Title: Endoglucanase gene promoter upregulated by nematodes
Abstract: The present invention provides a nucleic acid construct comprising a cyst and root knot nematode responsive promoter, preferably the Nicotiana Ntcel7 promoter or promoters that hybridize thereto, operatively associated with a heterologous nucleic acid segment that encodes a product disruptive of nematode attack. Plants and plant cells using the same and methods of use thereof are also disclosed.
Patent Number: 6,906,241 Issued on 06/14/2005 to Davis, et al.
| Inventors:
|
Davis; Eric L. (Raleigh, NC);
Goellner; Melissa (Raleigh, NC)
|
| Assignee:
|
North Carolina State University (Raleigh, NC)
|
| Appl. No.:
|
970367 |
| Filed:
|
October 2, 2001 |
| Current U.S. Class: |
800/287; 435/320.1; 435/419; 435/430.1; 435/468; 536/23.4; 800/278; 800/279; 800/298; 800/301; 800/317; 800/320 |
| Intern'l Class: |
C12N 015/09; C12N015/82; A01H005/00; A01H005/10 |
| Field of Search: |
800/279,278,298,301,320,317,287,295
435/320.1,468,419,430.1
536/234,232,236,241
|
References Cited [Referenced By]
U.S. Patent Documents
Other References
Keller et al . The Plant Cell, vol. 3, pp. 1051-1061, 1991.
Kim et al. Plant Molecular Biology, vol. 24, pp. 105-117, 1994.
|
Primary Examiner: Nelson; Amy J.
Assistant Examiner: Ibrahim; Medina A.
Attorney, Agent or Firm: Myers Bigel Sibley & Sajovec, P.A.
Claims
1. A nucleic acid construct which comprises:
(a) the isolated promoter of SEQ ID NO:9 ; and
(b) a heterologous nucleic acid operably linked to said isolated promoter, wherein
said heterologous nucleic acid encodes a nematocidal or insecticidal protein or
peptide.
2. The nucleic acid construct according to claim 1, wherein said heterologous
nucleic acid encodes an insecticidal protein.
3. The nucleic acid construct according to claim 2, wherein said heterologous
nucleic acid encodes a
Bacillus thuringiensis crystal protein toxic to insects.
4. The nucleic acid construct according to claim 1, wherein said heterologous
nucleic acid encodes a product toxic to plant cells.
5. The nucleic acid construct according to claim 1, wherein said nucleic acid
construct is a plasmid.
6. A plant cell transformed with a nucleic acid construct according to claim 1.
7. A method of producing a transformed plant, comprising regenerating a plant
from the plant cell according to claim 6.
8. An
Agrobacterium tumefaciens cell containing the nucleic acid construct
according to claim 5, wherein said nucleic acid construct is a Ti plasmid.
9. A method of producing a transformed plant, comprising infecting a plant cell
with the
Agrobacterium tumefaciens cell according to claim 8 to produce
a transformed plant cell, and regenerating a plant from said transformed plant cell.
10. A microparticle comprising the nucleic acid construct according to claim
1, wherein said microparticle is for plant transformation.
11. A method of making a transformed plant, comprising propelling the microparticle
according to claim 10 into a plant cell to produce a transformed plant cell, and
regenerating a plant from said transformed plant cell.
12. A plant cell protoplast comprising a nucleic acid construct according to
claim 1.
13. A method of making a transformed plant, comprising regenerating a plant from
the plant cell protoplast according to claim 12.
14. A transformed plant comprising transformed plant cells, said transformed
plant cells containing the nucleic acid construct according to claim 1.
15. The transformed plant according to claim 14, wherein said plant is a dicot.
16. The transformed plant according to claim 14, wherein said plant is a monocot.
17. The transformed plant according to claim 14, wherein said plant is a tobacco
(
Nicotiana tabacum) plant.
18. A transformed seed produced from the transformed plant according to claim 14.
19. A method of producing a cyst and root knot nematode resistant plant, comprising
the steps of:
(a) providing a DNA construct comprising the isolated promoter of SEQ ID NO:
9 operably linked to a heterologous nucleic acid encoding a nematocidal protein
or peptide, and
(b) transforming a plant with the nucleic acid construct to produce a cyst and
root knot nematode resistant plant.
20. The method according to claim 19, wherein said plant is a monocot.
21. The method according to claim 19, wherein said plant is a dicot.
22. A plant produced by the method of claim 19.
23. An isolated nucleic acid comprising the isolated promoter of SEQ ID NO: 9.
Description
FIELD OF THE INVENTION
This invention relates to tissue-specific gene promoters, and particularly relates
to a promoter which is responsive to the cyst and root knot nematodes.
BACKGROUND OF THE INVENTION
A promoter is a DNA sequence which flanks a transcribed gene, and to which RNA
polymerase must bind if it is to transcribe the flanking gene into messenger RNA.
A promoter may consist of a number of different regulatory elements which affect
a structural gene operationally associated with the promoter in different ways.
For example, a regulatory gene may enhance or repress expression of an associated
structural gene, subject that gene to developmental regulation, or contribute to
the tissue-specific regulation of that gene. Modifications to promoters can make
possible optional patterns of gene expression, using recombinant DNA procedures.
See, e.g., Old and Primrose, Principles of Gene Manipulation (4th Ed., 1989).
U.S. Pat. No. 5,459,252 to Conkling and Yamamoto describes a root specific promoter
designated RB7, which was identified in tobacco. U.S. Pat. No. 5,837,876 to Conkling
et al. describes a root cortex specific gene promoter designated the RD2 promoter,
which was also identified in tobacco.
Rather than use a promoter that is constitutively active, it is desirable
to have promoters that are responsive to particular stimuli. In particular, if
a promoter is responsive to a particular pathogen, then that promoter could be
used to impart selective disease resistance to that pathogen through expression
of a transgene that disrupts that pathogen.
U.S. Pat. No. 5,750,386 to Conkling, Opperman and Taylor describes pathogen
resistant transgenic plants in which a nematode-responsive element is operatively
associated with a nucleotide of interest (in this case, a gene encoding a product
toxic to plant cells). One nematode responsive element was a deletion fragment
of the RB7 root specific promoter described above.
U.S. Pat. No. 5,589,622 to Gurr et al. suggests nematode resistant transgenic
plants in which cells of the plant contain a heterologous construct comprising
a nematode responsive promoter operatively associated with a product disruptive
of nematode attack. However, the DNAs disclosed by Gurr et al. as nematode responsive
promoters do not appear to represent such promoters, and instead appear to represent
extraneous or irrelevant DNA.
To impart useful traits to plants by the expression of foreign genes using genetic
engineering techniques, a variety of pathogen-responsive promoters will be required
to allow traits to be expressed selectively, in the appropriate plant tissues,
and at the appropriate times. Accordingly, there is a continued need for pathogen
responsive elements that operate in plant cells.
