Cloning, expression and localization of mycorrhizal responsive CyENT3 from Cymbidium hybridum

Abstract

The ecological and physiological significances of orchid mycorrhizae (OM) are well documented. However, very little is known about the molecular regulation of these associations, especially for adult green orchids. The equilibrative nucleoside transporters (ENTs) contains a group of newly characterized active proteins which may involve in nucleoside and nucleoside-type cytokinin transport, and H+-ATPases are considered to be essential to sustain arbuscular mycorrhizal colonization. In this paper, we studied the expression profiles of three OM-associated candidate genes, one equilibrative nucleoside transporter 3-like gene (CyENT3) and two H+-ATPase-like genes (CyHA1 and CyHA2). The transcript levels for CyENT3 increased gradually after 6dpi and reached a significant peak at 15dpi when treated with mycorrhizal fungi compared to that of mock-inoculated control and non-mycorrhizal fungi (NMF) treatment. The expression of CyHA1 and CyHA2 was also significantly up-regulated in Cymbidium roots in response to mycorrhizal fungi at 15 and 20dpi. The highest transcript levels of these three genes were synchronized with the time point of active peloton formation in orchid mycorrhizae. The full length cDNA of CyENT3 is 1642bp long with an ORF of 1254bp encoding a 418 amino acid polypeptide which shows the highest similarity (70%) to AtENT3 from Arabidopsis. The CyENT3 protein is further localized at plasma membrane. Our results revealed that the CyENT3 was membrane bound protein and probably a proton-coupled transporter. The data also suggest a potential role of the CyENT3 in meeting the drastically increased demands for nucleosides in OM formation.