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• W2083150981 abstract "Article1 December 1998free access RPE, a plant gene involved in early developmental steps of nematode feeding cells Bruno Favery Bruno Favery INRA, Laboratoire de Biologie des Invertébrés, 123 bd F. Meilland, 06600 Antibes, France Search for more papers by this author Philippe Lecomte Philippe Lecomte INRA, Laboratoire de Biologie des Invertébrés, 123 bd F. Meilland, 06600 Antibes, France Search for more papers by this author Nathalie Gil Nathalie Gil INRA, Laboratoire de Biologie des Invertébrés, 123 bd F. Meilland, 06600 Antibes, France Search for more papers by this author Nicole Bechtold Nicole Bechtold INRA, Laboratoire de Génétique et d'Amélioration des Plantes, Route de Saint Cyr, 78026 Versailles, Cedex, France Search for more papers by this author David Bouchez David Bouchez INRA, Laboratoire de Biologie Cellulaire, Route de Saint Cyr, 78026 Versailles, Cedex, France Search for more papers by this author Antoine Dalmasso Antoine Dalmasso INRA, Laboratoire de Biologie des Invertébrés, 123 bd F. Meilland, 06600 Antibes, France Search for more papers by this author Pierre Abad Corresponding Author Pierre Abad INRA, Laboratoire de Biologie des Invertébrés, 123 bd F. Meilland, 06600 Antibes, France Search for more papers by this author Bruno Favery Bruno Favery INRA, Laboratoire de Biologie des Invertébrés, 123 bd F. Meilland, 06600 Antibes, France Search for more papers by this author Philippe Lecomte Philippe Lecomte INRA, Laboratoire de Biologie des Invertébrés, 123 bd F. Meilland, 06600 Antibes, France Search for more papers by this author Nathalie Gil Nathalie Gil INRA, Laboratoire de Biologie des Invertébrés, 123 bd F. Meilland, 06600 Antibes, France Search for more papers by this author Nicole Bechtold Nicole Bechtold INRA, Laboratoire de Génétique et d'Amélioration des Plantes, Route de Saint Cyr, 78026 Versailles, Cedex, France Search for more papers by this author David Bouchez David Bouchez INRA, Laboratoire de Biologie Cellulaire, Route de Saint Cyr, 78026 Versailles, Cedex, France Search for more papers by this author Antoine Dalmasso Antoine Dalmasso INRA, Laboratoire de Biologie des Invertébrés, 123 bd F. Meilland, 06600 Antibes, France Search for more papers by this author Pierre Abad Corresponding Author Pierre Abad INRA, Laboratoire de Biologie des Invertébrés, 123 bd F. Meilland, 06600 Antibes, France Search for more papers by this author Author Information Bruno Favery1, Philippe Lecomte1, Nathalie Gil1, Nicole Bechtold2, David Bouchez3, Antoine Dalmasso1 and Pierre Abad 1 1INRA, Laboratoire de Biologie des Invertébrés, 123 bd F. Meilland, 06600 Antibes, France 2INRA, Laboratoire de Génétique et d'Amélioration des Plantes, Route de Saint Cyr, 78026 Versailles, Cedex, France 3INRA, Laboratoire de Biologie Cellulaire, Route de Saint Cyr, 78026 Versailles, Cedex, France *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:6799-6811https://doi.org/10.1093/emboj/17.23.6799 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Sedentary plant-parasitic nematodes are able to induce the redifferentiation of root cells into multinucleate nematode feeding sites (NFSs). We have isolated by promoter trapping an Arabidopsis thaliana gene that is essential for the early steps of NFS formation induced by the root-knot nematode Meloidogyne incognita. Its pattern of expression is similar to that of key regulators of the cell cycle, but it is not observed with the cyst nematode. Later in NFS development, this gene is induced by both root-knot and cyst nematodes. It encodes a protein similar to the D-ribulose-5-phosphate 3-epimerase (RPE) (EC 5.1.3.1), a key enzyme in the reductive Calvin cycle and the oxidative pentose phosphate pathway (OPPP). Quantitative RT–PCR showed the accumulation of RPE transcripts in potato, as in Arabidopsis NFS. Homozygous rpe plants have a germination mutant phenotype that can be rescued in dwarf plants on sucrose-supplemented medium. During root development, this gene is expressed in the meristems and initiation sites of lateral roots. These results suggest that the genetic control of NFSs and the first stages of meristem formation share common steps and confirms the previous cytological