SUMMARY OF THE INVENTION
The present invention is based on the discovery that the endo-1,4-β-glucanases,
Ntcel2 (SEQ ID NO:1), Ntcel7 (SEQ ID NO:3), and Ntcel8 (SEQ ID NO:5), of
Nicotiana
tabacum are upregulated in cyst and root-knot nematode feeding cells (i.e.,
giant cells). Plant parasitic nematodes cause approximately 100 billion dollars
annually in crop loses worldwide. The root knot nematode has a host range of over
2000 plant species, and is one of the most damaging nematodes.
Accordingly, a first aspect of the present invention is an isolated
nucleic acid, particularly DNA, molecule which directs cyst and/or root knot nematode
responsive transcription of a downstream heterologous nucleic acid/DNA segment
in a plant cell (i. e., a promoter), and the use thereof in providing or imparting
nematode resistance to plants and plant cells. Preferably the promoter is responsive
to, or activates transcription in response to, both cyst and root knot nematodes.
A further aspect of the present invention is a construct comprising a promoter
as described above and a heterologous nucleic acid/DNA segment (i. e., a DNA segment
not naturally associated with that promoter) positioned downstream from, and operatively
associated with, the promoter. The heterologous nucleic acid/DNA segment preferably
encodes a product disruptive of nematode attack (i. e., a product that hinders
or interferes with the ability of a nematode to feed upon a plant cell, or establish
a feeding site in relationship to a plant cell, when that product is expressed
in a plant cell).
Further aspects of the present invention are plant cells containing the above
described constructs, methods of making transformed plants from such plant cells,
the transformed plants comprising such transformed plant cells, and the use of
the foregoing to impart resistance to root knot nematodes to plants.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the immunolocalization of EGases during parasitism of tobacco roots.
(A) Longitudinal section through a tobacco root 24 h after inoculation with second-stage
juveniles (J2) of
Globodera tabacum (N) probed with mouse pre-immune serum.
(B) Longitudinal section through a tobacco root 24h after inoculation with
G.
tabacum J2 and probed with GR-ENG-1 antiserum. Binding of the GR-ENG-1 antiserum
(green fluorescence) is observed along the migratory path (MP) of multiple migratory
juveniles within the root cortical tissue. An arrow points to a nematode tail (N).
(C) Brightfield view of a longitudinal section through a tobacco root 24 h after
inoculation with second-stage juveniles of
G. tabacum (N) (D) Same section
as C showing binding of GR-ENG-1 antiserum on the cell wall just outside the head
of the nematode and within the nematode's subventral secretory gland cells (SvG).
(E) Longitudinal section through a tobacco root containing a sedentary parasitic
J2 of
G. tabacum during early syncytia (Syn) development. (F) Same section
as E showing specific binding of GR-ENG-1 antiserum to the SvG, but not within
the developing syncytium. (G) Section through a tobacco root containing a sedentary
parasitic J2 feeding from a well-developed syncytium. (H) Same section as G showing
slight binding of GR-ENG-1 antiserum to the surface of the nematode, but not within
the syncytium. Scale bars=50 μm. Sty=nematode stylet.
FIG. 2 shows an amino acid sequence comparison of Ntcel2 (SEQ ID NO:2), Ntcel7
(SEQ ID NO:4), and Ntcel8 (SEQ ID NO:6). Amino acid sequences were aligned using
programs of the Wisconsin Package Version 10.0, Seqweb Version 1.1 (Genetics Computer
Group, Madison, Wis.). Dots were introduced by the program to optimize the alignment.
Black boxes depict identical amino acids among the three sequences. An arrowhead
designates the putative secretion signal peptide cleavage sites of the proteins
as determined by the SignalP V 1.1 program (Nielson, et al. (1997)
Protein Engin.
10:1-6). Arrows designate two conserved amino acid domains used to amplify
tobacco EGases from nematode-infected root tissue. An extra 124 amino acid sequence
encoding a putative cellulose-binding domain at the C-terminus of Ntcel8 is underlined.
FIG. 3 illustrates a phylogenetic comparison of plant EGases. The evolutionary
relationships among mature plant EGase amino acid sequences were calculated using
programs of the Wisconsin Package Version 10.0, Seqweb Version 1.1 (Genetics Computer
Group, Madison, Wis.) and the Kimura distance correction method (Kimura, 1983).
The tree was constructed using the UPGMA method. Sequences used for the analysis
(Genbank accession numbers in parentheses) were:
Arabidopsis Cel1 (X98544);
Bean Bac1 (M57400); Orange Celb1 (AF000136); Pea Egl1 (L41046); Pepper Cel3 (X97189);
Tobacco Pistal EGase (AF128404); Tobacco Cel1 (AF362949), Cel2 (AF362948), Cel4
(AF362950), Cel5 (AF362951), Cel7 (AF362947); Tomato Cel1 (U13054), Cel2 (U13055),
Cel3 (U78526), TPP18/Cel4 (U20590), Cel5 (AF077339), Cel7 (Y1 1268), Cel8 (AF098292);
Strawberry Eg3 (AJ006349).
FIG. 4 shows DNA gel blot analysis of Ntcel2, Ntcel7, and Ntcel8 genes in tobacco.
Genomic DNA (5 μg) was digested with BamHI, EcoRI, and HindIII, electrophoresed
on a 0.7% agarose gel, blotted to nylon membranes, and probed with a 1 kb fragment
spanning the conserved amino acid domains, CWERPED (SEQ ID NO:7) and YINAPL (SEQ
ID NO:8), of Ntcel2, Ntcel7, and Ntcel8. Blots were hybridized in 5×SSC at
65° C. and washed twice in 0.5×SSC at 68° C. and twice in 0.1×SSC
at 68° C. B=BamHI, E=EcoRI, and H=HindIII.
FIG. 5 shows the relative RT-PCR analysis of tobacco EGase transcripts in uninfected
and nematode-infected root tissue. RT-PCR products were amplified from uninfected
(U), tobacco cyst nematode-infected (T), or root knot nematode-infected (R) tobacco
root tissue 7-9 days post-infection using tobacco EGase gene-specific primers.
A DNA gel blot of the RT-PCR products was probed with tobacco EGase digoxigenin-labeled
DNA probes. L=1 kb DNA ladder (GibCo BRL, Rockville, Md.).
