Screening for differentially expressed genes in Anoectochilus roxburghii (Orchidaceae) during symbiosis with the mycorrhizal fungus Epulorhiza sp.

Mycorrhizal fungi promote the growth and development of plants, including medicinal plants. The mechanisms by which this growth promotion occurs are of theoretical interest and practical importance to agriculture. Here, an endophytic fungus (AR-18) was isolated from roots of the orchid Anoectochilus roxburghii growing in the wild, and identified as Epulorhiza sp. Tissue-cultured seedlings of A. roxburghii were inoculated with AR-18 and co-cultured for 60 d. Endotrophic mycorrhiza formed and the growth of A. roxburghii was markedly promoted by the fungus. To identify genes in A. roxburghii that were differentially expressed during the symbiosis with AR-18, we used the differential display reverse transcription polymerase chain reaction (DDRT-PCR) method to compare the transcriptomes between seedlings inoculated with the fungus and control seedlings. We amplified 52 DDRT-PCR bands using 15 primer combinations of three anchor primers and five arbitrary primers, and nine bands were re-amplified by double primers. Reverse Northern blot analyses were used to further screen the bands. Five clones were up-regulated in the symbiotic interaction, including genes encoding a uracil phosphoribosyltransferase (UPRTs; EC 2.4.2.9) and a hypothetical protein. One gene encoding an amino acid transmembrane transporter was down-regulated, and one gene encoding a tRNA-Lys (trnK) and a maturase K (matK) pseudogene were expressed only in the inoculated seedlings. The possible roles of the above genes, especially the UPRTs and matK genes, are discussed in relation to the fungal interaction. This study is the first of its type in A. roxburghii.