Positional cloning of a gene for nematode resistance in sugar beet

Science

Volumn 275, Issue 5301, 1997, Pages 832-834

  Cai, Daguang ^a   Kleine, Michael ^a   Kifle, Sirak ^a   Harloff, Hans Joachim ^a   Sandal, Niels N ^b   Marcker, Kjeld A ^b   Klein Lankhorst, René M ^c   Salentijn, Elma M J ^c   Lange, Wouter ^c   Stiekema, Willem J ^c   Wyss, Urs ^a   Grundler, Florian M W ^a   Jung, Christian ^a  

^a UNIVERSITY OF KIEL   (Germany)

^b AARHUS UNIVERSITY   (Denmark)

^c WAGENINGEN UNIVERSITY   (Netherlands)

Author keywords

[No Author keywords available]

Indexed keywords

COMPLEMENTARY DNA;

AGRICULTURE; AMINO ACID SEQUENCE; ARTICLE; CROP; INFESTATION; MOLECULAR CLONING; NEMATODE; NONHUMAN; PRIORITY JOURNAL;

AMINO ACID SEQUENCE; ANIMALS; CELL MEMBRANE; CLONING, MOLECULAR; DNA, COMPLEMENTARY; GENES, PLANT; GENETIC COMPLEMENTATION TEST; LEUCINE; MEMBRANE PROTEINS; MOLECULAR SEQUENCE DATA; NEMATODA; PLANT DISEASES; PLANT PROTEINS; PLANT ROOTS; TRANSFORMATION, GENETIC; VEGETABLES;

EID: 14444268507     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.275.5301.832     Document Type: Article

References (39)

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32. The full-length cDNA 1832 was cloned into the Eco RI site of the binary vector pAM194 [provided by J. Kraus, PLANTA GmbH, Einbeck, Germany] under the transcriptional control of the 35S promoter from cauliflower mosaic virus and was transformed into the A. rhizogenes strain C58C1 with the plasmid pRi15834. Hairy roots of the susceptible sugar beet line 93161p were induced after co-infection of leaf segments with the A. rhizogenes strain containing the recombinant pAM194 plasmid and the A. tumefaciens strain LBA4404. The transgenic nature of the hairy roots evolving from the infection site was verified by β-glucuronidase assay [R. A. Jefferson, T. A. Kavanagh, M. W. Bevan, EMBO J. 6, 3901 (1987)] and subsequent DNA blot analysis (D. Cai et al., data not shown). The hairy roots from the resistant line A906001 and from the susceptible line 93161p and the hairy roots from the susceptible line 93161p that were transformed with the nonrecombinant pAM194 vector were used as controls. After 3 weeks of cultivation at 24°C on MS medium [ T. Murashige and F. Skoog, Physiol. Plant 15, 473 (1962)], hairy roots were dissected from the primary transformants and transferred to new petri dishes. After 1 week of cultivation under the same conditions, 300 sterile nematode juveniles (J2) were inoculated to each culture plate. The penetration sites and the developed females were counted under a stereo microscope 3 weeks after inoculation. The in vitro testing experiments were repeated at least twice. Each time, about 6 to 40 hairy root segments of one primary transformant were examined. The transgenic root culture 100B was retransformed with a pAM194 vector carrying the full-length cDNA 1832 in antisense orientation and tested for nematode development as described.
33. The full-length cDNA 1832 was cloned into the Eco RI site of the binary vector pAM194 [provided by J. Kraus, PLANTA GmbH, Einbeck, Germany] under the transcriptional control of the 35S promoter from cauliflower mosaic virus and was transformed into the A. rhizogenes strain C58C1 with the plasmid pRi15834. Hairy roots of the susceptible sugar beet line 93161p were induced after co-infection of leaf segments with the A. rhizogenes strain containing the recombinant pAM194 plasmid and the A. tumefaciens strain LBA4404. The transgenic nature of the hairy roots evolving from the infection site was verified by β-glucuronidase assay [R. A. Jefferson, T. A. Kavanagh, M. W. Bevan, EMBO J. 6, 3901 (1987)] and subsequent DNA blot analysis (D. Cai et al., data not shown). The hairy roots from the resistant line A906001 and from the susceptible line 93161p and the hairy roots from the susceptible line 93161p that were transformed with the nonrecombinant pAM194 vector were used as controls. After 3 weeks of cultivation at 24°C on MS medium [ T. Murashige and F. Skoog, Physiol. Plant 15, 473 (1962)], hairy roots were dissected from the primary transformants and transferred to new petri dishes. After 1 week of cultivation under the same conditions, 300 sterile nematode juveniles (J2) were inoculated to each culture plate. The penetration sites and the developed females were counted under a stereo microscope 3 weeks after inoculation. The in vitro testing experiments were repeated at least twice. Each time, about 6 to 40 hairy root segments of one primary transformant were examined. The transgenic root culture 100B was retransformed with a pAM194 vector carrying the full-length cDNA 1832 in antisense orientation and tested for nematode development as described.
34. Abbreviations for the amino acids are as follows: A, Ala; R, Arg; N, Asn; D, Asp; C, Cys; Q, Gln; E, Glu; G, Gly; H, His; I, Ile; L, Leu; K, Lys; M, Met; F, Phe; P, Pro; S, Ser; T, Thr; W, Trp; Y, Tyr; and V, Val.
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39. Technical assistance of B. Wohnsen, V. Petersen, M. Gagyi, N. Pallisgaard, Y. Gregersen, W. Dirkse, and M. J. B. Arens-DeReuver is gratefully acknowledged. Supported by grants from the European Community (grant AIR2-CT93-1206), from the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (grant 0319092A) and from the Danish Biotechnology Programme.