observations which indicate that root cells undergo metabolic reprogramming when they turn into NFSs. Introduction Among plant pathogens, sedentary endoparasitic nematodes interact with their hosts in a most fascinating way. They are able to induce the redifferentiation of root cells into nematode feeding sites (NFSs). Nematode growth and reproduction depend on the establishment of these NFSs. Nematodes withdraw food from NFSs until the completion of their life cycle, causing a tremendous threat to crop production worldwide (Sasser and Freckman, 1987). It is not yet understood how these nematodes cause such alterations, but it is suspected that glandular secretions injected into plant cells interact directly or indirectly with the plant nuclear genome (Hussey, 1989). Cytological observations have indicated that NFSs are multinucleated with an enlargement of the nucleus and the nucleolus. Compared with normal cells, NFSs also show an increase in cytoplasmic density, a loss of normal vacuolation and a proliferation of cell organelles. Another characteristic feature of these structures is the development of cell-wall ingrowths, typical of transfer cells (Jones, 1981). These cell-wall ingrowths increase the surface area of the associated membrane and thus facilitate the import of elaborated photosynthates, minerals and other metabolites. Depending on the nematode species, the initial feeding cell develops into either a syncytium (for cyst nematodes such as Heterodera spp. and Globodera spp.) or a system of giant cells (for the root-knot nematodes Meloidogyne spp.) (Jones, 1981). Syncytia result from cell fusions after cell-wall dissolutions between the initial cell on which the nematode starts feeding and an increasing number of neighboring cells. Up to 200 cells can be incorporated in a large syncytium. Conversely, giant cell formation is the result of repeated nuclear divisions of the initial feeding cell without cytokinesis (Huang, 1985). Each root-knot nematode triggers the development of five to seven giant cells, each containing as many as 100 nuclei, which have undergone vast endoreduplication (Wiggers et al., 1990). Because Meloidogyne species can induce similar giant cells in several thousand host species, they probably interact with some fundamental key steps of the plant cell cycle (Niebel et al., 1996). In addition, root-knot nematode development is accompanied by divisions of cortical cells around the NFS, giving rise to a typical root-knot or gall. These complex morphological and physiological changes during the establishment of NFSs are reflected by altered gene expression in affected root cells (Gheysen et al., 1996; Williamson and Hussey, 1996). Approaches based on differential gene expression between healthy and infected roots have allowed the identification of cDNA clones with homology to several known plant defense genes (Niebel et al., 1993, 1995; Lambert, 1995). In parasitized cells, there is upregulation of cDNAs homologous to a key component of the protein ubiquitination pathway (E2 enzyme), a large subunit of RNA polymerase II, a Myb-type transcription factor and a plasmalemma H+-ATPase (Bird and Wilson, 1994). There is also an upregulation of a late embryogenesis-abundant protein (Van der Eycken et al., 1996). Sequencing of differentially expressed genes and computer searching of molecular data banks might indicate a putative function for the products they encode. This approach, however, must be coupled with biochemical and physiological investigations if their actual function is to emerge. More direct evidence for the role of known plant genes in the establishment or maintenance of NFSs came from a variety of promoter–gusA fusion constructs introduced into Arabidopsis and tobacco (Goddijn et al., 1993; Niebel et al., 1996). It has thereby been shown that the root-specific promoter TobRB7 (Conkling et al., 1990), which encodes a presumed water channel expressed in root meristematic and immature vascular cylinder regions, is reactivated in tobacco giant cells induced by Meloidogyne incognita (Yamamoto et al., 1991; Opperman et al., 1994a). Similarly, transcriptional activation of cell cycle markers such as the cyclin-dependent kinase CDC2a and the mitotic cyclin CYC1At is observed during