Note: Descriptions are shown in the official language in which they were submitted.
WO95/03690 2 1 6 8 4 3 0 PCT~S94/08722
HMG2 PROMOTER EXPRESSION SYSTEM AND
POST-HARVEST PRODUCTION OF GENE PRODUCTS
IN PLANTS AND PLANT CELL CULTURES
This application is a continuation-in-part of co-
5 pending application Serial No. 08/100,816 filed August 2,
1993, which is hereby incorporated by reference in its
entirety.
1. INTRODUCTION
The present invention relates in part to HMG2
10 promoters, active fragments thereof, and their use to drive
the expression of heterologous genes. Expression vectors may
be constructed by ligating a DNA sequence encoding a
heterologous gene to an HMG2 promoter or a promoter modified
with an HMG2 cis-regulatory element. Such constructs can be
15 used for the expression of proteins and RNAs in plant cell
expression systems in response to elicitor or chemical
induction, or in plants in response to wounding, pathogen
infection, pest infestation as well as elicitor or chemical
induction.
The present invention also relates to a method of
producing gene products in harvested plant tissues and
cultures. The "post-harvest" production method utilizes
tissues or cultures from transgenic plants or plant cells,
respectively, engineered with expression constructs comprising
25 inducible promoters operably linked to sequences encoding
desirable protein and RNA products. Treatment of the
harvested tissues or cultures with the appropriate inducer
and/or inducing conditions initiates high level expression of
the desired gene product. The post-harvest production method
30 may advantageously be used to produce gene products and
metabolites of gene products that are labile, volatile or
toxic or that are deleterious to plant growth or development.
This aspect of the invention is demonstrated by way
of examples which describe the production of transgenic plants
35 engineered with expression constructs comprising inducible
promoters operably linked to coding sequences of interest, and
21 68430
W09~/03690 PCT~S94/08722
-- 2
which show that days-old and weeks-old harvested tissues from
such transgenic plants remain capable of induced production of
high levels of the gene product of interest.
s 2. BACKGROUND OF THE INVENTION
Plant diseases caused by fungal, bacterial, viral
and nematode pathogens cause significant damage and crop loss
in the United States and throughout the world. Current
approaches to disease control include breeding for disease
10 resistance, application of chemical pesticides, and disease
management practices such as crop rotations. Recent advances
in plant biotechnology have led to the development of
transgenic plants engineered for enhanced resistance to plant
pests and pathogens. These successes indicate that
15 biotechnology may significantly impact production agriculture
in the future, leading to improved crops displaying genetic
resistance. This mechanism of enhanced disease resistance has
the potential to lower production costs and positively impact
the environment by reducing the use of agrichemicals.
Recent advances in plant biotechnology have led also
to the development of transgenic plants engineered for
expression of new functions and traits. Such engineering has
produced transgenic plants having new synthetic capabilities.
These successes indicate that biotechnology may enhance the
25 role of plants as a producer of chemicals, pharmaceuticals,
polymers, enzymes, etc. The engineering of novel synthetic
capabilities has the potential to increase crop values as well
as create new uses for crops such as tobacco.
2.1. PLANT DEFENSE RESPONSES
Higher plants have evolved a variety of structural
and chemical weapons for protection against pathogens,
predators, and environmental stresses. Expression of
resistance toward pathogens and pests involves the rapid
35 induction of genes leading to accumulation of specific
defense-related compounds (Dixon et al., 1990, Adv. Genet.
28:165-234). These responses include accumulation of
2 1 68430 ~ O~ 7 22
J Z6 JUL '~
-- 3
phytoalexin antibiotics, deposition of lignin-like material,
accumulation of hydroxyproline-rich glycoproteins, and
increases in hydrolytic enzymes such as chitinase and
glucanase.
Phytoalexins are low molecular weight compounds
which accumulate as a result of pathogen challenge.
Phytoalexins have antimicrobial activity and have proved to be
of critical importance in disease resistance in several
plant:pathogen interactions (Darvill et al., 1984, Annu. Rev.
10 Plant Physiol. 35:243; Dixon et al., 1990, Adv. Genet. 28:165-
234; and Kuc and Rush, 1895, Arch. Biochem. Biophys. 236:455-
472). Terpenoid phytoalexins represent one of the major
classes of plant defense compounds and are present across a
wide range of plant species including conifers, monocots and
15 dicots. The regulation of terpenoid phytoalexin biosynthesis
has been most extensively studied among the solanaceae (e.g.,
tomato, tobacco, potato), however, most important crop species
utilize this pathway in disease resistance.
In well-characterized plant:microbe interactions,
20 resistance to pathogens depends on the rate at which defense
responses are activated (Dixon and Harrison, 1990, Adv. Genet.
28:165-234). In an incompatible interaction, the pathogen
triggers a very rapid response, termed hypersensitive
resistance (HR), localized to the site of ingress. In the
25 susceptible host, no induction of defense responses is seen
until significant damage is done to host tissue.
Defense-responses, including phytoalexins
accumulation, can also be induced in plant cell cultures by
compounds, termed "elicitors", for example components derived
30 from the cell walls of plant pathogens (Cramer et al., 1985,
ENBO J. 4:285-289; Cramer et al., 1985, Science 227:1240-1243;
Dron et al., 1988, Proc. Natl. Acad. Sci. USA 85:6738-6742).
In tobacco cell cultures, fungal elicitors trigger a
coordinate increase in sesquiterpene phytoalexin biosynthetic
35 enzymes and a concomitant decrease in squalene synthetase
which directs isoprene intermediates into sterol biosynthesis
(Chappell and Nable, 1987, Plant Physiol. 85:469-473; Chappell
~0 ~
21 68430
WOgS/03690 PCT~S94108722
et al., 1991, Plant Physiol. 97:693-698; Threlfall and
Whitehead, 1988, Phytochem. 27:2567-2580).
2.2. PHYSIOLOGICAL ROLES OF HMGR IN PLANTS
s A key enzyme involved in phytoalexin biosynthesis
and, hence, plant defense against diseases and pests, is
3-hydroxy-3-methylglutaryl CoA reductase (HMGR; EC 1.1.134).
HMGR catalyzes the conversion of 3-hydroxy-3-methylglutaryl
CoA to mevalonic acid. Mevalonic acid is an intermediate in
10 the biosynthesis of a wide variety of isoprenoids including
defense compounds such as phytoalexin.
In addition to phytoalexin biosynthesis, the
mevalonate/isoprenoid pathway produces a diverse array of
other biologically important compounds (see Figure 1). These
lS include growth regulators, pigments, defense compounds,
W-protectants, phytotoxins, and specialized terpenoids such
as taxol, rubber, and compounds associated with insect
attraction, fragrance, flavor, feeding deterrence and
allelopathy. Many of these compounds have critical roles in
20 cellular and biological functions such as membrane biogenesis,
electron transport, protein prenylation, steroid hormone
synthesis, intercellular signal transduction, photosynthesis,
and reproduction.
The conversion of 3-hydroxy-3-methylglutaryl CoA to
25 mevalonic acid appears to be the rate-limiting step of
isoprenoid biosynthesis. Thus, the regulation of HMGR
expression serves as a major control point for a diversity of
plant processes including defense responses. Plant HMGR
levels change in response to a variety of external stimuli
30 including light, plant growth regulators, wounding, pathogen
attack, and exogenous sterols (Brooker and Russell, 1979,
Arch. Biochem. Biophys. 198:323-334; Russell and Davidson,
1982, Biochem. Biophys. Res. Comm. 104:1537-lS43; Wong et al.,
1982, Arch. Biochem. Biophys. 216:631-638 and Yang et al.,
35 1989, Mol. Plant-Microb. Interact. 2:195-201) and vary
dramatically between tissues and developmental stages of plant
growth (Bach et al., 1991, Lipids 26:637-648 and Narita and
W095/03690 2 1 6 8 4 3 0 PCT~S94,08722
Gruissem, 1991, J. Cell. Biochem l5A:102 (Abst)). Reflecting
this complexity, multiple HMGR isozymes exist in plants which
are differentially regulated and may be targeted to distinct
organellar locations. In addition to microsomal locations,
5 plant HMGR isozymes have been localized to mitochondrial and
chloroplast membranes (Brooker and Russell, 1975, Arch.
Biochem. Biophys. 167:730-737 and Wong et al., 1982, Arch.
Biochem. Biophys. 216:631-638).
2.3. PLANT HMGR GENES
HMGR genomic or cDNA sequences have been reported
for several plant species including tomato (Narita and
Gruissem, 1989, Plant Cell 1:181-190; Park, 1990, Lycopersicon
esculentum Mill. Ph.D. Dissertation, Virginia Polytechnic
15 Institute and State University, Blacksburg, VA), potato (Choi
et al., 1992, Plant Cell 4:1333-1344 and Stermer et al., 1991,
Physiol. Mol. Plant Pathol. 39:135-14S), radish (Bach et al.,
1991, American Oil Chemists Society, Champaign, IL paqe 29-
49), Arabidopsis thaliana (Caelles et al., 1989, Plant Mol.
20 Biol. 13:627-638; Learned and Fink, 1989, Proc. Natl. Acad.
Sci. USA 86:2779-2783), Nicotiana sylvestris (Genschik et al.,
1992, Plant Mol. Biol. 20:337-341), Catharanthus roseus
(Maldonado-Mendoza et al., 1992, Plant Physiol. 100:1613-
1614), the Hevea rubber tree (Chye et al., 1991, Plant Mol.
25 Biol. 16:567-577) and wheat (Aoyagi et al., 1993, Plant
Physiol. 102:622-638). Cloning of additional HMGR genes from
rice (Nelson et al., 1991, Abstract 1322, 3rd Intl. Cong.
Intl. Soc. Plant Mol. Biol., Tucson, AZ), and tomato (Narita
et al., 1991, J. Cell. Biochem 15A:102 (Abst)) also has been
30 reported but no DNA sequence information has been published
for these HMGR genes. In all plants thus far investigated,
HMGR is encoded by a small gene family of two (Arabidopsis) or
more members (tomato, potato, Hevea). See Table 1 for a
summary of published information pertaining to plant HMGR
3S genes.
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TABLE 1 (continued)
Footnotes
a. G, genomic clone; cDNA, complementary DNA generated from
mRNA (lacks promoter, introns); F, full-length clone
(contains entire coding sequence); P, partial sequence;
GenEMBL accession numbers are listed
b. Many of the HMGR sequences were not tested for induction
by any of the following indicated inducing conditions and
factors. These sequences are indicated as (?). Of the
tested HMGR sequences, the indicated inducing conditions
or factors are not meant to be exclusive, as a "tested"
HMGR sequence may not have been tested with all of the
following indicated inducing conditions and factors. For
example, the potato hmg2 gene was not tested for
induction by fungal pathogen infection or nematode
infestation. Induced by bacterial pathogen (B), fungal
pathogen (F), virus pathogen (V), parasitic nematode (N),
or defense elicitors (E).
c. Promoter has undergone sequence analysis (S), has been
analyzed in transgenic plants by expression of
promoter:reporter gene fusions (GUS), has undergone
deletion analyses (D).
References and Gen~MRT Accession Numbersa
1. This disclosure and tM63642]
2. Narita et al, 1989, Plant Cell 1:181-190.
3. Choi, et al., 1992, Plant Cell 4:1333-1344. tL01400;
L01401; L01402]
4. Stermer et al., 1992, Phytopathology 82:1085 (Abstract).
5. Genschik et al., 1992. Plant Mol. Biol. 20:337-341.
[X63649]
6. Learned and Fink, 1989, Proc. Natl. Acad. Sci. USA
86:2779-2783.
tJ04537]
7. Caelles et al., 1989, Plant Mol. Biol. ~3:627-638.
tXl5032 ]
8. Chye et al., 1991, Plant Mol. Biol 16:567-577. tX54657;
X54658; X54659]
9. Chye et al., 1992, Plant Mol. Biol. 19:473-484.
10. Maldonado-Mendoza et al., 1992, Plant Physiol. 100:1613-
1614.
[M96068]
11. Bach et al., 1991, In Physiology and Biochemistry of
Sterols (Patterson,G. and W. Nes, eds.), Am. Oil Chemists
Soc., Champaign, IL, pp. 29-49.
12. Aoyagi et al., 1993, Plant Physiol. 102:623-628
a GenEMBL Accession Number are in brackets.
'} ~ T
- - 2 1 68430 . J.,~ 4 ~ 08 7 22
o J~L 95
2.4. TOMATO HMGR GENES
Tomato HMGR genes have been isolated by direct
probing of a tomato genomic library with yeast HMGl cDNA
sequences tCramer et al., 1989, J. Cell. Biochem. 13D:M408;
5 Park, 1990, Lycopersicon esculentum Mill. Ph.D. Dissertation,
Virginia Polytechnic Institution and State University,
Blacksburg, VA). One of these genes, designated tomato HMG2,
has been characterized further and the sequence of the coding
region reported (Park et al., 1992, Plant Nol. Biol. 20:327-
10 331; GenBank M63642). The nucleic acid sequence of tomatoHMG2 has been compared to those of other plant HMGRs.
Sequence comparisons within regions encoding the HMGR protein
show identities in the range of 69 to 96%, with the most
closely related sequences being a partial cDNA sequence of
15 potato HMG2 (96% sequence identity for 744 bases at the
C-terminus coding region) and an HMGR cDNA from Nicotiana
sylvestris (89% sequence identity; entire coding region). The
tomato HMG2 gene is distinct from tomato HMGl showing 78%
sequence identity within the region encoding the N-terminus
20 (1067 bases compared; see Figure 2). Comparisons of sequences
outside the coding region (e.g., the 5'-untranslated leader
sequence) reveal very little sequence conservation between
divergent species and only limited homology among HMGR genes
within the Solanaceae. Tomato HMG2 shows less than 7 5%
25 sequence identity over an 81 base stretch with the N.
Sylvestris leader sequences. Comparisons between the
5'-leader sequences of tomato HMGl and HMG2 are included in
Figure 2.
The coding region of the tomato HMG2 gene is
30 interrupted by three introns and encodes a protein with a
calculated molecular mass of 64,714 (Park et al., 1992, Plant
Mol. Biol. 20:327-331). Table 2 shows a comparison of the
derived amino acid sequence of the tomato HMG2 to HMGRs of
other organisms. The N-terminal membrane domain of the tomato
35 HMGR2 (the protein encoded by the HMG2 gene) is significantly
smaller, shows no sequence similarity to the yeast or animal
transmembrane domain, and contains 1-2 potential membrane
spanning regions compared to
h~D
2 1 6 8 4 30 ~ ,4~ 2Z
g ~ Z 6 JULt95
7-8 postulated in non-plant HMGRs (Basson et al., 1988, Mol.
Cell Biol. 8:3797-3808; Liscum et al., 1985, J. Biol. Chem
260:522-530). This transmembrane domain is also highly
divergent among plant species and among HMGR isogenes within a
species although the actual membrane spanning residues are
quite conserved.
Amino acid sequence comparisons between the
N-terminal two hundred residues of tomato HMGR2 and other
plant HMGRs yield sequence identities in the range of 44-85%
(Table 2). The amino acid sequences of tomato HMGRl and HMGR2
show only 75% sequence identity in this region. The catalytic
domain of tomato HNGR2, located within the C-terminal 400
amino acids, shows significant sequence homology with the
yeast (56% identity; 75% similarity) and human (55% identity;
75% similarity) HMGRs. This domain is highly conserved
between plant species: comparisons between tomato HMGR2
(residues 200-602) and other plant HMGR2 yield amino acid
identities ranging from 74-90% (see Table 2).
In contrast to the information available on plant
HMGR coding sequences, relatively little is known of the
structure of plant HMGR promoters. Indeed, to date, only one
plant HMGR promoter sequence has been reported (Chye et al.,
1991, Plant Mol. Biol. 16:567-577).
21 68430
WO 95/03690 10 PCTIUS94/08722
Table 2. Amino acid identity (%) of the membrane and catalytic
domains of pl~nt HMGRs.
