Takken F L, Luderer R, Gabriëls S H, Westerink N, Lu R, de Wit P J, Joosten M H
Laboratory of Phytopathology, Wageningen University, Binnenhaven 9, 6709 PD Wageningen, The Netherlands.
Plant J. 2000 Oct;24(2):275-83. doi: 10.1046/j.1365-313x.2000.00866.x.
We have devised a novel, high-throughput functional cloning method to isolate cDNAs from plant pathogens of which the products elicit a hypersensitive response (HR) in plants. Copy DNA, made from RNA isolated from the tomato pathogen Cladosporium fulvum grown under nutrient-limiting conditions in vitro, was cloned into a binary, potato virus X (PVX)-based expression vector and transformed to Agrobacterium tumefaciens. 9600 colonies were individually toothpick-inoculated onto leaflets of tomato plants resistant to C. fulvum. Four cDNAs were identified whose expression induced formation of a necrotic lesion around the inoculation site. One of these clones, specifically inducing HR on tomato plants carrying the Cf-4 resistance gene, encodes race-specific elicitor AVR4. The other three cDNAs, inducing a non-genotype-specific HR, encode a protein highly homologous to bZIP, basic transcription factors. To determine whether this approach has general applicability, part of the library was also inoculated onto Nicotiana tabacum var. Samsun NN, which is not a host for C. fulvum. Four independent HR-inducing cDNAs were identified which all encode ECP2, an extracellular protein of C. fulvum known to induce necrosis in certain Nicotiana species. These observations confirm that this functional screening method is a versatile strategy to identify cDNAs of pathogens that encode (race-specific) elicitors and other HR-inducing proteins.
我们设计了一种新颖的高通量功能克隆方法，用于从植物病原体中分离cDNA，这些病原体的产物能在植物中引发超敏反应（HR）。从在体外营养限制条件下生长的番茄病原体番茄叶霉（Cladosporium fulvum）中分离的RNA合成的cDNA，被克隆到基于马铃薯X病毒（PVX）的二元表达载体中，并转化到根癌农杆菌（Agrobacterium tumefaciens）中。用牙签将9600个菌落分别接种到对番茄叶霉有抗性的番茄植株的小叶上。鉴定出四个cDNA，其表达诱导接种部位周围形成坏死斑。其中一个克隆，在携带Cf-4抗性基因的番茄植株上特异性诱导HR，编码种属特异性激发子AVR4。另外三个cDNA，诱导非基因型特异性HR，编码一种与bZIP（碱性转录因子）高度同源的蛋白质。为了确定这种方法是否具有普遍适用性，文库的一部分也接种到了普通烟草（Nicotiana tabacum var. Samsun NN）上，普通烟草不是番茄叶霉的寄主。鉴定出四个独立的诱导HR的cDNA，它们都编码ECP2，一种已知能在某些烟草物种中诱导坏死的番茄叶霉细胞外蛋白。这些观察结果证实，这种功能筛选方法是一种通用策略，可用于鉴定编码（种属特异性）激发子和其他诱导HR蛋白的病原体cDNA。
