Methods Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol160.Issue1.2000:

prising leghemoglobins and enzymes of nodule carbon and nitrogen metabolism, early nodulins are mainly structural proteins involved in infection or nodule organogenesis [32]. One of the first early nodulin genes specifically activated after infection of legume roots by rhizobia are the Enod12 genes. Up to now, Enod12 sequences were isolated from Pisum sati6um [13,31], Medicago sati6a [1], Medicago truncatula [26] and Vicia sati6a [41]. Enod12 genes code for proline-rich proteins char- acterized by different numbers of PPX 3 pentapep- tide repeats preceded by a signal peptide. The two similar Enod12 genes encoded in P. sati6um PsEnod12a and-b and M. sati6a MsEnod12a and-b differ in the number of PPX 3 repeats. The occurence of PPX 3 pentapeptide repeats defines a category of structural cell-wall proteins designated hydroxyproline rich glycoprotein HRGPs, [35]. Hence, Enod12 proteins are assumed to be struc- tural components of plant cell walls in root nod- ules involved in the reaction of the plant to an infection by rhizobia [31]. In addition to the Enod12 proteins, several proline-rich early nodulins were identified [32], e.g. the well-known Enod5 and Enod2 nodulins as well as the Enod10 [21] and MtPRP4 proteins [44]. Csanadi et al. [6] identified an alfalfa line that does not contain any Enod12 gene. In such plants nodule formation was not impaired, nor was nitrogen fixation reduced. Obviously, at least in M. sati6a root nodule organogenesis and function is not dependent on Enod12 proteins. This might be explained by the existence of similar PRPs that substitute for Enod12 proteins. In infected roots, PsEnod12 transcripts were localized in cells containing an infection thread and cells placed in front of the growing infection thread leading to the suggestion that Enod12 might be involved in the infection process [31]. Later it was demonstrated that the Enod12 gene from M. truncatula is activated in root hairs as early as 3 h after infection [26]. In mature root nodules, Enod12 transcripts were localized in the prefixing zone II [2,31,41]. In contrast to the PsEnod12a and PsEnod12b genes, which are ex- pressed comparably, for the two Enod12 genes from M. sati6a markedly different expression char- acteristics were found. MsEnod12a was activated exclusively in the proximal part of the prefixing zone II of root nodules dependent on the presence of an active meristem, whereas MsEnod12b was detected in root hairs within few hours after appli- cation of NOD factors [2] as it was already demonstrated for PsEnod12a [17] and MtEnod12 [26]. Enod12 genes are also activated in spontanu- ous nodules [27] or by phytohormones [3] indicat- ing that Enod12 gene expression is part of the preexisting plant programme underlying nodule formation. Using fusions of Enod12 promoters from M. sati6a and M. truncatula to the gusA reporter gene, regions mediating activity were identified [3,26]. In case of the PsEnod12 promoters, essen- tial regions were localized within 200 bp upstream of the transcription start sites [42]. Recently, a promoter element was identified in the PsEnod12b promoter that was able to specifically interact with the transcription factor ENBP1 from Vicia sati6a [5]. Interestingly, mutations in this element com- pletely abolished PsEnod12b promoter activity in transgenic root nodules of Vicia hirsuta [14]. The characteristic expression properties of Enod12 genes made them valuable tools to analyse early aspects of legume-Rhizobium interactions. To investigate organ-specific gene expression in broad bean Vicia faba L. nodules, we constructed a nodule-specific cDNA library by differential hy- bridization [24]. Sequence analysis of a cDNA from clone group VfNDS-X7 V6icia f6aba n6odule d6ifferential s6creening, group X7 of this library revealed that this incomplete cDNA encoded a broad bean Enod12 protein. We here report on the expression properties of a Vicia faba Enod12 gene and analyze the promoter in transgenic hairy roots and nodules of Vicia hirsuta. We show that the VfEnod12 promoter fragment isolated is active although it contains a binding site for the tran- scription factor ENBP1 that is altered in a subele- ment that is exactly conserved in all other Enod12 genes identified so far.