Solanaceae EuPhorb. ADO Brassicaceae
Toml Tom2 Potl Pot3 Nic Hevl Hev3 Car Aral Radl Rad2
Toml 100 75 98 - 77 46 53 64 57 48 57
Tom2 1007~ - 85 4~ 52 66 S0 48 52
10090 90 95 85 7~ 8~ 81 80 80
Potl 100 - 76 45 54 63 58 49 57
100 90 93 88 85 88 83 83 83
Pot3
100 92 86 85 84 82- 81 82
Nic 100 42 49 63 54 49 57
100 87 86 87 83 83 82
Hevl 100 44 38 76 Sl 55
100 86 87 86 86 85
Hev3 100 46 56 48 48
100 85 82 82 82
Car 100 48 44 49
100 81 82 82
Aral 100 73 78
100 94 95
Radl 100 72
100 93
Rad2 100
100
Upper row: .,.e...brdne do-~ ?- ~S (1 to 200 amino acids); Lower row: catalytic domain ~201 to 600 amino
acids). Plant species and hm~r iso~ene (where desi~nated) are indicated: L. esculentum ltomato hm~ 1
(incomplete sequence) and hm~2; Tom1, Tom21, S. tuberosum [potato; Potl, Pot3 ~incomplete seq.~l, N.
sy/ve~b,s (Nic, iso~ene unknown), A. thal;ana hm~1 ~Ara1~, H. br s 'ie7sis (rubber tree; Hev1, Hev3~, C.
roseus (Car, iso~ene unknown), and R. sativus (radish; Rad l, Rad2). Comparisons are based on the
sequences available from GenBank.
Euphorb.: fuphoriaceae, Apo.: Apocynaceae.
wo 95~036go 2 1 6 8 4 3 0 PCT/US94/08722
2.5 THE FUNCTIONS AND EXPRESSION PATTERNS
OF DIFFERENT HMGR GENES
Specific plant HMGR isogenes show very distinct
patterns of regulation during development and in response to
5 pathogen infection or pest infestation (Caelles et al., 1989,
Plant Mol. Biol. 13:627-638; Choi et al., 1992, Plant Cell
4:1333-1344; Chye et al., 1992, Plant Mol. Biol. 19:473-484;
Narita and Gruissem, 1989, Plant Cell 1:181-190; Park et al.,
1992, Plant Mol. Biol. 20:327-331; Park, 1990, Lycopersicon
10 esculentum Mill. Ph.D. Dissertation, Virginia Polytechnic
Institute and State University, Blacksburg, VA; Yang et al.,
1991, Plant Cell 3:397-405).
In the Solanaceae, the pathogen-induced synthesis of
phytoalexin antibiotics is due to the induction of microsome-
15 associated HMGR (Shih and Kuc, 1973, Phytopathology 63:826-829
and Stermer and Bostock, 1987, Plant Physiol. 84:404-408). In
cultured cells, the induction occurs with the addition of
elicitors isolated from the cell walls of pathogenic microbes
(Chappell et al., 1991, Plant Physiol. 97:693-698 and Stermer
20 and Bostock, 1987, Plant Physiol. 84:404-408). Typically,
elicitor treatment results in a marked, transient increase in
HMGR mRNA level that is typical of transcriptionally regulated
defense genes in plants (Cramer et al., 1985, EMBO J. 4:285-
289; Dixon and Harrison, 1990, Adv. Genet. 28:165-234; Yang et
25 al., 1991, Plant Cell 3:397-405).
The rapid pathogen-induced HMGR expression appears
to be encoded by the HMG2 HMGR isogene. This has been
established in tomato cells treated with elicitors (Park,
1990, Lycopersicon esculentum Mill. Ph.D. Dissertation,
30 Virginia Polytechnic Institute and State University,
Blacksburg, VA) and in potato tubers infected with the soft-
rotting bacterium Erwinia carotovora (Yang et al., 1991, Plant
Cell 3:397-405). In potato tuber, HMG2 mRNA levels increased
20-fold within 14 hr following bacteria inoculation but was
35 not induced by wounding in the absence of pathogen (Yang et
al., 1991, Plant Cell 3:397-405).
wo 95/03690 2 1 6 8 4 3 0 PCT/USg4/08722
- 12 -
HMG2 expression has been shown to be critical to
disease resistance. An elicitor, arachidonic acid, induces
defense responses including increases in ~MG2 mRNA and thus,
phytoalexin synthesis in potato tubers (Stermer and Bostock,
5 1987, Plant Physiol. 84:404-408 and Yang et al., 1991, Plant
Cell 3:397-405). Tubers treated with arachidonic acid 48 hr
prior to inoculation with Erwinia carotovora were completely
resistant to rotting. Tubers inhibited in HMGR activity by
mevinolin (an HMGR-specific competitive inhibitor) showed
10 significantly increased rotting (Yang et al., 1991, Plant Cell
3:397-405).
Homologs to the tomato HMG2 HMGR isogene, based on
analogous expression patterns, exist in other plants. For
example, potato HMG2 and HMG3 mRNAs also are induced in
15 response to wounding, elicitors and bacterial pathogens (Choi
et al., 1992, Plant Cell 4:1333-1344 and Yang et al., 1991,
Plant Cell 3:397-405). An HMGR isogene isolated from
Nicotiana sylvestris is induced by virus inoculation and
defense elicitors (Genschik et al., 1992, Plant Mol. Biol.
20 20:337-341). Similarly, an HMGR isogene of rice also is
induced by wounding and elicitors (Nelson et al., 1991, Abst
1322, 3rd Intl. Cong. Intl. Soc. Plant Mol. Biol., Tucson, AZ)
suggesting that defense-specific HMG2 homologs are also
present in monocotyledonous plants. However, the three HMGR
25 cDNAs isolated from wheat apparently are not wound-inducible
(Aoyagi et al., 1993, Plant Physiol. 102:623-628). Although
DNA sequences have not been isolated, increases in HMGR enzyme
activity in response to bacterial or fungal pathogens have
been documented in a number of other plant species suggesting
30 that the HMG2 isogene is widely represented among plants.
Tomato HMG2 is quite distinct from the tomato HMGl
isogene at the nucleic acid level (Figure 2). The HMGl HMGR
isogene appears to be expressed under circumstances that are
different from those that induce the expression of the HMG2
35 gene. Narita and Gruissem have shown that tomato HMGl is
expressed at low levels in all tomato tissues analyzed and is
highly expressed in immature fruit during the period of rapid
WO95/03690 2 1 6 8 4 3 0 PCT~S94/08722
- 13 -
growth (Narita and Gruissem, 1989, Plant Cell 1:181-19O;
Narita et al., 1991, J. Cell. Biochem. 15A:102 (Abst)). Thus,
tomato HMG1 may function in sterol synthesis and membrane
biogenesis. Choi et al. have shown that potato HMG1
5 expression is induced in potato tubers by wounding but is
suppressed by defense-elicitors or bacterial pathogens (Plant
- Cell 4:1333-1344). These data suggest that the HMGR encoded
by the HMG1 is associated with the biosynthetic branch
responsible for sterol production. Based on similarities in
lO sequence or expression patterns (e.g., constitutive low level
expression, suppression by elicitors or microbes, high
expression during rapid cell division), Hevea HMGR3, potato
HMG1, Arabidopsis HMG1, and radish HMG2 probably belong to
this HMG1 HMGR isogene class.
2.6. PLANT HMGR PROMOTERS
Little is known of the promoters of plant HMGR
genes. To date, there have been no known publications of
studies analyzing the structure of plant HMGR promoters (e.g.,
20 deletion analyses, DNaseI footprint analysis, DNA-protein
binding/gel retardation analyses). One article (Chye et al.,
1991, Plant Mol. Biol. 16:567-577) documents approximately 1.5
kb of the upstream sequence of the Hevea HMG1 gene. This
sequence shows no significant homology to the analogous region
25 of the tomato HMG2 promoter disclosed herein. Another report
cursorily mentioned expression of potato HMGR13 (presumably an
HMG1 homolog) promoter:~-glucuronidase (GUS) fusions in
transgenic tobacco (Stermer et al., 1992, Phytopathology
82:1085), but revealed no details concerning the promoter used
30 nor the construction of the fusion.
2.7. PRODUCTION OF DESIRABLE GENE
PRODUCTS IN PLANTS
The concept of using transgenic plants to produce
35 desirable gene products is by no means new. Virtually all
examples reported to date of such engineering of transgenic
plants have utilized "constitutive" promoters, especially the
wo 95/03690 2 1 6 8 4 3 0 PCT/USg4/08722
caulifloWer mosaic virus (CaMV) 35S promoter or enhanced
versions thereof, to drive production of transgenic products.
The following is a representative list of the many mammalian
therapeutiC proteins that have been produced in plants using
5 the CaMV 35S promoter or derivatives. Expression of mouse
immunoglobulin chains was achieved by transforming tobacco
leaf segments with gamma- or kappa-chain cDNAs derived from a
mouse hybridoma cell line (Hiatt et al., 1989, Nature 342:76-
78; Hein et al., 1991, Biotechnol. Prog. 7:455-461).
10 Transformed plants expressing either gamma or kappa chains
were genetically crossed resulting in the production of
assembled, functional antibodies (termed "plantibodies").
Transgenic tobacco producing human serum albumin (Sijmons et
al., 1990, Bio/Technology 8:217-221), rabbit liver cytochrome
15 P4S0 (Saito et al., 1991, Proc. Natl. Acad. Sci. USA 88:7041-
7045), hamster 3-hydroxy-3-methylglutaryl CoA reductase
(Chappell et al., 1991, Plant Physiol. 96:127), and the
hepatitis B surface antigen (Mason et al., 1992, Proc. Natl.
Acad. Sci. USA 89:11745-11749) have also been reported. HSA
20 produced as a fusion protein with plant prepro-signals
resulted in a secreted HSA protein that was indistinguishable
from authentic mature human protein (Sijmons et al., 1990,
Bio/Technology 8:217-221). The hepatitis B antigen-expressing
plants accumulated spherical HBsAg particles with physical and
2S antigenic properties similar to human serum-derived HBsAg
particles and to those generated in yeast (current source of
human vaccine). Plant virus-mediated expression of human
proteins in plants, including human interferon, ~- and ~-
hemoglobin, and melanin has also been demonstrated (Fraley,
30 R., 1992, Bio/Technology 10:36-43; de Zoeten et al., 1989,
Virology 172:213-222).
The constitutive expression of desirable gene
products, however, is disadvantaged whenever the desirable
gene product is 1) labile, 2) deleterious to the growth or
35 development of the transgenic plant or culture line, 3) toxic
to humans, livestock, animals, or insects that could
inadvertently eat the transgenic plant, 4) a real or potential
W095/03690 2 1 6 8 4 3 0 PcT~s94/08722
_ - 15 -
environmental hazard, or 5) a security risk due to the
extremely high value of the gene product. Moreover, some
constitutive promoters, such as the CaMV 35S, decline in
activity as plants matures. Such losses of activity can
- 5 result in inefficient exploitation of the host plant as a
production system, since mature or maturing plants have
greater biomass and, often, greater excess biosynthetic
capacity for the production of the "extraneous" desirable gene
product than younger plants which are programmed to devote
10 much of their biosynthetic capacities to normal growth and
development functions.
Compared to the aforementioned conventional
approach, the post-harvest production method of the instant
invention can be a much more efficient and productive means of
15 producing desirable gene products in plants. Using the post-
harvest production method, the desired gene product is not
produced until after the plant tissue or cell culture has been
harvested and induced. This obviates any problems that might
arise from the presence of the desired gene product during the
20 growth of the plant or culture line.
3. SUMMARY OF THE I-NVI':N1'10N
One aspect of the present invention relates to the
use of plant promoter sequences responsive to wounding,
25 pathogen infection, pest infestation as well as to elicitors
or chemical inducers. This aspect of the invention generally
relates to the use of promoter sequences of 3-hydroxymethyl-3-
glutaryl coA reductase (HMGR) genes, and specifically to the
tomato HMG2 (HMGR2) promoter element and its homologs from
30 other plant species, to control the expression of protein and
RNA products in plants and plant cells. The invention further
relates to the use of sequences from the tomato HMG2 promoter
element or its homologs to modify or construct plant active
promoters, which in turn may be used to control the expression
35 of proteins and RNAs in plants and plant cells.
The tomato HMG2 promoter element and its homologs
have a variety of uses, including but not limited to
W095/03690 2 1 6 8 4 3 0 PcT~s94/08722
- 16 -
expressing or overexpressing heterologous genes in a variety
of plant cell expression systems, plant cultures, or stably
transformed higher plant species. Expression of the
heterologous gene product may be induced by elicitor or
5 chemical induction in plant cell expression systems, or in
response to wounding, pathogen infection, pest infestation as
well as elicitor or chemical induction in plants.
The use of the promoter elements described herein to
engineer plants may have particular value. By way of
lO illustration, and but by limitation, an agronomically
important plant may be stably transformed with an HMG2 or HMG2
homolog promoter element or a promoter derived therefrom
controlling a gene which confers resistance to pathogen
infection or pest infestation. When such a plant is invaded
15 by a pathogen or pest, the invasion will trigger the
expression of the H~G2 or HMG2-homolog promoter element-
controlled resistance gene and cause the plant to become
increasingly resistant to the pathogen or pest at the site of
invas ion .
The second aspect of the present invention relates
to a method of producing gene products in harvested plant
tissues and plant cell cultures. The method utilizes tissues
of transgenic plants or transformed plant cells engineered
with expression constructs comprising inducible promoters
25 operably linked to the coding sequence of genes of interest.
Tissues or cultures harvested from engineered plants or
culture lines, respectively, may be induced to produce the
encoded proteins or RNAs by treatments with the appropriate
inducers or inducing conditions.
As demonstrated by the examples described herein,
plants may be engineered with expression constructs comprising
an inducible promoter, such as the HMG2 promoter, operably
linked to a sequence encoding a protein or RNA. Tissues
harvested from the engineered plants may be induced to express
35 the encoded protein or RNA by treatment with the appropriate
inducer or inducing condition. With the post-harvest
production method, the amount of encoded gene product produced
21 68430
W095/03690 PCT~S94108722
- 17 -
from an expression construct utilizing an inducible promoter
may surpass that produced by an expression construct utilizing
a strong constitutive promoter. Moreover, such harvested
plant tissues can maintain, for up to two weeks after
S harvesting, their capability for strong induced production of
the gene product encoded in the expression construct.
- The post-harvest production method of the invention
may be advantageously used to produce desired gene products
that are adverse to plant or plant cell growth or development.
lO In plants or plant cells engineered with expression constructs
comprising inducible promoters operably linked to sequences
encoding the desired gene product, the expression of the
desired but plant-deleterious gene product would remain
dormant during the growth of the plant or culture line, thus
lS allowing for productive and efficient cultivation or culturing
of the engineered plant or culture line. When the engineered
plant or culture line has attained optimal condition for
harvest or processing, the expression of the desired gene
product may be artificially triggered by treatment with the
20 appropriate inducer or inducing condition, which, in the case
with expression constructs comprising the HMG2 promoter, is
mechanical wounding or elicitor or chemical induction. The
desired product, either encoded by the induced gene or a
compound produced by the induced gene product, may then be
2S extracted from the induced plant, harvested plant tissue, or
culture line after allowing for a short expression period.
The post-harvest production method of the invention
also may be advantageously used to produce a labile gene
product or a gene product that synthesizes a labile compound.
30 In plants or plant cells engineered with expression constructs
comprising inducible promoters operably linked to sequence
encoding the desired gene product, the expression of the
labile gene product or compound is deferred until the optimal
condition is attained for harvesting of the plant or culture
3s or processing of the desired product or compound. The
synthesis of the desired product may then be induced in the
plant or culture by treatment with the appropriate inducer or
21 68430
W095/03690 PCT~S94/08722
- 18 -
inducing condition, and the desired gene product or
synthesized compound expeditiously extracted from the induced
plant or culture after allowing for a short expression period.
This aspect of the invention is based in part on the
5 surprising findings that harvested plant tissues maintain
significant capacity for expressing inducible genes after
excision from the plant and that such capacity may actually
increase with some storage time.
3.1. DEFINITIONS
The terms listed below, as used herein, will have
the meaning indicated.
35S = cauliflower mosaic virus promoter for
the 35S transcript
CAT = chloramphenicol acetyltransferase
cDNA = complementary DNA
cis-regulatory A promoter sequence 5'
element = upstream of the TATA box that confers
specific regulatory response to a
promoter containing such an element.
A promoter may contain one or more
cis-regulatory elements, each
responsible for a particular
regulatory response.
