Investigating the proteome of barley powdery mildew extra-haustorial complexes to identify modulators of immunity.

Aim
The proteome of powdery mildew haustoria will be explored to unravel proteins involved in virulence and susceptibility during infection by obligate fungal pathogens.

Background :Pathogenic fungi and oomycetes are responsible for substantial crop yield losses, thus constitute a constant threat to food security. The obligate biotrophic fungal pathogen Blumeria graminis is responsible for the powdery mildew disease in cereals. As an obligate biotroph, it is only able to grow on living tissue, and therefore not amenable to genetic modifications for functional genomics studies. Instead, we devised a new gene silencing method to validate effector function in planta (Orman et al, in revision). Like other biotrophic microbes, Blumeria forms specialized feeding structures exclusively within the plant epidermis called haustoria. These are required for effector delivery, virulence, and nutrient uptake from the host. Haustoria are surrounded by a host extrahaustorial membrane (EHM) which is in continuum to but distinct from the plant plasma membrane. Host proteins localized to the EHM are likely to modulate the infection process, but the EHM proteome remains unknown. Proteins localised to the EHM include plant proteins involved in susceptibility, such as a remorin required for potato-Phytophthora (oomycete) interaction (Bozkurt et al, 2014, 2015). So far proteomic approaches have been fruitful for identifying Blumeria virulence factors such as the "RNAse-like proteins in haustoria"- (RALPH) effectors (Bindschedler et al, 2009, 2011a, Spanu et al, 2010, Pedersen et al, 2012, Pliego et al, 2013, Pennington et al, 2016). However, there is no large-scale proteome investigation to describe the plant EHM part of the haustoria of either powdery mildews or any haustoria-bearing biotrophic fungi or oomycetes. This project investigates the haustorial and the EHM proteomes of Blumeria-barley during powdery mildew infection to elucidate any potential re-localisation of plant proteins which are likely modulators of infection.

Experimental workflow
To achieve this, a differential proteomics approach was undertaken to identify barley proteins more abundant in barley epidermis and enriched haustorial structures during infection. These proteins are putatively associated to haustorial structures and possibly to the EHM. The role of these proteins in modulation infection was then validated in barley via a transient induced gene silencing assay (TIGS).

Main outcomes
Several pathogenesis-related proteins were more abundant in infected epidermis when compared to the healthy counterpart: cysteine-rich venom secreted proteins (PR1), peroxidases, chitinases, and several thaumatin-like proteins (TLP or PR5). One identified TLP shared high homology to TLP5, a suggested interactor of the Blumeria effector BEC1054. In some experimental growth conditions, transient gene silencing of TLP5 unexpectedly enhanced barley susceptibility to Blumeria, suggesting that TLP5 plays a role in host susceptibility (Lambertucci et al, 2019). This will be investigated further via transient overexpression of TLP5 in the model plant Nicotiana benthamiana to query the potential of TLP5 in promoting N. benthamiana susceptibility to the haustorium bearing, oomycete pathogen, Phytophthora infestans.

In addition, several membrane proteins were found more abundant in enriched haustoria samples when compared to infected epidermis. Of these, the membrane proteins Aquaporin PIP2;3 and Early nodulin like protein 9 are both likely to be implicated in host susceptibility, since their gene expression knockdown via the TIGS assay led to substantial reduced barley powdery mildew infection. In this way, coupling of a large-scale proteomics approach combined to transient gene silencing has allowed the identification of new key mediators of barley susceptibility to its powdery mildew.