The promoter of the Vicia faba L. VfENOD-GRP3 gene encoding a glycine-rich early nodulin mediates a predominant gene expression in the interzone II-III region of transgenic Vicia hirsuta root nodules

Abstract

We recently reported on the broad bean gene VfENOD-GRP3 encoding a glycine-rich early nodulin. This gene was predominantly expressed in the interzone II-III region of Vicia faba root nodules. The VfENOD-GRP3 promoter contained several sequence motifs potentially involved in the regulation of gene expression. To investigate the molecular basis for the specific VfENOD-GRP3 expression, defined VfENOD-GRP3 promoter fragments were fused to an intron-containing gusAint gene. Agrobacterium rhizogenes ARqual strains carrying these fusions integrated into the TL DNA were used to generate hairy roots on Vicia hirsuta, which subsequently were nodulated. Histochemical analysis of transgenic nodules indicated that a strong gusAint expression in the interzone II-III region was mediated by the -1252/+10 VfENOD-GRP3 promoter region. This reporter gene expression in V. hirsuta was comparable to the location of VfENOD-GRP3 transcripts in V. faba nodules. An analysis of defined promoter fragments revealed that a strong gusAint expression in the interzone II-III region was also mediated by the -737/+10 promoter, whereas the -239/+10 promoter only mediated a weak gusAint expression in the interzone II-III region. Since the -239/+10 promoter fragment did not resemble published nodulin gene promoters, we propose that it contains new sequence motifs involved in mediating gene expression in the interzone II-III region of Vicia nodules.

Knock‐down of the MEP pathway isogene 1‐deoxy‐d‐xylulose 5‐phosphate synthase 2 inhibits formation of arbuscular mycorrhiza‐induced apocarotenoids, and abolishes normal expression of mycorrhiza‐specific plant marker genes

“…, 2002). For the localization of MtDXS2‐1 promoter activity, a 35S‐GUS‐int construct was generated from pGUS‐INT (Küster et al. , 1995) using primers carrying Nco I– Spe I restriction sites (Table S2), and was inserted into pRNAi (Limpens et al.…”

“…An M. truncatula genomic library in the phage vector Lambda Fix II (Stratagene, http://www.stratagene.com; kindly provided by M. Harrison, Boyce Thompson Institute, http://bti.cornell.edu) was screened by standard methods using the MtDXS2 cDNA probe (Walter et al, 2002). For the localization of MtDXS2-1 promoter activity, a 35S-GUS-int construct was generated from pGUS-INT (Kü ster et al, 1995) using primers carrying NcoI-SpeI restriction sites (Table S2), and was inserted into pRNAi (Limpens et al, 2004). In order to create pPMtDXS2-1:GUS-int 2723 bp of the MtDXS2-1 promoter was amplified with primers including the XhoI-NcoI restriction sites (Table S2), and was inserted into p35SGUS-int by replacement of the double CaMV-35S promoter.…”

Two genes encoding different truncated hemoglobins are regulated during root nodule and arbuscular mycorrhiza symbioses of Medicago truncatula

“…The MtTrHb1 (À1405/À15, in relation to the ATG) and the MtTrHb2 (À2620/À80, in relation to the ATG) promoters were inserted as XhoI/EcoRI and XhoI/SmaI fragments, respectively, into the plasmid pGUS-INT (Ku¨ster et al 1995). Both MtTrHb-gusAint fusions were subsequently cloned as XbaI/StuI fragments (filled in using Klenow polymerase) into the SmaI site of the binary Vector pRedRoot (Limpens et al 2004), and the resulting binary vectors were transformed into Agrobacterium rhizogenes Arqua1 (Quandt et al 1993).…”

The mycorrhiza-dependent defensin MtDefMd1 of Medicago truncatula acts during the late restructuring stages of arbuscule-containing cells

“…Fragments were cleaved by Sma I and Eco RI/ Hin dIII, respectively, and were cloned into pk18 [ 50 ]. The promoters were then subcloned into pGUS-INT [ 51 ], containing the gusA int reporter gene. Using Spe I, the transcriptional fusion was cleaved out, Klenow-blunted, and ligated into the Sma I-digested binary vector pRedRoot [ 52 ].…”