Final Report Summary - BLUMERIA EFFECTORS (FUNCTIONAL analysis of the Blumeria haustoria - Barley interactome)
Blumeria graminis f. sp hordei is an economically important pathogen of cereals that causes barley powdery mildew. This obligate biotrophic pathogen is able to penetrate the cell wall barrier and develop a highly complex intracellular structure known as 'haustorium'. This structure has long been known to actively take up nutrients, but it is now also believed to control host perception and defence, enabling the invading pathogen to survive, avoid and suppress rejection responses. Current thinking postulates that this control is mediated by protein effectors secreted by the pathogen into the host cells. In this research, a detailed discovery of the molecular mechanisms by which B. graminis establishes control of the host barley cell through the action of the effectors was attempted. To accomplish this, a panel of 50 genes encoding candidate effectors proteins based on Blumeria graminis proteomics and proteogenomics was defined in collaboration with Dr Laurence V. Brindschedler from Reading University. I focused on genes encoding small proteins, with preferential expression in haustoria and predicted to be secreted. These genes were amplified and cloned into destination vectors. Then, their ability to contribute to powdery mildew virulence was tested. For this, I used the host induced gene silencing (HIGS) technique in B. graminis growing on barley c.v Golden Promise in collaboration with Dr Patrick Swcheizer (IPK, Gatersleben, Germany). For this aim, a transient assay system based on biolistic bombardment of single barley epidermal cells with ribonucleic acid interference (RNAi) constructs following challenge inoculation with B. graminis was performed. Seven (BEC1005, BEC1011, BEC1016, BEC1019, BEC1038, BEC1054, BEC1061) out of 50 BECs (14 %), targeted messenger RNAs (mRNAs) of B. graminis resulting in a significant reduction of the percentage of fungal conidia able to form haustoria upon introduction of the RNAi construct into the host cell. Effector action of some of the selected Blumeria effectors candidates (BECs) is being confirmed by our collaborators, Dr Roger Wise from Iowa, University, using different functional analysis techniques such as: Xanthomonas infiltration on monocot plants and the virus induced gene silencing system. In addition, transgenic barley plants, expressing RNAi targeting some of the selected candidates, are being obtained to test whether observed HIGS effect can protect from fungal invasion. Amongst the selected effectors, three of them (BEC1005, BEC1019, BEC1054) had sequence domains of a glucosidase, a ribonucleiase (RNAse) such as protein and a protease, respectively. Two of them (BEC11 and BEC54) belong to a small family and share a high percentage of identity at the nucleotide level. To prove that there was no possible cross silencing in HIGS experiments between these two related effectors, I performed a complementation analysis by transient expression of synthetic genes, which are resistant to RNA interference due to silent point mutations at the codon level. This study revealed that these two effectors act independently and that they both have a function inside the host cell. An effective strategy of effectors to modulate host defencee circuitry is to suppress their programmed cell death (PCD). In this regard, I wanted to test whether the enhanced resistance to B. graminis f. sp. hordei observed for the 7 selected BECs, after silenced, was associated with cell death. For this, I carried out an anthocyanin assay in which barley epidermal cells are co-bombarded with the silencing construct together with a vector containing two transcription factors for anthocyanin production. Anthocyanin pigments will only be present in intact vacuoles of viable cells. This system was applied to the seven selected BECs; results obtained showed that the number of cells accumulating anthocyanin pigments drastically drops for BEC1011 silenced cells after Bgh inoculation, revealing that BEC11 might be involved in suppressing pathogen-induced host cell death. The most promising effectors identified in this work, BEC1054 BEC1011 and BEC1019, are being used to 'fish' Blumeria and Barley proteins they bind to and interact with them. This part of the research activity is being carried out in collaboration with Dr Cramer and Dr Bindschedler from University of Reading. For this, I cloned these three effectors into different expression vectors, and they were expressed as tagged recombinant proteins using the centrifugal ultrafiltration-dialysis method developed by this group. Two different and complementary strategies to identify proteins interacting with fungal effectors and barley targets are being carried out: (a) identification of the in vitro interactome by affinity chromatography and mass spectrometry and (b) identification of the in vivo interactome by tandem affinity purification (TAP)-tagging. All of these results will be published in different high impact factor journals. Collaboration was established with three groups, with expertise in the field, from UK (1), Germany (1) and the United States of America (USA) (1) to achieve the objectives of this project, leading to the identification and characterisation of seven novel effectors involved in Blumeria / Barley interaction. The understanding gained on this project from the details of how a significant biotrophic pathogen deals with plant immunity will help to identify effective resistance genes and, eventually, to design new combinations of successful resistance. It is therefore incumbent on us to continue discovering the fundamental drivers for pathogenicity, compatibility and disease resistance to increase our chances of dealing with these very real and dramatic threats - particularly those that threaten our major crops such as the cereal mildews. The results of this project will be of interest for cereal farmers, plant and fungal geneticist as well as for private companies involved in chemical and biological control of the cereal mildews.