the early stages of NFS formation (Niebel et al., 1996). Moreover, many other genes are downregulated in NFSs. For example, promoters of the bacterial nopaline synthase and the plant phenylalanine ammonia-lyase I gene, which are highly active in non-infected roots, are silenced within a few days of nematode infection (Goddijn et al., 1993). To identify new genes and obtain a more comprehensive view of the molecular mechanisms underlying the induction and maintenance of NFSs, a promoter-trapping strategy was developed, with a promoterless β-glucuronidase (GUS) construct being introduced randomly into the Arabidopsis genome via Agrobacterium T-DNA transformation (Kertbundit et al., 1991; Topping et al., 1991; Goddijn et al., 1993). This ‘tagging’ approach has been used in several laboratories, and tagged lines have recently been identified. However, despite interesting expression patterns in NFSs, none of these T-DNA flanking regions have presented homology with known genes (Barthels et al., 1997). Here we report the molecular characterization of a strongly upregulated nematode-responsive gene in the early steps of giant cell formation induced by M.incognita in Arabidopsis. This gene encodes for the D-ribulose-5-phosphate 3-epimerase (RPE), an enzyme involved in the pentose phosphate pathway. This upregulation is also observed in syncytia induced by Heterodera schachtii, but at a lower level and later after nematode infection. Moreover, we demonstrated that this gene is similarly regulated in potato after infection by both cyst and root-knot nematodes. Finally, the implication of this enzyme in root apex and lateral root initiation sites indicates that the genetic control of NFSs and root formation share common steps and suggests that root cells undergo metabolic reprogramming when turning into NFSs. Results Characterization of the RPE-tagged line To isolate molecularly tagged lines showing GUS induction or repression in galls, we screened a collection of T-DNA-tagged Arabidopsis lines (ecotype WS) obtained by in planta transformation (Bechtold et al., 1993; Bouchez et al., 1993). These lines were screened in a GUS assay 7 days after infection with M.incognita. Of 3000 T-DNA tagged lines tested, 25 transgenic lines showed increased GUS expression, and three transgenic lines showed a repressed GUS expression in the galls. In other tissues, specific GUS activity was detected in 7% of the transformants. None of the tagged lines showed GUS expression that was exclusively restricted to the galls. Expression was observed in other parts of the plants and was always found in the root apex. One of these lines, named RPE, showed early and strong GUS expression in the galls. It presented an abnormal segregation (2:1 instead of 3:1) of the kanamycin marker carried by the T-DNA, suggesting mutant-impaired seed germination (embryo-lethal, seed viability or germination mutant). Of the 435 T2 seeds tested in bulk, 287 grew on kanamycin medium and displayed a normal phenotype compared with the wild type. This frequency fitted well with the hypothesis of the ratio of kanamycin-resistant (Kr) to sensitive (Ks) being 2:1 (χ2 = 0.09; P >0.05), suggesting also that the T-DNA was inserted in one nuclear locus. To analyze precisely at which stage of the interaction the GUS gene is activated, we infected the RPE line in vitro. Time-course experiments pointed out the presence of an early induced plant promoter that activates the transgene in young galls less than 3 days post-infection (d.p.i.), that is to say, 24–48 h after giant cell initiation (Jones, 1981; Wyss et al., 1992). No GUS expression was detected at the penetration site (in the elongation zone) nor during nematode migration. The RPE line showed a strong GUS expression at 7 d.p.i. in galls induced by M.incognita (Figure 1A). This expression was maintained until sexual maturity of the females. To determine the localization of GUS expression, we made thin cryosections of the galls. Cross sections of 10-day-old galls clearly showed GUS staining in the giant cells of the gall (Figure 1B). No significant GUS activity was seen in cortical cells surrounding the feeding cells. Similar results were obtained with other Meloidogyne species such as M.javanica and M.hapla, and with