Download Pathogenesis-Related Proteins Are Developmentally

The Plant Cell, Vol. 1, 881-887,September 1989 O 1989 American Society of Plant Physiologists
Pathogenesis-Related Proteins Are Developmentally
Regulated in Tobacco Flowers
Tamar Lotan, Naomi Ori, and Robert Fluhr’
Department of Plant Genetics, The Weizmann lnstitute of Science, Rehovot 76100, Israel
The accumulation of pathogenesis-related proteins (PR) in tobacco leaves has been casually related to pathogen
and specific physiological stresses. The known enzymatic function of some of these proteins is potentially
antimicrobial. By using antibodies specific to three classes of pathogenesis-relatedproteins, we examined tobacco
plants during their normal growth. The pathogenesis-related proteins accumulated during the normal development
of the tobacco flower. The PR-1 class of proteins (biological function unknown) is located in sepal tissue. PR-P,Q
polypeptides are endochitinases and are present in pedicels, sepals, anthers, and ovaries. A glycoprotein serologically related to the PR-2,N,O class is a (1,3)-P-glucanase and is present in pistils. Differential appearance during
flower development, in situ localization, and post-translational processing of floral pathogenesis-related proteins
point to a hitherto unsuspected function these classes of pathogenesis-related proteins play in the normal process
of flowering and reproductive physiology.
INTRODUCTION
The de novo synthesis of pathogenesis-related (PR) proteins (Van Loon, 1985; Matsuoka et al., 1987; Kombrink,
Schroder, and Hahlbrock, 1988; Nasser et al., 1988; Rigden and Coutts, 1988) is a ubiquitous reaction of monocot
and dicot plants to pathogen attack. The PR proteins were
originally discovered in relation to the hypersensitive response induced by tobacco mosaic virus (TMV) (Gianinazzi, Martin, and Vallee, 1970; Van Loon and Van Kammen, 1970). They have been associated with systemic
acquired resistance and incipient antipathogen effects (Van
Loon, 1982; Richardson, Valdes-Rodriguez, and BlancoLabra, 1987). Best characterized in tobacco, PR proteins
fall into five major groups that are coordinately regulated
in response to infection by fungi, bacteria, and viruses.
Distinct enzymatic functions recently have been described
for two groups of PR proteins. One group, consisting of
hydrolytic acidic proteins PR9,N,O, has been shown to
have (1,3)-@-glucanaseactivity (Kauffmann et al., 1987).
Another group, consisting of two acidic protein isoforms
PR-P,Q, has endochitinase activity (Legrand et al., 1987).
Both groups of hydrolases can attack the main components of fungal and bacterial cell walls (Schlumbaum et al.,
1986; BOI and Van Kan, 1988; Mauch, Mauch-Mani, and
Boller, 1988). The function of a third group of three acidic
proteins, viz., PR-la, PR-1b, and PR-lc, remains enigmatic. Ethylene, a plant hormone, may be a natural mediator of the response (Van Loon, 1982, 1985; Vogeli, Meins,
and Boller, 1988), which can be brought about by both
’ To whom correspondence should be addressed.
biotic and chemical elicitors (White, 1979; Asselin, Grenier,
and Cote, 1985) and physiological stress induced by aging
(Fraser, 1981) or aberrant hormonal levels (Memelink,
Hoge, and Schilperoort, 1987; Shinshi, Mohnen, and
Meins, 1987).
The pathogenesis-related response and the in vitro demonstration of direct inhibition of fungal growth by some of
the proteins have highlighted their defensive functions in
the plant. Here we present evidence showing that, in
addition to their pathogen-related induction in leaves, at
least three classes of these proteins are present in healthy
tobacco flowers, irrespective of microbial attack or other
stress.
RESULTS
Distributionof PR Proteins in Tobacco Flowers
We prepared polyclonal antibodies to polyacrylamide gelpurified, extracellular (Parent and Asselin, 1984), acidic
proteins of TMV-infected tobacco leaves and used them
to quantify the amounts of PR protein using immunoblots.
Extracts from leaves of healthy tobacco plants contained
very low to nondetectable levels of these proteins. However, upon TMV infection of hypersensitive plants, these
proteins accumulated massively, as can be seen by comparing lanes 1 and 2 in Figures 1A to 1C. Analysis of fully
developed tissues of healthy flowers revealed an unusual
882
The Plant Cell
stressed with an elicitor, all three PR classes appeared in
concert (Figures 1A to 1C, lanes 11).
