Functional characterisation of the flax rust AvrP effector protein

Abstract

AvrP is an effector protein from the flax-rust fungus (Melampsora lini) that is translocated into host plant (Linum usitatissimum) cells to facilitate infection. AvrP is a zinc-binding protein that possess an elongated zinc-finger-like structure. The aim of this research was to characterise the function of AvrP. To investigate the role of AvrP, interacting proteins were identified via a yeast two hybrid (Y2H) cDNA library screen. The putative interactors included flax phosphoglucomutases (LuPGM1, LuPGM2 and LuPGMcp), peptidyl-prolyl cis-trans isomerase (LuFKBP12) and DEAD-box RNA helicase 56 (LuDRH56). These were further tested for interaction with AvrP using full-length proteins. AvrP was shown to interact with full-length flax PGM1 in planta by co-immunoprecipitation and bimolecular fluorescence complementation. The interacting complex was localised in the cytosol of the cell. Y2H analysis was used to show that AvrP targets the phosphate-binding domain of LuPGM1. AvrP interacts with other isoforms of phosphoglucomutase, and other alleles of AvrP, including AvrP123, interact with LuPGM1. In an in vitro enzymatic assay, purified AvrP protein was able to modulate the activity of purified LuPGM1 protein to increase the conversion of glucose-1-phosphate to glucose 6-phosphate. The interaction between AvrP and full-length LuFKBP12 appeared to be weak in yeast but an in planta bimolecular fluorescence complementation assay confirmed this interaction. Purified flax FKBP12 protein showed little or no prolyl isomerase activity in an in vitro enzymatic assay, which made it impossible to test for any effect of AvrP on its activity. AvrP was shown to interact with full-length flax DRH56 in planta by co-immunoprecipitation and bimolecular fluorescence complementation. The interacting complex was localised inside the plant cell nucleus. Co-expression of AvrP and flax DRH56 in leaves of Nicotiana benthamiana resulted in cell death. Mutational disruption of LuDRH56 ATPase activity did not abolish its interaction with AvrP nor their combined ability to induce cell death. However, mutational disruption of the RNA-binding ability of LuDRH56 abolished the cell death response and changed the localisation of the interacting complex within the plant cell nucleus. Y2H analysis showed that AvrP interacts with conserved motifs involved in the RNA binding and helicase activities of LuDRH56. In in vitro enzymatic assays, purified AvrP protein was able to inhibit the ATPase and helicase activities of purified LuDRH56 protein. An RNA sequencing experiment was performed to test what effect the modification of sugar and RNA metabolism would have on the flax transcriptome. Many differentially expressed genes were identified in agroinfiltrated flax leaves expressing AvrP, indicating that it had a highly disruptive effect on the flax transcriptome. Overall, these results suggest that AvrP is a multifunctional effector able to affect both glucose and RNA metabolism by manipulation of flax cytosolic phosphoglucomutases and inhibition of LuDRH56, respectively. Interaction between AvrP and LuFKBP12 may also contribute to a disruptive effect on RNA metabolism and translation by potentially interfering with RNA methylation. AvrP may therefore be able to affect multiple metabolic pathways by interfering with both sugar metabolism and RNA processing. Show more