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

研究大麦白粉病吸器外复合物的蛋白质组以确定免疫调节剂。

• 批准号: 1954388
• 金额: --
• 依托单位: Royal Holloway University of London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1954388
• 关键词: Investigating; proteome; barley; powdery; mildew

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.

目的通过对白粉病吸器蛋白质组的研究，揭示与致病性和易感性相关的蛋白质。背景：致病性真菌和卵菌是造成农作物产量损失的主要原因，对粮食安全构成持续威胁。专性生物营养型真菌病原体小麦白粉病菌（Blumeria graminis）是导致谷物中的白粉病的原因。作为一种专性生物营养型，它只能在活组织上生长，因此不适合用于功能基因组学研究的遗传修饰。相反，我们设计了一种新的基因沉默方法来验证植物中的效应子功能（Orman等人，修订版）。与其他活体营养微生物一样，Blumeria专门在植物表皮内形成专门的摄食结构，称为吸器。这些是效应子传递、毒力和从宿主摄取营养所必需的。吸器被寄主吸器外膜（EHM）所包围，该膜与植物质膜连续但不同。定位于EHM的宿主蛋白可能调节感染过程，但EHM蛋白质组仍然未知。定位于EHM的蛋白质包括参与易感性的植物蛋白质，例如马铃薯-疫霉（卵菌纲）相互作用所需的蛋白质（博兹库尔特等人，2014，2015）。到目前为止，蛋白质组学方法已经在鉴定布氏菌属毒力因子如“吸器中的RNA酶样蛋白”-（RALPH）效应子方面取得了丰硕成果（Bindschedler等人，2009，2011 a，Spanu等人，2010，Pedersen等人，2012，Pliego等人，2013，彭宁顿等人，2016）。然而，没有大规模的蛋白质组研究来描述植物EHM部分吸器的白粉病或任何吸器轴承的活体营养真菌或卵菌。本项目调查吸器和EHM蛋白质组的Blumeria大麦在白粉病感染阐明任何潜在的重新定位的植物蛋白质，这可能是调制器infection.Experimental workflowTo实现这一点，差异蛋白质组学方法进行鉴定大麦蛋白质更丰富的大麦表皮和丰富的吸器结构在感染过程中。这些蛋白质与吸器结构相关，可能与EHM相关。通过瞬时诱导基因沉默试验（TIGS）验证了这些蛋白在大麦中的调节感染的作用。主要结果与健康对应物相比，感染表皮中的几种发病机制相关蛋白更丰富：富含半胱氨酸的毒液分泌蛋白（PR 1）、过氧化物酶、几丁质酶和几种奇异果毒蛋白样蛋白（TLP或PR 5）。一种鉴定的TLP与TLP 5具有高度同源性，TLP 5是Blumeria效应子BEC 1054的一种建议的相互作用物。在一些实验生长条件下，TLP 5的瞬时基因沉默出乎意料地增强了大麦对布氏病的易感性，表明TLP 5在宿主易感性中发挥作用（Lambertucci et al，2019）。这将通过在模式植物本氏烟草（Nicotianabenthamiana）中瞬时过表达TLP 5来进一步研究，以询问TLP 5在促进N.此外，在富集的吸器样品中，发现几种膜蛋白比感染的表皮更丰富。其中，膜蛋白水通道蛋白PIP 2;3和早期胰蛋白酶样蛋白9都可能与宿主易感性有关，因为它们的基因表达通过TIGS测定敲低导致大麦白粉病感染显著减少。以这种方式，耦合的大规模蛋白质组学的方法相结合的瞬时基因沉默，允许鉴定新的关键介质大麦的白粉病易感性。