The intrinsic, programmed nature of PR protein accumulation in flowers was ascertained by studying the behavior of homeopathic, cytoplasmic, male-sterile mutants
prevalent in certain alloplasmic lines of tobacco (Aviv and
Galun, 1986). In Nicotiana tabacum plants containing N.
undulata cytoplasm, the stamens are malformed and differentiate into atrophied stigmatoid filaments, as illustrated
in Figures 2A and 2B. The PR proteins that accumulated
in the mutant pistillate structures were of the PR-2,N,O
type (Figure 1B, lane 12) and not the PR-P type seen in
normal anthers (Figure 1C, lane 9). The accumulation of all
other organ-specific flower PRs was unchanged in this
cytoplasmic male-sterile mutant (data not shown). Evidently, PR protein accumulation is determined by the final
organ structure and not its ontogenic origin.
The possibility of a cryptic, nonsymptomatic, infective
process being present as a result of flower opening and
exposure to the environment was eliminated following
examination of the temporal mode of PR-2,N,O-like protein
accumulation in developing flower buds. As shown in
Figure 3A, PR-2,N,O immunoreactivity during flower differentiation was already detected in unopened flower buds
about 2 days before opening and increased markedly at
the time of anthesis.
pattern of PR protein accumulation. A polypeptide that
reacted to PR-1 antisera appeared in sepal tissue and had
PR-1 class mobility, as shown in Figure 1A, lane 4. A
polypeptide of apparent higher molecular weight reacting
to PR-2,N,O antisera was observed in pistils shown in
Figure 1B, lanes 6 and 7. Polypeptides reacting to antiPR-P.Q were abundantly present in pedicels and sepals
but present at lower levels in anthers and ovaries, as
shown in Figure 1C, lanes 3, 4, 9, and 10. Note that,
whereas mobilities identical to both PR-P and PR-Q were
detectable in TMV-treated leaf and untreated sepals (Figure 1C, lanes 2 and 4), PR-P polypeptide was the only
type present in pedicels and ovaries (Figure 1C, lanes 3
and 10). No immunoreactive material specific for the three
acidic PR protein classes was detected in the other flower
parts, i.e., petals and filaments.
Accumulation of PR Proteins Is Related to Flower
Development
The following observations illustrate that the accumulation
of PR proteins in flower tissues is developmentally controlled and unrelated to a pathogenesis response. The
concerted induction of all three PR protein classes in
pathogenesis is the general rule (Parent and Asselin, 1984;
Asselin, Grenier, and Cote, 1985; Van Loon, 1985; Bol
and Van Kan, 1988; Rigden and Coutts, 1988). Hence, the
differential appearance in flower parts suggests an alternative inductive pathway. Indeed, the potential for concerted PR protein accumulation, as found in leaves, can
also be found in flowers. When stylar structures were
kD
94-
The 41-kD Stylar Polypeptide Is a Glycoprotein
The higher apparent molecular weight of PR-2,N,O immunoreactive material (41 kD) in styles compared with its
PR-lo, Ib.lc
PR-2,N,0
PR-P.O
67-
43-
41 kD-
30-
PR-2.N.O-
.
PR-
I
2
3 5 7 9 I
4 6 8 10
I
3
2
5
4
6
7 9 II I2
8 10
I
3 5 7 9
2
4 6 8
10
B
Figure 1. Presence of Pathogenesis-Related Proteins in Leaf and Flower Organs.
(A) Immunoblot of an SDS-polyacrylamide gel on which mature flower and leaf protein extracts were fractionated and developed with
anti-PR-la, 1b, 1c antibody. Lane 1, normal leaf; lane 2, TMV-infected leaf; lane 3, pedicel; lane 4, sepal; lane 5, petal; lane 6, style; lane
7, stigma; lane 8, filament; lane 9, anther; lane 10, ovary; lane 11, style pretreated with pathogenesis elicitor.
(B) Immunoblot as in (A) but immunolabeled with anti-PR-2,N,O. Lane 12 is protein extracted from stigmatoid-like structures of an N.
tabacum mutant containing the N. undulata cytoplasm line MS92 (Aviv and Galun, 1986).
(C) Immunoblot as in (A) but immunolabeled with anti-PR-P,Q.
