Cloning and expression of a down-regulated gene (TrEnodDR1) of white clover responded by the nod genes derived from Rhizobiumleguminosarum bv. trifolii strain 4S

Abstract

The nodulation genes of Rhizobiumleguminosarum bv. trifolii 4S (strain 4S) were cloned into cosmid vector pLAFR1 named pC4S8 which was contained nodNMLFEDABCIJ and a part of nodT as an insert. The pC4S8 was transferred to strain H1, Sym plasmid (pRt4Sa) cured strain of strain 4S, and isolated as Tc resistant and nodulation restored mutant, strain H1(pC4S8). During infection process of this strain, visible symbiotic features, such as root hair curling (Hac), root hair deformation (Had) and infection thread formation (Inf) were also restored. The nodule forming ability of strain H1(pC4S8) was increased 3–4 times in nodule number than that of strain 4S. Then, to investigate the effect of Rhizobiumnod genes on the host plant (Trifoliumrepens L.) gene expression, cDNAs which were responded to the inoculation of rhizobia were differentially screened based on the presence or absence of nod genes treated with strains H1(pC4S8) or H1, respectively. The cDNA, TrEnodDR1 (Trifoliumrepensearlynodulindownregulation1) gene was isolated from cDNA library prepared from white clover seedlings treated with nod− strain H1, but didn't exhibit in nod+ treated cDNA library, as a down-regulated gene. Expression analysis of TrEnodDR1 was performed in various tissues of white clover, it is suppressed in root nodule and also strongly suppressed by the inoculation of rhizobia in the seedlings. It is discussed that TrEnodDR1 gene is suppressed when the white clover comes into symbiosis with rhizobia.