Annals of Botany 81 : 779–781, 1998
SHORT COMMUNICATION
The Angiospermous Root Parasite Orobanche L. (Orobanchaceae) Induces
Expression of a Pathogenesis Related (PR) Gene in Susceptible Tobacco Roots
D A N I E L M. J O E L* and V I T A L Y H. P O R T N O Y
Department of Weed Research, Agricultural Research Organization, Newe-Ya’ar Research Center, P.O. Box 1021,
Ramat-Yishay 30095, Israel
Received : 19 January 1998
Returned for revision : 22 February 1998
Accepted : 15 March 1998
Parasitic plants develop a haustorium that intrudes host tissues. In roots of transgenic PRb-1b-GUS tobacco the
expression of the chimeric gene was prominent near Orobanche infection. The expression of the pathogenesis related
(PR) protein gene in Orobanche-infected roots indicates that Orobanche releases appropriate elicitors, and that the
susceptible plant does sense Orobanche invasion. The Orobanche-responsive promoter may be a useful tool in
engineering resistances to this parasitic weed.
# 1998 Annals of Botany Company
Key words : Haustorium, Nicotiana tabacum, Orobanche aegyptiaca, parasitic plants, PR proteins, tobacco.
INTRODUCTION
All parasitic plants develop a haustorium which is a
multicellular organ that invades host tissues and serves as a
bridge between host and parasite (Kuijt, 1969 ; Musselman
and Dickison, 1975 ; Stewart and Press, 1990 ; Press and
Graves, 1995 ; Riopel and Timko, 1995). Theoretically,
when a parasite penetrates host tissues, it can behave either
as a compatible partner or as an alien. In the latter case,
which applies to the majority of plant pathogens, the
parasite forces its way through host tissues, by mechanical
or enzymatic means. But the parasite may also act in
another way : it may mimic host cells so that its advance
through host tissues is facilitated by co-ordination with the
host that identifies it, mistakenly, as a compatible partner.
The relations between host and parasite would then be
similar to those between a pollen tube and the compatible
stigmatic tissue or may resemble the ‘ intrusive growth ’
(Fahn, 1982) of certain fibre cells and laticifers that elongate
in mature tissues of higher plants. The mode of penetration,
by which a parasite would stimulate changes in the host
facilitating its intrusion, can be termed co-ordinated penetration. In a recent study we presented evidence for the
enzymatic nature of the invasion of the Orobanche haustorium into host tissues (Losner-Goshen et al., 1998), but until
now the basic question of whether parasitic haustoria grow
in co-ordination with host tissues has not been addressed.
This question is relevant because at later developmental
stages, the development of the host at the infection zone is
indeed co-ordinated with that of the parasite : cambium of
the parasite becomes closely aligned with that of the host,
and, at least in certain Orobanchaceae, these cambia
function in a co-ordinated manner (Baird and Riopel,
1986).
* For correspondence. Fax ­972 4 9836 936, e-mail dmjoel!netvision.net.il
0305-7364}98}060779­03 $25.00}0
The difference between resistant and susceptible Orobanche hosts may be in their ability to sense the presence of
the parasite. If this is the case, only resistant hosts would
recognize Orobanche intrusive cells as alien. Whereas there
are clear indications that some resistant hosts have the
ability to sense the intrusion of parasite cells in their tissue,
as indicated by their hypersensitive response (Cubero,
1991), there is, as yet, no indication of whether susceptible
hosts have the same ability.
In this paper we address this question experimentally
using a molecular approach based on the expression of
pathogenesis-related (PR) proteins, a group of host-encoded
proteins accumulating in response to various pathogens
(Bol, Linthorst and Cornelissen, 1990 ; Eyal and Fluhr,
1991).
MATERIALS AND METHODS
Orobanche aegyptiaca Pers. was grown on transgenic
tobacco plants that served as hosts. These host plants were
grown in two different systems : (a) the ‘ sandwich method ’
(Losner-Goshen et al., 1998), i.e. between two layers of
glass-fibre paper lying inside sand in 10 cm pots. Pots were
opened at different times after planting and infected roots
were harvested for β-glucuronidase (GUS) localization ; (b)
in Šitro, in polyethylene bags (modified from Parker and
Dixon, 1983) : seeds of O. aegyptiaca were spread over one
side of an 11¬28 cm GFA paper, inserted into a stationary
polyethylene bag containing 10 ml of half strength Hoagland
nutrient solution, and hung in dark boxes for a month in a
growth chamber at 25 °C. Transgenic tobacco plants,
containing the PRB-1b promoter fused to the β-glucuronidase (GUS) reporter gene (Eyal et al., 1991) were grown
first on agar, then transferred into the bags (1 week after
sowing Orobanche). The bags were placed in a growth
chamber at 25 °C, 16 h light. Non-transgenic tobacco plants,
bo980629
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Joel and Portnoy—Expression of a Pathogenesis-related Gene is Induced by Orobanche
used as control, were grown as above and transferred into
similar bags and pots. Frequent observations were made
under a dissecting microscope to follow tobacco development and Orobanche germination and development in the
polyethylene bags. Tobacco plants became infected with
Orobanche within 2 weeks. Each root system carried between
one and 17 Orobanche infections. When young Orobanche
tubercles that developed on both transgenic and nontransgenic tobacco roots were seen in the transparent
system, they were harvested with the roots to which they
were attached, for GUS localization. The tobacco plants
grown in pots were similarly harvested at about the same
time. Infection sites from ten polyethylene bags and ten pots
were examined for GUS expression.
