Li D, Chung K R, Smith D A, Schardl C L
Department of Plant Pathology, University of Kentucky, Lexington 40546-0091, USA.
Mol Plant Microbe Interact. 1995 May-Jun;8(3):388-97. doi: 10.1094/mpmi-8-0388.
Among the antimicrobial phytoalexins produced by Phaseolus vulgaris (French bean) is the prenylated isoflavonoid, kievitone. The bean pathogen, Fusarium solani f. sp. phaseoli, secretes a glycoenzyme, kievitone hydratase (EC 4.2.1.95), which catalyzes conversion of kievitone to a less toxic metabolite. Among F. solani strains, those that are highly virulent to P. vulgaris also produce kievitone hydratase constitutively, suggesting that the enzyme is a virulence factor. Based on the N-terminal amino acid sequence of purified enzyme, the kievitone hydratase cDNA and gene (khs) were cloned. The identities of khs and the cDNA were confirmed by their expression in transgenic Neurospora crassa and Emericella nidulans. Based on the gene and cDNA sequences, khs is predicted to encode a preprotein of 350 amino acids, from which a 19 amino acid N-terminal transit peptide is removed during maturation and secretion. The predicted mass of the mature polypeptide, 37 kDa, contrasts with the 47 to 49 kDa size estimated by electrophoresis of purified enzyme, confirming that the enzyme is extensively glycosylated. The inferred polypeptide sequence has seven canonical sites for N-glycosylation. Southern blot-hybridization analysis of F. s. f. sp. phaseoli DNA indicates one khs locus and an additional locus with weak hybridization to the khs probe. Sequences related to khs were also detected in several isolates of F. solani and the related teleomorph, Nectria haematococca. However, strains of F. oxysporum known to exhibit inducible kievitone hydratase activity (but not pathogenic to bean) did not have detectable khs homology. Nevertheless, all isolates known to cause severe disease on bean possessed khs sequence.
菜豆（法国豆）产生的抗微生物植物抗毒素中有一种异戊烯基化异黄酮——基维酮。菜豆病原体菜豆镰孢菌分泌一种糖酶——基维酮水合酶（EC 4.2.1.95），该酶催化基维酮转化为毒性较小的代谢产物。在茄腐镰孢菌菌株中，那些对菜豆具有高毒力的菌株也组成型地产生基维酮水合酶，这表明该酶是一种毒力因子。基于纯化酶的N端氨基酸序列，克隆了基维酮水合酶cDNA和基因（khs）。通过在转基因粗糙脉孢菌和构巢曲霉中的表达，证实了khs和cDNA的身份。根据基因和cDNA序列预测，khs编码一个350个氨基酸的前体蛋白，在成熟和分泌过程中会去除一个19个氨基酸的N端转运肽。预测的成熟多肽质量为37 kDa，与纯化酶电泳估计的47至49 kDa大小形成对比，证实该酶被广泛糖基化。推断的多肽序列有七个N-糖基化的典型位点。对菜豆镰孢菌f. sp. phaseoli DNA的Southern杂交分析表明有一个khs位点以及另一个与khs探针弱杂交位点。在茄腐镰孢菌的几个分离株以及相关有性型血红色丛赤壳中也检测到了与khs相关的序列。然而，已知表现出可诱导的基维酮水合酶活性（但对菜豆无致病性）的尖孢镰孢菌菌株没有可检测到的khs同源性。尽管如此，所有已知对菜豆造成严重病害的分离株都具有khs序列。
