Note: Descriptions are shown in the official language in which they were submitted.
~ WO 96/28561 2 1 8 9 3 6 ~ F~_ll-.l ,', .
Ch1meric genes comprislng a fungus-responsive element
This invention relates to the use, in a transgenic plant, of newly-idéntified
fungus-responsive elements of the Drc1-1 promoter (fungus-responsive Ep~-1
elements) to induce, in response to a fungus infection of the plant, the expression
of a DNA fragment substantially selectively in cells oF the plant around the site of
the fungal infection. The use of the fungus-responsive oro1-1 elements of this
invention is especially valuable in transgenic plants for controlling a foreign DNA
fragment that is to be e%pressed selectively in the cells of the plant which
i" " "edid~ly surround the fungal infection site.
This invention further relates to a hrst or fungus-responsive chimeric gene
that can be used to transform a plant and that comprises a first foreign DNA that:
a) encodes a product which, when expressed in cells of the plant il l l~ didl~lysurrounding a fungal infection site, can either i) kill or at least disable the
plant cells illllll~didltlly surrounding the fungal infection site or ii) kill, disable
or repel one or mcre fungi in the fungal infection site; and
b) is under the control of a promoter comprising at least one fungus-
responsive r~ro1-1 element.
This invention further relates to a cell of a plant, the genome of which is
1, c,, ,~r~,", l~d to contain the first chimeric gene and optionally a second or restorer
rhimerjc cene; the second chimeric gene contains a second promoter that
controls a second foreign DNA encoding a product which allows the inhibition or
inactivation of the first foreign DNA or its encoded produd at least in cells of the
plant other than those immediately surrounding a fungal infection site, particulariy
when the first foreign DNA encodes a product that can kill or adversely disturb
such other plant cells.
This invention yet further relates to: a) the fungus-resistant transgenic plant,such as a Solanaceae (e g., tomato or potato) or B~ (e.g., oilseed
SU~STITUTE SHEET ~ULE 2~)
wos6/2s~6l 218q 3~1 p ll~r~ ~
rape) plant, which is ~ ~y~ , dll~d from the plant cell of this invention 11 dl la~ul 1 l ,ed
with the first and optionally the second chimeric gene of this invention, b) fungus-
resistant transgenic plants derived from the r~:y~ dL~:d transgenic plant and
seeds of such plants, and c) plant cell cultures comprising the ~, dl la~ul Ill~d plant
cells of this invention.
The plants of this invention are cl Idl dCt~l i~d by the fungus-responsive
e~ siu" of the hrst chimeric gene of this invention in plant cells surrounding,
preferably immediately surrounding, the fungal infectiûn site and either:
a) the substantial, preferably cûmplete, absence of expression of the first
chimeric gene in all other plant cells; or
b) the substantial absence and preferably the complete absence, e.g., by
expression of the second chimeric gene of this invention, of the effects of any
expression of the hrst chimeric gene in all other plant cells --thereby
rendering the plants resistant to fungal infections.
Backqround of the Invention
The fungi are a very old group of ll,icluulydlliallls. Harmful fungi cause
diseases of man, other animals, and especially plants. About 8ûOO species of
fungi can cause plant diseasesr and all plants are attacked by some kind of fungi.
Some plant-pdll ,uyl~ fungi can attack many plant species, others attack only
one.
In general, fungal plant diseases can be classified into two types: those
caused by soilbom fungi and those caused by airbom fungi. Soilbom fungi cause
some of the most ~kl~aul~ad and serious plant diseasesl such as root and stem
rot caused by Fusarium spp. and root rot caused by Phvtophthora spp.
Since airbom fungi can be spread long distances by wind, they can cause
devastating losses, particularly in crops which are grown over large regions. A
number of these pathogens have caused v.~iJeaul~dd epidemics in a variety of
SII~STITUTE SHEET (RUI E 26)
Wos6/2856I 2=1 8 9 3 6 1 1~11~ 5
crops. Important diseases caused by airborn fungi are stem rust (Puccinia
~) on wheat, corn smut (~L~Q~ maydis) on oorn, and late blight disease
(Phvtophthora infestans) on potato and tomato.
Most of these fungal diseases are difficult to combat, and farmers and
growers must use- a culllL,illd~iull of practices, such as sanitary measures,
resistant cultivars, and effective fungicides, against such diseases. Hundreds of
million dollars are spent annually for chemical control of plant-pdLl,u~p"i" fungi.
As a result, there is today a real need for new, more effective and safe means to
control plant-ud~l luy~l1iC fungi.
It is known that plants possess defense " ,e~! Idl li~l l IS against fungal diseases.
When a plant It:cu,u,lli~s a fungal attack, it can respond by inducing several
reactions in its cells i"""adidl~ly surrounding the fungal infection site.
Resistance l,,e~,l,d,,i:,,,,~ are activated by the initial infection, so as to limit the
spread of the invading fungal pathogen (Ward et al, 1991). The resistance
~ hdlli~ s include a localized cell death known as a hypersensitiYe response,
the accumulation of phytoalexins, and liul ,iricd~iu" (De Wit, 1987). The specihcity
of these responses, which c-dn be very effective in limiting the spread of a fungal
infection, depends on the genetic make-up of the host and the pathogen.
C~ Idl dl,l~ dliUIl of the genetic cul, ~uu~ which control cultivar/race
specihc I~U:~UUdll lO~,t~l I il l~dldUIiUl IS is a goal of current molecular plant pathology
research. Tl dl 1~ iUl~dl activation of defense-related genes is part of the
complex defense system which enables plants to deal with contads with potential
pathogens (Collinge and Slusarenko, 1987; Hahlbrock and Scheel, 1989;
Bowles, 1 99û). The idt:"~ir~d~iul ~ of cis-acting elements regulating the expression
of defense-related genes has been sought in order to elucidate the process by
which signal transduction chains connect the initial recognition of a pathogen by
a plant host with its induction of defense reactions (Lamb et al, 1989). As found
for several other l~U~>UUd~l IOU~l I systems (van Loon, 1985; Hahlbrock and Scheel,
1989), infection of potato with the fungus Phvtophthora infestans, which is the
SU~STlTiJTE SHEET (RULE 26)
Wo 96/28561 2 ~ 8 9 3 6 I F~
causal agent of late blight disease, leads to ~IdllscliuLiulldl activation of genes
encoding enzymes of the phenylpropanoid Illdldbu'i~lll and PR-proteins
(Fritzemeier et al, 1987; Kombrink et al, 1988; Taylor et al, 1990). Tldll~ iul~of these genes is induced with similar kinetics in compatible and i~uu~udLible
lduLiulls of different potato cultivars with different PhytoDhthora infestans
races. The nucleotide and deduced amino acid sequences of one of the
",~Jd~ y~l ~e~is related" (or "PR")-protein genes in potato, i.e., prD1-1, which is a
member of the large DrD1 gene family (with 10-1~ very similar copies per haploidgenome), shows striking similarity to the ~ull~uolldilly sequences of a gene
encoding the HSP26 heat-shock protein in soybean (Taylor et al, 1990). In situ
hybridization e~e,i",~"l~ showed that the PRP1-1 transcript accumulates
around the site of fungal U~ ld~iUII but the function of this protein in the
defense strategy of potato is not yet clear The homologous soybean HSP26
protein represents a unique member within a group of low molecular weight heat-
shock proteins of plants, appearing in an unusually high relative CUI ~ dLlul l
under a broad variety of stress oonditions (Czarnecka et al, 1984; Vierling, 1991 )
but also having no known role in cell Ill~ldL~ul~ No sequence similarity has
been found between the protein encoded by the orr~1-1 gene and several known
PR-proteins from other Solanaceous species (Taylor et al, 1990). In PCT patent
publication WO 93/19188 and Martini et al. (1993), which are both ill~uluuldled
herein by reference, a 273 basepairs (bp) fragment of the DrD1-1 promoter was
found to still induce local expression of a DNA sequence upon fungal infection,
but, in contrast to the native Prp1-1 promoter, this promoter element was found
not to be induced by heavy metal salts.
Most plant genes enooding proteins related to pathogen defense, analyzed to
date on the level of cis-acting elements, are also activated by several other stress
stimuli like I~ ,hdlli~,dl wounding, light and/or elevated WllC~ ldliull~ of heavy
metals (Oshima et al, 1990; Schmid et al, 1990; Stemler et al, 1990; Douglas et
al, 1991; Joos and Hahlbrock, ~992). In a plant-nematode interaction, a part of
the tobacco RB7 promoter was found to confer selective and local expression in
SU~STITUTE SHEET (RU~E 26)
~ Wo 96128561 2 1 8 9 3 6 1 ~ ~ s3c r
the nematode feeding stnuctures induced in the roots upon infection by certain
l l~l l Id~Od~s (Oppemman et al., 1994).