2. Methods

2 . 1 . Plant material Vicia faba L. cv. Kleine Thu¨ringer plants were grown in the greenhouse in sand or Seramis clay granules. Two days after sowing, the seedlings were inoculated with Rhizobium leguminosarum bv. 6 iciae VF39 [28] to obtain infected plants. To harvest uninfected roots, the plants were grown in sterile soil as described [24]. Vicia hirsuta hairy tare seeds were obtained from John Chambers Wild Flower Seeds, Kettering, UK. 2 . 2 . cDNA libraries, genomic libraries and cloning of reporter gene fusions A nodule cDNA library and a nodule-specific cDNA library were constructed in lgt11 [18] from polyA + mRNA isolated from root nodules of V. faba L. cv. Kleine Thu¨ringer [24]. A ge- nomic library of V. faba L. cv. Kleine Thu¨ringer was prepared in lEMBL3 [10] according to Sambrook et al. [30]. The complete -1955-41 VfEnod12 promoter region was PCR-amplified using the M13 reverse primer in conjunction with an oligonucleotide spanning the -36-60 re- gion of the VfEnod12 promoter where positions -36-41 were modified to form an EcoRI site. As a template, plasmid pUC18:16-69 was used that contained the 3.3 kb genomic VfEnod12 frag- ment cloned in the EcoRI site of plasmid pUC18 [45]. The -692-41 region was subse- quently released as EcoRVEcoRI fragment and was cloned into the SmaIEcoRI sites in front of the gusAint gene in plasmid pGUS-INT [19]. From the resulting plasmid, the pVfEnod12-gu- sAint fusion was subcloned as SphISalI frag- ment into the TL DNA integration vector pIV2 [19] and the resulting plasmid was transferred to A. rhizogenes ARqua1 using E. coli S17-1 medi- ated conjugation [36]. Correct integration in the TL DNA was verified by Southern hybridiza- tions. 2 . 3 . DNA sequencing and sequence analysis Sequencing reactions were carried out accord- ing to Zimmermann et al. [46] using the ‘Au- toRead Sequencing Kit’ Pharmacia. Sequencing gels were run on the ‘A.L.F. DNA Sequencer’ Pharmacia using sequencing gel mixes of stan- dard composition. All sequences reported here were determined from both strands. Nucleic acid sequences were read using the ‘A.L.F. MAN- AGER V3.0’ software Pharmacia. Deduced amino acid sequences were analysed according to von Heijne [43] and Straden [37] and by using the PCGene software package Intelligenetics, release 6.80. 2 . 4 . Isolation of RNA, northern, cDNA-cDNA and tissue-print hybridizations Total RNA was isolated from nodules 32 days after sowing, uninfected roots 32 days af- ter sowing, leaves 32 days after sowing, seeds 90 days after sowing, epicotyls 8 days after sowing, stems 12 days after sowing and flow- ers 60 days after sowing of broad bean accord- ing to Perlick and Pu¨hler [24]. For time-course hybridizations, total RNA was prepared accord- ingly from infected roots harvested in 2 day in- tervals from day 3 after inoculation with R. leguminosarum bv. 6iciae VF39. For Northern blotting, the amount of 30 mg of total RNA from different tissues was separated elec- trophoretically and blotted onto Hybond – N ny- lon membranes Amersham according to standard protocols [30]. Fifty nanograms of VfEnod12 probe DNA were labelled with 50 mCi of [a 32 P]dATP and hybridizations were carried out as described [24]. Stringent washes were car- ried out at room temperature using 2 × SSC, 0.1 wv SDS 5 min and at 68°C using 0.2 × SSC, 0.1 wv SDS twice for 30 min. cDNA – cDNA hybridizations were carried out according to Fru¨hling et al. [11]. Tissue print hybridizations were carried out as described by Schro¨der et al. [33]. To relate hybridizing regions of the print to distinct nodule zones, nodule sec- tions corresponding to the print were stained for starch in a solution containing 1 wv KI and 1 wv I 2 in distilled water. Subsequently, stained sections were rinsed in distilled water and photographed at the same magnification as the tissue print filter. 2 . 5 . Induction and analysis of transgenic hairy roots and root nodules of V. hirsuta V. hirsuta seeds were surface-sterilized and plants were grown in petri dishes in a growth chamber as described [29]. Transgenic hairy roots of V. hirsuta were generated using A. rhi- zogenes Arqua1 derivatives and nodulated using R. leguminosarum bv. 6iciae Vh5eSm as reported previously [29]. After 2 – 4 weeks, roots and root nodules were analysed histochemically as re- ported by Ku¨ster et al. [19].

3. Results and discussion