Rust diseases are a serious threat to cereals and other crops throughout the world. Significant advances in understanding the molecular basis of rust fungus pathogenicity and rust disease resistance in plants have been achieved using the flax/flax rust pathosystem. Flax has at least 30 resistance  (R) genes able to confer resistance to flax rust. Each resistance gene enables flax to recognise and respond to isolates of the flax rust fungus carrying a corresponding Avr gene.  Many of these resistance genes have been isolated and some (e.g. L6 and M) have been studied at the protein level. Several Avr genes have also been isolated and studied at the protein level (e.g. AvrL2, AvrL567, AvrM, AvrM14, AvrP and AvrP123). These Avr genes encode small secreted proteins that serve as disease effectors in susceptible flax plants and are expressed early in infection at the interface between the fungus and the plant in a specialised feeding structure called the haustorium, common to many plant pathogenic fungi. Four of these Avr genes encode proteins with plant homologues. AvrM14 encodes a putative nudix hydrolase. AvrP and AvrP123 are allelic genes encoding proteins with structural homology to the plant homeodomain (PHD) motif. PHD proteins are often involved in transcriptional regulation. Initial results from a yeast two-hybrid screen using AvrP as bait suggest a possible interaction with a DEAD-box RNA helicase. AvrL2 encodes a protein with a signal peptide for protein secretion and a chloroplast transit peptide for chloroplast uptake. It shows chloroplast localisation when expressed in plant cells without its signal peptide and it has homology to a subdomain of a DYW family nucleic acid deaminase involved in chloroplast RNA editing. This project will examine the functional roles of the AvrP and AvrL2 effectors during infection of flax by the fax rust fungus. The aims of this project are therefore as follows:

AvrP

Aim 1: Confirm the yeast two-hybrid interaction between AvrP and the DEAD-box RNA helicase, and test for interaction with its nearest family members, by isolating, cloning and testing the corresponding full-length flax cDNAs for yeast two hybrid interaction with AvrP.Aim 2: Confirm the interaction between AvrP and the DEAD-box RNA helicase by an independent method such as bimolecular fluorescence complementation or co-immunoprecipitation (or both).

Aim 3: Investigate the predicted functional activity of the DEAD-box RNA helicase by RNA binding and ATP hydrolysis assays.

Aim 4: Investigate the subcellular localisation of the DEAD-box RNA helicase by citrine tagging and confocal fluorescence microscopy.

Aim 5: Generate a knockout of the flax DEAD-box RNA helicase gene by CRISPR/Cas9 genome editing and look at the effect on plant morphology and on AvrP-mediated resistance to flax rust and on rust fungus infection in susceptible flax.

Aim 6: Generate DEAD-box RNA helicase over-expression lines of flax and look for phenotypic effects as in Aim 5.

AvrL2

Aim 1: Conduct a yeast two-hybrid interaction experiment using the prey cDNA library already available in the host laboratory and AvrL2 as bait. Analyse positive clones for multiple independent hits and conduct a bioinformatic analysis to determine likely interactors versus likely artefacts.

Aim 2: Confirm the yeast two-hybrid interaction between AvrL2 and any putative interactor, and test for interaction with its nearest family members, by isolating, cloning and testing the corresponding full-length flax cDNAs for yeast two hybrid interaction with AvrL2.

Aim 3: Confirm the interaction between AvrL2 and the putative interactor(s) by an independent method such as bimolecular fluorescence complementation or co-immunoprecipitation (or both).

Aim 4: Investigate the possible effect of AvrL2 on chloroplast RNA editing by RNAseq analysis of chloroplasts isolated from plant tissue expressing AvrL2 minus its signal peptide. This experiment will be conducted in two ways:

1. fluorescence-activated cell sorting of chloroplasts from plant tissues expressing either AvrL2-citrine or chloroplast-targeted citrine (as a negative control)
2. generation and dexamethasone (DEX) induction of transgenic flax lines expressing DEX-inducible AvrL2 or a DEX-inducible negative control gene

Aim 5: If AvrL2 interacts with a nuclear-encoded protein, generate a knockout of the interactor gene by CRISPR/Cas9 genome editing and look at the effect on plant morphology and on AvrL2-mediated resistance to flax rust and on rust fungus infection in susceptible flax.

Aim 6: Generate interactor over-expression lines of flax and look for phenotypic effects as outlined in Aim 5.

Aim 7: If AvrL2 interacts with a nuclear-encoded protein that is not targeted to the chloroplast, investigate the subcellular localisation of the interactor by citrine tagging and confocal fluorescence microscopy.