Infected root portions were removed from the tobacco
plants and incubated for 24 h at 40 °C in X-gluc (Stomp,
1992), then examined under light microscopy to localize
expression of the reporter gene.
RESULTS
Orobanche aegyptiaca developed normal tubercles when
infecting transgenic tobacco plants both in Šitro and in
soil. In pots, normal Orobanche flowering also occurred,
comparable to that occurring on non-transformed tobacco
that served as control ; thus parasite growth on transformed
hosts can be considered entirely normal, and the transgenic
tobacco plant typically susceptible.
Incubation of infected tobacco roots in X-gluc resulted in
blue coloration in all infection sites (Fig. 1), indicative of
GUS activity. The colouring was pronounced mainly in
tissues surrounding the parasitic haustorium. Blue colouring
was sometimes also found in adjacent root tissues, in the
region extending a few millimeters above the infection zone,
mainly around the vascular system, but not further away
from the infection site.
GUS activity also occurred in transgenic plants at the
sites of lateral root emergence (data not shown). Apart from
F. 1. Light micrograph of a root of a transgenic tobacco plant (R)
containing the PRb-1b-GUS gene, parasitized by Orobanche aegyptiaca
(P). The blue colour, indicative of GUS expression, is seen in the
infection site. ¬25.
that, no GUS activity could be found in any other noninfected root region of transgenic tobacco, nor was it found
in either infected or non-infected roots of non-transgenic
tobacco plants.
DISCUSSION
A significant expression of the reporter gene was found at
infection sites in all transgenic roots, indicating that
Orobanche penetration of the roots is capable of eliciting the
expression of the PR promoter in a susceptible host. The
expression of GUS in emergence sites of lateral roots, where
they force their way out of the main root, serves as a
‘ positive control ’, since wounding is known to occur at such
sites. In some cases the expression of the chimeric gene was
also prominent a few millimeters above the infection zone,
along the vascular system, but not further away from the
infection site, indicating that this is a restricted local event.
The present study was conducted in order to examine
whether parasitic haustoria of Orobanche force their way
into host roots, or grow in co-ordination with host tissues.
A ‘ co-ordinating ’ parasite would avoid eliciting host defense
reactions that may jeopardize its development. It has
recently been shown that Orobanche releases wall degrading
enzymes during the penetration of its haustorium into host
tissues (Ben-Hod et al., 1993 ; Losner-Goshen et al., 1998).
Oligomeric fragments released by such cell wall degrading
enzymes are known to act as defense elicitors in resistant
host plants during their interactions with intruding fungi
(Ryan and Farmer, 1991). Defense reactions are indeed seen
in some resistant Orobanche hosts during infection by
Orobanche (Panchenko and Antonova, 1974 ; Kirollos and
El-Hafees, 1985 ; Ish-Shalom Gordon, Cohen and Jacobsohn, 1990 ; Wegmann et al., 1991), but the actual elicitors
of these reactions are so far unknown. The PRb-1b belongs
to the basic-type PR-1 genes that are also known to be
activated in correlation with pathogenic micro-organisms or
elicitors, as part of the systemic-acquired resistance (SAR)
response in plants (Eyal and Fluhr, 1991 ; Ward et al., 1991).
The expression of the PR gene promoter in the Orobancheinfected roots indicates that Orobanche releases appropriate
elicitors, and that the transgenic tobacco plants do sense
Orobanche invasion, in spite of the fact that they do not
resist haustorial invasion. The results of this study clearly
indicate that Orobanche does not behave as a compatible
partner in susceptible host tissues and that there is no coordination with the host during invasion. It seems that
susceptible hosts do not prevent haustorial penetration
because they lack the appropriate genes for resistance, or
they develop a response that is too slow or ineffective.
The promoter of the PRb-1b gene that was shown to be
activated by Orobanche attack may serve as a powerful tool
in engineering Orobanche resistant crops if it is used in
conjunction with appropriate genes for resistance against
this parasite, such as genes encoding the synthesis of antiOrobanche toxins.
A C K N O W L E D G E M E N TS
The kind donation of the PRB-1b transformed tobacco
seeds by R. Fluhr is gratefully acknowledged. This research
Joel and Portnoy—Expression of a Pathogenesis-related Gene is Induced by Orobanche
was supported by grant No. IS-2170-92RC from BARD,
the United States-Israel Binational Agriculture Research
and Development Fund. Contribution from the Agricultural
Research Organization, Institute of Plant Protection, BetDagan, Israel, No. 511}98.
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