the beet cyst nematode H.schachtii Schmidt (data not shown). Nevertheless, the latter species induced GUS expression later in the feeding cell (15 d.p.i.). During plant development, the GUS gene was expressed in the root meristem and in part of the elongation zone, in which cells divide and expand (Figure 2A). GUS activity was also visible early in the lateral root primordia, before visible evidence of root formation (Figure 2B). High levels of expression were also observed in aerial parts of the plant such as flowers (Figure 2C). In mature embryos from dry seeds, GUS activity was observed through more-intense staining in the zone corresponding to the root apical meristem and in cotyledons (Figure 2D), young tissues with active DNA synthesis for cell polyploidization (Brown et al., 1991). Figure 1.GUS expression in galls of RPE-tagged line induced by M.incognita. (A) Localized GUS expression in root gall (arrow) 7 days after infection. (B) Cross-section of a gall 10 days after infection. Galls were excised from histochemical β-glucuronidase- (GUS) stained plants, fixed and cryosectioned. Sections, examined under dark-field light, clearly showed GUS expression (pink precipitate) limited to giant cells. The head of the feeding nematode (N) can be seen at the edge of the giant cells (*). Bar, 100 μm. Download figure Download PowerPoint Figure 2.Histochemical localization of GUS activity in the RPE T-DNA tagged Arabidopsis line. (A) Root apex. The meristematic (MZ), elongation (EZ) and specialization zones (SZ) are indicated.(B) Lateral root primordia. (C) Flower. (D) Mature embryo from dry seed. RAP, root apical meristem; C, cotyledon. Download figure Download PowerPoint To ascertain whether the T-DNA insertion was closely linked to the rpe mutation, we analyzed cosegregation of the mutant phenotype with the T-DNA insertion. Fifty T4 progenies resulting from each of the 40 selfed Kr T3 plants segregated with the expected ratios of 1/4 non-germinating seeds (mutant homozygous phenotype) and 3/4 germinating seeds of which 2/3 were Kr and 1/3 Ks (data not shown; χ2 = 0.10; P >0.05). Thus there was no recombination between the rpe mutation and the T-DNA cassette. These results suggested that rpe mutation was recessive; it prevented germination when homozygous and was actually linked to the T-DNA. Molecular cloning of the RPE gene DNA hybridization analysis of RPE plants through internal fragments of the T-DNA, GUS (Figure 3A) and left border (data not shown), confirmed that the RPE line carries a single intact T-DNA insert. A 515 bp genomic DNA fragment adjacent to the right border of the T-DNA was isolated by kanamycin plasmid rescue with PstI restriction sites (Bouchez et al., 1996) (Figure 3B). Using an inverse PCR strategy (IPCR; Earp et al., 1990) we cloned 657 bp of DNA flanking the left border (LB) of the T-DNA (BglII site from the IPCR primers T4 and T5) (Figure 3B). These two flanking sequences were used as probes on a Southern blot and indicated that the cloned fragments corresponded to a unique genomic fragment (data not shown). To analyze the insertion point of the T-DNA, we designed the oligonucleotides LA2 and RA2 (Figure 3B). Comparison of the nucleotide sequences of the transformed line with wild-type sequence revealed that a deletion of 77 bp and an insertion of a 5 bp filler sequence at the end of the right border resulted from the insertion of the T-DNA. In addition, typical deletions in the 24 bp repeat of the left and right borders were observed; they resulted in the presence of only one nucleotide from the left border at the insertion junction (Gheysen et al., 1987; Mayerhofer et al., 1991). Figure 3.Molecular analysis of the insertional mutation at the RPE locus. (A) Southern blot analysis of digested DNA from RPE line carrying a single T-DNA copy. The blot was hybridized with GUS probe. B, BglII; E, EcoRV; P, PstI; S, SmaI; X, XbaI. (B) Partial restriction map of the rpe gene mutated by insertion of the T-DNA. Arrows indicate coding sequences and black boxes indicate promoter and terminal regions. RB and LB correspond to the right and leftT-DNA borders, respectively. uidA, coding region of the β-glucuronidase (reporter gene) from E.coli; 3′nos and P nos, 3′ region and promoter regions of