Studies of flax rust effector gene expression, and effector protein localisation and interactions
Creators: name: Wu, Wenjie
Publisher: name: The Australian National University
Year: 2016
Subjects: M. lini
effectors
Y2H
split luciferase assay
co-IP
immunolocalisation
qPCR
RNA-Seq

description: During infection, rust fungi secrete effector proteins that are believed to play important roles in nutrient acquisition and infection facilitation. To increase our understanding of the production and deployment of fungal effectors in the process of plant infection, the main aims of the experiments described in this thesis were to investigate the previously suggested role of the fungal protein SIA2 in effector secretion or uptake, to obtain direct evidence of flax rust effector localisation in rust-infected plant cells and to determine the expression profiles of genes in the flax rust secretome, including rust effectors, during plant infection. SIA2 was previously identified in a cohort of proteins that bound to the effector protein, AvrL567, of the flax rust pathogen, Melampsora lini. In the experiments described in Chapter 2 of this thesis, three different assays of protein-protein interactions were employed to further characterise the binding of SIA2 with flax rust effectors. The Yeast-two-hybrid (Y2H) assay provided evidence that SIA2 bound to the M. lini effector proteins, AvrL567, AvrP and AvrP123 and the binding to AvrL567 preferentially occurred at the N-terminal uptake domain. No evidence for a specific interaction between SIA2 and any of the effectors in planta was obtained in split luciferase or co-immunoprecipitation assays. To understand the localisation of SIA2 and flax rust effectors in rust-infected plant cells, SIA2, AvrP and AvrP123 proteins were purified and used to generate polyclonal antisera, as described in Chapter 3. With extensive efforts, purified polyclonal antisera specific for AvrP were obtained and used in immunofluorescence and immunogold labelling experiments. The AvrP labelling was first detected in germ tubes and later at the periphery of hypha and within the extrahaustorial matrix. Cytoplasmic labelling of AvrP inside the plant cell was also detected, indicating that AvrP enters the plant cell during infection. Transient expression of SIA2, AvrP and AvrP123 that lacked their secretion signals and were tagged with the yellow fluorescent protein (YFP) in Nicotiana tabacum leaf cells showed different distributions of these proteins. YFP-AvrP and YFP-AvrP123 were observed within the plant cell nucleus and cytoplasm,whereas YFP-SIA2 accumulated inside the plant cell nucleus in intensely fluorescent spots. Patterns of expression of SIA2 and flax rust effector genes in spores and during an infection time-course up to 10 days post-inoculation (dpi) of flax leaves were assessed in Chapter 4. Results of real time-quantitative polymerase chain reaction (qPCR) and RNA sequencing (mRNA-Seq) showed that all known M. lini effector genes that produce proteins with avirulence (Avr) activities had similar expression patterns during infection. The expression profile of SIA2 differed from that of the Avr genes, but was similar to that of genes homologous to fungal genes encoding appressorial and membrane-anchored proteins. Evaluation of the data obtained in the current research led to the hypothesis that SIA2 might function in appressorium development and/or regulation of effector uptake into plant cells. The importance of the results obtained from studies presented in this thesis and their significance for future research are discussed in Chapter 5.