DNA = deoxyribonucleic acid
functional portion = a functional portion of a promoter is
any portion of a promoter that is
capable of causing transcription of a
linked gene sequence, e.g., a
truncated promoter
gene fusion = a gene construct comprising a
promoter operably linked to a
heterologous gene, wherein said
promoter controls the transcription
of the heterologous gene
3s gene product = the RNA or protein encoded by a gene
sequence
GUS = 1,3-~-Glucuronidase
21 68430
~ . 3; ~ 9 4 / 08 7 2 2
- 19 - ~ J~ ~6JUL'95
heterologous gene = In the context of gene constructs, a
heterologous gene means that the gene
is linked to a promoter that said
gene is not naturally linked to. The
heterologous gene may or may not be
from the organism contributing said
promoter. The heterologous gene may
encode messenger RNA (mRNA),
antisense RNA or ribozymes. --
HMGR = 3-hydroxy-3-methylglutaryl CoA
Reductase
10~MG2 homolog = A plant promoter sequence which
selectively hybridizes to a known
HMG2 promoter (e.g., the tomato HMG2
promoter element disclosed herein),
or the promoter of an HMGR gene that
displays the same regulatory
responses as an HMG2 promoter tie.
promoter activity is induced by
wounding, pathogen-infection, pest-
infestation, or elicitor treatment).
homologous promoter = a promoter that selectively hybridize
to the sequence of the reference
promoter
mRNA = messenger RNA
product of
gene product = a product produced by a gene product,
e.g., a secondary metabolite
5 operably linked = A linkage between a promoter and gene
sequence such that the transcription
of said gene sequence is controlled
by said promoter.
RNA = ribonucleic acid
30RNase = ribonuclease.
4. DESCRIPTION OF THE FIGURES
Figure 1. The mevalonate/isoprenoid pathway in plants.
PGR, plant growth regulators.
D ~IEET
21 68430
08 ~ 2 2 .
- 20 - I~ ~U~ 2 6 iUL 95
Figure 2. Nucleic acid sequence comparison of tomato
NMG2 ( SEQ ID NO:l) and tomato HMG1 (SEQ ID NO:2). Sequences
are aligned by the algorithm of Smith and Waterman (Devereux
et al., 1984, Nucl. Acid Res. 12:387-395) which inserts gaps
indicated by dots to optimize alignment. The transcriptional
initiation sites are indicated by +1 and arrow. The
translational start codons and TATAA boxes are underlined.
The comparison ends at the first intron of each gene.
Ambiguous bases are indicated by lower case letters.
Figure 3. Schematic of tomato HMG2 promoter.
Panel A The tomato HMG2 genomic DNA insert of pTH295.
Panel B The 2.5 kb EcoRI fragment of pTH295 used to general
pDW101. pTH295 contains an approximately 7 kb HindIII
fragment from the original lambda genomic clone inserted into
the HindIII site of the multiple cloning region of bacterial
plasmid pSP6/T7 (BRL). pDW101 contains a 2.5 kb EcoRI fragment
of pTH295 inserted at the EcoRl site of plasmid bluescript
SK-(Stratagene). Restriction endonucleases: Ac, AccI; Av,
AvaI; Bg, BglII; E, EcoRI; Ev, EcoRV; H, HindIII; K, KpnI; P,
PstI; T, TaqI; X, XbaI. The solid boxes represent the four
exons encoding HMG2 protein. The fragments below pDW101
indicate the location of the HMG2 promoter sequences described
in Figures 4, 5, and 6. The parentheses around HMG2 Sequence
2 indicate that the exact location within this region has not
yet been determined.
Figure 4. Nucleic acid sequence of HMG2 Promoter
Sequence I (SEQ ID N0:3). This 1388 bp sequence (SEQ ID
NO:3) is from the 3'-end of the 2.5 kb EcoRI insert of pDW101.
The transcriptional (+ 1) and translational (ATG) start sites
are indicated. The TATAA box, PCR primer locations, and
relevant restriction enzyme sites are underlined. Lower case
letters indicate ambiguous bases.
Figure 5. Nucleic acid sequence of HMG2 Promoter
Sequence II (SEQ ID NO:4). The 480 bp sequence (SEQ ID N0:4)
2 1 68430
W095/03690 PCT~S94/08722
- 21 -
is from an internal region of the 2.5 kb EcoRI insert of
pDW101. This region spans approximately -1,039 to -2,000 base
pairs upstream of the HMG2 translation start site. The exact
location of the sequence within this region remains to be
5 determined. Two AT repeat motifs are underlined. An X
indicates unknown bases; lower case letters are ambiguous
bases.
Figure 6. Nucleic acid sequence of HMG2 Promoter
10 Sequence III (SEQ ID N0:5). The 415 bp sequence (SEQ ID N0:5)
is from the 5'-end of the 2.5 kb EcoRI insert of pDW101. A 23
base palindromic sequence is underlined. Lower case letters
are ambiguous bases.
Figure 7. Schematic representations of the promoter
deletion clones, pDW201, pDW202 and pDW203, in pBI101. The
open bars indicate the insert fragments from the tomato NMG2
promoter. RB and LB are T-DNA border sequences. The 5'
HindIII and 3' BamHI sites flanking each HMG2 promoter region
20 were generated within the sequence of the primers used to PCR-
amplify these inserts.
Figure 8. Restriction map of plasmid pS W330.1.
HMGA,EcoRI/BglII HMG2 promoter fragment from pDW101; GUS, beta
25 glucuronidase coding sequence; ocs 3', 3' region and
polyadenylation site derived from A. tumefaciens octopine
synthetase gene; p3SS, promoter of the cauliflower mosaic
virus 35S transcript; NPT, NPTII gene encoding neomycin
phosphotransferase conferring kanamycin resistance; LB,RB,
30 left and right border sequences of Ti plasmids required for
transfer of DNA from Agrobacterium to plant cell.
Figure 9. Restriction map of plasmid pS W 1911. This
plasmid was used to transform plants with a 35S:GUS construct
35 whose activity served as a benchmark to the various HMG2:GUS
constructs. Abbreviations are as described in Figure 8.
2 1 68430
4/O~ 722
- 22 - 'f~ J'v~- 2 6 JJL~9
Figure 10. Tissue specificity of HMG2 Promoter
expression in transgenic tobacco and tomato plants. Blue
color indicates regions where GUS is active.
Panel A. Anthers and pollen of HMG2: GUS compared to analogous
s tissues from 35S:GUS constructs (plants transformed with
pSLJ330.1 or pSLJ1911, respectively).
Panel B. Trichomes of fully-expanded tobacco leaf
(transformation vector pSLJ330.1).
Panel C. Root section of tomato seedling transformed with
10 pSLJ330.1. Seedling was grown axenically on agar medium.
Arrow indicates the location of lateral root initiation.
Figure 11. Defense-related HMG2 Promoter expression in
tobacco transformed with pSJL330.1.
15 Panel A. Leaf tissue 24 (2 left wells) and 48 hours after
wounding and inoculating with water (W, mock) or soft-rotting
bacterium Erwinia carotovora ssp. carotovora strain EC14 (EC).
Panel B. Tobacco seedlings (14 days post germination in
sterile potting mix) 24 and 48 hours following inoculation at
20 site indicated by arrow by placing Rhizoctonia infected oat
seed in direct contact with stem.
Panel C. Leaf section of field grown tobacco showing GUS
activity SU~ L O~ g necrotic regions due to natural insect
predation. Blue pigmentation at the outside edge of dis~ is
25 due to wound response.
Figure 12. Nematode response of HMG2: GUS transgenic
tomato (transformed with pSLJ330.1). Tomato seedlings were
inoculated with Meloidogyne hapla second stage juveniles. At
the indicated times after inoculation, roots were harvested,
30 incubated overnight in X-Gluc to stain for GUS activity, and
subsequently counter-stained with acid fucsin to visualize
nematodes.
Panel A. Squash of root tip 2 days after inoculation.
Panel B. Root region 3 days post-inoculation. Arrow
35 indicates region showing initial GUS activity.
~ ~D ~HE~
WO95/03690 2 1 6 8 4 3 0 PCT~S94/08722
_ - 23 -
Panels C, D, & E. Root tips showing characteristic nematode-
induced swelling 7 days post-inoculation. Note in Panel E
that uninfected root tips show no GUS activity.
s Figure 13. Comparison of post-harvest expression of HMG2
promoter-(hatched bars) and CaMV 35S promoter-(open bars)
driven GUS gene in transgenic tobacco leaf tissue (transformed
with pSLJ330.1 or pSLJ1911, respectively). Leaves were
removed from plants at day 0, wound-induced by scoring and
lO stored under moist conditions until harvest at the times
indicated. GUS activity was determined as described in the
text.
Figure 14: Post-harvest expression of an HMG2: GUS
15 expression construct. The graph shows a comparison of the
amount of GUS produced from freshly harvested leaves and that
from harvested leaves after two weeks of 4C storage. Leaves
of mature field-grown plants containing the ~MG2: GUS gene
construct were harvested by excising the leaves at the
20 petiole. The comparison was between the adjacent pairs of
leaves from the same plant in order to ensure developmental
similarity. One set of leaves, the "FRESH" samples, was
processed shortly after harvest by wounding (i.e., by heavily
scoring the leaf with a razor blade). The other set of
25 leaves, the "WEEK 2" samples, was stored in "zip-lock" bags at
4C for two weeks before wounding as described for the freshly
harvested leaves. The GUS activity in the wounded leaves was
determined immediately after wounding (the "Uninduced" sample)
or after 48 hr of incubation at room temperature (the "48hr.
30 Induced" sample). GUS activity is expressed as nM methyl-
umbelliferone (MUG) released per min. per ~g protein. The
induced GUS activity from the stored leaf tissue was
consistently equal to or greater than that from freshly
harvested leaf tissue. The GUS activity of equivalent fresh
35 leaf tissue of the untransformed parent cultivar is also shown
(the "UNT" sample).
21 68430
WOg5/03690 PCT~S94/08722
- 24 -
Figure 15: The effect of storage on the post-harvest
expression of an HMG2:GUS expression construct. The graph
shows a comparison of the amount of GUS produced from
harvested leaves that have been stored for 2 or 6 weeks at 4OC
5 or room temperature. Leaves of mature field-grown transgenic
tobacco plants containing the HMG2:GUS gene construct were
harvested by excising the leaves at the petiole. Adjacent
leaves from each plant were sorted into treatment groups,
i.e., storage in plastic bags at 4C (the "4C" samples) or
10 storage at room temperature in dry burlap bags (the "Room
Temp. Dry" samples). At the times indicated, a sample of the
stored leaf tissue was processed by wounding and the GUS
activity in the sample was determined at O and 48 hr. after
wounding. GUS activity is expressed as nM methyl-
15 umbelliferone (MUG) released per min. per ~g protein. Theinduced GUS activity from the stored leaves, even after 6
weeks of storage, was equal to, or higher than, that of
freshly harvested leaves.
5. DESCRIPTION OF THE INVENTION
One aspect of the present invention relates to the
HMG2 promoter and its homologs that are involved in the plant
response to pathogen infections, pest infestations or
mPch~n;cal wounding, and their use to drive the expression of
25 heterologous genes.
The tomato HMG2 promoter and its homologs control
the expression of a 3-hydroxy-3-methylglutaryl CoA reductase
(HMGR) isozyme in plants. HMGR catalyzes the production of
mevalonic acid, which is an intermediate in the biosyntheses
30 of various secondary metabolites that have critical roles in
plant defense responses and plant development. In most plant
tissues, tomato HMG2 promoter and its homologs remain dormant
until their activity is triggered by pathogen infections, pest
infestations or mechanical wounding. The induction of the
35 HMG2 and homologous promoters are mediated in part by so
called "elicitor" compounds derived from constituents of plant
(Darvill and Albersheim, 1984, Annu. Rev. Plant Physiol.
2 1 68430
W095/03690 - 25 - PCT~S94/08722
3s:234) or plant pathogen cell wall or surface components.
When induced, the ~MG2 promoter and its homologs effect rapid
and strong expression of the genes under their control. The
lack of significant constitutive expression and the rapidly
5 inducible, strong expression characteristics of the HMG2 and
homologous promoters make them ideal elements for controlling
the expression of various types of heterologous genes. Such
genes include those encoding pathogen and pest resistance
functions, products that are directly or indirectly
10 deleterious to plant growth, labile products, or products
involved in the biosyntheses of labile compounds, to name but
a few.
According to this aspect of the invention,
heterologous genes can be placed under the control of the
15 tomato HMG2 or homologous promoter elements or promoters
derived therefrom, all described herein, and used to engineer
cell culture expression systems, or transgenic plants.
Expression of the heterologous gene will be induced in
response to pathogen infection, pest infestation, mechanical
20 wounding or chemical or elicitor treatment. For example, the
induction of the heterologous gene in plant cell culture or
plant may be induced by treatments with cell wall extracts of
plant pathogens, purified elicitors contained within such
extracts, or synthetic functional analogs of those elicitors.
25 Alternatively, the expression of the heterologous gene in the
plant may be induced by various bacterial or fungal plant
pathogen infections. Similarly, the induction may also be
triggered by infestation of the plant by pests such as insects
and nematodes.
The second aspect of the invention relates to a
method of producing gene products in plant tissues and cell
cultures. The method utilizes the tissues of plants or
cultures of plant cells that have been engineered with
expression constructs comprising inducible promoters operably
35 linked to sequences encoding the desired gene products.
According to this aspect of the invention, the production of
the desired gene product takes place in the plant tissue and
21 68430
WOg5/03690 - 26 - PCT~S94/08722
culture after they have been harvested and induced by
treatment with the appropriate inducer or inducins condition.
The invention relates to the family of HMG2 and
HMG2-derived promoters and to the post-harvest production of
s gene products in plant tissues and cultures, and for the
purpose of description onlv, the description of the invention
will be divided into several stages: (a) inducible promoters
that may be used in engineering the plant and plant cells for
post-harvest production; (b) isolation, identification and
10 characterization of such promoter sequences, including that of
the HMG2; (c) identification and characterization of cis-
regulatory elements within the inducible promoters, including
those of the HMG2 promoter, that can regulate other plant
promoter sequences; (d) construction of expression vectors
15 comprising heterologous genes of interest operably associated
with an inducible promoters or derivatives thereof, including
expression vectors comprising HMG2 and derivative promoters;
(e) engineering of expression vectors into plants or plant
cells; (f) inducing the expression of said expression
20 construct and the production of the gene product of interest
in plant tissues or cell culture systems; and (g) the types of
heterologous gene products that can be advantageously produced
using the post-harvest production method, or more particularly
under the control of the HMG2 promoter elements and HMG2-
25 derived promoters.
The various embodiments of the claimed inventionpresented herein are by the way of illustration and are not
meant to limit the invention.
5.1. INDUCIBLE PROMOTERS
The inducible promoters that may be used in the
expression vectors of the post-harvest production method of
the invention can be any promoter whose activity is inducible
in the host plant. Useful promoters include, but are limited
35 to, those whose activities are induced by chemicals,
biological elicitors, heat, cold, salt stress, light,
wounding, desiccation, hormone, pathogen infection, or pest-
2 1 68430
W095/03690 PCT~S94/08722
_ - 27 -
infestation. Table 3 lists some examples of inducible plant
genes whose promoters may be used in the making cf the instant
invention.
Useful promoters are any natural or recombinant
5 promoter whose expression can be induced in harvested plant
tissue. Promoters with such a capability may be determined
- using any methods known in the art. For example, the promoter
may be operably associated with an assayable marker gene such
as GUS; the host plant can be engineered with the construct;
10 and the ability and activity of the promoter to drive the
expression of the marker gene in the harvested tissue under
various conditions assayed.
21 68430
WO 95/03690 2 PCT/US94/08722
-- 8
TABLE 3
Inducible Plant Genes with Potential for Post-harvest Induction and Accumulation of
Transgene Products. An asterisk designates those genes for which promoters have been isolated
and characterized. References represent one to two representative references or relevant review
article and are not intended to be ~ tive.