Floral-Specific Expression of PR Proteins
883
with the immunoreactive polypeptide (N. Ori, unpublished
results). Collectively, these data suggest that the stylar
41-kD protein is a glycoprotein and is related to a subclass
of PR-2,N,O polypeptides.
Cellular Localization of the 41-kD Glycoprotein
Figure 2. Normal and Mutant Flowers of N. tabacum on Day of
Anthesis.
Pathogenesis-related proteins of the PR-2,N,O class are
secreted from elicitor-induced cells (Parent and Asselin,
1984; Van Loon, 1985; Kauffmann et al., 1987) and thus
prevent pathogen spread in the extracellular spaces.
Therefore, it is of interest to examine the cellular localization of the related 41-kD stylar polypeptide. Histological
sections shown in Figure 4 were prepared from flowers at
anthesis and reacted with antisera to PR-2,N,O. The major
accumulation was detected in transmitting track cells starting well below the stigma surface and extending throughout the style (Figure 4A). The immunohistochemical spread
of the 41 -kD polypeptide is not continuous with the stigma
(A) Normal N. tabacum flower. Arrow points to anthers after
dehiscence.
(B) Mutant N. tabacum flowers containing N. undulata cytoplasm.
Arrows point to stigmatoid-like structures of the malformed
anthers.
4lkD-
apparent size in the leaf (34 kD) could indicate that a novel
immunoreactive polypeptide was synthesized, or that PR2,N,O-type protein has undergone post-translational modification. Experiments were designed to differentiate between these two alternatives. Concanavalin A (ConA),
which exhibits affinity for <*-D-mannopyranosyl and «-Dglucopyranosyl residues, was found to bind to many polypeptides from stylar extracts, as shown in Figure 3B. A
41-kD polypeptide that co-migrates with the immunoreactive band reacted strongly with ConA (Figure 3B, lanes 1
and 2), suggesting that it is glycosylated. When purified
immunoreactive material was digested with endoglycosidase F, an enzyme that removes N-linked glycans, and
then fractionated on denaturing gels, three new bands
were observed (Figure 3C, lane 1). The mobility of the
fastest moving band coincided with that of PR-2,N,O-size
protein found in leaves (Figure 3C, lane 4).
Due to their intrinsic acidity, PR proteins can easily be
fractionated on native polyacrylamide gels in which the
2,N,O polypeptides are resolved from each other. When
large amounts of immunoreactive stylar protein were fractionated on these gels, a minor fraction containing at least
two polypeptides corresponding to PR-N.O in mobility was
observed (Figure 3D, lanes 1 to 3). This minor fraction
probably results from nonuniform glycosylation of a PRIM,O-like polypeptide. In addition, a (1,3)-/J-glucanase activity, using laminarin as substrate, was observed to co-purify
*
2 3
A
r
I 2
B
I
2 3 4
C
Figure 3. Analysis of Temporal Accumulation and Processing of
the 41-kD Glycoprotein.
(A) Immunoblot of SDS-polyacrylamide gel-fractionated style extracts developed with anti-PR-2,N,O, as described in Figure 1.
Lane 1, flowers with 2-mm ovaries 10 days before anthesis; lane
2, flowers with 5-mm ovary 2 days before anthesis; lane 3, flowers
with 8-mm ovary on the day of anthesis.
(B) Immunoblot of SDS-polyacrylamide gel-fractionated style extracts. Lane 1, developed with ConA and horseradish peroxidase;
lane 2, autoradiograph of lane 1 developed with anti-PR-2,N,O as
described in (A).
(C) Immunoblot of SDS-polyacrylamide gel-fractionated, purified,
41 -kD polypeptide and extracts of TMV-induced leaves incubated
with endoglycosidase F and developed with anti-PR-2,N,O. Lane
1. purified 41-kD protein digested with endoglycosidase F; lane
2. purified 41-kD protein; lane 3, TMV-induced leaf extract digested with endoglycosidase F; lane 4, TMV-induced leaf extract.
(D) Native gel electrophoresis of style and TMV-induced leaf
extracts developed with anti-PR-2,N,O. Lane 1, 100 ^g of style
extracts; lane 2,100 ^g of stigma extracts; lane 3, 10 ^g of TMVinduced leaf extract. In nondenaturing gel electrophoresis, the
three isoforms of acidic (1,3)-/i-glucanase PR-2,N, and O present
in pathogen-induced leaves are resolved (Kauffmann et al., 1987).