FIG. 6 shows in situ hybridization of Ntcel7, Ntcel8, and Ntcel2 mRNA in root
knot kematode (RKN)-infected tobacco roots. Longitudinal serial sections through
a RKN-induced gall on tobacco roots 12-14 days post-infection (dpi) and hybridized
with a tobacco Ntcel7 sense (A) or antisense digoxigenin (DIG)-labeled riboprobe
(B). Longitudinal section through a RKN-induced gall on tobacco roots 7-9 dpi and
hybridized with a tobacco Ntcel7 antisense DIG-labeled riboprobe (C). Longitudinal
sections through tobacco roots infected with RKN and hybridized with a tobacco
Ntcel8 antisense DIG-labeled riboprobe at 7-9 dpi (D-F). Longitudinal serial sections
through a tobacco gall induced by RKN at 12-14 dpi and hybridized with a tobacco
Ntcel2 sense (G) and antisense (H) DIG-labeled riboprobe. N=nematode, GC=giant
cells, LR-lateral root. Scale bars=50 μm.
FIG. 7 shows in situ hybridization of tobacco Ntcel7 and Ntcel8 mRNA in tobacco
cyst nematode-infected tobacco roots. Longitudinal serial sections through tobacco
roots infected with tobacco cyst nematodes (TCN) and hybridized with a tobacco
Ntcel7 antisense (A) or sense (B) digoxigenin (DIG)-labeled riboprobe 7 days post-infection.
Longitudinal serial sections through tobacco roots infected with TCN and hybridized
with a tobacco Ntcel8 antisense (C) or sense (D) DIG-labeled riboprobe 7 days post-infection.
N=nematode, S=syncytia. Scale bars=50 μm.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Various preferred embodiments of the present invention are set forth below.
These embodiments are not intended to provide a detailed catalog of all manner
in which the instant invention may be carried out, as numerous variations will
be apparent to persons skilled in the arts to which the invention pertains. Accordingly,
the following is set forth for illustrative purposes, and is not intended to be
limiting of the invention.
1. Cyst and Root-Knot Nematodes
The invention may be carried out to protect plants from cyst (
Globodera and
Heterodera spp.) or root knot nematodes (
Meloidogyne spp.). Cyst
nematodes, like all plant-parasitic nematodes, are a microscopic roundworms very
simple animal, related to the animal-parasitic roundworms that infect livestock
and pets. The infective juvenile is the second-stage juvenile, so-called because
it molts once in the egg from first-stage to second-stage. The infective juvenile
is invisible to the naked eye. Its length is about {fraction (1/64)} inch. The
juveniles penetrate roots and cause the formation of specialized feeding cells
in the root's vascular system (veins). If the juvenile becomes a male, it leaves
the root and moves through the soil and probably does not contribute further to
plant damage. If the juvenile becomes a female, it loses the ability to move and
swells to a lemon-shape as it matures. The young adult female is referred to as
a white female. Plant damage is primarily due to the feeding of females. White
females become yellow as they age and then brown after they die. The brown stage
is the cyst for which the nematode is named. Each cyst can contain up to 500 eggs,
but under field conditions usually contain many fewer eggs. The cyst protects the
eggs from the soil environment.
Root-knot nematodes are sedentary endoparasites with an extremely intimate
and complex relationship to the host plant. The infective second stage juvenile
(J2) is free in the soil. Upon location of a host root, the J2 penetrates the root
intercellularly in the region just posterior to the root cap and migrates to the
developing vascular cylinder. The nematode then orients itself parallel to the
cylinder and injects glandular secretions into the plant cells surrounding its
head, resulting in the initiation of nematode feeding cells. These 5-7 cells undergo
rapid nuclear divisions, increase tremendously in size, and become filled with
pores and cell wall invaginations. The feeding site cells, or "giant cells", function
as super transfer cells to provide nourishment to the developing nematode. During
this time, the nematode loses the ability to move and swells from the normal eel
shaped J2 to a large, pear shaped adult female. As the nematode feeds on the giant
cells, parthenogenic reproduction results in the disposition of 300-400 eggs. This
entire process occurs over the span of 20-30 days, and root-knot nematodes may
complete as many as 7 generations during a cropping season. Thus, in addition to
delivering at the feeding site a product that is toxic to the nematode, it will
be seen that, by causing the plant itself to kill or disable the cells upon which
the pathogen must feed, the pathogen will be much less successful at infecting
the plant.
Feeding cells induced by cyst and root-knot nematodes (RKN), termed syncytia
and giant-cells, respectively, are formed from host root cells during parasitism
to sustain the growth and reproduction of the nematode (Hussey and Grundler (1998)
Nematode parasitism of plants. In The physiology and biochemistry of free-living
and plant-parasitic nematodes, ed. R. N. Perry, D. T. Wright, pp. 213-243, Wallingford
UK: CABI publishing). Motile second-stage juveniles (J2) of the nematode penetrate
plant roots and migrate to the vascular cylinder where the feeding cells serve
as the sole nutritive source for the subsequent sedentary parasitic life stages
of these nematodes. Evidence suggests that stylet (hollow feeding spear) secretions
originating from three large unicellular esophageal gland cells of RKN and cyst
nematodes play essential roles during parasitism of plant roots, including the
induction of feeding cells (Davis, et al. (2000)
Annu. Rev. Phytopathol. 38:341-372;
Hussey (1989)
Annu. Rev. Phytopathol. 27:123-141). Nematode secretions may
directly or indirectly alter the development of affected host plant cells (Hussey
(1989)
Annu. Rev. Phytopathol. 27:123-141). This modification of normal
plant cell development causes plant cells to re-differentiate into unique cell
types strictly for the benefit of the nematode and is accompanied by multiple changes
in plant gene expression (Bird, D. McK. (1996)
J. Parasitol. 82:881-888).
Giant-cells and syncytia have several characteristics in common. In both
cell types, there is an increase in metabolic activity and cytoplasmic density,
the large central vacuole is reduced to several smaller ones, organelles proliferate,
individual cells hypertrophy, cell walls thicken, and fingerlike protuberances
(ingrowths) form along walls adjacent to the xylem vessels to increase membrane
surface area for solute uptake (Hussey and Grundler (1998) Nematode parasitism
of plants. In The physiology and biochemistry of free-living and plant-parasitic
nematodes, ed. R. N. Perry, D. T. Wright, pp. 213-243, Wallingford UK: CABI publishing).
The nuclei within these cells enlarge, develop an amoeboid appearance, have a very
prominent nucleolus, and are polyploid. In giant-cells of RKN, the nuclei are stimulated
to divide in the absence of cell division resulting in enlarged plant root cells
containing hundreds of nuclei (Huang and Maggenti (1969)
Phytopathol. 59:447-455).
The syncytium of cyst nematodes is also multinucleate but arises via a different
mechanism than that of giant-cells. Within the initial syncytial cell, the plasmodesmatal
openings begin to gradually widen and wall degradation is initiated at pit fields
(Jones (1981)
Ann. Appl. Biol. 97:353-372; Grundler et al. (1998)
Eur.
