Apoplastic extracts from a transgenic wheat line exhibiting lesion-mimic phenotype have multiple pathogenesis-related proteins that are antifungal
Apoplastic extracts from a transgenic wheat line exhibiting lesion-mimic phenotype have multiple pathogenesis-related proteins that are antifungal
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DOI：
10.1094/mpmi.2004.17.12.1306
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发表时间：
2004-12-01
影响因子：
3.5
通讯作者：
Muthukrishnan, S
中科院分区：
文献类型：
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作者：
Anand, A;Lei, ZT;Muthukrishnan, S
组成型表达编码IV类酸性几丁质酶和酸性β-1，3-葡聚糖酶的基因的转基因小麦品系在温室条件下显示出镰刀菌头枯病（赤霉病）传播的显著延迟。在早期的工作中，我们观察到一个病变模拟表型在这个转基因株系时，纯合子的转基因位点。从病变模拟植物中提取的质外体液（AF）具有属于β-1，3-葡聚糖酶、几丁质酶和奇异果甜蛋白样蛋白（TLPs）家族的病程相关（PR）蛋白。AF对禾谷镰刀菌（Fusarium graminearum）和小麦全蚀病菌（Gaeumannomyces graminis var.）三个月。通过两步离子交换层析方案，我们回收了许多PR蛋白和一些未知蛋白。通过串联质谱法纯化并鉴定了对应于TLP（分子量，16 kDa）和两种β-1，3-葡聚糖酶（分子量，各32 kDa）的三个单独蛋白条带。我们测量了在体外抗真菌活性的三个纯化的酶和大麦II类几丁质酶（在我们的实验室较早纯化）在微量滴定板测定与大分生孢子或分生孢子梗的F。graminearum和Pyrenophora tritici-repentis。蛋白质的混合物显示协同或加和抑制活性对F。graminearum和P. tritici-repentis菌丝。观察到这些效应的PR蛋白浓度可能是在表现出过敏反应的细胞AF中达到的浓度。我们的研究结果表明，质外体PR蛋白是抗真菌的，它们的抗菌效力取决于在微生物攻击后植物中有效达到的浓度和组合。
A transgenic wheat line constitutively expressing genes encoding a class IV acidic chitinase and an acidic beta-1,3-glucanase, showed significant delay in spread of Fusarium head blight (scab) disease under greenhouse conditions. In an earlier work, we observed a lesion-mimic phenotype in this transgenic line when homozygous for transgene loci. Apoplastic fluid (AF) extracted from the lesion-mimic plants had pathogenesis-related (PR) proteins belonging to families of beta-1,3-glucanases, chitinases, and thaumatin-like proteins (TLPs). AF had growth inhibitory activity against certain fungal pathogens, including Fusarium graminearum and Gaeumannomyces graminis var. tritici. Through a two-step ion-exchange chromatography protocol, we recovered many PR proteins and a few uncharacterized proteins. Three individual protein bands corresponding to a TLP (molecular mass, 16 kDa) and two beta-1,3-glucanases (molecular mass, 32 kDa each) were purified and identified by tandem mass spectrometry. We measured the in vitro antifungal activity of the three purified enzymes and a barley class II chitinase (purified earlier in our laboratory) in microtiter plate assays with macroconidia or conidiophores of F. graminearum and Pyrenophora tritici-repentis. Mixtures of proteins revealed synergistic or additive inhibitory activity against F. graminearum and P. tritici-repentis hyphae. The concentrations of PR proteins at which these effects were observed are likely to be those reached in AF of cells exhibiting a hypersensitive response. Our results suggest that apoplastic PR proteins are antifungal and their antimicrobial potency is dependent on concentrations and combinations that are effectively reached in plants following microbial attack.