884
The Plant Cell
Figure 4. Immunohistochemical Localization of the 41-kD Glycoprotein in Median Longitudinal and Transverse Sections of Stigma and
Style Tissue.
(A) Median longitudinal section of mature stigma and style organs. Bar represents 100 Mm.
(B) Transverse section 1.5 mm below stigma surface. Bar represents 100 Mm.
(C) Magnification of style tissue examined in (A). Bar represents 100 Mm.
(D) Magnification of style tissue examined in (C). Bar represents 10 Mm.
Immunohistochemical stain (dark area) was developed with horseradish peroxidase linked to anti-PR-2,N,O. st, stigma; sv, style; e,
epidermis; co, cortex; tt, transmitting track; vb, vascular bundle; v, vacuole.
upper parts or surface; therefore, the heavily stained cells
must represent a specialized subset of the transmitting
track cells previously described for N. tabacum (Bell and
Hicks, 1976). The cells containing immunoreactive protein
were organized as a double-lobed structure as seen in a
transverse section 1.5 mm below the stigma surface (Figure 4B). Directly above this section, only a very low amount
of 41-kD polypeptide was detected, indicative of the discontinuity with the stigma surface. The dumbbell-shaped
population of transmitting cells reveals an inner core of
unstained cells that condenses further down the transmitting track to yield a concentric tube. Vascular bundles are
evident at right angles to the dumbbell axis and run alongside throughout the style (Figures 4B and 4C). The lack of
immunoreactive material accumulating in the vascular system shows that a major part of the 41-kD polypeptide is
not secreted extracellularly in contrast to the behavior of
the PR-2,N,O class of polypeptides induced in the leaf.
The vacuolar localization of the 41-kD glycoprotein becomes apparent at higher magnification of style tissue.
Each transmitting cell contains at least one large rectangular-shaped vacuole (Bell and Hicks, 1976) darkly stained
with immunoreactive material, giving a distinct mosaic
pattern (Figure 4D).
DISCUSSION
Pathogenesis-related proteins have been shown previously to be related to flowering in tobacco. Fraser (1981)
detected PR accumulation in the leaves of flowering plants,
and Memelink, Hoge, and Schilperoort (1987) showed
chitinase RNA transcripts in nucleic acid extracts of whole
Floral-SpecificExpressionof PR Proteins
flowers, particularly in hormone-stressed plants. We have
extended these observations to show spatial and temporal
accumulation of PR polypeptides in a developmentally
dependent manner. In addition to the spatial separation
between groups of PR proteins, which clearly underscores
the use of a pathogen-independent inductive pathway, we
observed the novel appearance of subsets of polypeptides
within the endochitinase and (1,3)-p-glucanase isoenzymes families. The simplest explanation is that the polypeptides are part of a larger gene family containing members with pathogen-induciblecharacters and members that
exhibit purely flower-specific developmental regulation. Alternatively, certain genes may have multifunctional promoter elements. PR-I polypeptides have been shown to
belong to a multigene family of 6 to 12 members (Cornelissen et al., 1987; Matsuoka et al., 1987; Pfitzner and
Goodman, 1987). However, the other acidic PR classes
have not been characterized at the gene level. The 41 -kD
glycoprotein might belong to an extended family of PR2,N,O-type polypeptides. If, in this case, identical genes
are involved, one must ask why the leaf-specific genes are
not similarly glycosylated. In this respect, it is interesting
that a basic (1,3)-p-glucanase expressed in tobacco cell
cultures has recently been shown to undergo transient
glycosylation (Shinshi et al., 1988). The possibility exists
that similar cryptic post-translationalprocessing may occur
in the acidic leaf-specific (1,3)-P-glucanases and that this
processing activity is inoperative in styles. It is intriguing
to speculate whether the glycolyzation of the 41-kD
polypeptide is a symptom or a cause of its intracellular
targeting.
This study suggests the existence of an intrinsic dual
functionality of the ubiquitous pathogenesis defense-related proteins. The function of pathogenesis-related proteins in the leaf is understood to be related to disease
resistance; however, a specific biological function for these
proteins in flowers is unknown. It should be kept in mind
that, although enzymatic activities have been defined for
the endochitinases and (1,3)-p-glucanases using polysaccharide substrate of nonplant origin, other enzymatic specificities and endogenous substrates may exist. As an example, normal pollen tube growth through the transmitting
tract tissue is accompanied by massive callose (1,3)-pglucose polymer deposition. The 41 -kD polypeptide may
play a role in stylar energy metabolism, enabling efficient
growth of the hundreds of competing pollen tubes as they
transverse the style to the ovary. However, a major endogenous substrate with (1,3)-P-glucosyl linkage has not
been defined in stylar tissue. Sirofluor, which binds to (1,3)P-glucans, shows distinct fluorescence of the pollen tube
walls and callose plugs, but only residual background
levels in the style tissues (Stone et al., 1984; T. Lotan,
unpublished data).
