Download Plant Physiology

Plant Physiol. (1995) 108: 1311-1312
Plant Cene Register
A 1.7-Kilobase Genomic Fragment of Alfalfa D N A
Contains the Lectin Gene Mslecl'
Laurence M. Brill, Viken R. Konyalian, and Ann M. Hirsch*
Department of Biology, University of California, Los Angeles, 405 Hilgard Avenue,
Los Angeles, California 90095-1 606
The lectin recognition hypothesis proposes that the specific
binding of a legume lectin to a carbohydrate of a compatible
Rhizobium (sensu luto) allows the two organisms to recognize
each other as symbionts (Bohlool and Schmidt, 1974). Although there is extensive evidence supporting the hypothesis,
other experiments contradict it. Thus, the controversy remains unresolved. As a first step to testing the lectin recognition hypothesis in the Medicugo sutivu-Rhizobium meliloti
symbiosis,we cloned and sequenced lectin genes from alfalfa.
We are now reporting the nucleotide sequence of the Mslecl
gene, so named because of its high sequence similarity to the
Mtlecl gene (Bauchrowitzet al., 1992).
Fragments from two overlapping genomic clones were sequenced and include promoter, ORF, and 3' untranslated
sequences (Table I). The 1716-bp sequence includes 817 bp of
sequence upstream of the putative start codon, an ORF of 819
bp, and 75 bp of sequence downstream of the putative stop
codon. There are no introns in Mslecl, a situation similar to
that in a11 legume lectin genes previously reported
(Bauchrowitz et al., 1992). The transcription start and stop
positions of Mslecl are unknown. There are two potential
TATA boxes. One is at positions 707 to 711 (TATAA) and the
other is at 738 to 745 (TATATAAA). The number of TATA
boxes can affect tissue-specific expression (Kloeckner-Gruissem et al., 1992). There is an undefined consensus promoter
element at positions 643 to 659 (Gatehouse et al., 1987;
Bauchrowitz et al., 19921, but no other highly significant features of the promoter were detected in a search of the data
base. The putative start codon, ATG, is at position 818, and
the ORF ends at 1637. A putative poly(A) addition signal
(AATAAA) is located at positions 1667 to 1673.
There is extensive homology at the nucleotide leve1 with
other legume lectin genes (Table I), consistent with results
showing high conservation of legume lectin sequence and
three-dimensional structure (Sharon and Lis, 1990; van Eijsden, 1994).Interestingly, there was also 74% homology of
66 bp of the putative Mslecl promoter with the third intron
of the alfalfa gene Msnod25 (Nms-25; Vegh et al., 1990),
which seems to be involved in nodulation. The significance
of this homology is unclear. The deduced MsLECl protein
is 92% identical with and 96% homologous to the deduced
MtLECl protein, and MsLECl is highly homologous to
other legume lectins. Especially highly conserved amino
acid residues in legume lectins are known to be important
in sugar binding (van Eijsden, 1994). These residues in
MsLECl are Aspsl (or Asps6), Glyio9,AsnlZ3,GlyZo8,IleZo9,
and Asn210.They are identical with residues in comparable
positions of PHA-L from Pkuseolus vulguris (van Eijsden,
1994),a lectin that binds only oligosaccharides (Sharon and
Lis, 1990), and are nearly identical with residues in comparable positions of other legume lectins. However, only
the first three of these six amino acid residues are identical
with those of MtLECl, which is believed to be a Glc/Manbinding protein (Bauchrowitz et al., 1992). The sequence
suggests that MsLECl may be an oligosaccharide-binding
protein, but binding studies are necessary to define the
specificity of MsLECl.
Genomic southern blots indicate that alfalfa contains a
small lectin gene family. Also, we have partially sequenced
Mslec2 (based on its homology to Mtlec2) (Bauchrowitz et
al., 1992) and another putative lectin gene (L.M. Brill, V.R.
Konyalian, and A.M. Hirsch, unpublished results). Medicago truncatulu, a close relative of alfalfa, also has a small
lectin gene family (Bauchrowitz et al., 1992). Further work
on alfalfa lectin genes is needed to resolve their importance
in the interaction between the plant and R. meliloti.
ACKNOWLEDCMENTS
We are grateful to Martina Bauchrowitz for graciously providing us with a Mtlecl plasmid clone. We thank Dr. Keith Wycoff
and other members of our group for valuable advice during the
course of this work. Rob Satterthwaite is thanked for assistance
with the DNA sequencing.
Received December 19, 1994; accepted January 24, 1995.
Copyright Clearance Center: 00324889/95/108/1311/02.
The GenBank accession number for the sequence reported in this
article is U18296.
Support was provided by National Research Initiative Competitive Grant Program grant No. 93-37305-9144 from the Nitrogen Fixation/Metabolism Program to A.M.H. L.M.B. was
supported in part by a National Research Service Award Biotechnology training grant to the University of California, Los Angeles.
* Corresponding author; e-mail [email protected].
edu; fax 1-310-206-5413.