Recent reports show that certain levels of resistance towards fungal
pathogens can be obtained by expressing antifungal proteins in transgenic
plants. Examples include the expression of a chitinase either alone (Benhamou etal., 1993) or in r u,, l~i, IdliUIl with a glucanase (Zhu et al., 1994), the expression of
osmotin (Liu et al., 1994), or the expression of certain PR proteins (Alexander et
al., 1993).
Summarv of the Invention:
In d~,~.UI ddl IC~ with this invention are provided portions of the e~ -1
promoter region, which can still induce expression of a chimeric gene upon
fungal ir~..Section but have a substantial lower expression in roots and/or are
:~iy~ ly less induced by ph~lull~ lul~ application when compared to the
DNA sequence of SEQ ID No. 1, preferably portions of the r~rr~1-1 promoter
region, which can still induce expression of a chimeric gene upon fungal infection
but which have substantially lost their expression in roots and have a siy,-ir,~d, ILly
~ower responsiveness to phyLul ,o" "une application.
Further in dCL~ul~dllut~ with this invention are provided fungus-responsive
EE 1-1 elements, derived directly or indirectly from the DNA sequence of SEQ ID
No. 1, but lacking the nucleotides from positions 1 to 1ûû in SEQ ID No. 1, or
lacking the nucleotides from positions 239 to 273 in SEQ ID No. 1., or fungus-
responsive EE 1-1 elements, derived directly or indirectly from the DNA sequenceof SEQ ID No. 1, and:
a) ~",~ i"g a nucleotide sequence from a position between nucleotide
position 1û0 and 176 to nucleotide position 273 of SEQ ID No. 1, provided that
said fungus-responsive element is not the DNA sequence of SEQ ID No. 1, or
b) colll~ g the DNA sequence of nucleotide position 1 to a position
between nucleotide positions 1~3 and 239 in SEQ ID No. 1;
Sll~STITUTE SHEET (RU~E 26)
WO 96/28561 2 1 8 9 3 6 1 p~l/r l
provided that said fungus-responsive element is not the DNA sequence of SEQ
ID No. 1.
Most preferred fungus-responsive r~rp1-1 elements are those seler~ted from
the group of the DNA sequence of SEQ ID No. 1 from nucleotide position 1 to
239, the DNA sequence of SEQ ID No.1 from nucleotide position 1 to 153, the
DNA sequence of SEQ ID No. 1 from nucleotide position 100 to 273, the DNA
sequence of SEQ ID No. 1 from nucleotide position 140 to 273 and the DNA
sequence of SEQ ID No. 1 from nucleotide position 176 to 273, as well as any
fungus-responsive element with substantially the same nucleotide sequence, so
that the DNA sequence has essentially the same promoter activity.
Also provided herein i5 a fungus-responslve promoter, comprising any of the
newly identifled fungus-responsive e~_1-1 elements, provided this fungus-
responsive promoter does not comprise the DNA sequence of Fig. 1.
Further ~ assed in this invention are fungus-responsive chimeric
genes, which comprise, besides the fungus-responsive promoter of this invention,a first foreign DNA that encodes a hrst RNA and/or protein or polypeptide which,when produced or overproduced in the cells of the plant which sumound,
preferably illlllle:~idl~ly surround, said fungus-infedion site, a) kills, disables or
repels said fungus, or b) kills, or at least disturbs signihcantly the Ill/~l~dbU.i.~
functioning and/or development of the plant oells surrounding, preferably
immediately surrounding, said fungus-infection site, so as to limit further spread
of said fungus; and suitable 3' Ll dl lS~,l iJLiUI ~ l l l lil IdLi~JI I signals for expressing
said first foreign DNA in the cells of the plant which surround, preferably
illlllledid~t:ly surround, said fungus infection site.
Also in d~w,~d"~e with this invention is provided a cell of a plant, in which
the nuclear genome has been tldll:,rul~ d to contain the first chimeric gene of
this invention and optionally - especially when the hrst foreign DNA is of type b)
above - to contain also the second or restorer chimeric gene, preferably in the
SU~STITUTE SHEET (RULE 26)
WO 96/28561 2 18 9 3 6 1 r~l,~ ,51~ iif
same genetic locus; the second chimeric gene comprises the following, operably
linked, DNA sequences:
1) a second promoter, such as a fungus-repressed promoter, which can
direct lldllsuli~uliull of a foreign DNA in at least celis of the plant other than
those surrounding, preferably other than those i~ lddidl~ly surrounding, the
fungus infection site;
2) a second foreign DNA that encodes a second RNA and/or protein or
polypeptide which, when produced or overproduced in at least such other
cells of the planl, inhibits or inactivates the first foreign DNA or the first RNA
or protein or polypeptide in at least said other cells of the plant; and
3) suitable 3' ~ldll::~UliU~iOl~ llllilld~iUII signals for expressing the secondforeign DNA in at least such other celis of the plant.
Further in a~u~dd~ with this invention are provided: the fungus-resistant
plant l~yd~ ldl~d from the ~Idll~rUlllled plant cell of this invention, fungus-
resistant plants and seeds derived therefrom, and plant cell cultures, each of
which comprises the ~, dl l~rUI 1~ ,ed plant cells of this invention.
Still further in auuuludll~d with this invention is provided a process for
rendering a plant resistant to one or more fungi, particularly plant-pathogenic
fungi such as Phvtophthora (e.g., P infestans) and CladosPorium (e.g.,
Cladosoorium fulvum), Pvthium spp, Fusarium spp, Sclerotinia spp, Puccinia spp,
Ustilaqo spp, Alternaria spp, I l~ii"i, 1~l ,u~u~, ium spp., SePtOria spp, PvrenoDhora
spp, Botrvtis spp, ErvsiPhe spp., as well as P~" tl ~uue~ i~d brassicae,
CV'il Idl u~uul iùm concentricum. Phoma linqam, Leu~u:,.vl ~ae~ id maculans,
Sclerotinia sclerotoirum, Botrvtis cinerea, Ervsiphe cruciferorum. r~.u~ uuld
Parasitica, Pld~l I lodiuul lul d brassicae, and Pse~uce, ~u~uul ~lld caPsella,
c~mprising the step of lldll~rullllill~ the plant's nuclear genome with the first or
fungus-responsive chimeric genes of the invention and optionally with the secondchimeric gene of this invention.
Detailed D~ , iulioll of the Invention
SUBSTITUTE SHEET (RULE 26
w0 96/28561 2 1 8 9 3 6 ~
Throughout this Description and the Claims, the following definitions apply:
"Fungus-infected plant", as used herein, is a plant v~hich is infeded by at
least one fungus species, particularly a plant-pdLI lO~dl li~, fungus species, such as
Phvtophthora spp, Cladosporium spp. Pvthium spp, Fusarium spp, Sclerotinia
spp, Puccinia spp, Ustilaao spp, Alternaria spp, I I~I",i, lll l- avul ium spp., sePtoria
spp, PvrenoPhora spp, Ustilaao spp, Botrvtis spp, Ervsiphe spp., as well as
P~l dll~ d brassicae. C~ dl U:~,UJI i~m concentricum, Phoma linaam,
Leptosphaeria m3culans. Sclerotinia sclerotiorum, Botrvtis cinerea. ErvsiPhe
cruciferorum, Pel~ nd parasitica, Pld~llludi~ lluld brassicae, and
Pse~u~,d,~ ltllld caPsella and the like.
IJ - - - -
"Promoter", as used herein, refers to a DNA sequence which is recagnized
and bound (directly or indirectly) by a DNA-dependent RNA polymerase during
initiation of lld~ IS~ lliUI 1. A promoter includes the ~Idl 1~,1 iJLiUI I initiation site, and
binding sites for lldll~ ll initiation factors and RNA polymerase, and c3n
comprise various other sites at v~hich gene regulatory proteins may bind. The
"prp1-1 promoter", as used herein, refers to the native promoter sequence of thePrP1-1 gene, also suggested to be named qs~1 (Hahn & Strittmatter, 1994),
~_hdld~dli~t:d by the partial sequence described in Fig.3 of Martini et al. (1993).
The "273 bp pro1-1 promoter", as used herein, refers to the 273 bp fragment of
the Ep1-1 promoter, described by Martini et al. (1993) as being suf~cient for
r3pid and strictly loc31ized Lldlls~ lldl activation at fungal infection sites. This
273 bp ~1-1 promoter is ~ 5e:l ILdd in SEQ ID No. 1.
"Fungus-responsive promoter", as used herein, refers to a promoter, v~hose
adion in controlling Ll dl 1~ kJl~ of a DNA sequence in a plant: 1 ) is induced (i.e.,
stimulated) by infection of the plant by a fungus, particular~y a plant-pathogenic
fungus; and 2) occurs substantially selectively, preferably exclusively, in plant
cells around the fungal infection site, preferably in plant cells i"""edidl~ly
surrounding the fungal infection site.