The organ specificity, developmental appearance, and
post-translational modification of this polypeptide are superficially similar to those seen for the 32-kD stylar glyco-
885
protein that was recently shown to be associated with the
phenomenon of gametophytic self-incompatibility in N.
alata (Anderson et al., 1986). However, when compared
with the reported distribution of the transcripts coding for
the 32-kD N. alata protein (Cornish et al., 1987), immunohistochemical localization of the 41 -kD polypeptide
showed no direct continuity between the stigma surface
and the tissue containing the 41-kD glycoprotein. Nevertheless, the presence of an abundant (1,3)-~-glucanase
activity may indicate a vestigial or active apparatus for
pollen selection. This scenario lends credence to the analogies drawn between the intrusion of funga1 hyphae in
race-specific pathogenicity and angiosperm pollen-compatibility systems (Heslop-Harrison, 1978). These findings
suggest a novel evolutionary origin for the PR proteins, in
which genes involved in the normal process of flowering
or reproductive physiology have adapted an additional
function in plant defense.
METHODS
Plant Material, Infection, and lnduction by Elicitor
Nicotiana tabacum cv Samsun NN plants were infected at 200
lesions per leaf with TMV-U2 strain, and leaves were harvested 3
days later. Mature flowers were harvested, and tissues were
individually ground in PBS buffer (PBS is 1O mM NaHPO,, pH 7.4,
150 mM NaCI, 3 mM KCI). Elicitor induction was achieved by
injecting a 10 pg/mL solution of tunicamycin (N. Ori, unpublished
data) or 5 mM salicylic acid (White, 1979; Van Loon, 1985) and
harvesting tissues 1 day to 2 days later. N. tabacum plants
containing N. undulata cytoplasm were provided by Drs. D. Aviv
and E. Galun.
Protein Gels and lmmunoblotting
SDS (12.5%)-PAGE was performed as described by Laemmli
(1970). The same system was used for native gels, with SDS
omitted. lmmunoblots were processed in the following manner:
SDS or native polyacrylamidegel-fractionated proteins were electrophoretically transferred to nitrocellulose sheets (Towbin, Staehelin, and Gordon, 1979) and blocked for 1 hr with 5% skim milk
in PBS containing 0.1% Triton X-100 (blocking buffer). Diluted
rabbit antiserumwas added for a 2-hr incubation,the nitrocellulose
was washed four times, 5 min each, with PBS containing 0.1%
Triton X-1 00, then '251-proteinA (affinity-purified;Amersham; 0.1
pCi/mL) in blocking buffer was added, and the blots were incubated for an additional 2 hr. The sheets were washed as above,
dried, and autoradiographed. Reactionof protein blots with ConA
and subsequent incubation with horseradish peroxidase were as
described by Clegg (1982).
Preparationof Sera
lntercellular fluid extracts were prepared from TMV-infected tobacco leavesaccording to Parentand Asselin (1984). The extracts
886
The Plant Cell
were fractionated on 12.5% native gel, and the protein bands
were excised and directly homogenized in PBS buffer containing
0.1% SDS. The homogenate was emulsified with an equal volume
of Freund’s complete adjuvant in a homogenizer. The solution (4
mL) containing about 100 Fg of protein was injected intradermally
into a 2- to 3-month-old rabbit. Boost injections (after 30 days, 45
days, and 60 days) were performed in a similar manner, using
Freund’s incomplete adjuvant. The sera were stored at -2OOC.
The sera prepared in this manner were specific for acidic PR
forms and did not react to basic (1,9)-P-glucanase or chitinase
isolated from mature senescing tobacco leaves (Shinshi, Mohnen,
and Meins, 1987; T. Lotan, unpublished data).
