Hill Erin H, Solomon Peter S
Division of Plant Sciences, Research School of Biology, The Australian National University, Canberra, 2601 Australia.
Fungal Biol Biotechnol. 2020 Sep 18;7:13. doi: 10.1186/s40694-020-00103-2. eCollection 2020.
The fungal pathogen is a significant constraint to wheat production in temperate cropping regions around the world. Despite its agronomic impacts, the mechanisms allowing the pathogen to asymptomatically invade and grow in the apoplast of wheat leaves before causing extensive host cell death remain elusive. Given recent evidence of extracellular vesicles (EVs)-secreted, membrane-bound nanoparticles containing molecular cargo-being implicated in extracellular communication between plants and fungal pathogen, we have initiated an in vitro investigation of EVs from this apoplastic fungal wheat pathogen. We aimed to isolate EVs from broth cultures and examine their protein composition in relation to the soluble protein in the culture filtrate and to existing fungal EV proteomes.
EVs were isolated from broth culture filtrates using differential ultracentrifugation (DUC) and examined with transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). EVs were observed as a heterogeneous population of particles, with most between 50 and 250 nm. These particles were found in abundance in the culture filtrates of viable cultures, but not heat-killed cultures incubated for an equivalent time and of comparable biomass. Bottom-up proteomic analysis using LC-MS/MS, followed by stringent filtering revealed 240 EV proteins. These proteins were distinct from soluble proteins identified in culture filtrates, but were similar to proteins identified in EVs from other fungi, based on sequence similarity analyses. Notably, a putative marker protein recently identified in EVs was also consistently detected in EVs.
We have shown EVs can be isolated from the devastating fungal wheat pathogen and are similar to protein composition to previously characterised fungal EVs. EVs from human pathogenic fungi are implicated in virulence, but the role of EVs in the interaction of phytopathogenic fungi and their hosts is unknown. These in vitro analyses provide a basis for expanding investigations of EVs to examine their involvement in the infection process of this apoplastic wheat pathogen and more broadly, advance understanding of noncanonical secretion in filamentous plant pathogens.
这种真菌病原体是全球温带种植区小麦生产的重大制约因素。尽管其对农艺学有影响，但在导致宿主细胞广泛死亡之前，该病原体能够无症状地侵入小麦叶片质外体并在其中生长的机制仍不清楚。鉴于最近有证据表明细胞外囊泡（EVs）——分泌的、含有分子货物的膜结合纳米颗粒——参与植物与真菌病原体之间的细胞外通讯，我们已启动对这种质外体真菌小麦病原体的细胞外囊泡的体外研究。我们旨在从肉汤培养物中分离细胞外囊泡，并检查其蛋白质组成与培养滤液中的可溶性蛋白质以及现有真菌细胞外囊泡蛋白质组的关系。
使用差速超速离心法（DUC）从肉汤培养滤液中分离出细胞外囊泡，并用透射电子显微镜（TEM）和纳米颗粒跟踪分析（NTA）进行检查。观察到细胞外囊泡是一群异质性颗粒，大多数在50至250纳米之间。在活的培养物的培养滤液中大量发现这些颗粒，但在同等时间培养且生物量相当的热灭活培养物中未发现。使用液相色谱-串联质谱（LC-MS/MS）进行自下而上的蛋白质组学分析，随后进行严格筛选，共鉴定出240种细胞外囊泡蛋白。根据序列相似性分析，这些蛋白质与在培养滤液中鉴定出的可溶性蛋白质不同，但与其他真菌细胞外囊泡中鉴定出的蛋白质相似。值得注意的是，最近在细胞外囊泡中鉴定出的一种假定标记蛋白也在细胞外囊泡中持续被检测到。
我们已经表明，可以从具有破坏性的小麦真菌病原体中分离出细胞外囊泡，并且其蛋白质组成与先前表征的真菌细胞外囊泡相似。人类致病真菌的细胞外囊泡与毒力有关，但植物致病真菌的细胞外囊泡在与其宿主相互作用中的作用尚不清楚。这些体外分析为扩大对细胞外囊泡的研究提供了基础，以检查它们在这种质外体小麦病原体感染过程中的作用，更广泛地说，有助于推进对丝状植物病原体中非经典分泌的理解。
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