Honée G, Melchers L S, Vleeshouwers V G, van Roekel J S, de Wit P J
Department of Phytopathology, Wageningen Agricultural University, Netherlands.
Plant Mol Biol. 1995 Dec;29(5):909-20. doi: 10.1007/BF00014965.
Three constructs were used to study the expression of the avirulence gene Avr9 from the fungal tomato pathogen Cladosporium fulvum in plants. They include pAVIR1, pAVIR2 and pAVIR21, encoding the wild-type AVR9 protein and two hybrid AVR9 proteins containing the signal sequences of the pathogenesis-related proteins PR-S and PR-1a, respectively. Transgenic tobacco plants obtained with the three constructs showed a normal phenotype and produced AVR9 elicitor with the same specific necrosis-inducing activity as the wild-type AVR9 elicitor produced in planta by isolates of C. fulvum containing the Avr9 gene. Level of expression was not correlated with number of T-DNA integrations, but plants homozygous for the Avr9 gene produced more elicitor protein than heterozygous plants. The amino acid sequence of the processed AVR9 peptide present in apoplastic fluid (AF) of pAVIR1 transformed plants producing the wild-type AVR9 elicitor was identical to that of the wild-type AVR9 peptide isolated from C. fulvum-infected tomato leaves. Transgenic Cf0 genotypes of tomato, obtained by transformation with construct pAVIR21, showed a normal phenotype. However, transgenic F1 plants expressing the Avr9 gene, obtained from crossing transgenic Cf0 genotypes with wild-type Cf9 genotypes, showed delayed growth, necrosis and complete plant death indicating that the AVR9 peptide produced in plants carrying the Cf9 gene is deleterious. The necrotic defence response observed in Cf9 genotypes expressing the Avr9 gene support the potential to apply avirulence genes in molecular resistance breeding.
利用三种构建体研究了番茄真菌病原体黄枝孢菌无毒基因Avr9在植物中的表达。它们包括pAVIR1、pAVIR2和pAVIR21，分别编码野生型AVR9蛋白和两种分别含有病程相关蛋白PR-S和PR-1a信号序列的杂合AVR9蛋白。用这三种构建体获得的转基因烟草植株表现出正常表型，并产生与含有Avr9基因的黄枝孢菌分离株在植物中产生的野生型AVR9激发子具有相同特异性坏死诱导活性的AVR9激发子。表达水平与T-DNA整合数量无关，但Avr9基因纯合的植株比杂合植株产生更多的激发子蛋白。在产生野生型AVR9激发子的pAVIR1转化植株的质外体液（AF）中存在的加工后的AVR9肽的氨基酸序列与从感染黄枝孢菌的番茄叶片中分离的野生型AVR9肽的氨基酸序列相同。用构建体pAVIR21转化获得的番茄转基因Cf0基因型表现出正常表型。然而，通过将转基因Cf0基因型与野生型Cf9基因型杂交获得的表达Avr9基因的转基因F1植株表现出生长延迟、坏死和植株完全死亡，这表明携带Cf9基因的植物中产生的AVR9肽是有害的。在表达Avr9基因的Cf9基因型中观察到的坏死防御反应支持了在分子抗性育种中应用无毒基因的潜力。