Purification of 41-kD Polypeptide
Stylar 41-kD polypeptide was purified as follows: 5 g of frozen
styles were ground to a fine powder in liquid nitrogen in 15 mL of
PBS buffer containing 0.5 mM phenylmethylsulfonyl fluoride, 1
mM DTT, 0.1% polyvinylpolypyrillidone, and 20 mM sodium ascorbate (added as powder). The slurry was centrifuged at 10,000
relative centrifugal force x 10 min, and the supernatant was
passed through a Sephadex G-25 column (2.5 x 25 cm; void
volume, 20 mL) equilibrated with 20 mM Tris, pH 8.0. Sixty mL of
post-void volume eluent was applied to a QAE-A25 (Pharmacia
LKB Biotechnology Inc.) column, and the unbound fraction was
adjusted to pH 7.4, 0.1 M NaCI, 1 mM CaCI,, 1 mM MgCI,, and
1 mM MnCI, (ConA buffer). The eluent was then reacted for 4 hr
with 5 mL of “washed ConA-Sepharose.” Washed ConA-Sepharose was prepared by equilibrating ConA with ConA buffer and
washing in a small column with 5 volumes of 0.1 M methyl-a-omannoside in ConA buffer, and finally washing in 10 volumes of
ConA buffer. The eluent and ConA-Sepharose slurry was transferred to a 5-mL column and washed with 10 volumes of ConA
buffer. The 41-kD polypeptide was eluted with 0.2 M methyl-a-Dmannoside in ConA buffer, dialyzed against 20 mM ammonium
acetate, pH 7.4, and concentrated by lyophilization. The 41-kD
polypeptidewas estimated to be 95% pure as assayed on stained
denaturing gels and exhibited approximately 400 nkat/mg specific
activity on laminarin substrate (N. Ori, unpublished data). This
activity is similar to that published for PR-2,N,O leaf-specific
glucanases(Kauffmann et al., 1987).
Enrymatic Deglycosylation
Enzymatic deglycosylation of the 41-kD polypeptide was carried
out in 0.1 M NaHP04, 50 mM EDTA, 1% Nonidet P-40, 0.1%
SDS, and 1% P-mercaptoethanol. The sample was boiled for 2
min and cooled, and the reaction was brought to 0.1 M ophenanthroline(Nageswara and Bahl, 1987). One unit of endoglycosidase F (DuPont-New England Nuclear) was added, and the
mixture was incubated for 1.5 hr at 37°C. Diaested
.. samdes were
denatured in sample buffer, loaded on SDS-polyacrylamidegels,
and analyzed by immunoblotting.
lmmunohistochemicalStaining
Flowers were separated into different tissues which were fixed in
5% formaldehyde, 5% acetic acid, and 63% ethanol for 24 hr at
room temperature. After dehydration with ethanol, the flowers
were embedded in paraffin. Sections were cut serially, 15 pm
thick, mounted on slides covered with 3% gelatin, deparaffinized
in xylene, hydrated in decreasingethanolconcentrationsin series,
and washed with PBS. To reduce endogenous peroxidaseactivity,
sections were incubated in absolute methanol containing 0.5%
HZ02for 10 min, and then washed three times with PBS. Slides
were pre-incubatedfor 10 min in 5% skim milk and then reacted
for 2 hr in the presence of 5% skim milk with a dilution of 150 to
1:200 of the immune serum. The sections were washed with PBS,
incubated for 1O min with 5% skim milk, and then incubated with
peroxidase conjugated to goat anti-rabbit antisera (BioMakor) at
a dilution of 1:300 for 1 hr. The substrate reaction included 0.5
pg/mL 3,3’-diaminobenzidine and 0.01% H,O, in PBS for 2 min
to 15 min. Reactions were stopped with a water rinse. Control
slides treated with pre-immune serum showed only very faint
backgroundstaining.
ACKNOWLEDGMENTS
Our thanks to Yossie Orly for help in preparing antisera, to Dvora
Aviv and Esra Galun for providing the materials in Figure 2, and
Autar Krishen Matoo and Marvin Edelman for critical reading of
the manuscript. This work was supported by the RockefellerWeizmann Rapid Fund and the National Council for Research and
Development Joint German-lsraeli Research program. R.F. holds
the Yigal Allon Career Development Award and T.L. a Levi Eshkol
Scholarship Grant.
Received May 11, 1989; revised June 26, 1989.
REFERENCES
Anderson, M.A., Cornish, E.C., Mau, S.-L., Williams, E.G., Hoggart, R., Atkinson, A., Bonig, I., Grego, B., Simpson, R.,
Roche, P.J., Haley, J.D., Penschow, J.D., Niall, H.D., Tregear,
G.W., Coghlan, J.P., Crawford, R.J., and Clarke, A.E. (1986).
