• The Monosaccharide Transporter Gene, AtSTP4, and the

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    • Abstract: The Monosaccharide Transporter Gene, AtSTP4, and theCell-Wall Invertase, At fruct1, Are Induced inArabidopsis during Infection with the Fungal BiotrophErysiphe cichoracearum1Vasileios Fotopoulos, Martin J. Gilbert2, Jon K. Pittman3, Alison C. Marvier4, Aram J. Buchanan,

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The Monosaccharide Transporter Gene, AtSTP4, and the
Cell-Wall Invertase, At fruct1, Are Induced in
Arabidopsis during Infection with the Fungal Biotroph
Erysiphe cichoracearum1
Vasileios Fotopoulos, Martin J. Gilbert2, Jon K. Pittman3, Alison C. Marvier4, Aram J. Buchanan,
Norbert Sauer5, J.L. Hall, and Lorraine E. Williams*
School of Biological Sciences, University of Southampton, Biomedical Sciences Building, Bassett Crescent
East, Southampton SO16 7PX, United Kingdom
Powdery mildew fungi are biotrophic pathogens that form a complex interface, the haustorium, between the host plant and
the parasite. The pathogen acts as an additional sink, competing with host sinks, resulting in considerable modification of
photoassimilate production and partitioning within the host tissue. Here, we examine the factors that may contribute to
these changes. We show for the first time in one biotrophic interaction (Arabidopsis/Erysiphe cichoracearum) all of the
following responses: Glc uptake in host tissues is enhanced after fungal infection; this coincides with the induction of
expression of the monosaccharide transporter gene, Arabidopsis sugar transport protein 4 (AtSTP4), in infected leaves; invertase
activity and transcript levels for a cell wall invertase, At fruct1, increase substantially in Arabidopsis during attack by this
pathogen. Before infection, Arabidopsis plants transformed with an AtSTP4 promoter- -glucuronidase construct show
expression mainly in sink tissues such as roots; after infection, AtSTP4 expression is induced in the mature leaves and
increases over the 6-d time period. Sections of infected leaves stained for -glucuronidase show that AtSTP4 expression is
not confined to infected epidermal cells but is also evident in a wider range of cells, including those of the vascular tissue.
The results are discussed in relation to the possible coordinated expression of hexose transporters and cell wall invertase in
the host response to powdery mildew infection.
Powdery mildews are widespread biotrophic essential solutes, particularly sugars, across the host/
pathogens that cause major losses in crop yield. They pathogen interface, or about the molecular responses
are usually restricted to the shoot epidermal cells induced in the host transport processes in response to
where they acquire essential nutrients from the living such infection. There is good evidence that Glc may
host tissues over a long period. Thus they form an play a key role in such biotrophic interactions. Glc
additional sink, and this can lead to considerable appears to be the major carbon energy source trans-
changes in carbon transport and partitioning within ferred from the host to cereal and pea powdery mil-
the plant (see Farrar and Lewis, 1987; Ayres et al., dew (Mendgen and Nass, 1988; Clark and Hall, 1998;
1996; Hall and Williams, 2000). There is still much to Sutton et al., 1999), although the route and mecha-
learn about the nutrition of these important biotro- nisms by which this occurs are not clear. A recent
phic pathogens. For example, little is known about study investigating the role of haustoria in sugar
the molecular mechanisms involved in the transfer of supply during infection of broad bean (Vicia faba) by
the biotrophic rust fungus Uromyces fabae demon-
1 strated that a fungal gene encoding a hexose trans-
This work was supported by the Biotechnology and Biological
Science Research Council (grant no. 51/P14477), by the European
porter was expressed abundantly in rust haustoria
Commission DGXII Biotechnology Program (contract no. BIO4 (Voegele et al., 2001). The protein was localized ex-
CT96 – 05831), and by The Royal Society. clusively to the haustorial plasma membrane, sug-
Present address: School of Biological Sciences, University of gesting that this hexose transporter may have a major
Exeter, Exeter EX4 4QQG, UK. role in carbon transfer into the fungus from the ex-
Present address: Baylor College of Medicine, Houston, TX trahaustorial matrix (Voegele et al., 2001).
77030. An important observation in particular powdery
Present address: School of Animal and Microbial Sciences, mildew/host compatible interactions is that infection
University of Reading, Reading RG6 6AJ, UK. can result in an increased capacity for sugar uptake
Present address: Department of Botany II/Molecular Plant
by the host tissue (Sutton, 1997; Clark and Hall,
Physiology, University of Erlangen/Staudtstrasse 5, D–91058
1998). However the molecular mechanisms leading to
* Corresponding author; e-mail [email protected]; fax 44 –23– 80 – such a response have not been defined. The increased
594319. capacity may be due to enhanced activity of existing
Article, publication date, and citation information can be found transporters or synthesis of new transporters possi-
at www.plantphysiol.org/cgi/doi/10.1104/pp.103.021428. bly at the epidermal cells, thus increasing the level of
Plant Physiology, June 2003, Vol. 132, pp. 821–829, www.plantphysiol.org © 2003 American Society of Plant Biologists 821
Fotopoulos et al.
