A single amino acid mutation affects elicitor and expansins-like activities of cerato-platanin, a non-catalytic fungal protein

Cerato-platanin (CP) is a non-catalytic, cysteine-rich protein, the first member of the cerato-platanin family. It is a single-domain protein with a double Ψ/β barrel domain resembling the D1 domain of plant and bacterial expansins. Similarly to expansins, CP shows a cell wall-loosening activity on cellulose and can be defined as an expanisin-like protein, in spite of the missing D2 domain, normally present in plant expansins. The weakening activity shown on cellulose may facilitate the CP-host interaction, corroborating the role of CP in eliciting plant defence response. Indeed, CP is an elicitor of primary defences acting as a Pathogen-Associated Molecular Patterns (PAMP). So far, structure-function relationship study has been mainly performed on the bacterial BsEXLX1 expansin, probably due to difficulties in expressing plant expansins in heterologous systems. Here, we report a subcloning and purification method of CP in the engineered E. coli SHuffle cells, which proved to be suitable to obtain the properly folded and biologically active protein. The method also enabled the production of the mutant D77A, rationally designed to be inactive. The wild-type and the mutated CP were characterized for cellulose weakening activity and for PAMP activity (i.e. induction of Reactive Oxygen Species synthesis and phytoalexins production). Our analysis reveals that the carboxyl group of D77 is crucial for expansin-like and PAMP activities, thus permitting to establish a correlation between the ability to weaken cellulose and the capacity to induce defence responses in plants. Our results enable the structural and functional characterization of a mono-domain eukaryotic expansin and identify the essential role of a specific aspartic residue in cellulose weakening.

角素-platanin（Cerato-platanin, CP）是一种非催化型富含半胱氨酸的蛋白质，为角素家族（cerato-platanin family）的首个成员。该蛋白属于单结构域蛋白质，携带有双Ψ/β桶结构域，其结构与植物和细菌膨胀素（expansin）的D1结构域高度相似。与膨胀素类似，CP可对纤维素展现细胞壁松弛活性；尽管缺少植物膨胀素通常具备的D2结构域，但仍可被归类为类膨胀素蛋白。其在纤维素上展现的弱化活性可能有助于CP与宿主的相互作用，佐证了CP在激发植物防御反应中的功能。事实上，CP作为一类病原相关分子模式（Pathogen-Associated Molecular Patterns, PAMP），是植物初级防御反应的激发子。迄今为止，结构-功能关系研究主要以细菌BsEXLX1膨胀素为对象，这可能是由于在异源表达系统中制备植物膨胀素存在较大困难。本研究报道了一种在工程化大肠杆菌SHuffle细胞中开展CP亚克隆与纯化的方法，该方法可获得正确折叠且具有生物活性的目标蛋白。利用该方法还成功制备了经理性设计失活的突变体D77A。研究人员对野生型与突变型CP的纤维素弱化活性以及PAMP活性（即活性氧合成与植保素生成的诱导能力）进行了系统表征。分析结果表明，D77位点的羧基基团对类膨胀素活性与PAMP活性均至关重要，由此确立了纤维素弱化能力与诱导植物防御反应能力之间的直接相关性。本研究成果实现了单结构域真核膨胀素的结构与功能表征，并明确了特定天冬氨酸残基在纤维素弱化过程中的关键作用。