Cloning of cDNA for a stylar glycoprotein associated with
expressionof self-incompatibility in Nicotianaalata. Nature 321,
38-44.
Asselin, A., Grenier, J., and Cote, F. (1985). Light-influenced
extracellular accumulation of b (pathogenesis-related)proteins
in Nicotiana green tissue induced by various chemicals or
prolonged floating on water. Can. J. Bot. 63,1276-1283.
Aviv, D., and Galun, E. (1986). Restoration of male fertile Nicotiana by fusion of protoplasts derived from two different cytoplasmic male-sterile cybrids. Plant MOI.Biol. 7, 411-41 7.
Bell, J., and Hicks, G. (1976). Transmitting t i ~ in~the
e Pistil of
tobacco: Light and electron microscopic observations. Planta
131,187-200.
BOI,J.F., and Van Kan, J.A.L. (1988). The synthesis and possible
functions of virus-induced proteins in plants. Microbiol. Sci. 5,
47-52.
Clegg, J.C.S. (1982). Glycoprotein detection in nitrocellulose
transfers of electrophoreticallyseparated protein mixtures using
Floral-SpecificExpressionof PR Proteins
887
concavalin A and peroxidase: Application to arenavirus and
flavivirus proteins. Anal. Biochem. 127, 389-394.
are chitinases. Plant MOI.Biol. 11, 529-538.
Parent, J.G., and Asselin, A. (1984). Detection of pathogenesis-
Cornelissen, B.J.C., Horowitz, J., Van Kan, J.A.L., Goldberg,
R.B., and BOI,J.F. (1987). Structure of tobacco genes encoding
relatedproteins (PR or b) and of other proteins in the intercellular
fluid of hypersensitiveplants infectedwith tobacco mosaic virus.
Can. J. Bot. 62, 564-569.
Pfitzner, U.M., and Goodman, H.M. (1987). lsolation and characterizationof cDNA clones encodingpathogenesis-relatedproteins from tobacco mosaic virus infected tobacco plants. Nucl.
Acids Res. 15,4449-4465.
pathogenesis-relatedproteins from the PR-1 group. Nucl. Acids
Res. 15, 6799-681 1.
Cornish, E.C., Pettitt, J.M., Bonig, L., and Clarke, A.E. (1987).
Developmentally controlled expression of a gene associated
with self-incompatibility in Nicotiana alata. Nature 326, 99-1 02.
Fraser, R.S.S. (1981). Evidence for the occurrence of the “pathogenesis-related” proteins in leaves of healthy tobacco plants
during flowering. Physiol. Plant Pathol. 19, 69-76.
Gianinazzi, S., Martin, C., and Vallee, J.C. (1970). Hypersensibilite aux virus, temperature et proteines solubles chez le Nicotiana Xanthi-nc. Apparition de nouvelles macromolecules lors
de Ia repression de Ia synthese virale. CR Acad. Sci. Paris
D270,2383-2386.
Heslop-Harrison, J. (1978). Cellular recognition in plants: Interaction in symbiosis parasitism and disease. In Studies in Biology
No. 1O0 (London: Edward Arnold Publishers),pp. 43-51.
Kauffmann, S., Legrand, M., Geoffroy, P., and Fritig, B. (1987).
Biological function of pathogenesis-related proteins: Four PR
proteins of tobacco have 1,3-p-glucanaseactivity. EMBO J. 6,
3209-3212.
Kombrink, E., Schroder, M., and Hahlbrock, K. (1988). Severa1
“pathogenesis-related” proteins in potato are 1,3-P-glucanases
and chitinases. Proc. Natl. Acad. Sci. USA 85, 782-786.
Laemmli, U.K. (1970). Cleavage of structural proteins during the
assembly of the head of a bacteriophageT4. Nature 227, 680685.
Legrand, M., Kauffmann, S., Geoffroy, P., and Fritig, B. (1987).
Biological function of “pathogenesis-related” proteins: Four tobacco PR-proteins are chitinases. Proc. Natl. Acad. Sci. USA
84,6750-6754.
Matsuoka, M., Yamamoto, N., Kamo-Muranami, Y., Tanaka, Y.,
Ozeki, Y., Hirano, H., Kagawa, H., Oshima, M., and Ohashi,
Y. (1987). Classification and structural comparison of full-length
cDNAs for pathogenesis-related proteins. Plant Physiol. 85,
942-946.
