Modification of phytohormone response by a peptide encoded by ENOD40 of legumes and a nonlegume

Science

Volumn 273, Issue 5273, 1996, Pages 370-373

  Van De Sande, Karin ^a,b   Pawlowski, Katharina ^a,b   Czaja, Inge ^a   Wieneke, Ursula ^a   Schell, Jeff ^a   Schmidt, Jürgen ^a   Walden, Richard ^a   Matvienko, Martha ^b   Wellink, Joan ^b   Van Kammen, Ab ^b   Franssen, Henk ^b   Bisseling, Ton ^b  

^a MAX PLANCK INSTITUTE FOR PLANT BREEDING RESEARCH   (Germany)

^b WAGENINGEN UNIVERSITY   (Netherlands)

Author keywords

[No Author keywords available]

Indexed keywords

ARTICLE; GROWTH REGULATION; HORMONE RESPONSE; LEGUME; PEPTIDE SYNTHESIS; PLANT GROWTH; PRIORITY JOURNAL; PROTEIN MODIFICATION;

NICOTIANA TABACUM;

EID: 9344220520     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.273.5273.370     Document Type: Article

References (29)

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12. The Eco RI fragment from pGmENOD40-2 (4) containing the first 448 bp of the cDNA was subcloned in the metothrexate resistance-transferring binary vector pMEX001 [B. Reiβ, C. Koncz, I. Moore, J. Schell, Plant Physiol. 13, 143 (1994)] containing the CaMV 35S promoter, yielding construct 40-2/1-448. Gm-ENOD40-2 was amplified by PCR from the original transgenic phage (4) with the use of primers 5′-TTTTGGATCCGAATTCCGCTAAACCAATCTATC-3′ and 5′-TTTTGTCGACGAAAGGACTCTGGAAACTTTTC-3′ and subcloned Bam HI-Sal I in pBlue-script KS+ and pRT105 (see below). The sequence of the inserts was confirmed after subcloning. Constructs 40-2/1-656, 40-2/1-90, 40-2/91-448, 40-2/ 91-656, Nt40/98-470, and 40-2/1-90 AAG were made by cloning of the PCR-amplified or excised DNA fragments into the CaMV 35S promoter containing vectors pRT105 or pRT106 [R. Töpfer, C. Maas, C. Höricke-Grandpierre, J. Schell, H.-H. Steinbiss, Methods Enzymol. 217, 66 (1993)] according to standard procedures. The tobacco ENOD40 cDNA clone was amplified by PCR with the use of primers 5′-GGC(A/T)(C/A)(A/G)(C/A)A(A/T)C(C/A)ATCCATGGTTCTT-3 and 5′-GGA(G/A)TCCATTGCCTTTT-3′. We isolated full-length clones by racing, using two specific primers (5′-GCTTTTGCCAACATCCTTTC-3′ and 5′-CTATTAGTGTGATTATCAATC-3′) and two universal primers (5′-CTCGAGGATCCGCGGCCGCTTTTTTTTTTTTTTTTTT-3′ and 5′-GCTCGAGGATCCGCGGC-3′) for the 5′ race. For the 3′ race, primers 5′-CAAGTTTGTTCATACTTTGCC-3′ and 5′-GCTAGAATTCCAGAAAATGC3′ were used. The sequence is given by the European Molecular Biology Laboratory database (accession number X98716). For amplifying the genomic clone, we used primers 5′-GACTAGCTTGTCTCAAGAAC-3′ and 5'-ATGACAATCTTAACAACTCT-3′. The genomic fragment was cloned in pGEM and from there was subcloned to pBlue-script KS+. From there it was excised with Kpn I-Sst I and cloned into pRT106.
13. The Eco RI fragment from pGmENOD40-2 (4) containing the first 448 bp of the cDNA was subcloned in the metothrexate resistance-transferring binary vector pMEX001 [B. Reiβ, C. Koncz, I. Moore, J. Schell, Plant Physiol. 