Abbreviation: ORF, open reading frame.
LITERATURE ClTED
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990)
Basic local alignment search tool. J Mo1 Biol 215: 403-410
Bauchrowitz MA, Barker DG, Nadaud I, Rouge P, Lescure B
(1992) Lectin genes from the legume Medicugo truncatulu. Plant
1311
1312
Brill et ai.
Plant Physiol. Vol. 108, 1995
Table 1. Characteristics of a 7.7-kb Eenomic seauence from M. sativa encoding Mslecl
~
~~
Organism:
Medicago sativa (L.), cv Chief.
Genome Location:
Nuclear genome; chromosomal location unknown.
Gene Product:
Presumed carbohydrate-binding lectin protein.
Cloning:
A A EMBL3 Sp6R7 genomic DNA library of M. sativa (L) cv Chief (Clontech Laboratories Inc., Palo Alto, CA) was
screened with an [c~-~~P]dCTP-labeled
Mtlec7 probe from M. truncatula (Bauchrowitz et al., 1992). Duplicate filters
were used for screening, and positive phage were cloned by plaque purification. Overlapping Haelll and Sstl fragments (124-bp overlap, positions 1-1 150 and 1026-1 709) were identified by restriction digestion, Southern blotting, and probing with the Mtlecl probe. Another Haelll fragment (positions 1151-1 71 6) was identified in the same
way. These three fragments were subcloned into pBluescriptll KS +/- (Stratagene).
Sequencing:
60th strands (positions 1-1 709) were sequenced using transposon-facilitated sequencing (Strathmann et al., 1991) with
the TnlOOO kit (Cold Biotechnology Inc., St. Louis, MO), according to the manufacturer’s protocol. The end sequence of the 565-bp Haelll fragment gave rise to positions 171O to 1716 and agreed perfectly with the overlapping
sequence from the Sstl fragment. Computer sequence analysis was done on the Vax computer in the Biology Department at the University of California, Los Angeles, using University of Wisconsin Genetics Computer Croup
programs.
Characteristics of the Putative Promoter Region:
A putative promoter, identified based on sequence analysis, was sequenced for 81 7 bp upstream of the putative start
codon. Elements of interest include two potential TATA boxes and a 17-bp element (positions 643-659) conserved
in some legume lectin promoters. The homology of this element, allowing insertion of two nucleotides in Mslecl, is
71 % with Mtlecl (Bauchrowitz et al., 1992) and 65% with Psl (Catehouse et al., 1987).
Structural Features of the Deduced Protein:
The ORF encodes a protein of 273 amino acid residues, with a putative signal sequence of 26 amino acid residues,
predicted using the method of von Heijne (1986). The presumed mature protein has 247 amino acid residues and a
molecular weight of 27,220 and is presumed to be a carbohydrate-binding protein.
Antibodies:
None available.
Method of Identification:
End sequences of both the Haelll and Sstl fragments were compared to the data base (Altschul et al., 1990). Matches
primarily with lectin genes were found. Homologies of the complete nucleotide sequence of Mslec7 included 95%
with Mtlec7 for 804 bp, 79% with Mtlec2 for 482 bp, 75% with Psl (Pisum sativum lectin) for 219 bp, and significant homology with the frythrina coralodendron and Phaseolus lunatus lectins and Arachis hypogea agglutinin (a
lectin). Homology of the deduced protein with other legume lectins at the amino acid level is more extensive than
homolony at the nucleotide level.
Mo1 Biol 1 9 1011-1017
Bohlool BB, Schmidt EL (1974) Lectins: a possible basis for specificity in Rhizobium-legume root nodule symbiosis. Science 185:
269-271
Gatehouse JA, Bown D, Evans IM, Gatehouse LN, Jobes D,
Preston P, Croy RRD (1987) Sequence of the seed lectin gene
from pea (Pisum sativum L.).Nucleic Acids Res 1 5 7642
Kloeckner-Gruissem B, Vogel JM, Freeling M (1992) The TATA
box promoter region of maize Adhl affects its organ-specific
expression. EMBO J 11: 157-166
Sharon N, Lis H (1990) Legume lectins-a large family of homologous proteins. FASEB J 4 3198-3208
Strathmann M, Hamilton BA, Mayeda CA, Simon MI, Meyerowi t z EM, Palazzolo M J (1991) Transposon-facilitated DNA sequencing. Proc N a t l Acad Sci USA 88: 1247-1250
van Eijsden R (1994) Mutational analysis of pea lectin. PhD thesis.
University of Leiden, Leiden, The Netherlands
Vegh Z, Vincze E, Kadirov R, Toth G, Kiss GB (1990) The
nucleotide sequence of a nodule-specific gene, Nms-25 of Medicago sativa: i t s primary evolution via exon-shuffling and retrotransposon-mediated DNA rearrangements. Plant Mo1 Biol 1 5
295-307
v o n Heijne G (1986) A new method for predicting signal sequence
cleavage sites. Nucleic Acids Res 1 4 46834690