Genew/gene products Fuoctions Sourcw of clonw
Defense-Response Genew~
PhYtoale~in b Ga~llthw;5
PL~,..~I".~F -;d phytoale~cin
~Pi ~ ~lalanine G.. ,.. ,O,, ~ Iyase (PAL)2 Enzyme, central pathway Bean, parsley, potato,
tomato
4~oumarate CoA ligæ (4CL)3 Enzyme, central pathway Parsley, potato~qlcon.~ synthase (CHS)~5 Enzyme, Isoflavanoid branch Bean, soybean,
parsley
l'h~lcl~ne .so ue.~e (CHI)6 Enzyme, Isoflavanoid branch Bean
Resveratrol (stilbene) synthase7 Enzyme, Isoflavanoid br_nch Gr_pevine, peanut
Isoflavone .~lu~,Lse (IFR)8 Enzyme, Isoflavanoid br_nch Alfalfa
Terpenoid phytoale~ins
~HMG-CoA .~Ju~,~ (HMG)9 ~ Enzymes, central pathway Tomato, tobacco,
potato,
Casbene sl.ltl.. ,~se~t Casbene b;G~yl.tl.~;s Castor bean
Cell wall cG,..,~nc~lt,
Lignin
~Phenylalanine ~ ni~ Iyase See above
Cinnamyl alcohol dehy-l,.,g~,ase (CAD)12 Lignin biosyn. Tobacco
Caffeic acid o .u~lhyl~ f~,.~l3 Lignin biosyn. Al&lfa, tobacco
Lignin-formingpero~idasel4 Lignin polymerization Tobacco, wheat
H~d.oAy~,.oline-rich ~;lycop.~ ~ (HRGP)15 16 Structural protein Bean, tomato
Glycine-rich proteins (GRP)15 Structural protein Bean, potato, pea,
nce
Thioninsl7 Antifungal Barley
H~d,~,l~es. Iytic enzymes
~hitinqcPs (PR-P, PR-Q)18-20
Class I chitinqcP~ basic Vacuolar, antifungal Tobacco, bean,
tomato
Class I and II rhitinqse. acidic E~tracellular, antifungal Bean
Class II chitinase Bifi~nrtionql Iysozyme, Cnc~mh~r, tobacco,
chitinase barley, petunia
~B-1,3-Glucanase2~ Antifungal, chitinase Bean, tobacco,
potato,
syner~ist pea, rice,
Arvqbidopci~
,B-fmc~osi~iqcP22 Antifungal invertase Tomato
wo 95/03690 2 1 6 8 4 3 0 PCT/USg4/08722
- 29 -
TA~iLE 3 COI~'TINUED
Genes/gene products Functions Sources of clones
Others
~P~Gte.,.~e inhibitors (P1-I, PI-II)23 24 Trypsin-, chymolrypsin- Potato, tomato
inhibitors
Supero~tide ~ e (SoD)25 Anti-o~idant enzyme Tobacco, maize,
tomato
L,~.~æ~ ~ Lipid pern~ P'ion. A, L d~r -
jqcmr~n~ biosyn.
A~ itit~nql ~pathogenesis-related~ prot.
~PRl &mily, PR2, PR327-29 Unknown Tobacco, be.n,
parsley,
pea
Ocn~jn PRs3~32 Antifungal, th~n.. ~ti.~-ljke Tobacco, maize
Ubiquitin33 Protein degradation Potato
Wound-Inducible Genes-
~winl, ~win2 ~hevein-like)34 Chitin-bindingprot. Potato (hevein,
rubber
tree)
wunl, wuo235 Unknown potato
~DOS, nopaline synthase36 Ag.~lb.. ~t~.iul,- nutr. Agrobacterium
l.-.. f-~c.~ ~
ACC synthase37 Ethylene biosynthesis Tomato, squash
HMG-CoA .~luu~e hmgl38 Sterol/alkaloid synth. Potato
3-deo~y--D-arabino ~p~ n~crnq~
7 }~l~O~ tr synthase39 Lignin biosyn. Potato, tomato
HSP7033 Heat-shock protein,
.,La~,. ,one Potato
Salicylic acid inrlulible40
acid pero~idasel4 Lignin-forming Tobacco
PR .~t -40.41 (see above) Tobacco
Glycinc-rich protein41 Cell wall protein Tobacco
Methyl jr in~cible
*vspB Vacuolar storage prot. Soybean
P,ote.,.s~ inhibitors 1 and 1143 Trypsin, cL~ vt~ a;n inhib. Potato, tomato
Heat-shock genes43
HSP7033 Cha~.on,n Potato
Ubiquitin (see above)
Cold-stress inducible44
Drought, salt stress45
21 68430
WO 95/03690 3 0 PCT/US94108722
TABLE 3 CONTlNUED
Genes/gene products Functions Sources of clones
Osmotin3~32 Decicc~tion tolerance Tobacco, maize
Hormone jnf11~r,;' 1~
Gibberellin
Starch de~... ~tion Barley
Abscisic acid4547
EM-I, RAB, LEA genes45 Unknown, e... .l,.jo~ .s Wheat, rice, maize,
cotton
Ethylene
~'hitir~qcP,, phytoale~in biosyn. genes (see above)
a. Genes are t,~sc,.~JI.onally activated in response to pdlhCig_~S~ defense elicitors, wounding and in
some cases methyl j~ ~on~e. salicylic acid, HgCI2 or H2O2.
R,f~,.~ces.
1. Cramer et al., 1993, I. Nematol. 25:507-518.
2. Lois et al., 1989, EMBO J. 8:1641-1648.
3. Becker-Andre et al., 1991, J. Biol. Chem. 266:8551-8559.
4. Arias et al., 1993, Plant Cell 5:485496.
5. Doerner et al., 1990, Bio/Technology 8:845-848.
6. Mehdy et al., 1987, EMBO J. 6: 1527-1533.
7. Hain et al., 1993, Nature 361:153-156.
8. Paiva et al., 1991, Plant Mol. Biol. 17:653-667.
9. Park et al., 1992, Plant Mol. Biol. 20:327-331.
10. Yang et al., 1991, Plant Cell 3:397405.
11. Lois et al., 1990, Arch. Biorh~m Biophys. 276:270-277.
12. Schuch et al., 1991, 3rd Int. Cong. Plant Mol. Biol., Tucson, Az., Abstract 1653.
13. Iaeck et al., 1992, Mol. Plant-Microbe Interact. 5:294-300.
14. T-qg imini et al., 1987, Proc. Natl. Acad. Sci. USA 84:7542-7546.
15. S~,.. ' et al., 1992, Plant Mol. Biol. 19:205-215.
16. Wycoff et al., 1992, in D. P. S. Verma, ed., Molecular Signals in Plant-Microbe C~
Boca Raton, FL:CRC Press. pgs. 407-422.
17. Bohlmann et al., 1988, EMBO J. 7:1559-1565.
18. Hedrick et al., 1988, Plant Physiol. 86:182-186.
19. Roby et al., 1990, Plant Cell 2:999-1007.
20. Sarnac and Sha_, 1991, Plant Cell 3: 1063-1072.
21. Legrand et al., 1987, Proc. Natl. Acad. Sci. USA 84:6750-6754.
22. Rf~nh~ n-~u et al., 1991, Plant Physiol. 97:739-750.
23. Ryan, C. A., 1990, Annu. Rev. Phytopathol. 28:425 449.
24. Keil et al., 1989, EMBO J. 8: 1323-1330.
25. Bowler et al., 1989, EMBO 1. 8:31 -38.
wo 95~036go 2 1 6 8 4 3 0 PCT/US94/08722
TABLE 3 CONTINUED
References con~inned:
26. Melan et al., 1993, Plant Physiol. 101:441-450.
27. Cornelissen et al., 1986, EMBO J. 5:37-40.
28. Meler et al., 1991, Plant Cell 3:309-315.
29. Sharma et al., 1992, Mol. Plant-Microbe Interact. 5:89-95.
30. Cornelissen et al., 1986, Nature 321:531-532.
31. Stinizi et al., 1991, Physiol. Mol. Plant Pathol. 38:137-146.
32. KOnO.. V.. ~,L et al., 1992, Pl_nt Cell 4:513-524.
33. Rickey and BeL~cnap, 1991, Plant Mol. Biol. 16:1009-1018.
34. Weiss and Bevan, 1991, Plant Physiol. 96:943-951.
35. Log et al., 1988, Proc. Natl. Acad. Sci. USA 85:1136-1140.
36. An et al., 1990, Plant Cell 2:225-233.
37. Li et al., 1992, Plant Mol. Biol. 18:477-487.
38. Chol et al., 1992, Plant Cell 4:1333-1344.
39. Dyer et al., 1989, Proc. Natl. Acad. Sci. USA 86:7370-7373.
40. Ward et al., 1991, Plant Cell 3:1085-1094.
41. Van de Rhee et al., 1990, Plant Cell 2:357-366.
42. Mason et al., 1993, Plant Cell 5:241-251.
43. Ho and Sachs, 1989, In: Stump and Conn, eds., The Bio~ r of Plants: A Co~.~,L.,~.ve
Treatis, Vol. 15, pgs. 347-377.
44. Il - ' , 1992, In: Scqn~slios and Wright, eds., Genornic p~ ~nc. c to Envilo- -- ~.l Stress,
Adv. in G~leties Vol. 28, pgs. 99-131.
45. Skriver and Mundy, 1990, Plant Cell 2:503-512.
46. Gubler and Is~Qbs~n, 1992, Plant Cell 4:1435-1441.
47. Chandler and Robertson, 1994, Annu. Rev. Plant Physiol. Plant Mol. Biol. 45: 113-142.
21 68430
W095/03690 - 32 - PCT~S94/08722
Useful promoters may be tissue-specific. Non-
tissue-specific promoters (i.e., those that express in all
tissues after induction), however, are preferred. More
preferred are promoters that additionally have no or very low
s activity in the uninduced state. Most preferred are promoters
that additionally have very high activity after induction.
In a particular embodiment, the inducible promoter
is the HMG2 promoter and derivatives thereof. The HNG2 and
derivative promoters, which in addition to having utility in
10 the production method of the invention, also have utility in
the HNG2 promoter expression system of the invention. This
aspect of the invention is also described herein in some
detail.
5.2. PROMOTER ISOLATION AND CHARACTERIZATION
According to the present invention, functional
portions of the promoters described herein refer to regions of
the nucleic acid sequence which are capable of promoting
transcription of an operably linked gene in response to an
20 inducer or inducing condition at some point in the life cycle
of the plant or cell culture, including after the tissue or
culture has been harvested. In the case of the HNG2 promoter,
the functional portions are those regions which are capable of
causing transcription of an operably linked gene in response
25 to wounding, pathogen infection, pest infestation or
chemical/elicitor treatment during the entire pattern of plant
development.
Homologous nucleotide sequences is used herein to
mean nucleic acid sequences which are capable of selectively
30 hybridizing to each other. Selectively hybridizing is used
herein to mean hybridization of DNA or RNA probes 50 bases or
greater in length from one sequence to the "homologous"
sequence under stringent conditions, e.g., washing in 0.lX
SSC/0.1% SDS at 68C for at least 1 hour (Ausubel, et al.,
35 Eds., 1989, Current Protocols in Molecular Biology, Vol. I,
Greene Publishing Associates, Inc. and John Wiley & Sons,
Inc., New York, at page 2.10.3)
2 1 68430 J ~ 3 7 22
26 JUL 9~
Nucleotide sequences homologous to the tomato HMG2
promoter described herein refers to nucleic acid sequences
which are capable of selectively hybridizing to the nucleic
acid sequence contained in the approximately 2 . 2 kb EcoRI-
S BqlII fragment of pDW101 in hybridization assays or which arehomologous by sequence analysis (containing a span of 100
basepairs in which at least 75 percent of the nucleotides are
identical to the sequences presented herein) and which has
promoter activity identical or very similar to that of HMG2,
10 i.e., wound, elicitor, pathogen, etc., inducibility. Such a
promoter may be precisely mapped and its activity may be
ascertained by methods known in the art, e.g., deletion
analysis, expression of marker genes, RNase protection, and
primer extension analysis of mRNAs preparations from plant
15 tissues containing the homologous promoter operably linker to
a marker gene.
Homologous nucleotide sequences refer to nucleotide
sequences including, but not limited to, natural promoter
elements in diverse plant species as well as genetically
20 engineered derivatives of the promoter elements described
herein.
Methods which could be used for the synthesis,
isolation, molecular cloning, characterization and
manipulation of the inducible promoter sequences, including
25 that of the HMG2 promoter, described herein are well known to
those skilled in the art. See, e.g., the techniques described
in Sambrook et al., Molecular Cloning: A Laboratory Manual,
2nd. Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,
New York (1989).
According to the present invention, the inducible
promoter sequences, including that of the HMG2 promoter, or
portions thereof described herein may be obtained from an
appropriate plant source, from cell lines or recombinant DNA
constructs containing the inducible promoter sequences or
35 genes sequences comprising the promoter sequences, and/or by
chemical synthetic methods where the promoter sequence is
known. For example, chemical synthetic methods which are well
21 68430
W095/03690 PCT~S94/08722
- 34 -
known to those skilled in the art can be used to synthesize
the HMG2 sequences depicted in Figures 4, 5 and 6 herein (SEQ
ID N0:3, SEQ ID No:4, SEQ ID No:5, respectively). The
synthesized sequences can be cloned and expanded for use as
5 promoter elements. Such synthetic sequences can also be used
as hybridization probes to identify and isolate the desired
promoter sequences from appropriate plant and cellular
sources.
Alternatively, a desired promoter element may be
10 identified and isolated by screening gene libraries. ~or
example, a genomic DNA library may be screened for clones
containing sequences homologous to known inducible genes,
e.g., HMGR genes or, alternatively, inducible promoter
sequences, e.g., HMGR promoters. For example, sequences from
15 genomic or cDNA clones encoding the inducible gene or
oligonucleotide probes corresponding to known amino acid
sequences of the inducible protein may be used as
hybridization probes to identify homologous clones in a
genomic DNA library using standard methods. Such methods
20 include, for example, the method set forth in Benton and Davis
(Science 196:180 (1977)) for bacteriophage libraries, and
Grunstein and Hogness (Proc. Nat. Acad. Sci. USA 72:3961-3965
(1975)) for plasmid libraries.
In instances where the species sources of the probe
25 and the library are widely divergent, such as between a
monocot and a dicot, the probe is preferably from a region
encoding highly conserved amino acid residues of the inducible
protein and the hybridization and wash are carried out at
moderate stringencies, e.g., washing in 0.2X SSC/0.1% SDS at
30 42C (Ausubel, et al., 1989, supra) . Homologous gene
sequences are those that are detected by the probe under such
conditions and that encode a protein with 75% or greater amino
acid homology with the protein encoded by the gene that is the
source of the probe. In isolating HMG2 homologous genes,
35 useful hybridization conditions have been described in detail.
See Park et al., 1992, Plant Mol. Biol. 20:327-331.
21 68430 ~ 72Z
2 6 5UL 9~
- 35 -
Clones containing homologous inducible gene
sequences may be distinguished from other isogenes by their
ability to detect, at high stringency hybridization
conditions, mRNA transcripts that are induced by the expected
5 inducing factor or condition. For example, HMG2 homologous
genes may be identified by the rapid appearance of hybridizing
mRNA transcripts following wounding, pathogen infection, pest-
infestation, or elicitor treatment (for an example of such an
analysis see Yang et al., 1991, Plant Cell 3:397-405). The
10 HMG2 clones may also be identified by examining their ability
to hybridize at moderate or high stringency conditions to 5'
untranslated region or 5' upstream region of known HMGl and
HMG2 (e.g., the 2.5 kb EcoRI fragment depicted in Figure 3)
genes. HMG2 clones can be identified as those that show a
15 stronger signal with the HMG2 probe than with the HMGl probe.
In other instances, where the species sources of the
probe and the library are not widely divergent, such as
between closely related plants, the probes used to screen such
libraries may consist of restriction fragments or nucleotide
20 sequences encoding the N-terminal 200 or so amino acids of the
inducible protein, e.g., in the case of tomato HMG2 HMGR see
Park et al., 1992, Plant Mol. Biol. 20:327-331, portions
thereof or nucleotide sequences homologous thereto. Retrieved
clones may then be analyzed by restriction fragment mapping
25 and sequencing t~chn;ques according to methods well known in
the art.
In another approach, restriction fragments or
nucleotide sequences of the inducible promoter or portions
thereof and sequences homologous thereto may be used to screen
30 genomic libraries to identify genomic clones containing
homologous promoter sequences using the standard techniques
described supra.
In an embodiment, the tomato HMG2 promoter elements
described in Figure 3, Figure 4, Figure 5, and Figure 6,
35 portions thereof and sequences homologous thereto may be used
to screen genomic libraries to identify genomic clones
containing homologous promoter sequences. For example, the
A~ ~D ~EET
21 68430
W095/03690 PCT~S94/08722
36 -
2.5 kb EcoRI fragment containing the tomato HMG2 promoter in
plasmid pDW101 or portions thereof may be used to such ends.
In the above two approaches, the hybridization and
wash conditions used to identify clones containing the
5 homologous promoter sequences may be varied depending on the
evolutionary relatedness between the species origins of the
probe and the genomic library screened. The more distantly
related organisms would require moderate hybridization and
wash conditions. See supra for two wash conditions that can
10 be used.