J. Plant Pathol. 104:545-551). As the initial syncytial cell enlarges, the
cell wall gaps expand and neighboring protoplasms fuse. Progressive cell wall dissolution
allows the syncytium to expand longitudinally along the length of the vascular
cylinder (extending as far as 2-3 mm) and can incorporate up to 200 plant cells
(Jones (1981)
Ann. Appl. Biol. 97:353-372).
2. Promoters
As used herein, a nematode responsive (or "nematode inducible") promoter refers
to a promoter that (a) does not normally drive transcription in a plant cell except
when that cell resides in tissue infected by a cyst or root knot nematode, or (b)
normally drives transcription in a plant cell, and which drives increased levels
of transcription when that cell resides in tissue infected by a cyst or root knot
nematode. The promoter may be a naturally occurring promoter, may comprise a nematode
responsive element isolated from a naturally occurring promoter, or may be a synthetic promoter.
A preferred promoter for use in the present invention is the endo-1,4-β-glucanase
(Ntcel7) promoter of
Nicotiana tabacum described herein. This promoter is
referred to herein as a Nicotiana Ntcel7 promoter, and is set forth herein as SEQ
ID NO: 9. The Nicotiana Ntcel7 promoter and other promoters that may be used to
carry out the present invention are also disclosed in U.S. Pat. No. 6,005,092 to
Shoseyov and Z. Shani, issued Dec. 21, 1999, the disclosure of which is incorporated
by reference herein in its entirety.
Other DNAs that hybridize to a Nicotiana Ntcel7 promoter under high stringency
hybridization conditions as described below, and which encode a nematode responsive
promoter (particularly a cyst or root knot nematode responsive promoter) may also
be used to carry out the present invention.
High stringency hybridization conditions which will permit homologous DNA sequences
(e.g., other natural plant DNA sequences) to hybridize to a DNA sequence encoding
a Nicotiana Ntcel7 promoter are well known in the art. For example, hybridization
of such sequences to a DNA encoding a Nicotiana Ntcel7 promoter may be carried
out in 25% formamide, 5×SSC, 5×Denhardt's solution, with 100 μg/ml
of single stranded DNA and 5% dextran sulfate at 42° C., with wash conditions
of 25% formamide, 5×SSC, 0.1% SDS at 42° C. for 15 minutes, to allow
hybridization of sequences of about 60% homology. More stringent conditions are
represented by a wash stringency of 0.3M NaCl, 0.03M sodium citrate, 0.1% SDS at
60° or even 70° C. using a standard in situ hybridization assay. (See
Sambrook et al., Molecular Cloning, A Laboratory Manual (2d Ed. 1989)(Cold Spring
Harbor Laboratory)). In general, plant DNA sequences which code for nematode responsive
promoters and which hybridize to the DNA sequence encoding the nematode responsive
elements disclosed herein will be at least 75%, 80%, 85%, 90% or even 95% homologous
or more with the sequences of the DNA encoding the nematode responsive elements
disclosed herein.
It will be apparent that other sequence fragments from the promoter 5′
flanking region, longer or shorter sequences, or sequences with minor additions,
deletions, or substitutions made thereto, can be prepared which will also encode
a nematode responsive promoter, all of which are included within the present invention.
3. Heterologous DNAs and Expression Cassettes
DNA constructs, or "expression cassettes," of the present invention include,
5′-3′ in the direction of transcription, a nematode responsive promoter
of the present invention, a heterologous DNA segment operatively associated with
the promoter, and, optionally, transcriptional and translational termination regions
such as a termination signal and a polyadenylation region. All of these regulatory
regions should be capable of operating in the transformed cells. The 3′
termination region may be derived from the same gene as the transcriptional initiation
region or from a different gene.
The term "operatively associated," as used herein, refers to DNA sequences contained
within a single DNA molecule which are associated so that the function of one is
affected by the other. Thus, a promoter is operatively associated with a gene when
it is capable of affecting the expression of that gene (i.e., the gene is under
the transcriptional control of the promoter). The promoter is said to be "upstream"
from the gene, which is in turn said to be "downstream" from the promoter.
Heterologous DNAs used to carry out the present invention may encode
any product that is disruptive of nematode attack when that DNA is transcribed
(and, where applicable, translated) in a plant cell, including but not limited
to proteins, peptides, and non-protein products such as antisense RNAs, ribozymes,
other nucleic acids that suppress expression by sense strand suppression or triplex
formation, etc. (see, e.g., U.S. Pat. No. 4,801,540 (Calgene, Inc.)).
The heterologous DNA may encode a product that is toxic to the plant cells, as
described in U.S. Pat. No. 5,750,386 to Conkling et al. A wide variety of protein
or peptide products which are toxic to plant cells can be used, including (but
not limited to) enzymes capable of degrading nucleic acids (DNA, RNA) such as nucleases,
restriction endonucleases micrococcal nucleas, Rnase A, and barnase; enzymes which
attack proteins such as trypsin, pronase A, carboxypeptidase, endoproteinase Asp-N,
endoproteinase Glu-C, and endoproteinase Lys-C; ribonucleases such as RNase CL-3
and RNase T
1, toxins from plant pathogenic bacteria such as phaseolotoxin,
tabtoxin, and syringotoxin; lipases such as produced from porcine pancrease and
Candida cyclindracea, membrane channel proteins such as glp F and connexins (gap
junction proteins, and antibodies which bind proteins in the cell so that the cell
is thereby killed or debilitated. Genes which produce antibodies to plant cell
proteins can be produced as described in W. Huse et al. ((1989)
Science 246:1275-1281).
Proteins to which such antibodies can be directed include, but are not limited
to, RNA polymerase, respiratory enzymes, cytochrome oxidase, Krebs cycle enzymes,
protein kinases, aminocyclopropane-1-carboxylic acid synthase, and enzymes involved
in the shikimic acid pathway such as enolpyruvyl shikimic acid-5-phosphate synthase.
One preferred heterologous DNA is a structural gene encoding mature
Bacillus
amyloliquefaciens RNase (or Barnase). See, e.g., C. Mariani et al. ((1990)
Nature 347:737-741) and C. Paddon and R. Hartley ((1985)
Gene 40:231-39).
Note that the toxic product may either kill the plant cell in which it is expressed
or simply disable the cell so that it is less capable of supporting the pathogen.
It is preferred, particularly where the plant is a food plant, that the plant-toxic
product be non-toxic to animals, and particularly be non-toxic to humans.
The heterologous DNA may encode any other product disruptive of nematode attack,
including but not limited to those described in U.S. Pat. No. 5,589,622 to Gurr
et al. (e.g., products toxic to the nematode). Thus the heterologous DNA may encode
a
Bacillus thuringiensis crystal protein toxic to insects. Strains of
B.
thuringiensis which produce polypeptide toxins active against nematodes are
disclosed in U.S. Pat. Nos. 4,948,734 and 5,093,120 (Edwards et al.).
