Screen S, Bailey A, Charnley K, Cooper R, Clarkson J
Microbial Pathogenicity Group, School of Biology and Biochemistry, University of Bath, UK.
Gene. 1998 Oct 9;221(1):17-24. doi: 10.1016/s0378-1119(98)00430-2.
Attempts to improve the effectiveness of entomopathogenic fungi as biological control agents require a clear understanding of the pathogenicity determinants at both the biochemical and molecular level. Proteases play a key role in entomopathogenicity, allowing the fungus to penetrate the insect cuticle and rapidly invade the host. The most extensively studied of these protease activities, PR1A and PR2, are both subject to nitrogen derepression. The Metarhizium anisopliae nrr1 (nitrogen response regulator 1) gene was identified using a PCR-based strategy; it encodes a putative DNA-binding protein with a single zinc finger motif defined by the C-X2-C-X17-C-X2-C sequence. M. anisopliae NRR1 shows a significant sequence similarity to Neurospora crassa NIT2. Sequence analysis identified the presence of two introns, suggesting a greater degree of similarity to N. crassa nit2 than to the areA-like genes that have been identified. However, functional equivalence of nrr1 to areA was demonstrated, by co-transformation and complementation of an A. nidulans areA loss-of-function mutant (areA18 argB2 pabaA1 inoB2) with the M. anisopliae nrr1 gene. The areA-/nrr1+ Aspergillus transformants were able to grow on media with nitrate and glutamate as the sole nitrogen source, whereas the areA- strain is unable to grow under these conditions. The possible relevance of nitrogen regulation to pathogenicity is discussed.
提高昆虫病原真菌作为生物防治剂有效性的尝试需要在生化和分子水平上清楚地了解致病性决定因素。蛋白酶在昆虫致病性中起关键作用，使真菌能够穿透昆虫表皮并迅速侵入宿主。这些蛋白酶活性中研究最广泛的PR1A和PR2都受到氮去阻遏作用的影响。使用基于PCR的策略鉴定了绿僵菌nrr1（氮反应调节因子1）基因；它编码一种推定的DNA结合蛋白，具有由C-X2-C-X17-C-X2-C序列定义的单个锌指基序。绿僵菌NRR1与粗糙脉孢菌NIT2具有显著的序列相似性。序列分析确定存在两个内含子，这表明它与粗糙脉孢菌nit2的相似程度高于已鉴定的areA样基因。然而，通过用绿僵菌nrr1基因对构巢曲霉areA功能缺失突变体（areA18 argB2 pabaA1 inoB2）进行共转化和互补，证明了nrr1与areA的功能等效性。areA - /nrr1 + 曲霉转化体能够在以硝酸盐和谷氨酸作为唯一氮源的培养基上生长，而areA - 菌株在这些条件下无法生长。文中讨论了氮调节与致病性的可能相关性。