In yet another approach to the isolation of
inducible promoter elements, known oligonucleotide sequences
of inducible promoters can be used as primers in PCR
(polymerase chain reactions) to generate the promoter
15 sequences from any plant species. Similar to the
hybridization approaches, the amplification protocol may be
adjusted according to the evolutionary relatedness between the
target organism and primer-source organism. The adjustments
may include the use of degenerate primers and protocols well
20 known in the art for amplifying homologous sequences. For a
review of PCR techniques, see for example, Gelfind, 1989, PCR
TechnologY. Princi~les and Applications for DNA AmDlification,
Ed., H.A. Erlich, Stockton Press, N.Y., and Current Protocol
in Molecular Bioloqy, Vol. 2, Ch. 15, Eds. Ausubel et al.,
25 John Wiley & Sons, 1988.
In instances where only internal sequences of an
inducible promoter are known, sequences flanking such
sequences may be obtained by inverse PCR amplification
(Triglia et al., 1988, Nucl. Acids Res. 16:8186). The
30 flanking sequences can then be linked to the internal promoter
sequences using standard recombinant DNA methods in order to
reconstruct a sequence encompassing the entirety of the
inducible promoter.
The location of the inducible promoter within the
35 sequences isolated as described above may be identified using
any method known in the art. For example, the 3'-end of the
promoter may be identified by locating the transcription
r ~ 0 8 7 2 2
_ 37 _ ~r~ J~ 26JUL 9~
initiation site, which may be determined by methods such as
RNase protection (Liang et al., 1989, J. Biol. Chem.
264:14486-14498), primer extension (Weissenborn and Larson,
1992, J. Biol. Chem. 267:6122-6131), or reverse
5 transcriptase/PCR. The location of the 3 '-end of the promoter
may be confirmed by sequencing and computer analysis,
examining for the canonical AGGA or CAT and TATA boxes of
promoters that are typically 50-60 base pairs (bp) and 25-35
bp 5'-upstream of the transcription initiation site. The
10 5' -end of the promoter may be defined by deleting sequences
from the 5'-end of the promoter-containing fragment,
constructing a transcriptional or translational fusion of the
resected fragment and a reporter gene, and examining the
expression characteristics of the chimeric gene in transgenic
15 plants. Reporter genes that may be used to such ends include,
but are not limited to, GUS, CAT, luciferase, ~-galactosidase
and Cl and R gene controlling anthocyanin production.
According to the present invention, an inducible
promoter element is one that confers to an operably linked
20 gene in a plant or plant cell: 1) minimal expression in most
plant tissues; and 2) induced expression following treatment
by inducer or inducing condition known to trigger the
expression of the promoter or gene from which the element is
derived from.
In an embodiment, the HMG2 promoter element is one
that confers to an operably linked gene in a plant or plant
cell: 1) minimal expression in most plant tissues; and 2)
induced expression following wounding, pathogen infection,
pest infestation or chemical/elicitor treatment. An HMG2
30 promoter element may additionally confer specific
developmental expression as in pollen, mature fruit tissues,
and root tissues of the zone of lateral root initiation. See
Table 1, which compares the expression characteristics of
tomato HMG2 promoter to the other known plant HMGR promoters.
According to the present invention, a promoter
element comprise the region between -2, 500 bp and +1 bp
upstream of the transcription initiation site of inducible
~ iD ~HEET
2 1 68430 `-, -` Y4/ ~8.Z22
~ f~J'.J~ 2 6 JUL ~5
- 38 -
gene, or portions of said region. In a particular embodiment,
the NMG2 promoter element comprises the region between
positions -2,3 00 and +1 in the 5' upstream region of the
tomato HMG2 gene (see figure 3). Another embodiment of the
5 HMG2 promoter element comprises the region between positions
-891 and +1 in the 5' upstream region of the tomato HMG2 gene.
An additional embodiment of HMG2 promoter element comprises
the region between positions -347 and +1 in the 5' upstream
region of the tomato HMG2 gene. ~et another embodiment of
10 HMG2 promoter element comprises the region between positions
-58 and +1 in the 5' upstream region of the tomato HMG2 gene.
In further embodiments of the present invention, an HNG2
promoter element may comprise sequences that commence at
position +1 and continue 5' upstream up to and including the
15 whole of the nucleotide sequence depicted in Figure 4, Figure
5 or Figure 6.
5.3. CIS-REGULATORY ~T~M~NTS OF PROMOTERS
According to the present invention, the cis-
20 regulatory elements within an inducible promoter may beidentified using any method known in the art. For example,
the location of cis-regulatory elements within an inducible
promoter may be identified using methods such as DNase or
chemical footprinting (Meier et al., 1991, Plant Cell 3:309-
25 315) or gel retardation (Weissenborn and Larson, 1992,J. Biol. Chem. 267-6122-6131; Beato, 1989, Cell 56:335-344;
Johnson et al., 1989, Ann. Rev. Biochem. 58:799-839).
Additionally, resectioning experiments may also be employed to
define the location of the cis-regulatory elements. For
30 example, an inducible promoter-cont~in;ng fragment may be
resected from either the 5' or 3'-end using restriction enzyme
or exonuclease digests.
To determine the location of cis-regulatory elements
within the sequence containing the inducible promoter, the 5'-
35 or 3'-resected fragments, internal fragments to the inducible
promoter containing sequence, or inducible promoter fragments
containing sequences identified by footprinting or gel
;,itj.N~D ~EET
WO95/03690 2 1 6 8 4 3 0 PCT~S94/08722
_ - 39 -
retardation experiments may be fused to the 5'-end of a
truncated plant promoter, and the activity of the chimeric
promoter in transgenic plant examined as described in section
5.2. above. Useful truncated promoters to these ends comprise
5 sequences starting at or about the transcription initiation
site and extending to no more than 150 bp 5' upstream. These
- truncated promoters generally are inactive or are only
minimally active. Examples of such truncated plant promoters
may include, among others, a "minimal" CaMV 35S promoter whose
lO 5' end terminates at position -46 bp with respect to the
transcription initiation site (Skriver et al., Proc. Nat.
Acad. Sci. USA 88:7266-7270); the truncated "-9O 35S" promoter
in the X-GUS-90 vector (Benfey and Chua, 1989, Science
244:174-181); a truncated "-101 nos" promoter derived from the
15 nopaline synthase promoter (Aryan et al., 1991, Mol. Gen.
Genet. 225:65-71); and the truncated maize Adh-1 promoter in
pADcat 2 (Ellis et al., 1987, EMBO J. 6:11-16).
According to the present invention, a cis-regulatory
element of an inducible promoter is a promoter sequence that
20 can confer to a truncated promoter one or more of the
inducible properties of the original inducible promoter. For
example, an HMG2 cis-regulatory element is an HMG2 promoter
sequence that can confer to a truncated promoter one or more
of the following characteristics in expressing an operably
25 linked gene in a plant or plant cell: 1) induced expression
following wounding; 2) induced expression following pathogen
infection; 3) induced expression following pest infestation;
4) induced expression following chemical elicitor treatment;
or 5) expression in pollen, mature fruit, or other
30 developmentally defined tissues identified as expressing HMG2.
Further, an HMG2 cis-regulatory element may confer, in
addition to the above described characteristics, the ability
to suppress the constitutive activity of a plant promoter.
5.4. INDUCIBLE PROMOTER-DRIVEN EXPRESSION VECTORS
The properties of the nucleic acid sequences are
varied as are the genetic structures of various potential host
WO9~/03690 2 1 6 8 4 3 0 PCT~Sg4/08722
- 40 -
plant cells. The preferred embodiments of the present
invention will describe a number of features which an artisan
may recognize as not being absolutely essential, but clearly
advantageous. These include methods of isolation, synthesis
5 or construction of gene constructs, the manipulation of the
gene constructs to be introduced into plant cells, certain
features of the gene constructs, and certain features of the
vectors associated with the gene constructs.
Further, the gene constructs of the present
10 invention may be encoded on DNA or RNA molecules. According
to the present invention, it is preferred that the desired,
stable genotypic change of the target plant be effected
through genomic integration of exogenously introduced nucleic
acid construct(s), particularly recombinant DNA constructs.
15 Nonetheless, according to the present inventions, such
genotypic changes can also be effected by the introduction of
episomes (DNA or RNA) that can replicate autonomously and that
are somatically and germinally stable. Where the introduced
nucleic acid constructs comprise RNA, plant transformation or
20 gene expression from such constructs may proceed through a DNA
intermediate produced by reverse transcription.
The present invention provides for use of
recombinant DNA constructs which contain inducible promoter
fragments, functional portions thereof, and nucleotide
25 sequences homologous thereto. As used herein a functional
portion of a promoter and a promoter homologous sequence are
both capable of functioning as an inducible promoter. The
functionality of such sequences can be readily established by
any method known in the art. Such methods include, for
30 example, constructing expression vectors with such sequences
and determining whether they confer inducible expression to an
operably linked gene. In particular embodiments, the
invention provides for the use of tomato HMG2 promoter
fragments or sequences as depicted in Figures 3, 4, 5, and 6,
35 functional portions thereof, and nucleotide sequences
homologous thereto.
2 1 68430 ~; ~ 94 / 08 722
- 41 ~ 2 ~ JUL
The inducible promoter elements of the invention may
be used to direct the expression of any desired protein, or to
direct the expression of an RNA product, including, but not
limited to, an "antisense" RNA or ribozyme. Such recombinant
5 constructs generally comprise a native inducible promoter or a
recombinant inducible promoter derived therefrom, ligated to
the nucleic acid sequence encoding a desired heterologous gene
product.
A recombinant inducible promoter is used herein to
10 refer to a promoter that comprises a functional portion of a
native inducible promoter or a promoter that contains native
promoter sequences that is modified by a regulatory element
from a inducible promoter. For example, in particular
embodiments, a recombinant inducible promoter derived from the
15 HMG2 promoter may comprise the approximately 0.17 kb, 0.46 kb
or 1.0 kb HindIII-BamHI tomato ~MG2 promoter fragment of
pDW201, pDW202 and pDW203, respectively (see Fig. 7).
Alternatively, a recombinant inducible promoter derived from
the NMG2 promoter may be a chimeric promoter, comprising a
20 full-length or truncated plant promoter modified by the
attachment of one or more HMG2 cis-regulatory elements.
The manner of chimeric promoter constructions may be
any well known in the art. For examples of approaches that
can be used in such constructions, see section 5.3. above and
25 Fluhr et al., 1986, Science 232:1106-1112; Ellis et al;, 1987,
EMB0 J. 6:11-16; Strittmatter and Chua, 1987, Proc. Nat. Acad.
Sci. USA 84:8986-8990; Poulsen and Chua, 1988, Mol. Gen.
Genet. 214:16-23; Comai et al., 1991, Plant Molec. Biol.
15:373-381; Aryan et al., 1991, Mol. Gen. Genet. 225:65-71.
According to the present invention, where an
inducible promoter or a recombinant inducible promoter is used
to express a desired protein, the DNA construct is designed so
that the protein coding sequence is ligated in phase with the
translational initiation codon downstream of the promoter.
35 Where the promoter fragment is missing 5'- leader sequences
DNA fragment encoding both the protein and its 5' RNA leader
sequence is ligated immediately downstream of the
,~, ~;, `~ r:---
21 68430
WO9S/03690 PCT~S94/08722
- 42
transcription initiation site. Alternatively, an unrelated 5'
RNA leader sequence may be used to bridge the promoter and the
protein coding sequence. In such instances, the design should
be such that the protein coding sequence is ligated in phase
S with the initiation codon present in the leader sequence, or
ligated such that no initiation codon is interposed between
the transcription initiation site and the first methionine
codon of the protein.
Further, it may be desirable to include additional
10 DNA sequences in the protein expression constructs. Examples
of additional DNA sequences include, but are not limited to,
those encoding: a 3' untranslated region; a transcription
termination and polyadenylation signal; an intron; a signal
peptide (which facilitates the secretion of the protein); or a
15 transit peptide (which targets the protein to a particular
cellular compartment such as the nucleus, chloroplast,
mitochondria, or vacuole).
5.5. RECOMBINANT DNA CONSTRUCTS
The recombinant construct of the present invention
may include a selectable marker for propagation of the
construct. For example, a construct to be propagated in
bacteria preferably contains an antibiotic resistance gene,
such as one that confers resistance to kanamycin,
25 tetracycline, streptomycin, or chloramphenicol. Suitable
vectors for propagating the construct include plasmids,
cosmids, bacteriophages or viruses, to name but a few.
In addition, the recombinant constructs may include
plant-expressible, selectable, or screenable marker genes for
30 isolating, identifying or tracking plant cells transformed by
these constructs. Selectable markers include, but are not
limited to, genes that confer antibiotic resistance, (e.g.,
resistance to kanamycin or hygromycin) or herbicide resistance
(e.g., resistance to sulfonylurea, phosphinothricin, or
35 glyphosate). Screenable markers include, but are not be
limited to, genes encoding B-glucuronidase (Jefferson, 1987,
Plant Molec Biol. Rep 5:387-405), luciferase (Ow et al., 1986,
21 68430
J/~ 94/08 722
2 6 JUL ~g5
Science 234:856-859), or B protein that regulates anthocyanin
pigment production (Goff et al., 1990, EMBO J 9:2517-2522).
In embodiments of the present invention which
utilize the Agrobacterium tumefacien system for transforming
5 ~lants (see infra), the recombinant constructs may
additionally comprise at least the right T-DNA border
sequences flanking the DNA sequences to be transformed into
the plant cell. Alternatively, the recombinant constructs may
comprise the right and left T-DNA border sequences flanking
10 the DNA sequence. The proper design and construction of such
T-DNA based transformation vectors are well known to those
skilled in the art.
5.6. PRODUCTION OF TRANSGENIC PLANTS AND PLANT CELLS
According to the present invention, a desirable
plant or plant cell may be obtained by transforming a plant
cell with the nucleic acid constructs described herein. In
some instances, it may be desirable to engineer a plant or
plant cell with several different gene constructs. Such
20 engineering may be accomplished by transforming a plant or
plant cell with all of the desired gene constructs
simultaneously. Alternatively, the engineering may be carried
out sequentially. That is, transforming with one gene
construct, obtaining the desired transformant after selection
25 and screening, transforming the transformant with a second
gene construct, and so on.
In an embodiment of the present invention,
Agrobacterium is employed to introduce the gene constructs
into plants. Such transformations preferably use binary
30 Agrobacterium T-DNA vectors (Bevan, 1984, Nuc. Acid Res.
12:8711-8721), and the co-cultivation procedure (Horsch et
al., 1985, Science 227:1229-1231). Generally, the
Agrobacterium transformation system is used to engineer
dicotyledonous plants (Bevan et al., 1982, Ann. Rev. Genet
35 16:357-384; Rogers et al., 1986, Methods Enzymol. 118:627-
641). The Agrobacterium transformation system may also be
used to transform as well as transfer DNA to monocotyledonous
D SH~ET
21 68430
W095/03690 PCT~S94/08722
44 -
plants and plant cells. (see Hernalsteen et al., 1984, EMBO J
3 3039-3041 ; Hooykass-Van Slogteren et al., 1984, Nature
311:763-764; Grimsley et al., 1987, Nature 325:1677-179;
Boulton et al., 1989, Plant Mol. Biol. 12:31-40.; Gould et
5 al., 1991, Plant Physiol. 95:426-434).
In other embodiments, various alternative methods
for introducing recombinant nucleic acid constructs into
plants and plant cells may also be utilized. These other
methods are particularly useful where the target is a
10 monocotyledonous plant or plant cell. Alternative gene
transfer and transformation methods include, but are not
limited to, protoplast transformation through calcium-,
polyethylene glycol (PEG)- or electroporation-mediated uptake
of naked DNA (see Paszkowski et al., 1984, EMBO J 3:2717-2722,
~5 Potrykus et al. 1985, Molec. Gen. Genet. 199:169-177; Fromm et
al., 1985, Proc. Nat. Acad. Sci. USA 82:5824-5828; Shimamoto,
1989, Nature 338:274-276) and electroporation of plant tissues
(D'Halluin et al., 1992, Plant Cell 4:1495-1505). Additional
methods for plant cell transformation include microinjection,
20 silicon carbide mediated DNA uptake (Kaeppler et al., 1990,
Plant Cell Reporter 9:415-418), and microprojectile
bombardment (see Klein et al., 1988, Proc. Nat. Acad. Sci. USA
85:4305-4309; Gordon-Kamm et al., 1990, Plant Cell 2:603-618).
According to the present invention, a wide variety
25 of plants and plant cell systems may be engineered for the
desired physiological and agronomic characteristics described
herein using the nucleic acid constructs of the instant
invention and the various transformation methods mentioned
above. In preferred embodiments, target plants and plant
30 cells for engineering include, but are not limited to, those
of maize, wheat, rice, soybean, tomato, tobacco, carrots,
peanut, potato, sugar beets, sunflower, yam, Arabidopsis, rape
seed, and petunia.