Again note that the toxic product may either kill the nematode attempting to
feed on the plant cell in which it is expressed or simply disable the nematode
so that it is less capable of feeding on the plant cell or establishing a feeding
site. For example, the heterologous DNA may encode a peptide, antibody or the like
that disrupts feeding by interacting with the ingestion or digestion of food such
as one of the antibodies described for soybean cyst nematode including that against
the dorsal pharyngeal gland (Atkinson et al, (1988)
Annals of Applied Biology
112:459-469), modified as necessary for specificity to the root knot nematode,
using the procedures for transgenic expression of antibodies in plants described
by Hiatt, A. Gafferkey, R. C. & Bowdish, K. ((1989) Production of Antibodies in
Transgenic Plants,
Nature 342:76-78).
Again it is preferred, particularly where the plant is a food plant, that the
nematode-toxic product be non-toxic to other animals, and particularly be non-toxic
to birds, reptiles, amphibians, mammals and humans.
Where the expression product of the gene is to be located in a cellular compartment
other than the cytoplasm, the structural gene may be constructed to include regions
which code for particular amino acid sequences which result in translocation of
the product to a particular site, such as the cell plasma membrane, or secretion
into the periplasmic space or into the external environment of the cell. Various
secretory leaders, membrane integration sequences, and translocation sequences
for directing the peptide expression product to a particular site are described
in the literature. See, for example, Cashmore et al.,
Biotechnology (1985)
3:803-808, Wickner and Lodish,
Science (1985) 230:400-407.
The expression cassette may be provided in a DNA construct which also has at
least one replication system. For convenience, it is common to have a replication
system functional in
Escherichia coli, such as ColE1, pSC101, pACYC184,
or the like. In this manner, at each stage after each manipulation, the resulting
construct may be cloned, sequenced, and the correctness of the manipulation determined.
In addition, or in place of the
E. coli replication system, a broad host
range replication system may be employed, such as the replication systems of the
P-1 incompatibility plasmids, e.g., pRK290. In addition to the replication system,
there may be at least one marker present, which may be useful in one or more hosts,
or different markers for individual hosts. That is, one marker may be employed
for selection in a prokaryotic host while another marker may be employed for selection
in an eukaryotic host, particularly the plant host. The markers may provide protection
against a biocide, such as antibiotics, toxins, heavy metals, or the like; may
provide complementation by imparting prototrophy to an auxotrophic host; or may
provide a visible phenotype through the production of a novel compound in the plant.
Exemplary genes which may be employed include neomycin phosphotransferase (NPTII),
hygromycin phosphotransferase (HPT), chloramphenicol acetyltransferase (CAT), nitrilase,
and the gentamicin resistance gene. For plant host selection, non-limiting examples
of suitable markers are beta-glucuronidase (GUS) (providing indigo production),
luciferase (providing visible light production), NPTII (providing kanamycin resistance
or G418 resistance), HPT (providing hygromycin resistance), and the mutated aroA
gene (providing glyphosate resistance).
An advantage of the present invention is that two or more promoters can be "daisychained"
to a single structural gene. Where each promoter is responsive to a different pathogen,
the plant is then provided with resistance to a plurality of promoters. For example,
a second promoter may be positioned upstream from the structural gene and operatively
associated therewith so that the structural gene is associated with a plurality
of promoters, with each of the promoters activated by a different plant pathogen.
Still more promoters can be included if desired. Other promoters that may be used
in conjunction with the instant promoter are described in U.S. Pat. No. 5,750,386
to Conkling et al.
The various fragments comprising the various constructs, expression cassettes,
markers, and the like may be introduced consecutively by restriction enzyme cleavage
of an appropriate replication system and insertion of the particular construct
or fragment into the available site. After ligation and cloning, the DNA construct
may be isolated for further manipulation. All of these techniques are amply exemplified
in the literature. See, e.g., Maniatis et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982).
4. Plant Transformation Vectors and Techniques
A vector is a replicable DNA construct. Vectors which may be used to transform
plant tissue with DNA constructs of the present invention include both
Agrobacterium
vectors and ballistic vectors, as well as vectors suitable for DNA-mediated
transformation.
Agrobacterium tumefaciens cells containing a DNA construct
of the present invention, wherein the DNA construct comprises a Ti plasmid, are
useful in methods of making transformed plants. Plant cells are infected with an
Agrobacterium tumefaciens to produce a transformed plant cell, and then
a plant is regenerated from the transformed plant cell.
Numerous
Agrobacterium vector systems useful in carrying out the
present invention are known. For example, U.S. Pat. No. 4,459,355 discloses a method
for transforming susceptible plants, including dicots, with an
Agrobacterium
strain containing the Ti plasmid. The transformation of woody plants with an
Agrobacterium vector is disclosed in U.S. Pat. No. 4,795,855. Further, U.S.
Pat. No. 4,940,838 to Schilperoort et al. discloses a binary
Agrobacterium vector
(i.e., one in which the
Agrobacterium contains one plasmid having the vir
region of a Ti plasmid but no T-DNA region, and a second plasmid having a T-DNA
region but no vir region) useful in carrying out the present invention.
Microparticles carrying a DNA construct of the present invention,
which microparticle is suitable for the ballistic transformation of a plant cell,
are also useful for making transformed plants of the present invention. The microparticle
is propelled into a plant cell to produce a transformed plant cell and a plant
is regenerated from the transformed plant cell. Any suitable ballistic cell transformation
methodology and apparatus can be used in practicing the present invention. Exemplary
apparatus and procedures are disclosed in Sanford and Wolf, U.S. Pat. No. 4,945,050,
and in Agracetus European Patent Application Publication No. 0 270 356, titled
"Pollen-mediated Plant Transformation". When using ballistic transformation procedures,
the expression cassette may be incorporated into a plasmid capable of replicating
in the cell to be transformed. Examples of microparticles suitable for use in such
systems include 1 to 5 μm gold spheres. The DNA construct may be deposited
on the microparticle by any suitable technique, such as by precipitation.
Plant species may be transformed with the DNA construct of the present invention
by the DNA-mediated transformation of plant cell protoplasts and subsequent regeneration
of the plant from the transformed protoplasts in accordance with procedures well
known in the art.
5. Plants for Transformation and Propagation of Transformants
Plants that may be used to carry out the present invention are typically vascular
plants (including angiosperms and gymnosperms, monocots and dicots).
Cells used to carry out the present invention may be vascular plant cells,
which may reside in vitro or in vivo in a plant tissue or intact plant, but other
cell types such as bacterial cell may be employed to carry out intervening steps
involved in preparing the DNA constructs employed in carrying out the present invention.
