207 Gene, 141 (1994) 207-210 Q 1994 Elsevier Science B.V. All rights reserved. 037%1119/94/~07.~ GENE 07815 Cloning and sequence of a cDNA encoding a novel hybrid proline-rich protein associated with cytokinin-induced haustoria formation in Cuscu zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA ta reflexa (Parasitic plants; free hanging vines; Tecoma; cDNA library; differential screening) K. Subramaniama, Jamuna Ranieb, B.R. Srinivasab, Achyut M. Sinhab and S. Mahadevan” zyxwvutsrqponmlkjihgf ‘Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India; and bAstra Research Centre India, Post Box 359, 18th Cross Road, Malleswaram, Bangalore 560 003, India. Tel. (91-80) 344-41 l/340-372 Received by M. Belfort: 16 September 1993; Revised/Accepted: 10 November/l2 November 1993; Received at publishers: 3 January 1994 SUMMARY A complete cDNA encoding a novel hybrid Pro-rich protein (HyPRP) was identified by differentially screening 3 x 10” recombinant plaques of a Cuscuta refrexa cytokinin-induced haustorial cDNA library constructed in hgtl0. The nucleo- tide (nt) sequence consists of: (i) a 424-bp S-non coding region having five start codons (ATGs) and three upstream open reading frames (uORFs); (ii) an ORF of 987 bp with coding potential for a 329-amino-acid (aa) protein of M, 35 203 with a hydrophobic N-terminal region including a stretch of nine consecutive Phe followed by a Pro-rich sequence and a Cys-rich hydrophobic C terminus; and (iii) a 17%bp 3’-UTR (untranslated region). Comparison of the predicted aa sequence with the NBRF and SWISSPROT databases and with a recent report of an embryo-specific protein of maize [Jose-Estanyol et al., Plant Cell 4 (1992) 413-4231 showed it to be similar to the class of HyPRPs encoded by genes preferentiaIly expressed in young tomato fruits, maize embryos and in vitro-cultured carrot embryos. Northern analysis revealed an approx. 1.8-kb mRNA of this gene expressed in the subapical region of the C. reflexa vine which exhibited maximum sensitivity to cytokinin in haustorial induction. INTRODUCTION A number of so-called ‘organ-specific’ genes associated with the formation and function of plant organs like stem, root, pollen, floral organs and embryo have been reported in recent years (de Pater and Schilperoort, 1990; Conkling Correspaladence to: Dr. S. Mahadevan, Department of Biochemistry, Indian institute of Science, Bangalore 560 012, India. Tel. (91-80) 344-411; Fax (91-80) 341-683; e-mail: cuscuta@biochem.iisc.ernet.in Abbreviations: aa, amino acid(s); BA, benzyladenine; bp, base pair(s); C., Cuscuta; cDNA, DNA complementary to RNA; CH, C. haustoria; cpm, counts per min; HyPRP, hybrid Pro-rich protein; hyprp, gene encoding HyPRP; kb, kilobase or 1000 bp; nt, nucleotide(s); ORF, open reading frame; pfu, plaque forming unit(s); SDS, sodium dodecyl sulfate; SSC, 0.15 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA M NaC1/0.015 M Na,zitrate pH 7.5; SSPE, 0.15 M NaCl/IO mM NaH,PO,jl mM EDTA pH 7.5; uORF, upstream ORF; UTR, untranslated region(s). SSDI 0378-l 119(94)00026-O et al., 1990; Mascarenhas, 1990; Gasser, 1991; Jose- Estanyol et al., 1992). Current interest in identifying such genes and determining the function(s) of their products stems from the prevalent belief that these genes exist and understanding the control of their expression will help understand plant development at the molecular level (Gasser, 1991; Mahadevan, 1992, and references therein). The simple morphology of the rootless and leafless an- giospermic parasite C. rejlexa (dodder) makes it a useful system to study plant development (Mahadevan, 1983). Successive stages of cellular growth and differentiation can be studied in successive basipetal segments of an essentially linear, cylindrical vine, as these segments com- prise cell populations that represent in space a develop- mental sequence in time. Using this approach we have ‘mapped’ the responsiveness of successive regions of the free-hanging vine of C. reflexa to various applied plant