2 1 68430
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tv~ - 2 6 JbL'95-
5.7. SELECTION AND IDENTIFICATION OF
TRANSFORMED PLANTS AND PLANT CELLS
According to the present invention, desired plants
and plant cells may be obtained by engineering the gene
constructs described herein into a variety of plant cell
types, including but not limited to, protoplasts, tissue
culture cells, tissue and organ explants, pollen, and embryos --
as well as whole plants. In an embodiment of the present
invention, the engineered plant material is selected or
screened for transformants (i.e., those that have incorporated
or integrated the introduced gene construct(s)) following the
approaches and methods described below. An isolated
transformant may then be regenerated into a plant.
Alternatively, the engineered plant material may be
regenerated into a plant or plantlet before subjecting the
derived plant or plantlet to selection or screening for the
marker gene traits. Procedures for regenerating plants from
plant cells, tissues or organs, either before or after
selecting or screening for marker gene(s), are well known to
those skilled in the art.
A transformed plant cell, callus, tissue or plant
may be identified and isolated by selecting or screening the
engineered plant material for traits encoded by the marker
genes present on the transforming DNA. For instance,
selection may be performed by growing the engineered plant
material on media containing inhibitory amounts of the
antibiotic or herbicide to which the transforming marker gene
construct confers resistance. Further, transformed plants and
plant cells may also be identified by screening for the
activities of any visible marker genes (e.g., the B-
glucuronidase, luciferase, B or Cl genes) that may be present
on the recombinant nucleic acid constructs of the present
invention. Such selection and screening methodologies are
well known to those skilled in the art.
Physical and biochemical methods also may be used to
identify a plant or plant cell transformant containing the
gene constructs of the present invention. These methods
21 68430r~ 7 2.2
- 46 - " ~ o JUL 95
include but are not limited to: 1) Southern analysis or PCR
amplification for detecting and determining the structure of
the recombinant DNA insert; 2) northern blot, S-1 RNase
protection, primer-extension or reverse transcriptase-PCR
5 amplification for detecting and examining RNA transcripts of
the gene constructs; 3) enzymatic assays for detecting enzyme
or ribozyme activity, where such gene products are encoded by
the gene construct; 4) protein gel electrophoresis, Western
blot techniques, immunoprecipitation, or enzyme-linked
10 immunoassays, where the gene construct products are proteins;
5) biochemical measurements of compounds produced as a
consequence of the expression of the introduced gene
constructs. Additional techniques, such as in situ
hybridization, enzyme staining, and immunostaining, also may
15 be used to detect the presence or expression of the
recombinant construct in specific plant organs and tissues.
The methods for doing all these assays are well known to those
skilled in the art.
5.8. EXPRESSION OF HETEROLOGOUS GENE
PRODUCTS IN TRANSGENIC PLANTS
The present invention may be advantageously used to
direct the expression of a variety of gene products. These
gene products include, but are not limited to, proteins, anti-
25 sense RNA and ribozymes.
In embodiments of the present invention, a inducible
promoter or a recombinant inducible promoter may be used, in
post-harvest production, to express in plants and plant cell
cultures a variety of high valued protein products, including,
30 but not limited to, pharmaceutical and therapeutic enzymes and
proteins. Such protein products may include, for example,
various peptide-hormones, cytokines, growth factors,
antibodies, blood proteins and vaccines. Further, these
promoter elements may also be used to express in plants and
35 cell cultures multiple enzymes of complex biosynthetic
pathways, the induction of which would confer to host plant or
plant cell the ability to produce complex biochemicals and
A~N~ED ~HEET
2 1 68430
W095/03C90 PCT~S94/08722
_ - 47 -
biologicals. Examples of such products include secondary
metabolites such as alkaloids, antibiotics, pigments, steroids
and complex biological structures such as intact or defective
viruses or viral particles. Additionally, these promoter
5 elements may also be used to express various types of lytic
and processing enzymes that can convert what would be
otherwise unusable or low quality plant compounds or
constituents into useful or high quality compounds or chemical
feedstocks. Examples of such products include cellulases,
10 lignases, amylases, proteases, pectinases, phytases, etc.
Furthermore, a inducible promoter or a recombinant inducible
promoter may also be used to express RNA products such as
antisense RNA and ribozymes. In particular embodiments, HNG2
or HNG2-derived promoter elements which confer wound-inducible
15 and/or elicitor-inducible gene expression are used to express
the above-mentioned products.
In the above embodiments, the inducible promoter or
recombinant inducible promoter, including the wound- and/or
elicitor-specific HMG2 and HMG2-derived promoters, may be
20 advantageously used to direct the "post-harvest" production
and accumulation of the desired direct or indirect gene
products. That is, the production of the desired products
does not occur during normal growth of the plant or the cell
culture, but only occurs after the plant or the cell culture
25 (e.g., callus culture) is mechanically macerated and/or
elicitor-treated shortly before, during, or shortly after,
harvesting the plant or cell cultures. (For a general
reference describing plant cell culture techniques that could
be used in conjunction with the "post-harvest" production
30 approach disclosed here, see Handbook of Plant Cell Culture,
Vol. 4, Techniques and Applications, etc., Evans, D.A., Sharp,
W.R. and Ammirato, P.V., 1986 Macmillan Publ., New York, New
York).
Any plant tissue or culture lines of plants or plant
35 cells engineered with the expression construct described
herein may used in the production of desired direct or
indirect gene products. Useful plant tissue (and organs)
21 68430
WO95/03690 PCT~S94/08722
- 48 -
include, but are not limited to, leaf, stem, root, flower,
fruit and seed.
Where the production of the desired indirect gene
product, e.g., a secondary metabolite, requires two or more
s gene functions, it may be advantageous to engineer the host
plant or plant cell with several expression constructs,
wherein each construct comprises the same inducible promoter
controlling the expression of each required gene function, so
as to enable the coordinate expression of all required gene
10 functions.
The induction of the harvested tissues and cultures
from the engineered plants and cells may be by any means known
in the art for the particular inducible promoter used in the
expression construct. For example, to induce expression from
lS an expression construct vector comprising an HMG2 or HMG2-
derived promoter, the harvested tissue or culture may be
physically wounded by maceration, treated with biological
elicitors, infected with an appropriate pathogen, etc.
The induction of the harvested plant tissue or
20 culture may be done immediately after harvesting or the
harvested tissue or culture may be stored and then
subsequently induced. The storage of the harvested plant
tissue or culture may be by any procedure or method known in
the art that optimally preserves the gene expression
25 capability of the stored material.
In particular embodiments, harvested leaves can be
stored at 4C in sealed containers or at room temperature in
air permeable containers for up to 6 weeks before inducing the
expression of the desired gene products. Preferably, the
30 stored leaves are induced at about two weeks after harvest.
The induced tissue or culture may be incubated at
room temperature for up to a week to allow for the expression
of the induced gene(s) and accumulation of the desired direct
or indirect gene product before the tissue or culture is
35 processed for isolating the desired direct or indirect gene
product. In particular embodiments, induced leaf tissue is
2 1 68430
WOg5/03690 PCT~S94/08722
- 49 -
incubated at room temperature for approximately 48 hr. before
the leaf tissue is processed.
The application of inducible promoter or recombinant
inducible promoters to produce the above mentioned types of
5 products is particularly significant given that many such
products may be either labile or deleterious to cellular
metabolism or plant growth. Thus, their efficient and optimal
production, in many instances, may be best achieved by the use
of inducible, particularly wound-inducible or elicitor-
10 inducible, promoters coupled with a post-harvest induction
protocol. As explained previously, harvesting of plants or
plant parts for later use does not normally kill or harm plant
tissues if they are maintained in an environmentally suitable
condition.
In another embodiment, an HMG2 or HMG2-derived
promoter which confers pathogen-induced expression may be used
to express a variety of disease resistance genes during a
pathogen infection. Examples of such resistance genes include
virus coat protein, anti-sense RNA or ribozyme genes for anti-
20 virus protection (Gadani et al., 1990, Arch. Virol 115:1-21);
lysozymes, cecropins, maganins, or thionins for anti-bacterial
protection; or the pathogenesis-related (PR) proteins such as
glucanases and chitinases for anti-fungal protection.
In a further emboAi~?nt, an HMG2 or HMG2-derived
25 promoter which confers pest infestation-induced expression may
be used to express a variety of pest resistance genes during
an insect or nematode infestation. Examples of useful gene
products for controlling nematodes or insects include Bacillus
thuringiensis endotoxins, protease inhibitors, collag~nAc~s,
30 chitinase, glucanases, lectins, glycosidases, and neurotoxins.
The HMG2 and HMG2-derived promoters have a number of
characteristics that make them particularly useful for
expressing pathogen and pest resistance genes. These
characteristics include these promoters' very low background
35 activity in most uninduced tissues of the plant, their rapid
induction and strong activity once induced and the relatively
site-specific nature of the induced response. These combined
21 68430
WOg5/036~ PCT~S94/08722
-- 50 --
characteriStiCs make the HMG2-controlled resistance functions
highly efficient by limiting the resistance response to the
time and place that such plant responses would be the most
effective (i.e., the site of the pathogen or pest ingress).
s In yet another embodiment, an HMG2 or HMG2-derived
promoter which confers pollen-specific expression may be used
to engineer male-sterile plants. A pollen-specific HMG2 or
HMG2-derived promoter may be used to express gene functions
that interfere with vital cellular processes such as gene
10 expression, cell division or metabolism. Examples of such
functions include RNases, DNases, anti-sense RNAs and
ribozymes. The use of pollen-specific HMG2 or HMG2-derived
promoters would limit the expression of such deleterious
function to pollen tissue without affecting other aspects of
15 normal plant growth development.
6Ø EXAMPLE: ISOLATION AND
CHARACTERIZATION OF THE TOMATO HMG2
PROMOTER AND HMG2 GENE FUSIONS
The isolation of the tomato HMG2 promoter is
20 described here. The disclosed approach is generally
applicable to the isolation of homologs of any cloned
promoter, and is particularly applicable to the isolation of
homologs of the tomato HMG2 promoter, from other plant
species. The approach uses a gene sequence containing the
25 coding region of an HMGR to screen a genomic library under low
stringency hybridization conditions. In the present example,
the probe used was a fragment containing a region of the yeast
HMGR that shows a high degree of amino acid sequence
conservation with the hamster HMGR. Positive clones are
30 isolated and subcloned where necessary, and the hybridizing
region is sequenced along with the flanking sequences.
Sequences containing the HMG2 gene are identified based on
their nucleotide sequence comparisons with known HMGR genes
and their divergence from known HMGl genes (see Park et al.,
3 1992, Plant Mol. Biol. 20:327-331).
2 1 68430 ~ 94 / 08 7 22
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- 51 -
Promoter:reporter gene fusions are used here to
localize the HNG2 promoter elements within the cloned sequence
5' upstream of the HMGR coding sequence. One goal of the
analysis is to demonstrate the tissue-specificity and defense-
5 related and post-harvest inducibility that the 2.3 kb HMG2
promoter confers on operably linked heterologous genes. The
second goal of the analysis is to determine in transgenic
plants the smallest 5' upstream fragment that would confer the
complete array of regulatory responses of the HMG2 gene.
10 Thus, commencing with a 2. 5 kilo-base pair (kb) fragment 5'
upstream of the HMG2 transcription initiation site, smaller
promoter element fragments are generated using PCR leaving out
incrementally larger tracts of sequences from the 5'-end of
the, 2.5 kb fragment. Gene fusions are constructed by
15 ligating the 2.3 kb EcoRI/BglII fragment and each of the
smaller, PCR-generated fragments to ~-glucuronidase ~GUS)
reporter genes. The activities of the fusion genes are
examined in transgenic plants to determine the location of the
NMG2 promoter.
6.1. MATERIALS AND METHODS
6.1.1 PLANT AND FUNGAL MATERIAL
Tomato (Lycopersicon esculentum cvs. Gardener and
Vendor) and tobacco (Nicotiana tabacum cvs. Xanthi and NC95)
25 plants were grown under greenhouse conditions. Seedlings for
transformation experiments were grown axenically as described
below. For elicitor treatments of suspension cultured plant
cells, tomato EP7 cells were maintained in the dark in a
modified MS medium. Verticillium alboatrum (race 1) and
30 Fusarium oxysporum (race 1), provided by Dr. Martha Mutschler
(Cornell University, Ithaca, NY), were maintained on 2.4%
potato dextrose agar and grown in 2.4% liquid medium for cell
wall isolation. Fungal elicitor, the high molecular weight
material heat-released from isolated mycelial walls was
35 obtained and measured as described (Ayers et al., 1976, Plant
Physiol. 57:760-765). Rhizoctonia solani strains RS51 and
~ r! e~FrT
21 68430
W095/03690 PCT~S94/08722
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R992 were provided by Dr. Charles Hagedorn (Virginia
Polytechnic Institute and State University).
6.1.2. GENOMIC LIBRARY SCREENING
Recombinant clones (500,000) of a tomato genomic DNA
library (L. esculentum cv. VFNT Cherry) constructed in lambda
Charon 35 were screened by plaque hybridization.
Hybridization probe was the 1.75 kb EcoRI fragment of pJR326,
(provided by Dr. Jasper Rine of University of California,
10 Berkeley, CA) which contains the region of S. cerevisiae HMGl
most highly conserved with hamster HMGR (Basson et al., 1986,
Proc. Natl. Acad. Sci. USA 83:5563-5567). Initial screening
was at low stringency conditions (e.g., 30% formamide, 6X SSC,
5X Denhardt solution, 0.1% SDS, 100 ug/ml salmon sperm DNA at
15 37C for 24 hours; final wash conditions were 0.2X SSC at room
temperature). Plaques giving positive hybridization signals
were carried through at least three rounds of purification
prior to further characterization. A 7 kb HindIII fragment of
one clone (designated TH29) was subcloned into the HindIII
20 site of transcription vector pSP6/T7 (Bethesda Research
Laboratories (BRL), Gaithersburg, MD) and designated pTH295
(Figure 3).
6.1.3. NUCLEIC ACID ISOLATION
Total DNA was isolated from tomato leaves according
to the method of Draper and Scott, (Plant Genetic
Transformation and Gene Expression, 1988, Eds. Draper et al.,
Blackwell Scientific, Palo Alto, CA, pp211-214)). For RNA
isolation, suspension cultured tomato cells treated with
30 elicitors, healthy roots, stems, and leaves treated by
wounding (cut into l mm slices with a razor blade and
compressed with a pestle), or intact fruit at various stages
of development were stored at -70C. Total RNA was isolated
from 1 to 3 g fresh weight of tissue ground in liquid nitrogen
35 and homogenized directly in a phenol:0.1 M Tris (pH 9.0)
emulsion as described previously (Haffner et al., 1978, Can.
J. Biochem. 56:7229-7233).
- 2168430
, ~Ij '_ J 94 ~ 08 7 22
r i~ ~ 2 6 JUL ,- r
6.1.4. HYBRIDIZATION ANALYSIS
For genomic Southern analyses, 10 ~g/lane total DNA
was digested with restriction endonucleases, separated on 0.8%
agarose gels, and transferred to Nytran membranes using
conditions recommended by manufacturer (Schleicher and
Schuell, Keene, NH). For Northern analyses, total RNA (5 to
20 ~g/lane) was denatured by treatment with glyoxal prior to
electrophoresis in 1.2% agarose for gel analyses or
application directly to Nytran filters utilizing a slot
blotting apparatus. For hybridizations aimed at revealing all
members of the HMGR gene family, probes derived from the 3'
end of the gene which is most highly conserved between species
(Basson et al., 198~, Mol. Cell. Biol. 8:3797-3808) were used.
Either the 1.5 kb EcoRI fragment of pTH295 (Figure 3; Yang et
al., 1991, Plant Cell 5:397-405) or a 486 bp HMG2 cDNA clone
from this region derived from pCD1 were 32P-labeled by random-
primer methods (Multi-prime Labeling System, Amersham, U.K.).
Membranes were prehybridized overnight without labeled probe
and hybridized in the presence of 32P-labeled probe for 24 to
48 hr at 42C in solution containing 40% formamide, 6X SSC, 5X
Denhardt solution, 5 mM EDTA, 0.1% SDS, 100 ~g/ml salmon sperm
DNA (Sigma). Final wash conditions were 0.lX SSC, 0.1% SDS, 1
hr at room temperature. For analyses aimed at monitoring
hybridization of sequences specific only for the HMG2 isogene
encoded by pTH295, the 0.7 kb AvaI-EcoRI fragment encoding
5'-untranslated regions and the 5' end of the gene or a
smaller 340 bp subclone derived from this fragment and lacking
the upstream region, was utilized. Hybridization conditions
were as described above except that 50% formamide and 5X SSC
were used. Following hybridization, membranes were washed
(final wash used 0.lX SSC at 50C for 1 hr) to remove unbound
label prior to X-ray film exposure.
