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

目的探索白粉菌的蛋白质组学，以揭示专性真菌侵染过程中参与毒力和易感性的蛋白质。背景：病原菌和卵菌是造成农作物产量损失的主要原因，从而对粮食安全构成持续威胁。专性生养真菌白粉菌是引起谷物白粉病的主要病原菌。作为一种专性生物，它只能在活组织上生长，因此不能为了功能基因组学研究而进行基因修改。相反，我们设计了一种新的基因沉默方法来验证Planta的效应器功能(Orman等人，在修订中)。像其他生物营养微生物一样，白粉菌形成专门的摄食结构，专门在称为吸器的植物表皮内。它们是效应器传递、毒力和从宿主吸收养分所必需的。Haustoria被寄主吸器外膜(EHM)包围，EHM与植物质膜是连续的，但又不同。定位于EHM的宿主蛋白可能调节感染过程，但EHM蛋白质组仍不清楚。定位于EHM的蛋白质包括参与敏感性的植物蛋白，例如马铃薯-疫霉(卵菌)相互作用所需的remorin(Bozkurt等人，2014、2015)。到目前为止，蛋白质组学方法在鉴定布鲁氏菌毒力因子方面取得了丰硕成果，例如“吸器中的核糖核酸酶样蛋白”-(Ralph)效应因子(BindSchedler等人，2009,2011a，span u等人，2010年，Pedersen等人，2012年，Pliego等人，2013年，Pennington等人，2016年)。然而，还没有大规模的蛋白质组研究来描述白粉病或任何带有吸器的生物营养真菌或卵菌吸器的植物EHM部分。本项目研究了白粉菌-大麦在白粉病侵染过程中的吸器蛋白质组和EHM蛋白质组，以阐明可能是白粉菌侵染调控因子的植物蛋白的潜在重新定位。为实现这一目标，采用差异蛋白质组学方法鉴定大麦在感染过程中表皮中更丰富的大麦蛋白质和丰富的吸器结构。这些蛋白可能与吸器结构相关，也可能与EHM相关。通过瞬时诱导基因沉默分析(TIGS)验证了这些蛋白在大麦侵染中的调控作用。主要结果与健康大麦相比，几种与病程相关的蛋白质在大麦感染的表皮中含量更高：富含半胱氨酸的毒液分泌蛋白(PR1)、过氧化物酶、几丁质酶和几种类索马汀蛋白(TLP或PR5)。一个已鉴定的TLP与TLP5有很高的同源性，TLP5是一种建议的白僵菌效应器BEC1054的相互作用元件。在某些实验生长条件下，TLP5的瞬时基因沉默出人意料地增强了大麦对白粉菌的敏感性，这表明TLP5在寄主敏感性中发挥了作用(Lambertucci等，2019)。这将通过在模式植物本底烟草中瞬时过表达TLP5来进一步研究，以质疑TLP5在促进本底烟草对吸器、卵菌病原体和疫霉侵染的敏感性方面的潜力。此外，与感染的表皮相比，浓缩吸器样品中发现了更丰富的几种膜蛋白。其中，膜蛋白Aquaporin PIP2；3和早期结瘤蛋白样蛋白9都可能与寄主的敏感性有关，因为它们的基因表达通过TIGS检测而被下调，从而显著减少了大麦白粉病的侵染。通过这种方式，将大规模蛋白质组学方法与瞬时基因沉默相结合，可以识别大麦对白粉病易感的新的关键介体。

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 workflowTo 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 outcomesSeveral 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.