the nopaline synthase gene; 3′ocs, 3′ region of the octopine synthase gene; nptII, neomycin phosphotransferase II; P 35S, promoter region of the Cauliflower Mosaic Virus; bar, coding region of the basta resistance gene from Streptomyces hygroscopus; 3′g7, 3′ region of gene 7 from the T-DNA of pTi15955 (Bouchez et al., 1993). The positions of the probes corresponding to uidA (GUS) and Left Border (LB) of T-DNA are indicated by thick bars. The striped box designates the genomic DNA deletion after T-DNA integration. Oligonucleotides T4 and T5 used for IPCR, U1, RA2, LA2 and LA1 are indicated by small arrows. Download figure Download PowerPoint Sequence analysis of the RPE cDNA The RPE cDNA was cloned by rapid amplification of cDNA ends (5′ and 3′ RACE; Frohman et al., 1988) by using poly(A)+ RNAs from the wild-type Arabidopsis thaliana ecotype WS. Only one class of transcripts was detected. The RPE cDNA is 1259 nucleotides long with an open reading frame (ORF) of 281 amino acids, and with 5′- and 3′-untranslated regions (UTR) of 106 and 311 (268 + 43) nucleotides, respectively (Figure 4A). Translation was assumed to begin at nucleotide 107, the first ATG codon of the open reading frame. The context of this ATG does not match the plant consensus sequence (TAAACAATGGCTA; Joshi, 1987). Nevertheless, the nucleotide 3 bp upstream from the initiation codon is a purine, which is highly conserved in eukaryotic genes (Kozak, 1986). The 3′ UTR contains two putative polyadenylation signal sequences, AATAAA and a G/T cluster (GTGTTTTT) at 22 bp and 37 bp, respectively, upstream from the polyadenylation site (Dean et al., 1986). Blast search (Altschul et al., 1990) on the Stanford A.thaliana database revealed a strong sequence identity (98.6% at nucleotide level), with eight expressed sequence tags (EST) from the A.thaliana ecotype Columbia (Figure 4A). cDNA and genomic sequence alignments demonstrated that the RPE gene is interrupted by nine introns, including one in the 3′ UTR. The T-DNA was inserted into the third intron, and its resulting integration (deletion of 77 bp and insertion of 5 bp filler sequence) placed the ATG of the uidA gene in-frame with the rpe gene and allowed a functional translational β-glucuronidase gene fusion. Both introns and exons were characterized; they varied in size from 71 to 286 and from 60 to 268 nucleotides, respectively. The consensus sequences for the intron/exon splice junctions correspond to those reported for plants (White et al., 1992). A Southern blot analysis of the WS genomic DNA was performed using the cloned cDNA as a probe. The gene appears as a single copy per haploid genome of A.thaliana (Figure 4B), even at low stringency hybridization and wash conditions (data not shown). Figure 4.Organization of the RPE gene. (A) Structure of the RPE gene and its transcript. Solid black lines designate introns; open boxes designate exons (I–X, determinated from cDNA); shaded boxes designate untranslated sequences; the grey box designates putative signal peptide; and the striped box designates genomic DNA deletion after T-DNA integration. The T-DNA is inserted into the third intron. ATG (initiation codon) and TGA (stop codon) are indicated. Arabidopsis EST (Expressed Sequence Tagged) showing high homologies with the gene were obtained after a BlastN search on the Stanford Arabidopsis database (Altschul et al., 1990). (B) Southern blot analysis of 5 μg genomic DNA isolated from wild-type (WS) Arabidopsis. The blot was hybridized with 32P-labeled RPE cDNA. D, DraI cut once; while B, BamHI, and E, EcoRI, do not cut in the RPE gene. Download figure Download PowerPoint The predicted amino acid sequence of the RPE protein was compared to the NCBI database by the Blast network service. The protein is closely related to RPEs (EC 5.1.3.1) across species, with two conserved regions (Figure 5). RPE catalyzes the reversible interconversion of ribulose-5-phosphate and xylulose-5-phosphate in the pentose phosphate pathway. In particular 85, 81 and 80% of the resulting amino acids were identical to those of chloroplastic RPE from spinach (Nowitzki et al., 1995), potato (Teige et al., 1995) and rice (protein sequence deduced from rice RPE cDNA obtained by RACE PCR with primers localized on rice ESTs