6.1.5. HMG2 PROMOTER:REPORTER GENE FUSIONS
The 2.5 kb EcoRI fragment of the HMG2-containing
clone pTH295 was inserted into the EcoRI site of a Bluescript
N~D ~HET
21 68430 t~,l S94/08 722
i~ r,u~v~3 -i~6 JllL 95
- 54 -
SK-vector (Stratagene) and designated pDWl01 (Figure 3).
Digestion of this vector with EcoRI and BglII releases a
fragment (about 2. 3 kb) which contains sequences encoding the
first five N-terminal amino acids of the HNG2 HMGR and
5 extending approximately 2. 3 kb 5' upstream of these coding
sequences. This fragment was ligated to the GUS gene at an
NcoI site to create an in-frame fusion with the ATG start
codon of GUS and inserted into a modified pRK290 plasmid to
generate pSLJ330.1 (Figure 8). This plasmid was constructed
10 at the Sainsbury Laboratory, John Innes Institute (Norwich,
U.K.) in collaboration with Jonathan Jones using
transformation vectors provided by Dr. Jones. Subsequent NMG2
promoter constructs were inserted into plant
transformation/expression vectors of the pBI series (Clontech
15 Laboratories, Inc., Palo Alto, CA). The pSLJ and pBI plasmids
containing the fusion genes were introduced in Agrobacterium
tumefaciens strain LBA4404 (Clontech) by tri-parental matings
using the helper plasmid pRK2013.
6.1.6. SEOUENCING OF HMG2 PROMOTER DELETIONS
Progressively larger deletions from the 3' and 5'
ends of the 2. 5 kb promoter region of HMG2 contained in pDW101
were generated utilizing the Exonuclease III/Mung Bean
Nuclease reaction kit purchased from Stratagene (La Jolla,
25 CA). Double-stranded pDW101 was digested with BamHI and SacI
(both of which cut in the vector portion of the plasmid),
resulting in a linear piece of DNA with a 5' overhang and a 3'
overhang. After phenol:CHCl3 extraction to remove the
restriction enzymes, the DNA was precipitated with 100% EtOH
30 and dried. The digested DNA was then treated with 2 0 units
Exonuclease III/~g DNA to create a segment of single-stranded
DNA. Aliquots of the reaction were removed every 45 seconds
and added to tubes which contained Mung Bean Nuclease buffer.
Mung Bean Nuclease was then used to digest the single-stranded
35 portion of the DNA. This process was calculated to remove
approximately 300 bp for every time point. The enzymes were
extracted by treatment with lithium chloride and the DNA
~r~ ~ i~.~! .~FrT
2 1 68430
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2 6 JUL'g-5-
precipitated by the addition of sodium acetate and ethanol.
This process was followed by a fill-in reaction using the
Klenow fragment to ensure the presence of blunt ends for
subsequent ligation. The resulting plasmids were transformed
into Escherichia coli. Double-stranded DNA sequencing was
performed using Sequenase 2.0 (United States Biochemical,
Cleveland, OH) according to protocols provided by the
manufacturer. Electrophoresis was carried out on 5% HydroLink
Long Ranger acrylamide gels (AT Biochem, Malvern, PA).
6.1.7. GUS GENE FUSIONS WITH
HMG2 PROMOTER DELETIONS
Deletions in the HMG2 promoter were created using
PCR methodology. The primers used (designated in Figure 4),
and the approximate size fragments obtained were: primers #22
and #18, 1000 bp; primers #20 and #18, 460 bp; primers #19 and
#18, 170 bp. Primers #19, #20 and #21 generated a flanking
HindIII site; primer #18 generated a flanking BamHI site. The
PCR reactions contained 1.5 mM MgCl2, 200 ~M each dNTP, 2.5
units Taq polymerase, lX Taq polymerase buffer, 100 ng pDW101,
and approximately 40 pmol each primer. Three cycles were
used: Cycle 1 (lx): 95C-5 min, 72C-5 min, 58C-2 min,
72C-15 min, with the addition of the Taq polymerase after the
first 72C incubation; Cycle 2 (40x): 95C-1 min, 58C-2 min,
72C-3 min; Cycle 3 (lx): 72C-15 min. The DNA fragments
generated were electrophoresed on a 1.4% agarose gel to verify
the sizes. Polyacrylamide gel-purified fragments were
digested with the restriction enzymes HindIII and BamHI.
These fragments were then ligated into the binary vector
pBI101 which had been similarly digested (Figure 7). The
resulting plasmids, pDW201, pDW202, and pDW203 were
transformed into E. coli strain DH5~ and their sequences
verified. The binary plasmids were introduced into
Agrobacterium strain LBA4404 using tri-parental mating.
~..~I~D ~
2 1 68430 -- ~s~ 9~ 08-72-~
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6.1.8. TRANSFORMATION OF GENE
CONSTRUCTS INTO PLANTS
The HMG2 promoter:reporter gene fusions were
transformed into tobacco according to the leaf disk
transformation protocol of Horsch et al. (Horsch et al., 1985,
Science, 227:1229). Prior to co-cultivation with
appropriately engineered Agrobacterium tumefaciens, leaf --
disks excised from axenically grown tobacco seedlings were
incubated for 8 hours on sterile filter paper overlaying a
lawn of cultured tobacco "nurse" cells spread on a feeder
plate (modified MS medium cont~;n;ng Nitsch vitamins, 100 mg/L
myo-inositol, 30 gm/L sucrose, 1 mg/L 2,4-D, 0.4 mg/L BAP, 8
gm/L agar). Co-cultivation was accomplished by submersing the
leaf disks in a suspension of A. tumefaciens at a
concentration of 1 x 109 cells/ml, followed by vacuum
infiltration (3 x 1 min). The leaf disks were then returned
to the nurse plates and incubated for 48 hours at 25C with
indirect light. Disks were then transferred to
selection/regeneration plates (MS salts, Nitsch vitamins, 100
mg/L myo-inositol, 20 gm/L sucrose, 2 mg/L zeatin, 4 gm/L
agar) which contain carbenicillin (500 ~g/ml) and an
appropriate antibiotic for transformation selection. Plates
were returned to the growth chamber (25C, 18 hr light).
Resulting shoots were excised and transferred to rooting media
(MS salts, Nitsch vitamins, 100 mg/L myo-inositol, 30 g/L
sucrose, 4 gm/L agar, and IAA and kinetin at final
concentrations of 10 ~M and 1 ~M, respectively). Rooted
plantlets were then transferred to soil and moved to the
greenhouse.
Transformation of tomato was performed in a similar
manner with the exception of the source of tissue. Cotyledons
cut from aseptically grown tomato seedlings were cut under
water using a sterile scalpel into 0.5-1.0 cm explants.
Explants were then incubated on feeder plates for 8 hours
prior to transformation.
~ r~ F~
21 68430 ~ f~94/08 722
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6.1.9. EXAMINATION OF GUS EXPRESSION
IN TRANSGENIC PLANTS
Histochemical analysis of GUS activity in transgenic
plant tissues was carried out using standard techniques well
known in the art. For routine analyses, plant organs or
tissue sections were submerged in a solution of 1 mM X-gluc in
50 mM phosphate buffer (pH 7) and 0.1% Triton X-100. --
Infiltration of the substrate was facilitated by 3 x 1 minute
vacuum infiltration. The amount of vacuum infiltration was
varied based on the thickness and hardiness of the tissue to
be tested. Tissue was incubated 4-12 hours at 37C.
Following development of GUS staining, leaf tissue was
generally treated with ethanol (boil 2 minutes in 95% or
incubate overnight in 95%) to remove chlorophyll. GUS
activity was also monitored in cell-free extracts using the
fluorescent substrate MUG by standard protocols (Jefferson,
1987, Plant Mol. Biol. Rep. 5:387-405). GUS activity was
expressed as nmol MU/min/~g protein where protein was
determined by the method of Bradford (Pierce Coomassie Plus
Protein Assay Reagent) using BSA as the standard.
6.1.10 BACTERIAL INFECTION INDUCTION
OF HMG2 PROMOTER A~llVl~lY
A 5 ml culture of Erwinia carotovora ssp.
carotovora, strain EC14, causal agent of soft rot, was grown
overnight from a single colony in LB medium at 25C. The
culture was centrifuged and resuspended in 1 ml distilled
water (OD600 = 3.54-3.82). Leaves, 6-8 inches in length, of
greenhouse grown transgenic tobacco carrying the HMG2:GUS
construct were excised at the petiole and placed on water-
moistened filter paper in a large petri dish. The tip of amicropipettor was used to gently wound the top surface of the
leaf while depositing 2 ~l of distilled water (mock
inoculation) or EC14 suspension. The plates were closed and
placed in a plastic container with a tightly-fitting lid
(e.g., Tupperware~) that had been lined with water-saturated
paper towels and incubated at 28C in the dark. Samples were
~N~ED S~IEET
21 68430
9 4 / 0 8 Z 2
- - 58 - ~ ~ 26JUL 95
harvested at 24 hour intervals by excising a leaf section
surrounding the site of inoculation using a hole punch or
cork-borer and processed for histochemical analysis as
described above.
6.1.11 FUNGAL INFECTION INDUCTION
OF HMG2 PROMOTER ACllVllY --
Seeds of transgenic tobacco were surface sterilized
by soaking in 30% bleach, rinsed 4 times with sterile water
and germinated in autoclaved potting mix (ProMix BX; Premier
Brands, Inc, Stamford, CT) in 4 x 4 inch PlantCons (ICN
Biomedicals, Inc, Irvine, CA). Fungal inoculations were done
by placing a fungal-infested oat seed in contact with the
seedling hypocotyl. Rhizoctonia solani strains RS51 and R992
were inoculated as agar plugs into moist, sterilized oat seed
and grown for 8 days at room temperature prior to inoculation
on plants. At 24 hour intervals, seedlings were removed with
care taken to limit contact with leaves to minimize wounding
of the seeding near the site of inoculation, and the soil
removed by dipping the roots into water. The entire
seedlings, 1-2 inches in length, were then processed for
histochemical GUS determination as described above. Tomato
seedlings grown on agar (1/10 x MS salts plus 1% agar) were
also inoculated with a small agar plug of plate-grown
Rhizoctonia solani. This method of inoculation was less
effective than the soil-grown seedling method because the agar
medium supported vigorous fungal growth.
6.1.12 NEMATODE INFESTATION INDUCTION
OF HMG2 PROMOTER A~llVl~Y
Tl generation tomato seedlings were grown for 10
days on seed germination media (1/10 x MS salts, and 10 mg/L
myo-inositol, 3 gm/L sucrose, 6 gm/L agarose) in 4 x 4 inch
Plantcons. Approximately 2000 nematodes (either Melodigyne
incognita or M. hapla) were applied to the top of the media;
Plantcons were then placed in a 25C growth chamber.
Seedlings were harvested at 1, 2, 3, 5, and 7 days post-
~t~D9~E~r
PEOC - 4 0 r
21 68430
W095/03690 PCT~S94/08722
- 59 -
inoculation by slowly pulling seedlings from the agarose,
gently removing agarose from roots, and cleanly cutting top of
seedling. Roots were analyzed for GUS expression
histochemically.
s
6.1.13 POST-HARVEST WOUND INDUCTION
OF HMG2 PROMOTER ACTIVITY
Leaves approximately 8 inches in length were removed
from HMG2:GUS and 35S:GUS tobacco plants and were wounded by
10 heavily scoring with a razor blade. Sections of each leaf
were immediately removed, weighed, then frozen in liquid
nitrogen and stored at -70C. Additional sections of leaf
were removed, weighed, and frozen at 24 and 48 hours post-
harvest. Leaf extract was obtained by grinding in MUG
15 extraction buffer (Jefferson, 1987, Plant Mol. Biol. Rep.
5:387-405) with a mortar and pestle. Protein concentrations
were determined by the Bradford method. MUG assays were
performed according to the method of Jefferson (Jefferson,
1987, id.); activity was expressed as nmol MU/min/~g protein.
6.1.14 FIELD PERFORMANCE OF TRANSGENIC
PLANTS CONTAINING HMG2:GUS GENE
FUSIONS
Seeds of two independent transformants of each of
two cultivars (Xanthi and NC-95) showing high levels of wound-
25 inducible HMG2:GUS activity were used for initial field tests.Seedlings of transgenic tobacco carrying a 35S:GUS construct
(SJL1911, Figure 9) were also planted to use as controls.
Transgenic seedlings were grown in the greenhouse for
approximately 4 weeks prior to transfer to the field. Test I
30 involved 300 plants (seven genotypes) planted (automated
planter) at the Virginia Tech Southern Piedment Agricultural
Experiment Station in Blackstone, Virginia. Test II involved
175 plants (7 genotypes) planted as a randomized block in
experimental plots at Virginia Tech, Blacksburg, Virginia.
35 Leaf material is harvested at various times during the growing
season (including just prior to and 2 weeks following routine
topping of the plants) to determine field levels of basal HMG2
21 68430
WOg5/03690 PCT~S94/08722
- 60 -
promoter activity and post-harvest induction of transgene
activity. HMG2: GUS expression in response to natural pathogen
pressure (including, but not limited to, cyst nematodes,
aphids, beetle and hornworm predation, tobacco mosaic virus)
s is monitored by histochemical analysis.
6.2 RESULTS
6.2.1 NMG2 PROMOTER A~llVllY
In order to delineate regulation of HMG2 expression,
10 2. 3 kb of HMG2 upstream sequences were fused to the GUS
reporter gene in plasmid pSJL330.1 and used for Agrobacterium
tumefaciens-mediated plant transformation. More than 20
independent GUS-expressing tobacco transformants containing
1-4 copies of this construct or a 35S:GUS control construct
15 generated by transformation with plasmid pSJLl911 (Figure 9;
provided by Dr. Jonathon ~ones, John Innes Institute, Norwich,
U.K.) were generated. The 35S promoter is a high-level
constitutive plant promoter derived from the cauliflower
mosaic virus, (Benfey et al., 1989, EMBO J. 8:219S-2202; Fang
20 et al., 1989, Plant Cell 1:141-150).
6.2.2 TISSUE SPECIFIC A~llVllY
OF THE HMG2 PROMOTER
Plants expressing the HMG2: GUS constructs provide a
25 powerful tool for assessing tissue-specificity of H~G2
expression. Histochemical analyses of GUS activity indicated
that HMG2 is expressed in unstressed plants in the hypocotyl
region of seedlings (region of shoot which breaks through
soil), in trichomes (plant hairs on leaf surface important in
30 insect, pathogen-resistance, Gershenzon et al., 1992, Anal.
Biochem. 200:130-138), and in pollen (Figure 10). Expression
in these tissues could be defense-related. Previous reports
have shown that some defense-related genes are elevated in
pollen (Kononowicz, 1992, Plant Cell 4:513-524). However,
3S significant levels of GUS activity in the primary root of
young tomato seedlings were also observed at sites of lateral
root initiation. The significance of this expression is
2 1 68430
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currently unknown. It does not appear to be related to cell
division because no GUS expression is found in the zone of
cell division in the root tip. The activity of the HMG2:GUS
constructs has also been determined in ripening fruit of
5 transgenic tomato plants. Fruit at various stages of
development were harvested and tested for GUS activity in
cell-free extracts or analyzed for tissue specificity using
histochemical assays. Immature fruit (0.5 to 1.5 cm) showed
no HMG2:GUS expression. However, larger fruit (3-5 cm),
10 mature green fruit (full size but without carotenoids),
breaker fruit (undergoing carotenogenesis) and fully ripe
fruit showed GUS activity. Histochemical analyses identified
that the GUS activity was localized primarily to the
developing seeds and fruit vasculature. The fruit showed
15 dramatic wound-inducibility of the HMG2:GUS activity.
HMG2:GUS activity was also localized to the developing seeds
within the seed pods of tobacco.
Distinct regulatory circuits may mediate inducible
versus developmental control of HMG2 expression. During
20 anther (organ producing male gametophyte) development in
immature flowers, wounding induces high levels of GUS, but
immature pollen show no activity. As pollen matures, the
pollen expresses GUS but anthers no longer show wound-
inducible activity.
6.2. 3 DEFENSE-RELATED A~llvllY OF
THE HMG2 PROMOTER
Wounding by excision or crushing triggered rapid
increases in GUS activity within 1-12 hours. This response
30 was seen in all tissues tested with the exception of the
mature anthers of tobacco (discussed above). Tissues showing
marked wound-induced activation of the HMG2: GUS construct
include, but are not limited to, roots, hypocotyls, and leaves
of tobacco and tomato seedlings; roots, stems, petioles,
35 pedicles, all regions of expanding and mature leaves,
developing fruit and pods, mature fruit and pods, and petals
of mature tobacco and tomato plants.
