U.S. Department of Agriculture-Agricultural Research Service, Davis, California 95616, USA.
Appl Environ Microbiol. 2010 Dec;76(24):7990-6. doi: 10.1128/AEM.01049-10. Epub 2010 Oct 15.
Armillaria root disease is one of the most damaging timber and fruit tree diseases in the world. Despite its economic importance, many basic questions about the biology of the causal fungi, Armillaria spp., are unanswered. For example, Armillaria undergoes matings between diploid and haploid mycelia, which can result in a recombinant diploid without meiosis. Evidence of such somatic recombination in natural populations suggests that this reproductive mode may affect the pathogen's ecology. Investigations of the mechanisms and adaptive consequences of somatic recombination are, however, hampered by the lack of a method to reliably synthesize somatic recombinants. Here we report the first genetic transformation system for the genus Armillaria. We transformed A. mellea with selective markers for use in diploid-haploid matings to reliably synthesize somatic recombinants. This was accomplished with Agrobacterium tumefaciens carrying pBGgHg, which carries the hygromycin phosphotransferase gene (hph). hph was integrated into transformants, as evidenced by serial transfer to selective media, PCR, reverse transcription-PCR (RT-PCR), and Southern hybridization. Nuclear and mitochondrial markers were developed to genotype synthesized mycelia. In matings between a wild-type diploid and hygromycin-resistant haploids (transgenic), we identified recombinant, hygromycin-resistant diploids and, additionally, hygromycin-resistant triploids, all with the mitochondrial haplotype of the haploid partner. Our approach created no mycelium in which the haploid nucleus was replaced by the diploid nucleus, the typical outcome of diploid-haploid matings in Armillaria. This genetic transformation system, in combination with new markers to track chromosomal and cytoplasmic inheritance in A. mellea, will advance research aimed at characterizing the significance of somatic recombination in the ecology of this important fungus.
蜜环菌根腐病是世界上最具破坏性的木材和果树病害之一。尽管其具有重要的经济意义，但有关致病真菌蜜环菌属（Armillaria spp.）的许多基本生物学问题仍未得到解答。例如，蜜环菌经历二倍体和单倍体菌丝体之间的交配，这可能导致未经减数分裂的重组二倍体。在自然种群中存在这种体细胞重组的证据表明，这种生殖方式可能会影响病原体的生态。然而，由于缺乏可靠合成体细胞重组体的方法，对体细胞重组的机制和适应性后果的研究受到了阻碍。在这里，我们报告了第一个用于蜜环菌属的遗传转化系统。我们使用选择性标记物对 A. mellea 进行了转化，用于二倍体-单倍体交配，以可靠地合成体细胞重组体。这是通过携带潮霉素磷酸转移酶基因（hph）的根癌农杆菌 pBGgHg 来实现的。hph 整合到转化体中，这可以通过连续转移到选择性培养基、PCR、逆转录-PCR（RT-PCR）和 Southern 杂交来证明。开发了核和线粒体标记物来对合成菌丝体进行基因型分析。在野生型二倍体和潮霉素抗性单倍体（转基因）之间的交配中，我们鉴定了重组的、潮霉素抗性的二倍体，此外，还鉴定了潮霉素抗性的三倍体，它们都具有单倍体伙伴的线粒体单倍型。我们的方法没有产生单倍体核被二倍体核取代的菌丝体，这是蜜环菌属中二倍体-单倍体交配的典型结果。这种遗传转化系统与用于跟踪 A. mellea 中染色体和细胞质遗传的新标记相结合，将推进旨在描述体细胞重组在这种重要真菌生态学中的意义的研究。