Mauch, F., Mauch-Mani, B., and Boller, T. (1988). Antifungal
hydrolases in pea tissue. II. lnhibition of fungal growth by
combinations of chitinase and p-1,3-glucanase. Plant Physiol.
88,936-942.
Memelink, J., Hoge, J.H.C., and Schilperoort, R.A. (1987). Cytokinin stress changes the developmental regulation of severa1
defence-related genes in tobacco. EMBO J. 6, 3579-3583.
Nageswara, R.T., and Bahl, O.P. (1987). Enzymatic deglycosylation of glycoproteins. Methods Enzymol. 138, 350-359.
Nasser, W., de Tapla, M., Kauffmann, S., Montasser-Kouhsari,
S., and Burkard, G. (1988). ldentificationand characterization
of maize pathogenesis-related proteins. Four maize proteins
Richardson, M., Valdes-Rodriguez, S., and Blanco-Labra, A.
(1987). A possible function for thaumatin and a TMV-induced
protein suggested by homology to a maize inhibitor. Nature
327,432-434.
Rigden, J., and Coutts, R. (1988). Pathogenesis-relatedproteins
in plants. Trends Genet. 4, 87-89.
Schlumbaum, A., Mauch, F., Vogeli, U., and Boller, T. (1986).
Plant chitinase inhibitors of fungal growth. Nature 324, 365367.
Shinshi, H., Mohnen, D., and Meins, F., Jr. (1987). Regulationof
a plant pathogenesis-relatedenzyme: lnhibitionof chitinase and
chitinase mRNA accumulation in cultured tobacco tissues by
auxin and cytokinin. Proc. Natl. Acad. Sci. USA 84, 89-93.
Shinshi, H., Wenzler, H., Neuhaus, J.-M., Felix, G., Hofsteenge,
J., and Meins, F., Jr. (1988). Evidence for N- and C-terminal
processing of a plant defense-related enzyme: Primary structures of tobacco prepro-p-l.3-glucanase. Proc. Natl. Acad. Sci.
USA 85,5541 -5545.
Stone, B.A., Evans, N.A., Bonig, I., and Clarke, A.E. (1984). The
application of sirofluor, a chemically defined fluorochrome from
aniline blue for the histochemical detection of callose. Protoplasma 122,191 -1 95.
Towbin, H., Staehelin, T., and Gordon, J. (1979). Electrophoretic
transfer of proteins from polyacrylamide gels to nitrocellulose
sheets: Procedure and some applications. Proc. Natl. Acad.
Sci. USA 76,4340-4354.
Van Loon, L.C. (1982). Regulation of changes in proteins and
enzymes associated with active defense against virus infection.
In Active Defense Mechanisms in Plants, R.K.S. Woods, ed
(New York: Plenum Press), pp. 247-273.
Van Loon, L.C. (1985). Pathogenesis-relatedproteins. Plant MOI.
Biol. 4, 111-1 16.
Van Loon, L.C., and Van Kammen, A. (1970). Polyacrylamide
disc electrophoresis of the soluble leaf proteins from Nicotiana
tabacum var. “Samsun” and ”Samsun NN.” Changes in protein
constitution after infection with tobacco mosaic virus. Virology
40, 199-21 1.
Vogeli, U., Meins, F., Jr., and Boller, T. (1988). Co-ordinated
regulation of chitinase and (3-1,3-glucanase in bean leaves.
Planta 174, 364-372.
White, R.F. (1979). Acetylsalicylic acid (aspirin)induces resistance
to tobacco mosaic virus in tobacco. Virology 99, 410-41 2.
Pathogenesis-related proteins are developmentally regulated in tobacco flowers.
T Lotan, N Ori and R Fluhr
Plant Cell 1989;1;881-887
DOI 10.1105/tpc.1.9.881
This information is current as of August 3, 2017
Permissions
https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298
X
eTOCs
Sign up for eTOCs at:
http://www.plantcell.org/cgi/alerts/ctmain
CiteTrack Alerts
Sign up for CiteTrack Alerts at:
http://www.plantcell.org/cgi/alerts/ctmain
Subscription Information
Subscription Information for The Plant Cell and Plant Physiology is available at:
http://www.aspb.org/publications/subscriptions.cfm
© American Society of Plant Biologists
ADVANCING THE SCIENCE OF PLANT BIOLOGY