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Inoculation of seedlings or excised leaves with
compatible bacterial pathogens triggered specific HMG2:GUS
expression localized to the cells directly surrounding the
bacterial lesion. This response was significant at 24 hours
post-inoculation (the earliest time monitored) as shown in
Figure 11, Panel A. The fungal pathogen, Rhizoctonla solani,
also triggered dramatic increases in HMG2:GGS activity (Figure
11, Panel B) in inoculated seedlings of both tobacco and
tomato. Because these widely different microbial pathogens
both trigger very similar HMG2 activation events, it is likely
that other bacterial and fungal pathogens will elicit a
similar response. At later times during Rhizoctonia
infection, the fungus had spread down the vascular system of
the plants and was associated with significant HMG2:GUS
activity in these tissues. This suggests that the defense-
activation properties of HMG2 will function against vascular
pathogens (e.g., wilt-inducing pathogens) as well.
6.2.4 NEMATODE ACTIVATION OF THE
HMG2 PROMOTER
Transgenic plants containing the 2.3 kb HMG2
promoter fused to GUS were analyzed for GUS activity following
nematode inoculation. Tomato seedlings, germinated axenically
on agar medium, were inoculated by addition of second-stage
juveniles of either Meloidogyne incognita or M. hapla, causal
agents of root knot disease. The transgenic cultivars were
susceptible to both species. Prior to inoculation and at
various times following addition of juveniles, roots were
removed, histochemically assayed for GUS activity, and stained
for nematodes using acid fucsin. Uninoculated roots and roots
at 24 and 48 hours after inoculation showed no GUS activity at
root tips (the site of nematode penetration), but GUS activity
was evident at the zone of lateral root initiation and at the
wound site where the stem was removed. At 24 and 48 hours
post-inoculation, roots containing juveniles were observed
which showed no indication of GUS activity (Figure 12, Panel
A). By 72 hrs post-inoculation some roots showed low levels
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- - 63 - ;~ JiiJO- 26JUL-'95
of GUS activity in cells adjacent and proximal (toward the
stem) to the juvenile (Figure 12, Panel B). By five days
post-inoculation, high levels of GUS activity was evident
surrounding nematodes that had initiated feeding
(characterized by a change in nematode morphology). By days
5-7, infected root tips showed visible swelling. This galled
tissue expressed extremely high levels of GUS (Figure 12,
Panels C, D and E). The responses to M. incognita and M.
hapla did not differ significantly. Observation of multiple
roots and plant samples suggested that the onset of HMG2
activation was closely associated with the establishment of
feeding behavior. This suggests that utilization of this
promoter to drive expression of transgene proteins which are
toxic or inhibitory to nematode development would
significantly impact disease development. Both the temporal
and spacial expression patterns are optimal for rapid and
highly localized delivery of nematocides or nematostatic
agents.
6 . 2.5 ACTIVATION OF THE HMG2 PROMOTER
BY NATURAL PREDATORS
Transgenic tobacco plants grown in the field were
tested for HMG2:GUS expression in response to natural
predation. Leaf sections surrounding sites of localized
necrosis due to insect or microbial damage were excised and
tested for localized expression of HMG2:GUS. Leaf tissue that
had not been damaged showed no GUS activity. However, the
region immediately surrounding the lesion showed intense GUS
staining (Figure 11, Panel C). Transgenic tobacco plants were
planted in Blackstone, VA in fields known to provide high
levels of disease pressure due to the tobacco cyst nematode
(Globodera tobacum ssp. solanaceara) in order to assess HMG2
responses to natural nematode infection.
6.2.6 DISSECTION OF HMG2 PROMOTER
In order to localize elements mediating defense- and
tissue-specific expression, efforts were made to generate a
Ab~O ~EE~
W095/03690 2 1 6 8 4 3 0 PCT~Sg4/08722
- 64
series of 5' promoter deletions. In the central region of the
HMG2 promoter, delineated in Figure 5, are several stretches
of ATs which seemed to prevent exonuclease digestion through
this area. After repeated unsuccessful attempts at
s exonuclease strategies, an alternative strategy based on PCR
was used to generate deletions of the HMG2 promoter (see
Figure 7). These truncated promoters were fused to GUS
reporter genes in the plant expression plasmid pBI101
(Clontechj and introduced into tobacco. The promoter region
10 from -981 to +llO confers both wound and microbial pathogen
responses as well as trichome and pollen expression (Table 4).
However, analyses of multiple independently transformed
tobacco plants suggest that the levels of transgene expression
driven by this promoter are less than that of promoters that
~s are larger (e.g., to -2.3 kb) or smaller (e.g., -347 bp).
This suggests that this region may contain a "silencer" and
that the region upstream of 891 contains an additional
enhancer-type element. All deletions, including the construct
containing only 58 bp of H~G2 upstream of the transcriptional
20 start site continue to drive transgene expression in the
pollen suggesting that developmental regulation is distinct
from that associated with defense.
21 68430
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TABLE 4: DISSECTION OF TOMATO ~MG2 PROMOTER ~T-~M~NT
HMG2:GUS PROMOTERa INDUCED AND TISSUE EXPRESSIONb
CONSTRUCT REGION (bp) Wound Pathogens Trichomes Pollen
_______ ____________________________________________________
SLJ 330.1 -2.3k to +118 ++ +++ ++ ++
DW 203 -891 to +125 + + + ++
DW 202 -347 to +125 ++ ++ ++ ++
DW 201 -58 to +125 -- -- -- +
a Nu",beri"g based on the l,dnsc,iplional start site designaled as +1. The ATG start codon
begins at base 111. GUS fusions were l,ansldlionally in frame and added several bases of
HMG2 coding region to the N-terrninus of the GUS gene.
b GUS phenotype assayed in multiple independently-l,ansfo""ed plants for each construct.
c Based on leaf assays following inoculation with the soft-rotting bacterium Erwinia carotovora
ssp. carotovora.
~ 4~n ~HE~
21 68430
WO95/03690 - 66 - PCT~S94/08722
6.2. 7 HMG2 CIS-~EGULATORY ELEMENTS
Specific cis-regulatory elements within the HMG2
promoter are identified based on functional analyses and DNA-
protein interactions. For example, the region between bases -
S 58 and -347 contains one or more elements which direct strong
wound and pathogen induction. The -58 to -347 fragment is
generated by PCR using primer 21 and a primer complementary to
primer 19 (see Figure 4). Additional 3' and 5'
oligonucleotide primers synthesized based on sequences within
10 this region will generate subfragments for analyses of
regulatory activity. Functional analyses entail fusion of
these fragments to appropriate "minimal" plant promoters fused
to a reporter gene such as the minimal 35S promoter (discussed
in Sections 5. 2 and 5.3, above) and introduction and
15 expression of the resulting constructs in plants or plant
cells. Regions containing fully-functional HMG2 cis-
regulatory elements will confer HNG2-specific regulation on
this minimal promoter as determined by wound-, pathogen-,
pest-, or elicitor-inducibility or generation of HMG2
20 sequence-dependent developmental patterns of GUS expression.
Specific cis-regulatory elements are further delineated by gel
shift or footprint/DNase sensitivity assays. The -58 to -347
fragment or other HMG2 promoter fragments, generated by PCR
amplification, are incubated with nuclear extracts from
25 control and defense-elicited cells. For example, nuclei are
isolated (Elliot et al., 1985, Plant Cell 1:681-690) from
untreated tomato suspension cultured cells and cells 6-8 hours
after treatment with fungal elicitor or arachidonic acid
(Park, l99O, Lycopersican esculentum Mill., Ph.D.
30 Dissertation, Virginia Polytechnic Institute and State
University; Yang et al., 1991, Plant Cell 3:397-405). PCR
fragments which interact with nuclear components are
identified based on reduced mobility in polyacrylamide gel
electrophoresis or by digestion with DNase I and analysis of
35 DNase-insensitive areas on sequencing gels. Defense-
responsive elem~nts may or may not demonstrate differential
patterns upon interaction with control versus elicitor-treated
2 1 68430
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- 67 - ~ 26JUL 95
extracts. Promoter regions identified as having DNA-protein
interactions are further tested by creating chimeric
constructs with, for example, "minimal" 35S promoters to
determine effects of the cis-regulatory element on
heterologous gene expression in transgenic plants or plant
cells. This strategy may separate specific HMG2 promoter
functions (e.g., wound from pathogen induction, or defense
from developmental regulation) and delineate additional or
novel uses of HMG2 promoter elements.
6.2.8. USE OF NMG2 PROMOTER FOR DEVELOPING
NEMATODE RESISTANT PLANTS
Another important use of the present invention is
the development of namatode-resistant plants. Such resistant
plants are developed by using an HMG2 promoter element to
control the expression of a heterologous gene which is or
whose product is toxic or inhibitory to nematodes. Key
advantages of using the HMG2 expression systems for this
purpose are that 1) the heterologous gene product will be
limited primarily to tissues undergoing disease-related
stress, and 2) the heterologous gene will be strongly
activated directly in the tissue of ingress, thus delivering a
significant "dose" to the nematode.
The coding region of potato proteinase inhibitor I
or II (Johnson et al., 1989, Proc. Natl. Acad. Sci. USA
86:9871-9875) is excised by appropriate restriction enzymes
and inserted into a plant expression vector downstream of the
HMG2 promoter or a chimeric promoter containing active cis-
regulatory element(s) of the HMG2 promoter. Appropriate
enzyme sites or linker fragments are used to assure correct
positioning of the coding region with the transcriptional and
translational initiation sites. The plasmid pDW202 (Figure 7
is digested with SmaI and SstI and gel purified to obtain the
vector/HMG2 promoter fragment separate from the fragment
containing the excised GUS coding region. The appropriatel~
digested and processed potato proteinase inhibitor I gene i
then ligated into the SmaI/SstI site. The resulting plasmi~
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9 4 / 08 7 2 2
- 68 - ~ ~ ~ JUL'95
is transformed into E. coli strain DH5~ and sequenced to
assure correct insertion and orientation prior to introduction
into Agrobacterium strain LBA4404 by triparental mating. The
engineered LBA4404 is then used for tobacco leaf disk
co-cultivation and the transformed tobacco is subsequently
regenerated to mature plants.
Enzymatic or immuno-detection of proteinase ~~
inhibitor I in transgenic plants is done by described methods
(Johnson et al., 1989, Proc. Natl. Acad. Sci. USA 86:9871-
9875). The effect of the introduced HMG2:Proteinase inhibitor
constructs on plant:nematode interactions is tested in
seedlings inoculated, for example, with root knot (Meloidogyne
lncognita, M. halpa) or cyst (Globodera tobacum) nematodes or
by planting seedlings in fields of known disease pressure.
Plants are monitored for overall health, number of feeding
female nematodes, number and severity of galls, and nematode
egg production.
In another strategy, HMG2 promoter-driven expression
of an anti-sense RNA may be used to block production of plant
compounds required by parasitic nematodes. For instance, root
knot and cyst nematodes are dependent upon plant sterols for
growth and reproduction. Thus, sequences from the tomato HMG1
(sterol-specific) isogene (e.g., regions with the N-terminus
specific for HMG1 and not other HMGR isogenes) or the tomato
squalene synthetase gene are introduced into the SmaI/SstI
digested vector described above such that the complementary
RNA strand is produced. Expression of these gene constructs
in transgenic tomato decreases or blocks sterol production in
plant cells where HMG2 promoter is active and thus reduces or
prevents nematode growth and development.
7Ø EXAMPLE: POST-HARVEST PRODUCTION USING
AN EXPRESSION CONSTRUCT COMPRISING THE
TOMATO HMG2 OPERABLY LINKED TO GENE OF
INTEREST
Post-harvest production of desired gene products in
harvested plant tissues and cultures depends on the ability of
the harvested material to-remain competant for induced gene
ND~D ~HE~F
21 68430
W095/03690 PCT~S94/08722
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expression. The experiments described below demonstrate such
an ability in harvested leaf tissue. Moreover, in the
harvested leaf the HMG2 promoter activity superior to that of
the often used constitutive CaMV 35 promoter.
s The plants used in these experiments are from the
HMG2 :GUS or CaMV 3SS:GUS transformed tobacco plants described
- in section 6. 2 .1. above.
7 .1 . HMG2 PROMOTER IS SUPERIOR TO CaMV 35S
PROMOTER IN POST-HARVEST PRODUCTION
The yield of a specific transgene, ~-glucuronidase
(GUS), was compared for transgenic plants containing the
HMG2: GUS construct versus those containing a 35S:GUS
construct. The 35S promoter from the cauliflower mosaic virus
is widely used in transgenic plant research because it directs
high levels of transgene expression in most tissues (Benfey et
al., 1989, EMBO J. 8:2195-2202). Leaf material was harvested
from greenhouse-grown plants and wounded by scoring with a
razor blade. Tissue was extracted for GUS activity
20 immediately after harvest/wounding and after 24 and 48 hours
of room temperature storage in zip-lock bags to prevent
desiccation. As shown in Figure 13, the leaves containing the
35S:GUS construct showed greater that 50% loss of GUS activity
within 24 hours of harvest. For industrial utilization of
25 transgenic tobacco for bioproduction of a high-value protein,
this loss would be highly significant. In contrast, the
HMG2 :GUS construct was less active immediately after harvest,
but by 48 hours, directed wound-induced transgene product
accumulations to levels comparable to pre-storage 35S:GUS
30 levels.
A transgenic plant carrying a heterologous gene
encoding a valuable product, for example a human therapeutic
protein of pharmaceutical value, fused to the HMG2 promoter
would be expressed at relatively low levels throughout the
35 growing cycle of a transgenic plant (e.g., tobacco). Thus,
transgene expression is unlikely to affect biomass yield.
Upon optimal biomass production, the plants are harvested
21 68430O9~/03690 PCT~S94/08722
- 70 -
(e.g., by mowing) and the leaf material is transferred to thelaboratory or processing facility. HMG2-driven heterologous
gene expression triggered by mechanical wounding or wounding
plus chemical or elicitor treatment and the transgene product
s is then recovered for further processing accumulation. The
time range of product accumulation is generally (but not
limited to) 24 to 72 hours depending on the time and
conditions between harvest and induction and the stability
properties of the specific heterologous gene product.
Heterologous gene products whose production or high-
level accumulation is deleterious to the growth or vigor of
the plant can similarly be produced by utilizing the post-
harvest induction characteristics of the HMG2 promoter. For
highly toxic products it is advantageous to further dissect
15 the promoter to minimize developmental expression but retain
the wound and elicitor inducible responses. Promoter deletion
analyses (Table 4) indicate that multiple cis-regulatory
elements are present in the promoter and that the
developmental functions (e.g., pollen) are separable from the
20 defense-specific functions (e.g., wound, pathogen responses).
These analyses indicate that the wound and pathogen response
elements are distal to those elements mediating pollen
expression.
7.2. HARVESTED PLANT TISSUE MAINTAIN
INDUCIBLE GENE EXPRESSION
The usefulness of post-harvest production of desired
gene products depends in part on the ability of the harvested
plant tissue or culture to maintain inducible gene expression
30 for some time after harvest. Such an ability means that the
method of invention would have enhanced industrial utility in
as much as it may not always be practical to induce and
process the plant tissue or culture immediately after
harvesting the material. For example, the harvested material
35 may require transportation to a distant site for induction or
processing.
21 68430
W095/03690 PCT~S94/08722
- 71 -
Leaves from grown tobacco plants engineered with the
NMG2:GUS construct were harvested and either induced
immediately or after 2 weeks of storage at 4OC in sealed
containers. The results shown in Figure 14 indicate that the
- s stored leaves not only remained competant for the induced
expression of H~G2:GUS construct but actual expressed nearly
40-50% more activity than freshly harvested leaves. Figure 14
also shows that the induced material requires some incubation
time, up to 48 hr., at room temperature to allow for
10 expression of the induced gene product.
Figure 15 shows that the harvested leaves can be
stored either at 4C in sealed containers or at room
temperature in air permeable containers for up to 6 weeks and
still have ability equal to that of the freshly harvested
15 material for the expression of inducible genes.
Although the invention is described in detail with
reference to specific embodiments thereof, it will be
understood that variations which are functionally equivalent
30 are within the scope of this invention. Indeed, various
modifications of the invention in addition to those shown and
described herein will become apparent to those skilled in the
art from the foregoing description and accompanying drawings.
Such modifications are intended to fall within the scope of
35 the appended claims.
WO95/03690 2 1 6 8 4 3 0 PCT~S94,08722
Various publications are cited herein, the
disclosure of which are incorporated by reference in their
entireties.
