Transcriptional and posttranscriptional gene silencing are mechanistically related.

Two distinct gene-silencing phenomena are observed in plants: transcriptional gene silencing (TGS), which involves decreased RNA synthesis because of promoter methylation, and posttranscriptional gene silencing (PTGS), which involves sequence-specific RNA degradation. PTGS is induced by deliberate [1-4] or fortuitous production (R.v.B., unpublished data) of double-stranded RNA (dsRNA). TGS could be the result of DNA pairing [5], but could also be the result of dsRNA, as was shown by the dsRNA-induced inactivation of a transgenic promoter [6]. Here, we show that when targeting flower pigmentation genes in Petunia, transgenes expressing dsRNA can induce PTGS when coding sequences are used and TGS when promoter sequences are taken. For both types of silencing, small RNA species are found, which are thought to be dsRNA decay products [7] and determine the sequence specificity of the silencing process [8, 9]. Furthermore, silencing is accompanied by the methylation of DNA sequences that are homologous to dsRNA. DNA methylation is assumed to be essential for regulating TGS and important for reinforcing PTGS [10]. Therefore, we conclude that TGS and PTGS are mechanistically related. In addition, we show that dsRNA-induced TGS provides an efficient tool to generate gene knockouts, because not only does the TGS of a PTGS-inducing transgene fully revert the PTGS phenotype, but also an endogenous gene can be transcriptionally silenced by dsRNA corresponding to its promoter.

Cloning of sucrose:sucrose 1-fructosyltransferase from onion and synthesis of structurally defined fructan molecules from sucrose.

Sucrose (Suc):Suc 1-fructosyltransferase (1-SST) is the key enzyme in plant fructan biosynthesis, since it catalyzes de novo fructan synthesis from Suc. We have cloned 1-SST from onion (Allium cepa) by screening a cDNA library using acid invertase from tulip (Tulipa gesneriana) as a probe. Expression assays in tobacco (Nicotiana plumbaginifolia) protoplasts showed the formation of 1-kestose from Suc. In addition, an onion acid invertase clone was isolated from the same cDNA library. Protein extracts of tobacco protoplasts transformed with this clone showed extensive Suc-hydrolyzing activity. Conditions that induced fructan accumulation in onion leaves also induced 1-SST mRNA accumulation, whereas the acid invertase mRNA level decreased. Structurally different fructan molecules could be produced from Suc by a combined incubation of protein extract of protoplasts transformed with 1-SST and protein extract of protoplasts transformed with either the onion fructan:fructan 6G-fructosyltransferase or the barley Suc:fructan 6-fructosyltransferase.

Fructan of the inulin neoseries is synthesized in transgenic chicory plants (Cichorium intybus L.) harbouring onion (Allium cepa L.) fructan:fructan 6G-fructosyltransferase.

Fructan (polyfructosylsucrose) is an important storage carbohydrate in many plant families. fructan:fructan 6G-fructosyltransferase (6G-FFT) is a key enzyme in the formation of the inulin neoseries, a type of fructan accumulated by members of the Liliales. We have cloned the 6G-FFT from onion by screening a cDNA library using barley sucrose:fructan 6-fructosyltransferase (6-SFT) as a probe. The deduced amino acid sequence showed a high homology with plant invertases and 6-SFT. Incubation of protein extracts from transgenic tobacco plants with the trisaccharide 1-kestose and sucrose resulted in the formation of neokestose and fructans of the inulin neoseries with a degree of polymerization up to six. Introduction of the onion 6G-FFT into chicory resulted in the synthesis of fructan of the inulin neoseries, in addition to the synthesis of linear inulin.

A novel type of DNA-binding protein interacts with a conserved sequence in an early nodulin ENOD12 promoter.

