Wolpert T J, Navarre D A, Moore D L, Macko V
Department of Botany and Plant Pathology, Oregon State University, Corvallis 97331-2902.
Plant Cell. 1994 Aug;6(8):1145-55. doi: 10.1105/tpc.6.8.1145.
The fungus Cochliobolus victoriae, the causal agent of victoria blight of oats, produces the host-specific toxin victorin. Sensitivity of oats to victorin, and thus susceptibility to the fungus, is controlled by a single dominant gene. This gene is believed to also confer resistance to the crown rust pathogen Puccinia coronata. In the case of victoria blight, the gene has been hypothesized to condition susceptibility by encoding a toxin receptor. A 100-kD victorin binding protein (VBP) has been identified; it binds radiolabeled victorin derivatives in a ligand-specific manner and in a genotype-specific manner in vivo. The VBP may function as a toxin receptor. In vitro translation coupled with indirect immunoprecipitation was used to identify the mRNA for the 100-kD VBP, and fractionated mRNAs were used to prepare cDNA libraries enriched in the relative abundance of cDNA for the 100-kD VBP. A 3.4-kb cDNA clone was isolated that, when subjected to a 400-bp 5' deletion, was capable of directing the synthesis of a protein in Escherichia coli, which reacted to an antibody specific for the 100-kD VBP. Peptide mapping, by limited proteolysis, indicated that the protein directed by the cDNA is the 100-kD VBP. Nucleotide sequence analysis of the cDNA revealed extensive homology to a previously cloned cDNA for the P protein component of the multienzyme complex glycine decarboxylase. Glycine decarboxylase is a nuclear-encoded, mitochondrial enzyme complex. Protein gel blot analysis indicated that the 100-kD VBP copurifies with mitochondria. Based on analysis of in vitro translation products, nucleotide sequence homology, mitochondrial localization, and the widespread species distribution of the 100-kD VBP, we concluded that the 100-kD VBP is the P protein component of glycine decarboxylase.
燕麦维多利亚叶枯病的病原菌——维多利亚旋孢腔菌（Cochliobolus victoriae）可产生寄主特异性毒素维多利亚毒素。燕麦对维多利亚毒素的敏感性，进而对该真菌的易感性，由一个显性单基因控制。据信该基因还赋予了对冠锈病病原菌冠柄锈菌（Puccinia coronata）的抗性。就维多利亚叶枯病而言，已推测该基因通过编码毒素受体来决定易感性。已鉴定出一种100 kD的维多利亚毒素结合蛋白（VBP）；它在体内以配体特异性和基因型特异性的方式结合放射性标记的维多利亚毒素衍生物。VBP可能作为毒素受体发挥作用。采用体外翻译结合间接免疫沉淀法来鉴定100 kD VBP的mRNA，并使用分级分离的mRNA制备富含100 kD VBP cDNA相对丰度的cDNA文库。分离出一个3.4 kb的cDNA克隆，当对其进行400 bp的5'端缺失处理后，能够在大肠杆菌中指导合成一种与针对100 kD VBP的特异性抗体发生反应的蛋白质。通过有限蛋白酶解进行的肽图谱分析表明，由该cDNA指导合成的蛋白质就是100 kD VBP。对该cDNA的核苷酸序列分析显示，它与先前克隆的多酶复合物甘氨酸脱羧酶P蛋白组分的cDNA具有广泛的同源性。甘氨酸脱羧酶是一种核编码的线粒体酶复合物。蛋白质凝胶印迹分析表明，100 kD VBP与线粒体共纯化。基于对体外翻译产物、核苷酸序列同源性、线粒体定位以及100 kD VBP广泛的物种分布的分析，我们得出结论：100 kD VBP是甘氨酸脱羧酶的P蛋白组分。