SLLaSTlTUTE S~EET (RLILE 26)
~ W096/28561 2~89361 r~"~
"Fungus-repressed promoter"~ as used herein~ refers to a promoter, whose
action in controlling lldl 151,1 i,UIiUl I of a DNA sequence (e.g., a gene) in a plant is
locally repressed (i.e. partially or fully inhibited) upon infection of the plant by a
plant-udll,uu~"ic fungus; and this repression occurs substantially selectively,
preferably exclusively, in those plant cells around the fungal infection site,
particularly in those plant cells i"l",eiidI~ly surrounding the fungal infection site.
Preferably, this promoter is otherwise constitutively expressed throughout the
plant but locally repressed at the site of fungal infection.
"Plant promoter'` or "plant~,~,,d~ promoter", as used herein, refers to a
promoter sequence capable of driving Ildl~s~iu~iu,l in a plant cell. This includes
any promoter of plant origin, as well as any promoters foreign to plants but also
allowing tldlls~i,uliu~l in plant cells, i.e., certain promoters from viral or bacterial
origin such as the T-DNA and 35S or 19S promoters.
"Fungus-responsive eiement", as used herein in relation to a fungus-
responsive promoter, is that element or part of a promoter that is l~auul~Sii~i~ for
the fungus-responsiveness of the promoter. A promoter can have several
fungus-responsive elements, besides a minimal promoter element and enhancer
regions. Preferred fungus-responsive eiements, in a~cu, ~dll~ with this
invention, are the fungus-responsive ~L1-1 elements of Example 1, as
ne~StlILed in Figure 1.
"Artificial hypersensitive cell death" refers to a plant defense ",e~l,d"i:,",
v,/hich is conferred by a first chimeric gene of this invention on a plant
lldllarulilltld therewith and which involves necrosis of plant cells at a pathogen
infection site, thereby limiting further spread of the pathogen. This Ill~lldlli~lll is
analogous to a natural hypersensitive cell death occurring in i"w~ alii~JI~
~,la, li/i a~l IO,g~ l dUIiUl 1~.
SLI~STITUTE SHEET /RULE 26)
WO 96/28561 2 1 8 9 3 6 1
"Foreign" with regard to a DNA sequence, such as a first or second foreign
DNA of this invention, means that such a DNA is not in the same genomic
environment (e.g., not operably linked to the same promoter and/or 3' end) in a
plant cell, tld~rulllled with such a DNA in d~UlddllUtl with this invention, as is
such a DNA when it is naturally found in a cell of the plant, bacterium, animal,fungus, or in a virus or the like, from which such a DNA originates. Foreign DNA,
in au~,dd"~ with this invention, thus includes DNA that is onginally found in a
plant genome, but which has been inserted in a different r~enomic locus or site
compared to the endogenous DNA (see also the definition on page 5 of
European patent publication (EP) 0 344 029 in this respect).
"Fungus-resistant plant", as used herein, refers to a plant displaying
increased tolerance to infection with a fungal pathogen, as can be d~ " ,i, l~d by
routine fungal infection analysis, e g., the method described in WO 93119188. A
fungus-resistant plant typically has better dy,u"u",i~dl p~lfulllldllw~ e.g., yield,
under conditions of fungal attack when compared to the wild-type plants
"Selective e~l~siu, I'', as used herein in relation to the fungus-responsive
Prp1-1 elements of this invention, means expression with high specificity in t~epiant cells surrounding, preferably i,,,,lledid(~ly surrounding, the fungal infection
site. The term selective expression, as used herein, does not exclude that some
expression can oocur in other cells of a plant during a certain dev~lu~,,,,t,,l(dl
sta~e (e.g., in non-essential plant cells), nor does this definition require that the
promoter portions have to be exclusively induced by a fungal pathogen.
"Cells illllll~did~dly surrounding the fungal infection site", as used herein,
refers to those oe(ls that are located in the close vicinity of the fungus.
Preferably, the cells illllll~;lid(dly surrounding the fungal infection site are those
cells that, when killed or negatively affected by the local expression of a first
chimeric gene of this invention, (directly or indirectly) prevent the further growth
and spread of the fungus.
SiJ~STlTUTE SHEET (ftULE 2
W0 96/28561 2 1 8 9 3 6 ~ 5. 3~
11
"Essential plant cells'`, as used herein, refers to those cel~s of a plant that
negatively affect the yield or value of a plant as an agricultural crop when they
wouid be destroyed or when their function would be inhibited. ~ndeed, some cellsin a plant are not essential to the economic value of the plant, such as the oells in
a potato flower in a European potato tuber production held, or the pollen cells in
the anthers of plants sold as cut flowers, or some cells in a tissue whose death or
disfunctioning does not aflect the functioning of this tissue.
In a~culddll~ with this invention, portions of the 273 bp e~1-1 promoter
have been identified that are more selectively induced by fungal pathogens, i.e.,
that have substantially lost the root expression andlor are significantly less
induced by phylul~ ulle application, while still oonferring local induction of
expression upon fungal infection The term "fungus-responsive Ep1-1
elements", as used herein, refers to the newly identifled fungus-responsive
promoter elements, being portions of the 273 bp E!51-1 promoter of WO
93/18199 (l~ulusr~ d in SEQ ID No. 1) that retain significant fungus-
responsiveness but have improved ~_;hdld~ ~ such as a loss of non-target
e~p~ S:~;Ull andlor a lower responsiveness to phytohomlone application.
Included in this definition of fungus-responsive E~1-1 elements is any portion(s)
of the 273 bp EE~1-1 DNA of SEQ ID No. 1, provided that the 1, dl la~ i,u~io" from a
promoter, comprising this portion, is :~iul ,;r,cd, lily less induced upon
ph~/~ullul~ul~c application andlor is substantially lower in roots~ when compared
to the 273 bp E~e1-1 promoter. Preferably these fungus-responsive ~1-1
elements retain less than about 5 %, more preferably ~ess than about 2 to 4 /0 of
the phytohormone responsiveness of the 273 bp Pro1-1 DNA of SEQ ID No.1,
and preferably have a lli~u~,l1ul,,i~dlly non-detectable expression in roots, asidentified by GUS-linked marker gene analyses as shown in Example 2.
Deletion analyses of the 273 bp i ro1-1 promoter showed that the root
e~p,~s:,iu" of the 273 bp ~1-1 promoter resides in the portion of nucleotide 239to 273 of the partial ro1-1 DNA sequence of SEQ ID No. 1. Cullcullli
fungus-responsive promoters can be devised that are obtained from the E_1-1
SUnSTlTUTE SHEET (RULE 2~i)
WO g6,28561 2 1 8 9 3 6 ~ E~~
promoter but that lack significant expression in roots by deletion, substitution or
alteration of this part. This deletion analyses has also shown that a fungus-
responsive E~1-1 element still induced by fungal infec~ion but signihcantly lessinduced by phy~ollùr~u~s such as salicylate and indole acetate is situated
between nucleotide positions 176 and 273 in SEQ ID No. 1. Thus, preferred
fungus-responsive or,o1-1 elements of fhis invention are promoter elements
derived directly (i.e., by applying routine molecular biological techniques to the
DNA sequence It:ul~:a~ d in SEQ ID No. 1) or indirectly (i.e., h~u~ li~'ly
obtainable from the DNA sequence of SEQ ID No. 1; for example, in vitro DNA
synthesis) from the DNA sequence of SEQ ID No. 1, but lacking the nucleotides
from positions 1 to 1ûO in SEQ ID No. 1, preferably promoter elements obtained
or derived directly or indirectly from the DNA sequence of SEQ ID No. 1 but
lacking the nucleotides from positions 239 to 273 in SEQ ID No. 1. Altematively,the DNA sequence between nucleotide position 1 to 100 or between nucleotides
239 to 273 in SEQ ID No.1 can be rendered non-functional by deletion, addition
or , t:plact " ,~"l of nucleotides (e.g., site-directed mutagenesis) to obtain apreferred fungus-responsive Pro1-1 element derived from the DNA sequence of
SEQ ID No. 1 in dCCUl lidl ll,t7 with this invention.
Preferred fungus-responsive ~1-1 elements, in dl,WldCI l~ with this
invention, are those which comprise the DNA sequence of SEQ ID No. 1 from a
nucleotide position between nucleotide positions 100 and 176 to nucleotide
position 273, or which comprise the DNA sequence of SEQ ID No. 1 from
nucleotide position 1 to a position between nucleotide positions 153 and 239 in
SEQ ID No. 1 as their fungus-responsive element, provided that these fungus-
responsive promoters do not comprise the DNA sequence of SEQ ID No. 1 as its
fungus-responsive element or have a significantly lower phytohormone-
responsiveness and/or a substantially lower expression in roots when oompared
to the 273 bp Prp1-1 promoter.