D39200 and D48105), respectively. This high degree of conservation between monocotyledonous and dicotyledonous plant sequences is typical for enzymes of sugar phosphate metabolism (Nowitzki et al., 1995). The region where the encoded Arabidopsis protein is similar to other bacterial or eukaryotic cytosoles (Figure 5) is preceded by a 46-amino-acid sequence with typical properties of chloroplast transit peptides (Von Heijne et al., 1989; Gavel and Von Heijne, 1990). This region is rich in the hydroxylated amino acids serine (19.6%) and threonine (8.7%). We thus predict that RPE is a plastid-localized protein. Figure 5.Sequence alignment of the deduced amino acid sequence of Arabidopsis RPE (RPE_ATH) with RPE-related protein sequences. Sources of sequence are Solanum tuberosum, STPPEPIMR (Z50098), Teige et al., 1995); Spinacia oleracea, SPIR5P3E (L42328, Nowitzki et al., 1995); Oriza sativa, RPE_RICE; Synechocystis sp., RPE_SYN (D90911, Kaneko et al., 1996); Serratia marcescens, RPE_SERMA (P45455); Haemophilus influenza, RPE_HAEIN (P44756, Fleischmann et al., 1995); Alcaligenes eutrophus, RPEP_ALCEU (Q04539) and RPEC_ALCEU (P40117, Kusian et al., 1992); E.coli, RPE_ECOLI (P32661, Lyngstadaas et al., 1995), YJCU_ECOLI (P32719) and YLHK_ECOLI (P39362, Blattner et al., 1993); Rhodobacter capsulatus, RPE_RHOCA (P51012, Larimer et al., 1995); Rhodospirillum rubrum, RPE_RHORU (P51013, Falcone and Tabita, 1993); Mycobacterium tuberculosis, RPE_MYCOB (Z80108); and Saccharomyces cerevisiae, RPE_YEAST (P46969, Miosga and Zimmermann, 1996). Strictly conserved residues are indicated with an asterisk. Identical amino acid residues are colored red. The putative peptide signal and the two conserved regions selected as RPE signature patterns (PDOC00833, Prosite PS01085 and PS01086) are indicated in boxes. The dodecapeptide below the yeast sequence was set apart from the alignment since the corresponding DNA insertion relative to the other sequences is suspiciously intron-like (Nowitzki et al., 1995). The entire sequences of the RPE cDNA from A.thaliana and rice are available from DDBJ/EMBL/GenBank under accession Nos AF015274 and AF047444, respectively. Download figure Download PowerPoint Homologies were found also with the RPE from bacteria and yeast. Amino acid identity was 65% with its homolog from the cyanobacteria Synechocystis (Kaneko et al., 1996), and 50 and 48%, respectively, with the corresponding enzymes of Serratia marcescens and Haemophilus influenzae (Fleischmann et al., 1995). In addition, 42% amino acid identity was found with RPE_YEAST (Miosga and Zimmermann, 1996) and RPE_MYCOB (Figure 5). To determine the relationship between these enzymes, we carried out a phylogenetic analysis with a heuristic tree-building program. Plant RPE sequences clustered and formed a lineage distinct from that of yeast RPE. They are more similar to the cyanobacterial and eubacterial homologs than to the other eukaryotic sequence, RPE_YEAST (Figure 6). Figure 6.Unrooted neighbor-joining dendrogram of relationships among plant, yeast and bacterial RPE-related protein. The scale bar represents a genetic distance of 0.073 as the frequency of amino acid substitutions in the pairwise comparison of two sequences according to Kimura's two-parameter method (1980). Bootstrap support (data resampled 1000 times) for the apparent groupings is given. Database sources of sequences are given in the legend of Figure 5. Download figure Download PowerPoint Finally, the RPE gene was mapped on the A.thaliana CIC genomic library (Creusot et al., 1995). Four positive YAC clones carrying the RPE gene were identified by hybridization. These four clones (CIC3A3, CIC4D8, CIC9G7 and CIC10A10) have previously been anchored to chromosome 5 (contig 29) near the SEP5A and the LFY3 RFLP markers (Schmidt et al., 1997). Expression of RPE genes in plants To investigate the expression of the RPE gene, we isolated total RNAs and poly(A)+ RNAs from galls induced by M.incognita, and uninfected root tissues (root meristems, lateral root initiation sites and non-meristematic root fragments) of in vitro-grown wild-type Arabidopsis plants. RNA blots were not sufficiently sensitive to measure RPE gene expression because it was difficult to isolate enough tissue and/or