Laboratorio de Micología Molecular, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Roque Sáenz Peña 352, (B1876BXD) Bernal, Provincia de Buenos Aires, Argentina.
Microb Biotechnol. 2008 May;1(3):258-69. doi: 10.1111/j.1751-7915.2008.00029.x.
Ectomycorrhiza is a mutualistic symbiosis formed between fine roots of trees and the mycelium of soil fungi. This symbiosis plays a key role in forest ecosystems for the mineral nutrition of trees and the biology of the fungal communities associated. The characterization of genes involved in developmental and metabolic processes is important to understand the complex interactions that control the ectomycorrhizal symbiosis. Agrobacterium-mediated gene transfer (AMT) in fungi is currently opening a new era for fungal research. As whole genome sequences of several fungi are being released studies about T-DNA integration patterns are needed in order to understand the integration mechanisms involved and to evaluate the AMT as an insertional mutagenesis tool for different fungal species. The first genome sequence of a mycorrhizal fungus, the basidiomycete Laccaria bicolor, became public in July 2006. Release of Laccaria genome sequence and the availability of AMT makes this fungus an excellent model for functional genomic studies in ectomycorrhizal research. No data on the integration pattern in Laccaria genome were available, thus we optimized a plasmid rescue approach for this fungus. To this end the transformation vector (pHg/pBSk) was constructed allowing the rescue of the T-DNA right border (RB)-genomic DNA junctions in Escherichia coli. Fifty-one Agrobacterium-transformed fungal strains, picked up at random from a larger collection of T-DNA tagged strains (about 500), were analysed. Sixty-nine per cent were successfully rescued for the RB of which 87% were resolved for genomic integration sequences. Our results demonstrate that the plasmid rescue approach can be used for resolving T-DNA integration sites in Laccaria. The RB was well conserved during transformation of this fungus and the integration analysis showed no clear sequence homology between different genomic sites. Neither obvious sequence similarities were found between these sites and the T-DNA borders indicating non-homologous integration of the transgenes. Majority (75%) of the integrations were located in predicted genes. Agrobacterium-mediated gene transfer is a powerful tool that can be used for functional gene studies in Laccaria and will be helpful along with plasmid rescue in searching for relevant fungal genes involved in the symbiotic process.
外生菌根是树木细根与土壤真菌菌丝体之间形成的互利共生关系。这种共生关系在森林生态系统中对树木的矿物质营养和相关真菌群落的生物学起着关键作用。参与发育和代谢过程的基因的特征对于理解控制外生菌根共生关系的复杂相互作用非常重要。土壤农杆菌介导的基因转移（AMT）在真菌研究中开辟了一个新时代。随着几种真菌的全基因组序列的发布，需要研究 T-DNA 整合模式，以便了解所涉及的整合机制，并评估 AMT 作为不同真菌物种的插入诱变工具。第一个外生菌根真菌担子菌的基因组序列，栓菌 Laccaria bicolor，于 2006 年 7 月公布。栓菌基因组序列的发布和 AMT 的可用性使该真菌成为外生菌根研究中功能基因组研究的理想模型。栓菌基因组中没有整合模式的数据，因此我们为此真菌优化了质粒拯救方法。为此，构建了转化载体（pHg/pBSk），允许在大肠杆菌中回收 T-DNA 右边界（RB）-基因组 DNA 接头。从更大的 T-DNA 标记菌株（约 500 个）库中随机挑选了 51 个农杆菌转化的真菌菌株进行分析。RB 的成功率为 69%，其中 87%解析了基因组整合序列。我们的结果表明，质粒拯救方法可用于解析栓菌中的 T-DNA 整合位点。在该真菌的转化过程中，RB 得到了很好的保守，整合分析表明不同基因组位点之间没有明显的序列同源性。这些位点与 T-DNA 边界之间也没有发现明显的序列相似性，表明转基因的非同源整合。大多数（75%）整合位于预测基因中。土壤农杆菌介导的基因转移是一种强大的工具，可用于栓菌中的功能基因研究，并将有助于质粒拯救，以寻找参与共生过程的相关真菌基因。