A transformed plant or host cell is a plant or host cell which has been transformed
or transfected with DNA constructs as disclosed herein, using recombinant DNA techniques
such as those described above coupled with propagation techniques such as those
described below.
The promoter sequences disclosed herein may be used to express a heterologous
DNA sequence in any plant species capable of utilizing the promoter (i.e., any
plant species the RNA polymerase of which binds to the promoter sequences disclosed
herein). Examples of plant species suitable for transformation with the DNA constructs
of the present invention include both monocots and dicots, and include but are
not limited to tobacco, soybean, potato, cotton, sugarbeet, sunflower, carrot,
celery, flax, cabbage and other cruciferous plants, pepper, tomato, citrus trees,
bean, strawberry, lettuce, maize, alfalfa, oat, wheat, rice, barley, sorghum and
canola. Thus an illustrative category of plants which may be transformed with the
DNA constructs of the present invention are the dicots, and a more particular category
of plants which may be transformed using the DNA constructs of the present invention
are members of the family Solanacae.
Any plant tissue capable of subsequent clonal propagation, whether by organogenesis
or embryogenesis, may be transformed with a vector of the present invention. The
term "organogenesis," as used herein, means a process by which shoots and roots
are developed sequentially from meristematic centers; the term "embryogenesis,"
as used herein, means a process by which shoots and roots develop together in a
concerted fashion (not sequentially), whether from somatic cells or gametes. The
particular tissue chosen will vary depending on the clonal propagation systems
available for, and best suited to, the particular species being transformed. Exemplary
tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes,
callus tissue, existing meristematic tissue (e.g., apical meristems, axillary buds,
and root meristems), and induced meristem tissue (e.g., cotyledon meristem and
hypocotyl meristem).
6. Uses of the Invention
The present invention may be used in the manner described in U.S. Pat. No. 5,750,386
to Conkling et al. or U.S. Pat. No. 5,589,622 to Gurr et al. Thus, the present
invention provides a method of controlling nematodes, comprising: (a) providing
a cyst and root knot nematode-responsive promoter as described above, (b) preparing
a construct as described above by combining said promoter with a further region
which codes for a product disruptive of nematode attack, and (c) transforming plants
with the construct to obtain plants which are cyst and root knot nematode resistant.
The plants employed may be as described above, and transformation may be carried
out as described above. Once a first generation (F
O generation) of transformed
plants are obtained, plant seed that contains the aforesaid construct, and that
germinates into a cyst and root knot nematode resistant transgenic plant, may be
produced from the F
O plants by conventional breeding procedures. An
agricultural field infected with cyst or root knot nematodes, or susceptible to
cyst or root knot nematode infection, can then be planted with a crop of such plants
in accordance with standard techniques (e.g., by planting seed or plantlets) to
provide an agricultural field of crop plants that are resistant to cyst and/or
root knot nematode infection.
The present invention is explained in greater detail in the following non-limiting Examples.
EXAMPLE 1
Plant and Nematode Culture Maintenance
Plant Material. Tobacco (
Nicotiana tabacum ‘NC95’) seeds
were surface sterilized with 2.5% sodium hypochlorite for five minutes, followed
by several rinses with sterile water, and germinated in Petri plates containing
0.8% Noble agar (Fisher Scientific, Pittsburgh, Pa.) supplemented with Murashige
and Skoog ((1962)
Physiol. Plant Path. 15:473-497) minimal media, pH 5.8
and 3% sucrose. Tobacco seedlings were grown in a controlled temperature growth
chamber at 25° C. with a 14-hour photoperiod.
Nematode Cultures and Inoculations. The tobacco cyst nematode (TCN),
Globodera
tabacum subspecies solanacearum (Miller and Gray (1972)
Nematologica 18:404-413),
and the root-knot nematode (RKN),
Meloidogyne incognita Race 4 (Hartman
and Sasser (1985) Identification of Meloidogyne species on the basis of differential
host test and perineal—pattern morphology. In Advanced Treatise on Meloidogyne,
Vol.II (Biology and Control), ed. J. N. Sasser and C. C. Carter, pp. 69-77, Raleigh,
N.C.: NCSU Graphics) were propagated on greenhouse-grown tobacco (
Nicotiana
tabacum ‘NC95’) and tomato (
Lycopersicon esculentum cv.
Rutgers), respectively. To isolate cyst nematode eggs, TCN cysts were crushed gently
in a glass homogenizer and the eggs were rinsed onto a 25 μm sieve. Hatching
of TCN eggs was stimulated over filter-sterilized tobacco root diffusate (LaMondia
(1995)
J. Nematol. 27: 382-386) at 28° C. on a Baermann pan. RKN eggs
were isolated from egg masses on tobacco roots with 0.5% sodium hypochlorite then
rinsed with water and collected on a 25 μm sieve (Hussey and Barker (1973)
Plant Dis. Rep. 57:1025-1028). RKN eggs were set up to hatch over water
at 28° C. on a Baermann pan. After 3 days, hatched second-stage juveniles
(J2) of either TCN or RKN were collected on a 25 μm sieve and surface-sterilized
with 0.002% mercuric chloride, 0.002% sodium azide, and 0.001% Triton X-100 for
5 minutes followed by several washes with sterile water. Surface-sterilized J2
were resuspended in 50 μl of 2 mM Penicillin-G and 950 μl of 1.5% low
melting agarose at 37° C. at a concentration of approximately 50 J2/10 μl
for TCN and 5 J2/10 μl for RKN. Ten microliter aliquots of J2 were used to
inoculate two week-old tobacco root tips.
EXAMPLE 2
In Planta Localization of TCN EGases Tissue
Fixation and Embedding. For immunolocalizations, TCN-infected root pieces
were excised from Petri plates twenty-four to ninety-six hours after inoculation
and fixed in 1% paraformaldehyde in phosphate-buffered saline (PBS; 137 mM NaCl,
1.4 mM KH
2PO
4, 2.6 mM KCl, 1.8 mM Na
2HPO
4,
pH 7.4) for three hours at room temperature. After two 15 minute washes with PBS,
the fixed root pieces were dehydrated in a graded ethanol series (30%, 60%, 70%,
85%, 95%, 100%, 15 min. each) and then incubated sequentially in ethanol: Histoclear
(National Diagnostics, Atlanta, Ga.) 75:25, 50:50, 25:75 for 10 minutes each. After
two 15 minutes incubations in 100% Histoclear, the root pieces were transferred
to molten Paraplast plus (Fisher Scientific, Pittsburgh, Pa.) at 60° C. for
two hours and embedded in blocks. For in situ mRNA localizations, nematode-infected
tobacco root pieces were dissected from Petri plates 7-9 days or 12-14 days after
infection and infiltrated with 4% paraformaldehyde (PFA) in phosphate-buffered
saline (PBS; 130 mM NaCl, 7 mM Na
2H
2PO
4, 3 mM
NaH
2PO
4) using a short vacuum. The root pieces were then
transferred to fresh 4% PFA, incubated an additional 6 hours at room temperature
followed by seventeen hours in 4% PFA at 4° C. The root pieces were washed
twice in PBS for 20 minutes each time, dehydrated in a graded ethanol series (30%,
60%, 70%, 85%, 95%, 100%), incubated sequentially in Histoclear (National Diagnostics,
Atlanta, Ga.): ethanol 25:75, 50:50, 75:25, and then in 100% Histoclear twice for
30 minutes each time. The root pieces were incubated in Histoclear: Paraplast (Fisher
Scientific) 75:25 overnight at 60° C., and then overnight again in pure Paraplast
at 60° C. The Paraplast-embedded root pieces were sectioned to a thickness
of 12μm (TCN-infected tobacco root tissue) or 20 μm (RKN-infected tobacco
root tissue) using a rotary microtome (American Optical, Buffalo, N.Y.) and adhered
to Superfrost Plus microscope slides (Fisher Scientific) overnight at 40°
C. on a slide warmer. Three 15-minute incubations in Histoclear were used to remove
the Paraplast from the sections followed by an ethanol series up to water to rehydrate
the sections.
