Investigating the interactors of the Blumeria effector BEC1054

• Main Author: Pennington, Helen
• Other Authors: Spanu, Pietro
• Published: Imperial College London 2015
• Subjects: 572
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.684300

Obligate biotrophic pathogens, for example the powdery mildew Blumeria graminis f.sp. hordei, must counteract the host's defenses if infection is to be successful and maintained. Effectors are secreted by fungi in order to regulate host immunity. One such effector, BEC1054 possesses a structure similar to that of an RNase, but the key catalytic site for RNase activity is not conserved. A total of 247 putative protein interactors were identified solely with BEC1054 through in vitro affinity chromatography. Comparison of these sequences with an unrelated BEC and empty Ni-NTA columns demonstrated that a significantly higher percentage of ribosomal large subunit and elongation factor related proteins were found with BEC1054. In the literature, many of these proteins have also been found to occur with Ribosome Inactivating Proteins (RIP)s. I hypothesise that BEC1054 competes with ribosome inactivating proteins, for example JIP60, preventing them from cleaving host cell's ribosomes. This would prevent the host cell's death, and therefore rejection of the powdery mildew. In this investigation, I found that BEC1054 interacted with five proteins in yeast and in planta: a Pathogenesis Related protein 5 (PR5), PR10, Glutathione-S-Transferase (GST), eukaryotic Elongation Factor 1 Alpha (eEF1A) (1), eukaryotic Elongation Factor 1 Gamma (eEF1G), the latter two of which are associated with ribosomes. In addition, I identified an interaction between GST and PR10 in yeast and in planta, and a novel interacting protein was found for Jasmonate Induced Protein 60 (JIP60) in planta: eEF1a(3). Control genes were assayed, to determine the best combination for normalisation of barley and B. graminis genes during quantitative real-time PCR. The optimal B. graminis housekeeping genes were found to be act, gapdh and H3; and for barley gapdh, ubi and tuba2b. The housekeeping genes investigated were shown to vary significantly between species (B. graminis and barley) and tissues, demonstrating the necessity for appropriate controls for each qPCR assay. The control genes were used to normalise four members of Candidate Secreted Effector Protein (CSEP) family 21 across a B. graminis infection time course in both epidermal and epiphytic material. The four CSEPs showed an early peak in transcript abundance in epiphytic material, which had not previously been identified in the literature. In addition, the four CSEPs demonstrated peaks in abundance at 24-48h in epiphytic material. In epidermal material, the CSEPs showed more diverse patterns of expression, with transcript abundance peaks occurring at ca. 24 or 48 hpi. In vitro BEC1054 was found to interact with oligo(poly)nucleotides in a concentration-dependent manner. In addition, BEC1054 and JIP60 interacted with RNA in yeast; and the selective media assay indicated that this interaction may be specific for the region of the ribosome containing the ribosomal SRL. Expression of BEC1054 in N. benthamiana and wheat affected the resistance of the host plants to the biotrophic pathogens Peronospora tabacina and B. graminis f.sp. tritici respectively. Finally, we found that B. graminis f.sp. tritici prevented degradation of the ribosome by ribosome inactivating proteins; and that BEC1054 appeared to partially prevent ribosome degradation.