Identification of host proteins targeted by the flax rust effector protein AvrL567

Abstract

The flax rust fungus (Melampsora lini) and its flax host (Linum usitatissimum) have served as a successful model system for rust infection for many years. The experiments described in this thesis aim to identify cellular targets of the two M. lini effector proteins AvrL567 and AvrM and elucidate the function of these targets. To identify targets of the effector proteins, Yeast-2-Hybrid (Y2H) screens were performed with cDNA from rust infected flax leaves. This approach allowed for the identification of plant targets as well as potential fungal cofactors. Eight putative interactors (iA1 to iA8) were isolated for AvrL567, none could be identified for AvrM. The candidates identified in the Y2H screen were truncated proteins. Two of them showed low sequence homology to fungal proteins, three putative interactors had low sequence homology to plant proteins and three had no sequence homology to any known protein. At the time of this project, neither the flax nor the flax rust genome had been sequenced and thus additional experiments were conducted to obtain full length sequence information of the putative interactors. Using a lambda phage assay and Rapid Amplification of cDNA-Ends (RACE) full length sequence information was obtained for five putative interactors (iA1, iA2, iA3, iA4 and iA8). They were successfully tested for interaction with AvrL567 in Y2H assays and computational analysis revealed homology to known proteins and protein domains. Based on this analysis three interactors (iA1, iA2 and iA3) of AvrL567 were chosen for further investigation. iA1 is a plant protein that shows high sequence homology to the Arabidopsis thaliana cytokinin oxidase/dehydrogenase enzyme AtCKX7 and was termed L. usitatissimum CKX1. AtCKX7 is involved in plant hormone metabolism and two isoforms of the LuCKX1 were identified in flax. Both show interaction with AvrL567 in Bimolecular Fluorescent Complementation (BiFC) assays but differ in their enzymatic activity. Addition of purified AvrL567 in the enzyme assay significantly increased the maximum velocity (Vmax) of the reaction while lowering the Michaelis constant of LuCKX1. These results suggest that the fungus is actively manipulating the host's cytokinin hormone levels. iA2 is a fungal protein with an N-terminal secretion signal peptide and was termed Secreted Interactor of AvrL567 Two (SIA2). There are homologues of SIA2 in Melampsora larici-populina and Puccinia graminis f.sp tritici but their functions are unknown. SIA2 failed to show interaction with AvrL567 in BiFC experiments but it interacted with AvrL567 and other rust effectors in Y2H experiments. The importance of SIA2 for rust infection remains to be determined but the results suggest that it might be a potential cofactor for some rust effectors. iA3 is a plant protein with a protein kinase C-2 (C2) domain found in many lipid-binding proteins and was termed C2-domain containing Membrane targeting Protein (C2MP). It shows interaction with AvrL567 in BiFC assays, however, experiments to show lipid binding were inconclusive. One close homologue of C2MP can be found in tobacco (NaPCCP) and is involved in protein import of extracellular glycoproteins suggesting that C2MP could be involved in uptake if AvrL567 into plant cells. Show more