TCN-infected roots were sectioned to a thickness of 10 μm using
a rotary microtome (American Optical, Buffalo, N.Y.) and placed on Superfrost Plus
(Fisher Scientific) microscope slides. The sections were adhered to the slides
overnight on a 40° C. slide warmer. Three 10-minute incubations in Histoclear
at room temperature were used to remove the paraffin from the sections followed
by rehydration in a graded ethanol series up to water. Non-specific binding sites
in sections were blocked with 10% normal goat serum containing protease inhibitors
(10 μl/ml of Stock A=0.1 mM leupeptin, 100 mM Na
2EDTA, 20 mM iodoacetamide,
and Stock B=20 mM phenylmethylsulfonyl fluoride, 0.1 mM pepstatin A [all chemicals
from Sigma, St. Louis, Mo.]) for three hours at room temperature. Primary antibody
(GR-ENG1 mouse polyclonal sera; Smant, et al. (1998)
Proc. Natl. Acad. Sci.
USA 95:4906-491 1) diluted 1:100 with 10% goat serum in PBS was applied to
the sections and incubated overnight at 4° C. After three five-minute rinses
in PBS, secondary Alexa 488-goat anti-mouse IgG conjugate (Molecular Probes, Eugene,
Oreg.) diluted 1:500 was applied to the sections and allowed to incubate in the
dark for three hours at room temperature. The sections were rinsed three times
for five minutes each in PBS before mounting with antiquenching agent (0.2 M carbonate
buffer, pH 8.6, 50% glycerol, 0.02 mg/ml p-phenylenediamine). Sections were observed
and photographed on an epifluorescence microscope (Zeiss, Oberkochen, Germany).
Endo-β-1,4-Glucanases In Planta. Antiserum raised
against recombinant GR-ENG-1 (endoglucanase) of the potato cyst nematode (PCN),
Globodera rostochiensis (Smant, et al. (1998)
Proc. Natl. Acad. Sci.
USA 95:4906-4911), was confirmed to bind to both GT-ENG-1 and GT-ENG-2 of the
tobacco cyst nematode (TCN),
G. tabacum (Goellner, et al. (2000)
J. Nematol.
32:154-165). GR-ENG-1 antiserum did not bind to total protein preparations
from either uninfected or TCN-infected tobacco roots on protein gel blots (data
not shown). No staining was observed in plant tissue sections probed with mouse
pre-immune sera, nor did the pre-immune sera bind to the nematode cuticle (FIG.
1A). Sections of infected roots at 24 hours after inoculation with J2 of
TCN that were probed with GR-ENG-1 antiserum localized TCN EGases within the nematode
and in tobacco root cortical tissue (FIG. 1B). Within the nematode, EGases
were localized throughout the subventral esophageal gland cells including their
extensions and terminating in the subventral gland ampullae at the base of the
metacorpus (FIGS. 1C-1D). Within plant tissue, GR-ENG-1 antiserum
localized TCN outside the head of the nematode and along the migratory path of
the nematode through tobacco root tissue (FIGS. 1C-1D). Occasionally,
the antiserum bound to the surface of the nematode, which may indicate the binding
of EGases to the cuticle as the nematode migrated forward through root tissue (FIG. 1H).
To monitor the TCN EGases during the initiation of syncytia within host roots,
tobacco roots inoculated with infective J2 were fixed for sectioning at 48-96 hours
post-infection. The time of root penetration by J2 was monitored using an inverted
microscope, and the stage of nematode development and extent of syncytium formation
was determined in sections. EGases were not detected by GR-ENG-1 antiserum within
initial syncytial cells during the early stages of formation, even when EGases
were still detectable within the subventral gland cells of parasitic J2 (FIGS.
1E-1F). TCN EGases were also not detected within well-developed syncytia
(FIGS. 1G-1H).
