de Sá Patricia B, Havens Wendy M, Ghabrial Said A
Department of Plant Pathology, University of Kentucky, Lexington, KY 40546-0312, USA.
Phytopathology. 2010 Sep;100(9):880-9. doi: 10.1094/PHYTO-100-9-0880.
A broad-spectrum anti-fungal protein of approximately 10 kDa, designated victoriocin, was purified from culture filtrates of a virus-infected isolate of the plant-pathogenic fungus Helminthosporium victoriae (teleomorph: Cochliobolus victoriae) by a multistep procedure involving ultrafiltration and reverse-phase high-performance liquid chromatography (RP-HPLC). Amino acid sequences, obtained by automated Edman degradation sequencing of RP-HPLC-purified victoriocin-derived peptides, were used to design primers for degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) amplification from H. victoriae DNA and cDNA templates. An open reading frame coding for a victoriocin precursor of 183 amino acids with calculated molecular mass of approximately 20 kDa was amplified by PCR from H. victoriae genomic DNA but not from the control fungus Penicillium chrysogenum. Southern hybridization analysis confirmed the presence of the victoriocin gene in all H. victoriae strains tested. Sequence analysis indicated that victoriocin has a sequence motif similar to that found in scorpion short toxin/charybdotoxin and a consensus sequence similar to that found in defensins. Victoriocin, like some other antifungal proteins, including the totivirus-encoded killer proteins, is predicted to be expressed in vivo as a preprotoxin precursor consisting of a hydrophobic N-terminal secretion signal followed by a pro-region and terminating in a classical Kex2p endopeptidase cleavage site that generates the N terminus of the mature victoriocin. A putative cell wall protein of approximately 30 kDa (P30) co-purified with victoriocin from cultural filtrates. The potential role of P30 in the antifungal activity of H. victoriae culture filtrates is discussed.
从植物致病真菌维多利亚长蠕孢菌（有性型：维多利亚旋孢腔菌）的一个病毒感染分离株的培养滤液中，通过包括超滤和反相高效液相色谱（RP-HPLC）在内的多步程序，纯化出了一种约10 kDa的广谱抗真菌蛋白，命名为维多利亚毒素。通过对RP-HPLC纯化的维多利亚毒素衍生肽进行自动埃德曼降解测序获得的氨基酸序列，被用于设计引物，以便从维多利亚长蠕孢菌的DNA和cDNA模板进行简并寡核苷酸引物聚合酶链反应（DOP-PCR）扩增。通过PCR从维多利亚长蠕孢菌基因组DNA中扩增出了一个编码183个氨基酸的维多利亚毒素前体的开放阅读框，其计算分子量约为20 kDa，但从对照真菌产黄青霉中未扩增出。Southern杂交分析证实了在所有测试的维多利亚长蠕孢菌菌株中都存在维多利亚毒素基因。序列分析表明，维多利亚毒素具有与蝎短毒素/鲨毒素中发现的序列基序相似的序列基序，以及与防御素中发现的共有序列相似的共有序列。与其他一些抗真菌蛋白一样，包括全病毒编码的杀伤蛋白，维多利亚毒素预计在体内作为一种前原毒素前体表达，该前体由一个疏水的N端分泌信号、一个前区组成，并在一个经典的Kex2p内肽酶切割位点终止，该位点产生成熟维多利亚毒素的N端。一种约30 kDa的假定细胞壁蛋白（P30）与维多利亚毒素从培养滤液中共同纯化出来。讨论了P30在维多利亚长蠕孢菌培养滤液抗真菌活性中的潜在作用。