Particular~y preferred fungus-responsive prp1-1 elements of this invention are
those temmed delB34 (nucleotide positions 140 to 273 in SEQ ID No. 1), delB35
SLI~STITUTE SHEET (RULE 2~
~ Wo96/28561 1 89361 P ./~l sol
(nucleotide positions 100 to 273 in SEQ ID No. 1), and delB51 (nucleotide
positions 176 to 273 in SEQ ID No. 1), that are ~iy~ir~,dl~ly less responsive tosalicylic acid and indolyl acetate application (2- to 3-fold lower induction) when
compared to the 273 bp prp1-1 promoter, even more preferred fungus-responsive
~1-1 elements of this invention are those temmed delX4 (nucleotide positions 1
to 239 in SEQ ID ~o. 1 ) and delX5 (nucleotide positions 1 to 153 in SEQ ID No.
1), since these portions have substantially (i.e., drastically) lost the expression
observed in root tips with the 273 bp ~e1-1 promoter (W0 93/19188, e.g., no
u~l ,e" ,i~dlly detectable GUS protein by a delX4- or delX5-GUS chimeric gene
in roots), as well as being signiflcantly less responsive to salicylic acid and indole
acetate application (a 30- to 40-fold lower induction) when compared to the 273
bp Dro1-1 promoter, while still retaining a significant fungal responsiveness. Also
included in this definition are natural or artificial promoter elements with a DNA
sequence that is substantially similar to any of the delX4, delX5, delB34, delB35,
and delB51 DNA sequenoes defined above, i.e., having some nucleotides
deleted, replaced or added provided substantially the same promoter
i~ are retained.
In dac~ulddl ,~ with this invention, a fungus-resistant plant can be produced
from a single cell of a plant by lld~ u~ g the plant cell in a known manner to
stably insert, into its nuclear genome, the first chimeric gene of this invention
which comprises at least one first foreign DNA that is: under the control of a
fungus-responsive promoter comprising at least one fungus-responsive e~1-1
element of this invention, wherein said fungus-responsive promoter does not
contain the 273 bp ~1-1 promoter element descnbed in W0 93/19188 or
~,vherein this fungus-responsive promoter has a signihcantly lower lldll~
activation in roots and/or upon phytohommone application than the 273 bp pr~1-1
promoter of WO 93/19188; and fused at its ,i~ a", (i.e., 3') end (or fused
dc~ allt:dlll of a 3' non-translated trailer sequence) to suitable lldll::.UliU~iUII
d~iUI I (or regulation) signals, including a polyadenylation signal. Preferably, in the first chimeric gene, a fungus-responsive e~-~ element is used, in
r~",ujr,c,;;~ with a minimal promoter element such as the 35S minimal promoter.
SU~STITUTE SHEET (r~ULE 26)
wo g6/2ss61 2 1 8 9 3 6 ~ . 5 ~ ~
Thereby, the hrst RNA andlor protein or polypeptide is produced or
overproduced ,uladul~lilldlllly in those plant cells around, preferably i,~ làdidl~ly
surrounding, a fungal infection site. One or more of these fungus-responsive
prp1-1 elements can be present in a promoter, with an endogenous or foreign
TATA box, as well as enhancer regions or other pathogen-responsive promoter
elements, e.g., the nematode-responsive element described by Opperman et al.
(1994). Also, a fungus-responsive E~_1-1 element of this inventlon can also be
i,,~u,uu,dled in any promoter sequence so as to oonfer to this promoter the
capacity to respond to pathogen, e.g., fungal, infection: For instance, the sameor different fungus-responsive E~1-1 elements of this~invention can also be
linked in multiple consecutive copies to constitute a promoter comprising several
fungus-responsive pro1-1 elements of this invention (Benfey et al., 1990).
Optionally, the plant cell genome can also be stably ~d~rullllt:d with a
second chimeric gene comprising at least one second foreign DNA that is: under
the control of the second promoter which is capable of directing expression of the
second foreign DNA at least in cells of the piant where the first foreign DNA isexpressed, but preferably is repressed in the cells surrounding, more preferablyilll,ll~didl~ly surrounding, the fungal infection site. Alternatively, the seoond
promoter is capable of directing expression of the second foreign DNA
substantially selectively in plant cells where expression of the first foreign DNA
would result in damage to oells not around, preferably not illllll~:~idi~ly sur-rounding, the fungal infection site. The second chimeric gene further comprises
suitable 1, dl 1~ ,1 iUIiUl I ~ dl;UI I signals, including a polyadenylation signal. The
second chimeric gene is preferably in the same genetic locus as the first chimeric
gene, so as to guarantee with a high degree of certainty the joint seylaydliu,, of
both the first and second chimeric genes into offspring of the plant layt~lleldl~d
from the ildl,:,ru""ed plant cell. However in some cases, such joint Saulayaliull
is not always desirable, and the second chimeric gene could be in a different
genetic locus from the first chimeric gene. When the first foreign DNA, RNA or
protein affects plant cell viability (e.g., when the first foreign DNA encodes abamase protein), even when the fungusfesponsive promoter is shown to be
SUDSTITUTE SHEi-T (RULE 26~
Wo 96/2856~ 2 1 ~ 9 3 6 1 P~l/~ ' 1
selectively or exclusively induced in plant cells i~ did~ly surrounding the
fungal infection site, it is still preferred that a second DNA, RNA or protein is also
expressed in all essential piant cells (e.g., the second DNA will encode the
barstar protein when the first foreign DNA encodes the bannase protein),
preferabiy in all plant cells other than those cells surrounding the fungal infection
site, so as to prevent any expression of the promoter to negatively affect cells not
surrounding the fungal infection site at any moment during plant development.
In the second chimeric gene, the second foreign DNA can also be under the
control of a minimal promoter, such as the 35S minimal promoter. "Minimal
promoter", as used herein, is a DNA sequence capable of driving a basal level ofexpression of a coding region, so that some RNA, protein or polypeptide is
produced, and comprising at least a TATA-box region, preferably the 35S
minimal promoter from nucleotide position 48 to +8 of the 35S gene (Benfey et
al., 1990). Typically, a minimal promoter is a DNA sequence, ~ d by RNA
polymerase ll, e.g., a TATA-box, but can be any cis-acting DNA sequence
allowing minor expression of an RNA in the plant genome. Such a minimal
promoter can even be located in plant genomic DNA surrounding the inserted
gene, in this case the second chimeric gene even need not con~ain a plant-
s~ le promoter.
In a~,u~-ld~ce with this invention, the first foreign DNA in the first chimeric
gene is a DNA fragment that encodes a first RNA andlor protein or poiypeptide
which, when produced or overproduced in the plant cells surrounding, preferably
i"""edidlely surrounding, a site of a fungus infection, either: a) kills such
surrounding plant cells or at least disturbs significantly their Illt:Ld~ol;~,...
functioning andlor development so as to induce an artihcial hypersensitive cell
death in order to limit the further spread of the invading f~ngus; and/or b) kills,
disables or repels the fungus when it further infects such surrounding plant oells.
First foreign DNAs preferably encode, for example, the following which can kill
- the surrounding plant cells or at least disturb significantly their ,~ di rli~",,
functioning andlor development: RNases such as barnase, RNase T1, RNase SA
SUr'ST!TUT-. ~Hi-ET (RULE 26)
wo 96128561 ~ 1 8 9 3 6 ~
16
(SARNase), or binase; toxic proteins such as the Diphteria A toxin (e.g., Palmiter
et al., 1987), ricin, or botulin. Potential first foreign DNAs further include DNases
such as endonucleases (e.g, EcoRI); proteases such as a papain; enzymes
which catalyze the synthesis of ph~/~ul~ o~ , such as isopentenyl Lldll~ ld~
or the gene products of gene 1 and gene 2 of the T-DNA of Aqrobacterium:
glucanases; lipases, lipid ,.~ ds~, plant cell wall inhibitors, ribosome-
inactivating proteins (e.g., Stirpe et a~., 1992) and ribozymes . Other prefenred
examples of such first foreign DNAs are antisense DNAs encoding RNAs
~)I I I~JIt~l l lC:l l~dl y to genes encodi ng products essenti a l for the l l It~ldbO; ;~1 I I,
functioning and/or development of the surrounding plant cells Such an
antisense RNA could be ~ dly to the endogenous Drp1-1 RNA and
thus inhibit the actlon of the produced gluthathione-S-~Idlls~t:lds~ (Hahn and
Strittmatter, 1994) at the site of fungal infection.
