effector proteins can trigger plant host resistance in a gene-for-gene manner was determined partly by work with the model fungus Cladosporium fulvum and its tomato host. Comparative genome analysis of C. fulvum and D. septosporum genomes identified nine putative effector genes (DsAvr4, DsEcp2-1, DsEcp2-2, DsEcp2-3, DsEcp4, DsEcp5, DsEcp6, DsEcp13 and DsEcp14) in D. septosporum that are homologous to well-characterized C. fulvum effector genes. Other effector candidates were identified as small cysteine rich proteins that are highly expressed in planta, including DsHdp1 which is a hydrophobin gene and Ds69335 which belongs to the sperm-coating protein-like extracellular protein SCP/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) superfamily.

Transcriptome analysis showed that, except for DsEcp2-1 and DsEcp6, the in planta expression of D. septosporum effectors was low. Targeted gene replacement of DsAvr4, DsEcp2-1, DsEcp6 and DsHdp1 caused no observed changes in fungal physiology in vitro compared to wild type (WT) and also showed that DsAvr4, DsEcp6 and DsHdp1 are not virulence factors when infecting Pinus radiata. However deletion of DsEcp2-1 caused larger lesions compared to WT, suggesting that DsEcp2-1 may act to suppress a host target which is involved in necrosis induction during the biotrophic infection stage.

A domain swap experiment in this study showed that swapping the region between cysteine residues C6 (Cys102) to C7 (Cys114), which contains the chitin binding domain, caused loss of resistance (R) protein Cf-4 recognition of DsAvr4 (with CbAvr4) and gain of Cf-4 recognition of CbAv4 (with DsAvr4 or CfAvr4). Further experiments carried out in Wageningen University showed that a Pro residue located in the chitin binding domain in DsAvr4 is important for Cf-4 recognition, and may have a role in DsAvr4 stability. In this study, effector candidates DsEcp2-1 and DsEcp2-3 were able to trigger a non-host necrotic response in N. tabacum suggesting possible interaction with a N. tabacum protein. Polymorphism analysis showed that DsEcp4 and DsEcp5 have internal stop codons and encode pseudogenes in all the D. septosporum strains tested, except for DsEcp4 in strains from Guatemala and Columbia in which a functional gene is predicted. DsEcp4 and DsEcp5 are the only D. septosporum effectors tested that showed evidence of positive selection. Those results lead to the suggestion that R proteins that recognise DsEcp4 and DsEcp5 may be present in pine species. DsEcp13 appears to be absent from ten D. septosporum strains, suggesting that DsEcp13 is not important for virulence and can also be deleted to avoid an R protein mediated defence response such as a hypersensitive response. Infiltration of DsAvr4, DsEcp2-1 and DsEcp6 P. pastoris expression culture filtrates triggered necrosis in P. radiata needles suggesting that R proteins that directly or indirectly recognise those effectors may also be present in P. radiata.

The finding that D. septosporum has homologues of C. fulvum effectors allowed the first study of molecular pathogen-host interactions in this pathosystem. Targeted gene replacement studies identified genes that may have a virulence function and resistance against these effectors may be durable in the field. The pine needle infiltration assay provides a basic screening method to identify pine genotypes that carry resistance proteins and future work in this area is expected to impact on breeding strategies in the forest industry.