INRA, UMR1202 BIOGECO, F-33612 Cestas, France.
Electrophoresis. 2009 Dec;30(23):4118-36. doi: 10.1002/elps.200900415.
The secreted proteins (secretome) of fungi play a key role in interactions of pathogenic and symbiotic fungi with plants. Using the plant pathogenic fungus Leptosphaeria maculans and symbiont Laccaria bicolor grown in culture, we have established a proteomic protocol for extraction, concentration and resolution of the fungal secretome. As no proteomic data were available on mycelium tissues from both L. maculans and L. bicolor, mycelial proteins were studied; they also helped verifying the purity of secretome samples. The quality of protein extracts was initially assessed by both 1-DE and 2-DE using first a broad pH range for IEF, and then narrower acidic and basic pH ranges, prior to 2-DE. Compared with the previously published protocols for which only dozens of 2-D spots were recovered from fungal secretome samples, up to approximately 2000 2-D spots were resolved by our method. MS identification of proteins along several pH gradients confirmed this high resolution, as well as the presence of major secretome markers such as endopolygalacturonases, beta-glucanosyltransferases, pectate lyases and endoglucanases. Shotgun proteomic experiments evidenced the enrichment of secreted protein within the liquid medium. This is the first description of the proteome of L. maculans and L. bicolor, and the first application of liquid-phase IEF to any fungal extracts.
真菌的分泌蛋白（secretome）在病原真菌和共生真菌与植物相互作用中起着关键作用。我们使用植物病原真菌 Leptosphaeria maculans 和共生菌 Laccaria bicolor 进行培养，建立了一种真菌分泌蛋白提取、浓缩和分离的蛋白质组学方案。由于缺乏来自 L. maculans 和 L. bicolor 的菌丝体组织的蛋白质组学数据，我们研究了菌丝体蛋白；这些蛋白还帮助验证了分泌样本的纯度。最初通过 1-DE 和 2-DE 评估蛋白提取物的质量，首先使用宽 pH 范围进行 IEF，然后使用更窄的酸性和碱性 pH 范围进行 IEF，然后再进行 2-DE。与以前发表的协议相比，该协议仅从真菌分泌样本中回收了数十个 2-DE 斑点，我们的方法最多可解析约 2000 个 2-DE 斑点。沿几个 pH 梯度对蛋白质进行 MS 鉴定证实了这种高分辨率，以及存在主要的分泌蛋白标志物，如内切多聚半乳糖醛酸酶、β-葡聚糖转移酶、果胶裂解酶和内切葡聚糖酶。鸟枪法蛋白质组学实验证明了分泌蛋白在液体培养基中的富集。这是对 L. maculans 和 L. bicolor 蛋白质组的首次描述，也是液相 IEF 在任何真菌提取物中的首次应用。
