Identification of a domain of unknown function family gene involved in mediating the drought tolerance of Achnatherum inebrians colonised by an Epichloë endophyte

Abstract

The increasing frequencies of severe global droughts highlights the importance of cultivating high-yielding, drought-resistant crops. The Epichloë-Achnatherum inebrians symbiosis exhibits remarkable drought tolerance and investigating the molecular mechanism responsible for this trait would provide important information for drought tolerance in graminaceous crops. The transcriptome and proteome of A. inebrians and Epichloë-A. inebrians symbiosis was compared under the conditions of 100% and 40% soil moisture saturation, respectively. The expression of numerous genes exhibited differential patterns even at 100% soil moisture saturation, with the divergence intensifying significantly at 40% saturation. To detect key drought responsive genes, the top 20 genes exhibiting the most significant differences in expression levels were cloned. Utilizing a Agrobacterium-mediated transient expression system in Nicotiana benthamiana and a luciferase (LUC) gene drove by the promoter of a drought response marker gene as a reporter, the functions of these candidate genes in regulating drought response were detected. A DUF (Domain of Unknown Function) family gene, named AiDR6, significantly induced LUC expression under both of the control and PEG6000 treatment conditions. Intriguingly, subcellular localization analysis revealed that AiDR6 is specifically localized to chloroplasts of stomata. Furthermore, compared to the wild type, N. benthamiana overexpressing AiDR6 exhibited a very distinct stomatal closure phenotype. These results suggest that AiDR6 plays a critical role in regulating the Epichloë endophyte mediated A. inebrians drought tolerance. The functional analysis of AiDR6, along with further elucidation of its regulatory mechanisms, will advance the understanding of drought tolerance mechanisms in Epichloë-grass symbionts.