In a different strategy, wherein the pathogen is more directly killed or
negatively affected, the first foreign DNAs encode, for example, the following first
polypeptides or proteins which can kill, repel or disable fungi: Iytic enzymes, such
as chitinases and 13-1,3 glucanases, that catalyze the hydrolysis of fungal oellwalls; protease inhibitors (Ryan, 1990); fungus-inhibiting ribonucleases (WO
94/18335); and lectins (Broekaert et al, 1989); as well as other plant proteins with
antifungal activity, such as the small basic peptide, CMIII, isolated from corn (EP
465 009) and the osmotin-like proteins (EP 460 753 and WO 94/0810), as well as
the antifungal peptides from Amaranthus caudatus seeds described by Broekaert
et al. (1992), the antifungal peptides from Mirabilis ialaPa seeds described by
Cammue et al. (1992), the antifungal P14 proteins described in PCrpublication
WO 92/20800, the antifungal proteins described in PCT publication WO
94/15961, the antifungal peptides from Asperaillus qiaanteus described in PCT
publication WO 91/19738, the basic peptide CMIII from maize seed desibed in
EP 465 009, the Rs-AFP proteins obtained from radish described in PCT patent
publication WO 93/05153 and by Terras et al (1992), and genes encoding
phytoalexins (Hain et ai., 1993). The first foreign DNA can also be a DNA
sequence encoding an avinulence gene RNA (e.g., the avr9 gene) and/or the
SU~lTUTi ~H,ET (RULE 26)
WO 96/28561 ~ 9 3 6 1 , ~ 9
cull~a~ol~ lg resistance gene RNA (e.g., the Cf9 gene) as described in PCT
patent publication WO 91115585. In this strategy, a DNA sequence encoding the
Cf9 gene product (Jones et al., 1994) can be plaoed under the control of a
fungus-responsive prp1-1 element, such as delX4 of Example 1, and the avr9
coding region (Van Den Ackerveken et al., 1992) can be placed under the controi
of a fungus-responsive pro1-1 element, or under the control of a wound-induoed
or a constitutive promoter. In a Cf9-tomato plant, the avr9 coding region could be
linked to a promoter comprising the delX4 fungus-responsive element, thus
resulting in local necrosis at the fungal infedion site. Promoters comprising the
newly identified fungus-responsive e~1-1 elements can also drive ildll:~i,Uli
of an element of another plant-pathogen virulence/avirulence gene ~r,",L~ dLi~n,or can be applied in any other method for obtaining fungal resistance in plants,e.g., those communicated on the 7th ll ,~,, Id~iUI Idl Symposium on Molecular
Plant-Microbe IIlLtll~lio,~, University of Edinburgh (see e.g., The Plant Cell,
October 1994, pp. 1332-1341). First foreign DNAs can be naturally occurring or
r~an be fully or partially man-made (e.g., synthetic), provided the same protein as
originally encoded by the DNA sequence or a protein with substantially the same
activity, is encoded. Indeed, since it is known that DNA or RNA sequences can
vary siu, liiicdl ILly without altering the sequence of the encoded protein, it logically
follows that DNA or RNA sequences can differ in several r~rl~otid~s, without
changing siy"ir,cd, Illy the activiiy or function of that DNA or RNA beyond their
normally observed spectnum of biological activity. Also, when high expression ofa foreign DNA sequence is desired, certain nucleotides in the DNA may be
altered so as to prevent inefficient tl~l~s~,liu[iu~l or translation of that DNAsequence in the foreign host cell.
For some selected plant-pathogen i"L~, dL,liUI 15, the fungus-responsive pro1-1
elements of this invention can also drive selective L, dl ISI.,I iUliUI I of a DNA
enooding an RNA, protein or polypeptide inhibiting a toxin fommed by a
,udLI IUy~ , fungus, if the fungal pd~l ,o~"iuiL~ is largely depending on such toxin
production. In certain plant-fungus illLcld~Liull~, the production of such fungal
toxins is an essential element in pdllluy~lleai~ (Schafer, 1994). Similarly,
SUrSTiTUlE S~iEET (~ULE 26)
wos6/2ss6l 2 1 8~36 1 r~llr~ ~ ~
18
inhibitors of fungal enzymes, detoxifying natural plant antifungal toxins, can be
selectively expressed at the site of infection in a~u, ddl IC~ with this invention. A
pdlllo~ variety of Gaeumannomvces qraminis, is known to detoxify plant
avenacins by means of one fungal enzyme (Schàfer, 1994). Similarly, inhibitors
of such a fungal enzyme can be produced or secreted selectively by plant cells to
confer resistance to a fungal pathogen. Particularly interesting is the phomalide
toxin produced by LeuLu~l~l Iddl id maculans (the asexual stage of Phoma linqam)on canola plants (Soledade et al., 1993). Strategies could be devised inhibiting
toxic action of this ~ selectively at the site of fungal infection.
Alternatively, anti-fungal antibodies can be selectively expressed at the site
of fungal infection by the fungus-responsive ~1-1 elements of this invention.
Plants ~,d,,arul,lld~i with a first foreign DNA in a firs~ chimeric gene of thisinvention will be resistant to fungal infection either: because of the plants' fungus-
responsive breakdown, in a substantially selective manner, of the plant cells
which surround, preferably immediately surround, the fungal infection site,
thereby providing a hypersensitive response; or because fungi will be killed,
repelled or disabled by, for example, a fungal toxin produced in situ substantially
selectively by the plant cells surrounding, preferably immediately surrounding, the
fungal infection site.
A preferred fungus-responsive promoter of this invention comprises the
delX4 or delX5 fungus-responsive prD1-1 elements of Fig. 1, more preferably the
delX4 fungus-responsive ~1-1 element. It is beiieved that fungus-responsive
elements with substantial sequence homology to the fungus-responsive prp1-1
elements of this invention can be identlfied in the genomic DNA of other plants
(e.g., rapeseed, com, etc.) using the promoter fragments desuibed in Example 1
(and Figure 1 ) as hybridization probes in a conventional manner. The increased
specihcity of the deletion fragments delX4 and delX5 upon fungal infection
appears to make these improved promoter portions, as well as similar portions of other members of the prp1 gene family, suitable candidates for providing
SL"STiTLlTE SHEET (RULE 26~
~ W096/28S61 2 1 89 3 6 1 I~l,~ s~
19
cis-ading elements which can provide improved specificity of local ~, dl 1:~,1 i,UliUI Id
activation in plants upon fungal infection.
-
Examples of suitable plant-e,cp,t~iule second promoters are: the strong
consti~utive 35S promoters of the cauliflower mosaic virus of isolates CM 1841
(Gardner et al, 1981), CabbB-S (Franck et al, 1980) and CabbB-JI (Hull and
Howell, 1987); the relatively weaker constitutive nos promoter (De Picker et al,1982); and wound-inducible promoters, such as the TR1' and TR2' promoters
which drive the expression of the 1' and 2' genes, respectively, of the T-DNA
(Velten et al, 1984). Alternatively, a seoond promoter can be utili~ed which is
specific for one or more plant tissues or organs (such as leaves), particularly
specific tissues or organs (such as roots) not infected by a fungus where the first
foreign DNA is nevertheless expressed, whereby the second chimeric gene is
expressed only in such specific plant tissues or organs. Another alternative is to
use a promoter whose expression is inducible (e.g., by temperature or chemical
fadors). The second promoter, as used herein, is a promoter that is always
directing lldl 1~ iUI I at a level which is lower than the fungus-responsive or first
promoter of this invention in those plant cells around, preferabiy i"""edidlc:lysurrounding, the fungal infection site, upon fungal infection, so that the firstfûreign DNA is not fully inhibited or inactivated at the site of fungal infection.
In another d",uû.li",e,~l of the invention, the second promoter is a fungus-
repressed promoter, e.g., an otherwise oonstitutive plant promoter whose action
in oontrolling ~Idll~uliuliull is dov~n-regulated ~r inhibited upon fungal infection in
the cells surrounding the fungal infedion site, preferably in the cells illllll~didL~:ly
surrounding the fungal infedion site. An example of such a fungus-repressed
promoter is the 35S promoter in plants infeded with BotrYtis cinerea (Oral
s~l lldliull of Dr. R. Hain at the 4th Il ll~ dliul1al Congress of Plant Moiecular
Biology, Amsterdam, June 19-24,1994).