because of the low level of RPE mRNA expression, as observed in roots (Teige et al., 1995). Since no gene was available as a control in the feeding cell, we used competitive PCR to amplify and quantify cDNA copies from low-abundance RPE mRNAs (Gilliland et al., 1990; Kaneko et al., 1992). The target RPE cDNA was coamplified with primers RA2 and LA2 in the presence of a dilution series of a competitor DNA of known concentration, which differs from the RPE cDNA by a small deletion of 35 bp (Figure 7A). The quantification of RPE expression was performed on root tissues from 3 to 15 d.p.i. This time-course experiment showed that the quantity of RPE mRNA in galls is constant (data not shown). The results obtained at 15 d.p.i. showed that no RPE mRNAs were detected in the non-meristematic root fragments, and very few mRNAs were detected in the lateral root initiation sites. The highest quantity of the RPE mRNA was observed in the root meristems and giant cells of NFSs induced by M.incognita (Figure 7B and C). Figure 7.Competitive PCR for quantification of RPE mRNA in Arabidopsis and potato. (A) Target Arabidopsis RPE cDNA from galls competed against various amounts of competitor-deleted cDNA (10−2–10−6 ng). The primers RA2 and LA2 give a 573 bp fragment when RPE cDNA is amplified and a 538 bp fragment when the competitor is amplified. (B) Plot of the ratio of competitive template versus target cDNA after amplification. The inset shows an expansion of the 10−4–10−6 range. The point of equivalence (i.e. where there is a 1:1 ratio) represents the concentration of cDNA in the sample (arrow). (C) Quantification of RPE transcripts by competitive PCR in Arabidopsis and potato. Galls and syncytia were harvested at 15 d.p.i. Root tissues were obtained from uninfected plants. Download figure Download PowerPoint To test the RPE upregulation in plant NFSs, we infected potato plants with Meloidogyne chitwoodi and the cyst nematode Globodera rostochiensis, two very important pathogens on potato crops. Two-week-old galls and syncytia and root tissues from uninfected plants were analyzed for RPE mRNA expression. The competitive PCR was carried out with potato-RPE-specific primers and a competitor issued from potato cDNA. There was as much potato RPE mRNA in the galls as in the root meristems, whereas there was 30% less in the syncytia (Figure 7C). Therefore, the regulation of RPE in potato is similar to that found in Arabidopsis. Rescue of rpe mutant and complementation Because the homozygous rpe mutant is impaired in sugar phosphate and lethal, we investigated whether exogenous carbohydrate addition would lead to the rescue of the rpe mutant. When grown in vitro on medium that provides 2% sucrose, kanamycin-resistant dwarf plants appeared with a 1:2 ratio. These plants had light-green leaves and reduced root systems with few lateral roots (Figure 8A). These seedlings developed slowly and died when transferred to the soil. We used PCR assay to test for cosegregation of the rescued phenotypes with the rpe mutation. PCR experiments were carried out with two RPE primers (LA2 and RA2), which span the rpe T-DNA insertion site, and a third primer (U1) specific for the sequence of T-DNA right border (Figure 3C). When genomic DNA from RPE plants was used as a template, both a 437 bp and a 914 bp band were amplified, indicating the presence of both the mutant and wild-type alleles. In contrast, when DNA from dwarf rescue plants was used as a template, only the 437 bp product was obtained from amplifications with all three primers (Figure 8B). This analysis confirmed that all 200 dwarf plants were homozygous for the rpe allele. Figure 8.The rpe mutant phenotype. (A) The plant on the left is a heterozygous +/rpe-tagged line, and the two plants on the right show the homozygous rpe. rpe mutant phenotype when grown in vitro on selective (kanamycin) medium supplemented with sucro" @default.
• W2083150981 created "2016-06-24" @default.
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• W2083150981 date "1998-12-01" @default.
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• W2083150981 title "RPE, a plant gene involved in early developmental steps of nematode feeding cells" @default.
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