EXAMPLE 3
Isolation and Sequence Characterization of Tobacco EGases
To isolate poly A(+) RNA, 5 cm of infected or noninfected tobacco root pieces
(excluding root tips) were excised from Petri plates and ground in a small glass
homogenizer in 250 μl lysis-binding buffer (100 mM Tris-HCl, pH 7.5, 500
mM LiCl, 10 mM EDTA, pH 8.0, 5 mM dithiothreitol, 1% LiDS; Dynal, Lake Success,
N.Y.). After lysis, the homogenate was centrifuged for one minute at 13,000×g
and the supernatant was transferred to a clean tube. Twenty-five microliters of
Dynal magnetic oligo-(dT)
25 beads equilibrated with lysis-binding buffer
were added to the supernatant and placed on a rotator for 5 minutes to allow the
mRNA to anneal to the beads. Using a magnetic stand the beads were washed twice
in washing buffer with LiDS (10 mM Tris-HCl, pH 7.5, 0.15 M LiCl, 1 mM EDTA, 0.1%
LiDS) and three times in washing buffer without LiDS. For first strand cDNA synthesis,
the beads were washed several times in 1× first strand buffer (25 mM Tris-HCl,
pH 8.3, 37.5 mM KCl, 1.5 mM MgCl
2) and then resuspended in 12 μl
of RNase-free water. The following components were added to the bead suspension:
4 μl 5× first strand buffer, 2 μl 0.1 M DTT, 1 μl 10 mM
dNTP mix, 1 μl Superscript II reverse transcriptase (200U/μl; GibCo
BRL, Rockville, Md.). The reaction mixture was incubated on a rotator at 42°
C. for one hour. Following first strand cDNA synthesis, two units of RNase H were
added to the reaction and allowed to incubate at 37° C. for 20 minutes. The
beads were rinsed twice with TE (10 mM Tris-HCl, pH8.0, 1 mM EDTA) and then resuspended
in 25 μl of TE and stored at -20° C. Two degenerate primers to conserved
amino acid domains of known plant EGase sequences were designed as follows: CWERPED:
5′-TGTTGGGARAGRCCHGARGAY-3′ (SEQ ID NO:10) and YINAPL2: 5′-MACHADHGSWGCATTRAYRTAWGT-3′
(SEQ ID NO:11) where R=A+G, Y=C+T, M=A+C, S=G+C, W=A+T, H=A+T+C, D=G+A+T. A ten
microliter aliquot of first strand cDNA on the beads was washed with 1×PCR
buffer (20 mM Tris-HCl, pH 8.4, 50 mM KCl) before adding the following reaction
components: 5 μl 10×PCR buffer (200 mM Tris-HCl, pH8.4, 500 mM KC),
1.5 μl 50 mM MgCl
2, 1.0 μl 10 mM dNTP mix, 2.5 μl
10 μM 5′ CWERPED, 2.5 μl 10 μM 3′ YINAPL2, 27 μl
dH
20, and 2.5U Taq Polymerase). The PCR cycles were as follows: 1 cycle
at 94° C. 2 min; 5 cycles at 94° C. 1 min., 37° C. 1 min., 72°
C. 2 min with a ramp of 14° C./min between the annealing and elongation step
(Compton, 1990); 35 cycles at 94° C. 30 sec, 50° C. 50 sec, 72°
C. 1 cycle at 72° C. 10 min. A 1 kb amplified fragment was obtained and cloned
into the pCR2.1 TA cloning vector (Invitrogen, Carlsbad, Calif.). Plasmid DNA was
isolated from several individual transformants and the cDNA inserts were sequenced.
Sequencing was carried out by The Interdisciplinary Center for Biotechnology Research
(ICBR) DNA Sequencing Core Laboratory (DSEQ) located at the University of Florida,
Gainsville, Fla. Isolation of the full-length tobacco EGase cDNAs was accomplished
using 3′ and 5′ random amplification of cDNA ends systems (RACE,
GibCo BRL) according to the manufacturer's protocols. The five tobacco EGase cDNA
sequences were submitted to Genbank and have been designated with the following
names and accession numbers; Ntcel2=AF362948 (SEQ ID NO:1), Ntcel4=AF362950, Ntcel5=AF362951,
Ntcel7=AF362947 (SEQ ID NO:3), Ntcel8=AF362949 (SEQ ID NO:5).
FIG. 2 shows the 1 kb cDNA product amplified by RT-PCR from TCN-infected tobacco
root tissue (data not shown) using primers designed to two conserved amino acid
domains (CWERPEDM (SEQ ID NO:7) and YINAPL (SEQ ID NO:8)) present in plant EGases.
No observable products were amplified from identical uninfected root tissue. Five
distinct tobacco EGase cDNA sequences representing structurally divergent gene
family members were identified after sequencing random clones of the 1 kb product
and designated as Ntcel2, Ntcel4, NtCel5, Ntcel7, and Ntcel8 based on the nomenclature
described by del Campillo ((1999) Multiple endo-1,4-β-D-glucanase (Cellulase)
genes in
Arabidopsis. In Current Topics in Developmental Biology, Volume
46, ed. R. A. Pedersen and G. P. Schatten, pp. 39-61. New York: Academic Press).
Full-length coding sequences of three of these cDNA clones (Ntcel2, Ntcel7, and
Ntcel8) were obtained using 3′ and 5′ RACE PCR (FIG. 2). The
1 kb partial cDNA sequences of Ntcel4 and Ntcel5 showed the highest percentage
nucleotide and amino acid sequence identity with tomato Cel4 and Cel5, respectively
(Brummell, et al. (1997)
Plant Mol. Biol. 33:87-95; del Campillo and Bennett
(1996)
Plant Physiol. 111:813-820), and were not characterized further due
to their low expression levels (see below). A phylogenetic tree constructed using
mature amino acid sequences of Ntcel2 (SEQ ID NO:2), Ntcel7 (SEQ ID NO:4), and
Ntcel8 (SEQ ID NO:6), and 14 other plant EGase sequences depicts the relatedness
of the new tobacco EGases with selected members of the plant EGase gene family
(FIG. 3). The 1500 bp open reading frame (ORF) of the 1674 bp Ntcel2 cDNA
clone encodes a 500-amino acid polypeptide, including a 35-mer putative signal
peptide (FIG. 2). The mature protein has a deduced molecular mass of 51.3
kDa and a pI of 8.7. Ntcel2 has 54% nucleotide and amino acid sequence identity
to Ntcel7 and 55% nucleotide and 49% amino acid identity to Ntcel8. Ntcel2 shares
significant amino acid similarity with pepper Cel3 (89%; Trainotti, et al. (1998)
Hereditas 128:121-126), tomato Cel2 (86%; Lashbrook, et al. (1994)
Plant
Cell 6:1485-1493), and Arabidopsis Cell (73%; Shani, et al. (1997)
Plant
Mol. Biol. 34:837-842) (FIG. 3). The 1467 bp ORF of the 1723 bp Ntcel7
cDNA clone encodes a 489-amino acid polypeptide, including a putative signal peptide
corresponding to amino acids 1-24 (FIG. 2). The mature protein has a deduced
molecular mass of 51.8 kDa and pI of 8.7. Analysis of the predicted amino acid
sequence of Ntcel7 showed 52% nucleotide and 49% amino acid identity with Ntcel8.
Ntcel7 shares significant amino acid sequence similarity with tomato Cel7 (86%;
Catala, et al. (1997)
Plant Journal 12:417-426), orange Celb1 (71%; Burns,
J. K. et al., 1997, unpub.; Accession #AF000136 ), and pea Egl1 (68%; Wu, et al.
(1996)
Plant Physiol. 111:163-170) (FIG. 5). The 1872 bp ORF of the
2286 bp Ntcel8 cDNA clone encodes a 624-amino acid polypeptide, including a 28-mer
putative signal peptide (FIG. 2). The mature protein has a deduced molecular
mass of 65.7 kDa and a pI of 8.0. This tobacco EGase shares significant amino acid
similarity to tomato Cel8 (81%; Catala and Bennett (1998)
Plant Physiol. 118:1535)
and strawberry Eg3 (79%; Trainotti, et al. (1999)
Plant Mol. Biol. 40:323-332).
Amino acid sequence alignment of all three tobacco EGases depicts the extra 124
amino acids at the C-terminus of Ntcel8 that are absent from Ntcel2 and Ntcel7
(FIG. 2).
DNA Gel Blot Analysis. Tobacco genomic DNA was isolated fr