In d~,l,UlddllL~ with this invention, the second foreign DNA in the second
chimeric gene is a DNA fragment that encodes a second RNA and/or protein or
SU~STITUTE SHi-ET (RULE 26)
WO96~28561 2 ~ 8q36 1 ~ ~ sl ~
polypeptide which, when produced or overproduced in cells of a plant, inhibits or
preferably inactivates the first. foreign DNA or any hrst RNA, protein or
polypeptide expressed in such cells, particularly where the first RNA, protein or
polypeptide would kill or adversely disturb significantly the ~ Ld~ol;~
functioning or development of such cells. Second foreign DNAs preferably
encode, for example, the following: barstar which neutralizes the activity of
bamase (which degrades RNA molecules by hydrolyzing the bond after any
guanine residue), binstar which neutralizes the activity o~ binase, sarstar which
neutralizes the activity of samase; EcoRI methylase which prevents the activity of
the endonuclease EcoRI; or a protease inhibitor which neutralizes the activity of a
protease, such as a papain (e.g., papain zymogen and papain active protein), or
even an antibody or antibody fragment specifically inactivating a hrst foreign
protein. Another preferred example of a second foreign DNA encodes a strand of
an antisense second RNA (as described, for example, in EP 223 399) which
would be r,u"l,ule",~, ILdl y to a strand of a sense first RNA, such as an antisense
RNA inhibiting the activity of a Diphteria, rlcin or botulin RNA.
In the first an~ second chimeric 3enes of this invention, the 3~ ~Idll~l~llllilld~iUII signals or 3' ends r~an be selected from amongst those which arecapable of providing correct ~dl la~l iu~iu~ dliUI I and polyadenylation of
mRNA in plant cells The tldll~ iUII termination signals can be the natural
ones of the hrst and second foreign DNAs, to be ~Idl 1~ 1 ibt:'i, or can be foreign.
Examples of foreign 3' ~, dl 1~1_1 iU~iUI I termination signals are those of the octopine
synthase gene (Gielen et al, 1984; Ingelbrecht, 19~9), the 35S gene (Sanfacon etal, 1991 ) and of the T-DNA gene 7 (Velten and Schell, 1985).
Also, the chimeric genes of this invention can comprise a native or a foreign
intron. Plant introns have been described to increase expression of l~d~s~ es,
particularly in monocots (Callis et al, 1987). Introns can also be very useful since
they prevent proper expression of a gene during the bacterial cloning steps.
SU~STIT~TE SHEET (FWLE 2~)
~ WO9C/28561 2 i 8936 i r~l,~ r
The genome of a cell of a plant, particularly a plant capable of being infected
with Aarobacterium, can be l,d,,~u,ll,ed using a vector that is a disammed Ti-
plasmid containing the first chimeric gene and optionally the second chimeric
gene of this invention and carried by Aqrobacterium. This ~, dl l~rul IlldliUI l can be
carried out using the procedures described, for example, in EP 116718, EP
27û822, EP 604662 and Gould et al (1991). Preferred Ti-plasmid vectors contain
the first and second chimeric genes between the border sequences, or at least
located tû the left of the right border sequence, of the T-DNA of the Ti-plasmid.
Of course, other types of vectors can be used tû transfonm the plant cell, usingprocedures such as direct gene transfer (as described, for example, in EP
233,247), pollen mediated ~Idl)~rul,,ldIiu,l (as described, for example, in EP
27û,356, PCT publication W0 85/01856, and US patent 4,684,611), plant RNA
virus-mediated Ildll~rulllld~iull (as described, for example, in EP 67,553 and US
patent 4,4û7,956) and liposome-mediated tldllsrul,lldLiull (as desuibed, for
example, in US patent 4,536,475). in case the plant to be ~Idll~rullll~d is corn, it
is prefenred that more recently developed methods be used such as, for example,
the reoent method described in EP 6û4662 and the method described for certain
lines of corn by Fromm et al (199û) and Gordon-Kamm et al (1990) and the
method for cereals described in PCT patent publication W0 92/09696. The
resulting ~Idll~rul~ i plant cell can then be used to re~enerate a ~d"~ru""e i
plant in a conventional manner.
It is preferred that the first and second chimeric genes of this invention be
inserted in the same genetic locus in a plant genome, preferably in a
configuration where ill~lr~ c~ is minimized between cis-acting elements of the
fungus-responsive and second prûmoters~ Preferably, the first and second
chimeric genes are each inserted into a plant cell genome in the same genetic
locus as a conventional chimeric marker gene. The choioe of the marker DNA is
nût critical (see e.g., Plant Molecular r~iology Labfax (1993) for a list of knov~
marker genes). A marker DNA can also encode a protein that provides a
distinguishable color to ~Idll~ru""~ i plant cells, such as the A1 gene encodingdihydrûquercetin~reductase (Meyer et al, 1987).
SU~STITUTE S~iEET (~ULE 2
WO96/28561 2 1 8 9 3 6 1
22
The resulting tldl1~ru~ d plant can be used in a conventional breeding
scheme to produce more (I dl I~UI 11 l~d plants with the same .,l Idl d~ il~ or to
introduce the first chimeric gene and optionally the second chimeric gene in other
varieties of the same or related plant species or in hybrid plants resulting from a
cross with at least one ll dl~sru~ d plant of the present invention. Seeds
obtained from the ll d~ u~ ed plants contain the chimeric gene(s) of this
invention as a stable genomic insert.
In another ~IllLJudilll~lL of this invention, plants ~Idl~arulllled with chimeric
qenes comprising the fungus-responsive Dr,o1-1 elements and driving expression
of a selectable or screenable marker DNA, RNA or protein, such as the GUS
marker protein (see Examples), can be used to screen for molecules selectively
inducing the fungus-responsive ~1-1 elements, as is detected by marker gene
expression. These molecules can serYe as potential enhancers of the natural
resistance to pathogens (Doemer et al., 1990; Kessmann et al., 1994).
The following Examples describe the isolation and ~lldld~,Lt~ liull of the
fungus-responsive r,)rP1-1 elements of this invention and the use of such
promoter-effective portions for conferring fungus-resistance to plants. Unless
stated othenwise in the Examples, all nucleic acid manipulations are done by thestandard procedures described in Sambrook et al, Molecular Cloninq A
Laboratory Manual~ Second Edition. Cold Spring Harbor Laboratory Press, NY
(1989) and in volumes 1 and 2 of Ausubel et al., Current Protocols in Molecular
Bioloqv, Current Protocols, USA (1994). Standard materials and methods for
plant molecular work are described in Plant Molecular BioloqY Labfax by R.R.D.
Croy, jointly published by BIOS Scientific Publications Limited (UK) and Blackwell
Scientific Publications, UK (1993).
In the following Examples, reference is made to the following Figure and
Sequence Listing:
SLI~STITL~TE SHEET (RULE 2ti)
W0 96/28S61 2 1 8 9 3 6 7 r~
23
Fiqure 1
Schematical overview of the deletion fragments of the 273 bp PrP1-1
promoter. The nucleotide positions indicated refer to the positions in the 273
bp promoter, Co~ o~ to positions 402 to -130 of the native prp1-1
promoter of Martini et al. (1993).
Seauence Listina
SEQ ID no. 1 shows the 273 bp DNA sequence of the e~1-1 promoter. The
first and last nucleotide of this sequence correspond to positions 402 and -
130 of the 273 bp Prp1-1 promoter, respectively (Martini et al., 1993).
Exampies
ExamPle 1: Construction of prp1-1 promoter deletion fraaments
Piasmid pBS27-1, which has been desuibed in Martini et al. (1993), contains
the promoter sequence of the potato e~p1-1 gene covering positions -402 to -130
(presented in SEQ ID No. 1), inserted into the vector pBS(+) (purchased from
Stratagene, La Jolla, USA) via BamHI and Xbal restriction sites (the 5`-temminusof the ~1-1 sequence represents a Sau3A site which was ligated with the
BamHI site of the vedor, the Xbal site was added to the prp1-1 sequence with
the help of an Xbal linker, so that the 3' temminus of the prp1-1 sequence could t~e
ligated with the Xbal site of the vector). This plasmid was used as starting
material to ueate deletions of the prp1-1 sequence by treatment with
exonuclease lll (Double-Stranded Nested Deletion Kit, purchased from
Pharmacia, Uppsala, Sweden): for generation of 3' end-terminal deletions,
plasmid pBS27-1 was digested with Xbal and afterwards treated with
exonuclease lll, yielding prp1-1 promoter regions which covers positions 1 to 239
of SEQ ID No. 1 (delX4) and 1 to 153 of SEQ ID No 1 (delX5) (see Fig. 1); after
addition of Xbal linkers, the DNAs were recircularized producing plasmids
pBS27-11delX4 and pBS27-1/delX5; for aeneration of 5'-temminal deletions,
plasmid pBS27-1 was cut with BamHI and afterwards treated with exonuclease lll
treatment, yielding prp1-1 promoter regions covering positions 100 to 273
(delB35), 140 to 273 (delB34) and 176 to 273 (delB51 ) (Fig. 1 ); after addition of
S~STITUTE St IEET (~ULE ~6)
Wo 96128561 2 1 8 9 3 6 1 P~ c ~ : ~
BamHI linkers DNAs were recircularized resulting in plasmids pBS27-1/delB35,
pBS27-1/delB34 an~ pBS27-1/delB51. The ~ength of the prp1-1 inserts in all
plasmids was determined by sequencing (Sanger et al., 1977). Then, the ~1-1
promoter regions were isolated from the plasmids by cutting with EcoRI and Xbal;and inserting into vector pETVgus (Martini et al., 1993) digested with EcoRI andXbal; thereby, the Pro1-1 promoter sequences were located 5' temminal of the
CaMV35S TATA-box region (48 to +8), the coding region of the b-glucuronidase
(GUS) gene from E. coli and the polyadenylation signal of the pea rbsS-3C gene
(Benfey and Chua, 1990), giving plasmids pETVgus27-1/delB35, pETVgus27-
1/delB34, pETVgus27-~,/delB51, pETVgus27-1/delX4, and pETVgus27-1/delX5.