13, 143 (1994)] containing the CaMV 35S promoter, yielding construct 40-2/1-448. Gm-ENOD40-2 was amplified by PCR from the original transgenic phage (4) with the use of primers 5′-TTTTGGATCCGAATTCCGCTAAACCAATCTATC-3′ and 5′-TTTTGTCGACGAAAGGACTCTGGAAACTTTTC-3′ and subcloned Bam HI-Sal I in pBlue-script KS+ and pRT105 (see below). The sequence of the inserts was confirmed after subcloning. Constructs 40-2/1-656, 40-2/1-90, 40-2/91-448, 40-2/ 91-656, Nt40/98-470, and 40-2/1-90 AAG were made by cloning of the PCR-amplified or excised DNA fragments into the CaMV 35S promoter containing vectors pRT105 or pRT106 [R. Töpfer, C. Maas, C. Höricke-Grandpierre, J. Schell, H.-H. Steinbiss, Methods Enzymol. 217, 66 (1993)] according to standard procedures. The tobacco ENOD40 cDNA clone was amplified by PCR with the use of primers 5′-GGC(A/T)(C/A)(A/G)(C/A)A(A/T)C(C/A)ATCCATGGTTCTT-3 and 5′-GGA(G/A)TCCATTGCCTTTT-3′. We isolated full-length clones by racing, using two specific primers (5′-GCTTTTGCCAACATCCTTTC-3′ and 5′-CTATTAGTGTGATTATCAATC-3′) and two universal primers (5′-CTCGAGGATCCGCGGCCGCTTTTTTTTTTTTTTTTTT-3′ and 5′-GCTCGAGGATCCGCGGC-3′) for the 5′ race. For the 3′ race, primers 5′-CAAGTTTGTTCATACTTTGCC-3′ and 5′-GCTAGAATTCCAGAAAATGC3′ were used. The sequence is given by the European Molecular Biology Laboratory database (accession number X98716). For amplifying the genomic clone, we used primers 5′-GACTAGCTTGTCTCAAGAAC-3′ and 5'-ATGACAATCTTAACAACTCT-3′. The genomic fragment was cloned in pGEM and from there was subcloned to pBlue-script KS+. From there it was excised with Kpn I-Sst I and cloned into pRT106.
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24. 2 of 11S (termed 11S-F2) was used for further analysis (in protoplast experiments).
25. To construct in-frame and out-of-frame Gm-ENOD40-2-GFP translational fusions, a DNA fragment containing the 5' leader and the peptide-encoding sequences of GmENOD40-2 as well as the coding part of GFP [J. Haseloff and B. Amos, Trends Genet. 11, 328 (1995)] were generated by PCR. The DNA fragments were cloned into pMON999 [H. van Bokhoven, J. Verver, J. Wellink, A. van Kammen, J. Gen. Virol. 74, 2233 (1993)]. The nucleotide sequences of the inserts were determined, and two clones [pMON40-GFPΔM and pMON40-δXbaGFPΔM, representing in-frame and out-of-frame translational fusions of GmENOD40-2 and GFP (Fig. 2E) were used to transfect tobacco protoplasts (14). For a positive control, the GFP DNA fragment was cloned, into pMON999.
26. To construct in-frame and out-of-frame Gm-ENOD40-2-GFP translational fusions, a DNA fragment containing the 5' leader and the peptide-encoding sequences of GmENOD40-2 as well as the coding part of GFP [J. Haseloff and B. Amos, Trends Genet. 11, 328 (1995)] were generated by PCR. The DNA fragments were cloned into pMON999 [H. van Bokhoven, J. Verver, J. Wellink, A. van Kammen, J. Gen. Virol. 74, 2233 (1993)]. The nucleotide sequences of the inserts were determined, and two clones [pMON40-GFPΔM and pMON40-δXbaGFPΔM, representing in-frame and out-of-frame translational fusions of GmENOD40-2 and GFP (Fig. 2E) were used to transfect tobacco protoplasts (14). For a positive control, the GFP DNA fragment was cloned, into pMON999.
27. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
28. H. Franssen et al., data not shown.
29. We thank J. Haseloff for providing a GFP cDNA. J.S. was supported by a grant from the Bundesministerium für Forschung und Technologie (Germany). T.B., K.P., and K.v.d.S. were supported by the Dutch Organization for Scientific Research. K.v.d.S. received a European Commission Short-Term Training fellowship.