The pea genes PsENOD12A and PsENOD12B are expressed in the root hairs shortly after infection with the nitrogen-fixing bacterium Rhizobium leguminosarum bv. viciae or after application of purified Nod factors. A 199 bp promoter fragment of the PsENOD12B gene contains sufficient information for Nod factor-induced tissue-specific expression. We have isolated a Vicia sativa cDNA encoding a 1641 amino acid protein, ENBP1, that interacts with the 199 bp ENOD12 promoter. Two different DNA-binding domains were identified in ENBP1. A domain containing six AT-hooks interacts specifically with an AT-rich sequence located between positions -95 and -77 in the PsENOD12B promoter. A second domain in ENBP1 is a cysteine-rich region that binds to the ENOD12 promoter in a sequence non-specific but metal-dependent way. ENBP1 is expressed in the same cell types as ENOD12. However, additional expression is observed in the nodule parenchyma and meristem. The presence of three small overlapping ORFs in the 5'-untranslated region of the ENBP1 cDNA indicates that ENBP1 expression might be regulated at the translational level. The interaction of ENBP1 with a conserved AT-rich element within the ENOD12 promoter and the presence of the ENBP1 transcript in cells expressing ENOD12 strongly suggest that ENBP1 is a transcription factor involved in the regulation of ENOD12. Finally, the C-terminal region of ENBP1 shows strong homology to a protein from rat that is specifically expressed in testis tissue.

A 200 bp region of the pea ENOD12 promoter is sufficient for nodule-specific and nod factor induced expression.

ENOD12 is one of the first nodulin genes expressed upon inoculation with Rhizobium and also purified Nod factors are able to induce ENOD12 expression. The ENOD12 gene family in pea (Pisum sativum) has two members. A cDNA clone representing PsENOD12A [26] and a PsENOD12B genomic clone [7] have been previously described. The isolation and characterization of a PsENOD12A genomic clone is presented in this paper. By using a Vicia hirsuta-Agrobacterium rhizogenes transformation system it is shown that both genes have a similar expression pattern in transgenic V. hirsuta root nodules. Promoter analyses of both PsENOD12 promoters showed that the 200 bp immediately upstream of the transcription start are sufficient to direct nodule-specific and Nod factor-induced gene expression.

VsENOD5, VsENOD12 and VsENOD40 expression during Rhizobium-induced nodule formation on Vicia sativa roots.

We isolated ENOD5, ENOD12 and ENOD40 homologues from Vicia sativa and studied their expression pattern during Rhizobium-induced nodule formation. Comparison of the VsENOD40 nucleotide sequence with the pea, soybean and alfalfa ENOD40 sequences showed that the sequences contain two conserved regions, called region I and region II. Comparison of all the potential open reading frames (ORFs) showed that all the five different ENOD40 clones encode a highly conserved small polypeptide of 12 or 13 amino acids encoded by an ORF located in region I. Furthermore we studied with in situ hybridization the expression pattern of VsENOD5, VsENOD12 and VsENOD40 during Rhizobium-induced nodule formation. Although the expression of these genes is largely similar to that of the pea counterparts, differences where found for the expression of VsENOD12 and VsENOD40 in Vicia. VsENOD12 is expressed in the whole prefixation zone II, whereas in pea ENOD12 is only expressed in the distal part of this zone. VsENOD40 is expressed in the uninfected cells of interzone II-III, while in pea ENOD40 is expressed in both the uninfected and infected cells of this zone.

Early nodulin gene expression during Nod factor-induced processes in Vicia sativa.

Rhizobium leguminosarum bv. viciae-secreted Nod factors are able to induce root hair deformation, the formation of nodule primordia and the expression of early nodulin genes in Vicia sativa (vetch). To obtain more insight into the mode of action of Nod factors the expression of early nodulin genes was followed during Nod factor-induced root hair deformation and nodule primordium formation. The results of these studies suggested that the expression of VsENOD5 and VsENOD12 is not required for root hair deformation. In the Nod factor-induced primordia both VsENOD12 and VsENOD40 are expressed in a spatially controlled manner similar to that found in Rhizobium-induced nodule primordia. In contrast, VsENOD5 expression has never been observed in Nod factor-induced primordia, showing that the induction of VsENOD5 and VsENOD12 expression are not coupled. VsENOD5 expression is induced in the root epidermis by Nod factors and in Rhizobium-induced nodule primordia only in cells infected by the bacteria, suggesting that the Nod factor does not reach the inner cortical cells.