These piasmids were mobilized from E. coli strain S17-1 to Aqrobacterium
tumefaciens C58C1-GV3101 barboring helper plasmid pMP9ORK (Koncz et al.,
1989). Tldll~rulllldroll of pota~û rSolanum tuberosum L.) cultivar Desirée was
done according to the leaf disc tecbnique outlined by De Block (1988).
A,uuluulid~ly l,d"~rul",ed plants were identified by PCR analysis with
oligonucleotide primers speciflc for the GUS gene. Primary It:g~lleidll~ were
used to produce tubers, and 4 to 6 weeks old cuttings of tuber grown plants werehnally applied in expression studies. Transgenic plants were dt:~iyl Id~t:d based
on the chimeric constructs integrated into the plant genome: DelX4, DelX5,
DelB34, DelB35 and DELB51. The length of these fragments and their relative
position in the PrP1-1 promoter is Indicated in Figure 1.
Example 2: Expression of marker aene/PnP1-1 deletion constructs in transqenic
potato plants.
Leaves from transgenic potato plants (2 to 7 i, Idt~ d, :"~ lines per construct)were infected with Phvtophthora infestans race 1-11 as previously described
(Martini et al., 1993); as a control, leaves were treated with water. For stimulation
with ph~,~r,l ,u, l l IUI~S, discs of 0.5 cm diameter were punched out from transgenic
Ieaves with a cqrkborer and incubated in an aqueous solution containing 1mM
indole-3-acetic acid (IM) or 10 mM Na-~alicylate (SA), under constant white light
at 18 C; as a control, leaf discs were incubated in water. Leaf material was
SUrSTlTI~lTE SHEi--T (P~ULE 2~)
Wo 96/2856l 2 1 ~ 9 3 6 1
harvested three days after inoculation with fungal spores or 24 hours after
ini~iation of treatment with phy~ul ~o~ es. The ll dl la~ i,UllOl ldl activationmediated by the various e~1-1 promoter fragments was d~ il led by
measuring b-glucuronidase activity in total protein extracts from this leaf material,
according to Jefferson (1987).
Constitutive expression of the chimeric constructs in non-infected roots was
assayed by lliaLuull~llliudl GUS staining of axenically grown root material fromthe transgenic lines. Detached roots were vacuum infiltrated with a solution
consisting of 100 mM sodium phosphate (pH 7.0) and 0.5 mg/ml X-gluc (5-
bromo-4-chloro-3-indolyl b-D-glucuronide~ The enzymatic reaction was then
allPwed to proceed for 16 h at 37 C; afterwards roots were transferred to 70%
(v/v) ethanol and evaluated under the ~ l Uscop~.
The induction rates of GUS enzyme activity (measured according to
Jefferson, 1987) comparing fungus-infected and water-treated detached leaves,
or phytohormone-treated and water-treated leaf discs are listed in Table 1.
Additionally, detection of constitutive GUS activity in non-infected roots is
indicated in this table. Control constructs comprising only the 35S minimal
promoter (lacking a fungus-responsive element), fused to the GUS cPding region
of this invention, did not produce any detectable amount of GUS enzyme in
several tests, either upon infection with Phvtoohthora infestans or phytohonmone~, I ' " 1 Neither was there found any detectable amount of GUS enzyme in
roots. From Table 1, the most interesting line can be chosen, e.g., DelX4-4 or -3,
that have a cu" ,~i, IdliOIl of a high fungus-response and a low responsiveness to
phyIullu~llu~le application in culllbilldLiull with no detectable rPot expression.
Line DelB34-8 is probably defective, since all values are very low.
Example 3: Construction of plant ~I dl l~ UI 11 IdliUI I vectors
As desuibed in detail below, the identified fungus-responsive ~1-1
fragments are used to construct first chimeric genes of this invention v~hich are
SLI~STITUTE SHEET ~RULE 26)
wo96l28s6l ~ 1 8936 1
26
then used; e.g., with second chimeric genes of this invention, to constnud plant~Idll~rulllldLi~nl vectors. A preferred promoter comprises the Ep1-1 promoter
fragments delX4 or delX5 of Example 1 (Figure 1), operably linked to a CaMV
35S minimal promoter fragment from nucleotide 48 to nucleotide +8 of the CaMV
35S promoter (Benfey et al., 199û). Each of the promoter fragments is upstream
of, and in the same ~ dl l::>UI iU~iUI Idl unit as, a first foreign DNA encoding barnase
from Bacillus am~ Pf~iPns (Hartley et al., 1988). Downstream of the first
foreign DNA is the 3' ullLI~ ldl~d end of the nopaline synthase gene ("3'nos")
which is isolated as a 26û bp Taal fragment from the nopaline synthase gene
(Gielen et al, 1984). This results in a chimeric gene construd that is designated
"delX4/35S-barnase-3'nos'` and "delX5/35S-bannase-3' nos". These first chimeric
genes are introduced between the T-DNA border repeats of the vector pGV941
(Deblaere et al., 1987) as described in PCT publication WO 93/19188. This
vector contains a chimeric marker gene containing the nopaline synthase
promoter ("pnos"; Depicker et al, 1982), the neo coding region from Tn5 (Beck etal, 1982) and the 3' ~ dll~ldl~ end of the octopine synthase gene ("3'ocs"),
Cullt:~iOlldill9 to the 706 bp Pvull fragment from the odopine synthase gene
(Gielen et al, 1984). The construction of this chimeric"pnos-neo-3'ocs" gene is
described by Hain et ai (1985) and in EP 359 617.
In order to construd T-DNA piant 1, dl l~rul " IdliUI I vectors carrying also second
chimeric genes of this invention, a DNA fragment containing a Pnos-barstar-3'g7
gene construd is introduced in the above described plant vedors, as described
in WO 93119188.
Using the procedure described above, a plant ~Id~ ulllldl;ull vector is also
constructed containing a hrst chimeric gene, designated "delX4/35S-Rs_AFP2-
3'35S", using the DNA coding sequence of the anti-fungal Rs AFP2 protein
described in PCT patent publication WO 93105153 as first foreign DNA (without
"inhibiting" foreign DNA).
SU~STITUTE SHEET (RU~E 26
WO 96/28561 2 1 ~ 9 3 6 1 r~
Furthermore, following the above outlined procedures, a plant ll dl la~UI 1 l IdliUI I
vector, carrying the coding regions of the rice basic chitinase and the b-1,3-
glucanase gene of Zhu et al. (1994), each under the control of the delX4/35S
promoter construct described above and flanked by the 3' polyadenylation and
transcript ~ Id~iUI I region of the CaMV 35S gene, is also constructed.
Example 4: Tldll~UlllldliUII of potato arld oilseed raPe with Aqrobacterium
tumefaciens strains can yinq the plant ll dl l~UI 11 IdliUI I vectors of Example 3
To obtain ~I dl l~UI 11 Id~iUI I of, and major expression in, potato and oilseed rape
(Bnassica napus), the plant lldll:~Ulllld~iOII vectors of Example 3 are each
mobilized into the Aqrobacterium tumefaciens strain C58C1RifR carrying the
avirulent Ti plasmid pGV2260 as desuibed by Deblaere et al (1985). The
transconjugants are analyzed by Southern blotting. The respective
Aqrobacterium strains are used to transfomm potato plants (Solanum tuberosum
cvs. Binbe and Désiré) by means of tuber disc infection as described by De Blocket al (1987) and oilseed rape using the method described by De Block et al
(1989). T,d,,~ru,,,)ed calli are selected on medium wntaining 100mg/ml
kanamycin, and resistant calli are Itl~ ld~d into plants For each
lldll~r~ d~iu~ ,~,i",a"~, about 1û individual ~IdllSrulll~dllla are l~g~l~ldlud
and analyzed by Southern blotting for gene integration patterns.
Potato and oilseed rape plants tl dl 1~/111 ,ad with a first chimeric gene
w",~ ,i"g a first forei~n DNA sequence encoding bamase under the control of
the fungus-responsive delX4/35S of delX5/35S promoter of Example 3, and a
second chimeric gene w,,,~ ,i:,;"~ a second foreign DNA sequenoa encoding
barstar under the control of the nos promoter show a significantly higher degreeof resistance to fungus infection, particularly PhvtoPhthora infestans (potato) and
L~lU:~JIlde~lid maculans (oilseed rape) infection, than do non-l,d,,~u,,,,~d
control plants. As a result, the lld~ ulll~ed plants have significantly lower yield
Iosses than do the control plants upon statistical analysis of small scale fieldtrials, infected with fungal pathogen. When compared to control plants, fungal
SU~T~TUTE S~1EtT (RULE 26)
wo g6/2ss6l 2 1 8 ~ 3 ~ 1 r~ s ~
sporulation is ~i~"iri~, Illy inhibited even 7 days after infection. Sporulation is
followed by ~dlllilldLiUll under the stereoscope, after fungal infection, by
applying 2û ml droplets (when using either 2X106 to 5x105 spores/ml) to the
bottom side of the potato leaf. After l"ai"i~"d~ of the leaves in water, the
fungal growth and sporulation can be followed by visual inspection under a
a~ usw~e. The disease resistance phenotype of the tld~l~ru~ ed plants is
conhrmed by molecular analysis based on RNA qiJd~liirli d[iull and evaluation ofthe phenotype in the segregating progeny.
Similar significant reduction of fungal growth is observed on the potato and
oilseed rape plants, transformed with the chimeric genes of Example 3, encoding
the antifungal chitinase and glucanase, or the antifungai Rs/AFP2 protein
Needless to say, ~he use of the fungus-responsive fungus-responsive E~
elements and chimeric genes of this invention is not limited to the ll dl larUI I lldLiUI I
of any specific planS(s). Such promoters and chimeric genes can be useful in
tidllarulllllllu any crop, such as alfalfa, corn, cotton, sugar beet, brassica
vegetables, tomato, soybean, wheat or tobacco, where the promoters can control
gene expression, preferably where such expression is to occur abundantly in
plant cells which i"ll"edidl~ly surround fungal infection sites without major
induction by wounding, IM, SA and without signihcant expression in root tissue
Also, the use of the improved fungus-responsive promoters of this invention
is not limited to the control of particular hrst foreign DNAs but can be used tocontrol expression of any DNA fragment in a plant
Furthemmore, this invention is not limited to the specihc improved
fungus-responsive prp1-1 promoter fragments deâcribed in the foregoing
Examples. Rather, this invention ~I~WIIIUdSS~:~ promoter fragments, equivalent
to those of the Examples, such as equivalent promoter fragments of other r~o1
genes, which can be used to control the expression of a stnuctural gene, such asa first foreign DNA, at least substantially selectively in plant cells which
SU~STiTUTE SHEET (PIJLE 26)
Wo 96/28s61 1 ~ 9 3 6 i P~
ill,llle~id~ly surround a fungal infection site. Indeed, it is believed that the DNA
sequences of the prP1-1 promoter and promoter fragments of the Examples can
be modified by replacing some of their nucleotides with other r~ Irlp~ti~r-sl
provided that such I l luuiii~dliol ls do not substantially alter the ability ofpolymerase complexes. including lidll~uliuliull adivators, of plant cells, whichill,l"edidlely surround the fungal infection site, to recognize the promoters. Such
equivalent fungus-responsive prp1-1 elements preferably have 85 %, more
preferably 90 %, particularly 95 % nucleotide sequence similarity with the
sequences of the fungus-responsive elements derived from SEQ ID No. 1, such
as delX4 and delX5.
Nor is this invention limited to the use of the fungus-responsive chimeric
genes of this invention for proteding plants against a PhytoPhthora fungus such
as P infestans. Such chimeric genes can be used to proted plants against
plant-pdll l~ fungi, generally, particularly against Phvtophthora spp, Pvthium
spp, Fusarium spp, Sclerotinia spp, Puccinia spp, Ustilaqo spp, Alternaria spp,
o~uul i~m spp, Sclerotinia sclerotoxiorum, Pvrebioeriza brassicae.
CYI~ 1 U ,POI ium concentricum, Phoma lin~am and LeplU~ dttl id maculans.
All published documents and patent publications referred to herein are
hereby il~culuuld~d by reference, i.e. the cPntents of these documents referred
to should be considered as physically illcul,uuld~d into the above desuiption
and examples.
SLI~STiTUTE SHEET (P~JLE 26)
WO96/2~561 2 1 893 6 1
Tabie 1: Expression pattern of chimeric DrD1-1/GUS constructs. The fold-
induction of GUS enzyme activity comparing water-treated and stimulated
leaves is presented for various independent transgenic lines; values
represent averages of three to four independent experiments. GUS enzyme
activity in non-infected roots is indicated by "+". Potato line EG2706
harboring the 273-bp ~1-1 promoter portion 5'-terminal of the CaMV 3~S
TATA-box region (~8 to +8) and the GUS coding region (Martini et al., 1993)
was used as a positive control (Pi 1-11: Phytovhthora infestans race 1-11;
IM: 1 mM indole-3-acetic acid; SA: 10 mM sodium salicylate, nt: not tested)
Pi 1-11 IAA SA Root activi~y
De~X4-1 3.5 0.9 1.0
DelX4-2 2.4 1.0 1.0
DelX4-3 9.6 0.8 o.9
DelX4-4 8.4 0.8 0.9
DelX4-5 2 9 1 1 1.2
DelX5-1 2.7 0.9 o g
DelX5-2 3.3 0.~ 0.7
DelX5-4 6.0 0.9 0.8
DelB34-1 7.9 23.1 20.3 +
DelB34-6 7.0 21.2 17.9 +
DelB34-7 5.8 18.2 25.0 f
DelB34-8 1.5 1.0 0.9
DelB34-9 2.7 l3.3 8.5 nt
DelB34-10 3.1 18.8 14.0 n~
DelB35-1 9.3 25.3 12.6 +
DelB35-2 4.6 8.1 4.6 +
DelB51-1 8.0 10.6 6.6 +
DelB51-2 2.8 20.1 ~5.8 +
DelB51-3 4.2 21.8 18.8 nl
DelB51-4 3 4 16.7 16.9 nt
DelB51.5 1.9 1.1 o g n~
DelB516 2:1 1.0 1.1 n~
EG2708 14.7 38.9 31.7 +
SUnSTiTUTE S~IEET (PULE 26)
wos6/28s61 2 1 8 9 3 6 1 r~~
31
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1 d~ iUI 1~", pp. 1-25 in G. Pegg and P. Ayres (Eds.), Funqal Infection Os Plants
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Wos6/28s61 2 1 8 q 3 6 1 P~ . 9
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S~JeST~TUTE SltEET (RULE 26
WO 96/2f~561 2 ~ 8 9 3 6 1 r~
34
SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT: ~
(A) NAME: MAX-PLANCK-GESELLSCHAFT ZUR FORDERING DER WiSSEN-
SCHAFTEN E.V.
(B) STREET: I lu~u~ l. . C~r 2
(C) CITY: Munchen
(E) COUNTRY: GERMANY
(F) POSTAL CODE (ZIP): D-80~39
(ii) TITLE OF INVENTION: IMPROVED FUNGUS-RESPONSIVE CHIMAERIC GENE
(iii) NUMBER OF SEQUENCES: 1
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS~MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO)
(2) INFORMATION FOR SEO ID NO: 1:
(i) SEQUENCE CHARA~; I t~l~ ,S:
(A) LENGTH: 273 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY~ linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAMEIKEY: misc. feature
(B) LOCATION:1..273
(D) OTHER INFORMATlON:/note= ~273 bp ~2~-1 fra~ment, uu, Ir~uOrl~illu to
position
-402 to -130 of the orp1-1 promoter (Martini e~ al., 1993)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
GATCcMMTcTAAcMmMMGGI I I I MMmTTGTGu;l I I I I I I I AAATTMAM
TATGTCAAAT ATArTAAAAT ATATTTmA M I I I I ATAC rMMMACAT GTCACATGM
120
TAmGAAAT TATAAAATTA TCMAMTAA MAAAGMTA mCmMC AMTTMMT
180
TGMMTATG ATMMTAAAT TAMCTATTC TATCATTGAT I I I I CTAGCC
ACCAGAmG 240
ACCMMCAGT GGGTGACATG AGCACATAAG TCA
273
SU5TITUTE S~IEET (~ULE 2~i)