Glc available to the invading fungal haustoria. Alter- but significant increase in Glc uptake was observed
natively, the increased capacity may be a retrieval (Fig. 1). A sellotape treatment was carried out to
mechanism to support the increased demand for car- remove most, if not all, of the mycelium inoculum
bon in defense mechanisms. The monosaccharide from the surface of the leaf before uptake. Members
carrier AtSTP4 in Arabidopsis has been shown to be of the Erysiphales only colonize the outer surface of
stress regulated (Truernit et al., 1996). In the present the invaded tissue and do not produce hyphae that
study, we investigate its role in a biotrophic interac- penetrate within the host tissue, with the exception of
tion. a few species of powdery mildew fungi (Alexopoulos
Infection may also result in a general increase in et al., 1996). A similar level of enhanced Glc uptake
apoplastic Glc levels due to the activity of the cell was observed after removal of the mycelium by the
wall invertase, an enzyme catalyzing the cleavage of sellotape treatment, suggesting that this was a host
Suc to Glc and Fru (for review, see Hall and Williams, response rather than the fungus acting as an addi-
2000). Several types of invertases exist, differing in tional sink (Fig. 1). Glc uptake in noninfected and
their pH optima and their different cellular locations. infected material was inhibited by the protonophore
Neutral or alkaline invertases are thought to be lo- carbonylcyanide-m-chlorophenyl-hydrazone (Table
cated in the cytosol, whereas acid invertases are I). Uptake was inhibited by the permeable sulfydryl
found either in the vacuole or cell wall. An increase reagent, NEM, but showed low sensitivity to the
in the various types of invertases has been observed non-permeant sulfydryl reagent p-chloromercuri-
in a variety of biotrophic infections, including pow- benzenesulfonic acid. Diethyl pyrocarbonate, a His
dery mildew; this usually concerns acid invertase modifier, caused moderate inhibition, as did phlorid-
(Scholes et al., 1994; Wright et al., 1995; Clark and zin, a competitive inhibitor of Glc uptake in animal
Hall, 1998; Chou et al., 2000), although in some cases cells. Levels of inhibition were similar in infected and
increases in alkaline invertase have been observed noninfected material for all the reagents tested (Table
(Storr and Hall, 1992). However, it is not clear I). The inhibitor profile is consistent with a proton-
whether this is due to the synthesis of new isoforms coupled carrier mechanism mediating Glc uptake.
or to an increase in the level of existing isoforms. The
relationship between these changes clearly needs to Expression of the Monosaccharide Transporter, AtSTP4,
be studied to provide a fuller understanding of the Is Induced on Infection
processes involved. Glc and stress appear to indepen-
dently regulate source and sink metabolism and de- The monosaccharide transporter, AtSTP4, has been
fense mechanisms in cultured Chenopodium sp. cells implicated in responses to abiotic and biotic stress in
(Ehness et al., 1997); in particular, treatment with Glc Arabidopsis (Truernit et al., 1996). We investigated
induces the sink-specific extracellular invertase that whether this particular monosaccharide transporter
is thought to play a key role in Suc partitioning (Tang has a role in the response to infection by powdery
et al., 1999). mildew by comparing its expression in noninfected
To understand more fully the roles of Glc, monosac- and infected leaf tissue. Northern analysis was per-
charide transporters, and invertase activity in the re- formed with total RNA from infected and nonin-
sponse of plants to powdery mildew infection, we fected leaf material using the AtSTP4 probe (Fig. 2).
have examined the various related responses dis-
cussed above in the model plant Arabidopsis that is
now widely used in the study of plant-pathogen
interactions (Buell, 1998; Schulze-Lefert and Vogel,
2000). We have shown for the first time in one inter-
action that infection results in enhanced Glc uptake
in infected host tissue, an increase in the expression
of the host monosaccharide transporter gene, AtSTP4
in infected leaves, an increase in the host cell wall
invertase activity, and an increase in expression of
the cell wall invertase, At fruct1. The results are dis-
cussed in relation to the possible coordinated expres-
sion of hexose transporters and cell wall invertase in
the host response to powdery mildew infection. Figure 1. Glc uptake into Arabidopsis source leaves after infection
with E. cichoracearum. Glc uptake into infected (brushed with inoc-
ulum) and noninfected (brushed without inoculum) Arabidopsis leaf
RESULTS discs was measured using 0.5 mM [14C]Glc (1, noninfected; 2, in-
fected). Treatments 2 and 4 were 6 d post inoculation. Measurements
Effect of Infection on Glc Uptake were also taken after removal of the mycelium with sellotape before
uptake (3, noninfected; 4, infected). Results are the mean ( SE) of
When Glc uptake was measured in mature leaves three experiments, and the asterisk indicates significant difference
of Arabidopsis from healthy noninfected plants and from Glc uptake in corresponding control noninfected leaf discs (P
plants infected with Erysiphe cichoracearum, a small 0.001).
822 Plant Physiol. Vol. 132, 2003
Sugar Transport and Erysiphe cichoracearum Infection
Table I. The effect of inhibitors on Glc uptake into leaf discs from (Fig. 3a). Leaves inoculated with the incompatible
infected and noninfected tissue Blumeria graminis spores showed some increase in
Uptake of 0.5 mM Glc was measured at 25°C after 1.5 h following staining at d 1 postinfection, but this did not in-
incubation in reaction buffer containing 20 mM Mes-BTP (pH 5.8), 1 crease over 6 d as observed for the compatible in-
mM CaCl2, 500 M Glc and 1 Ci [14C] glucose. Inhibitors were teraction. Similar results were found with a semi-
added as indicated. Values are the mean % inhibitors calculated from quantitative RT-PCR analysis, where no increase in
three replicates. expression was observed between d 1 and 6 postinfec-
Inhibition of Glc Uptake tion after inoculation with B. graminis (V. Fotopoulos,
Noninfected Infected unpublished data).
10 M CCCP 67 63
1 mM NEM 70 66
1 mM PCMBS 17 20
1 mM DEPC 42 35
1 mM Phloridzin 42 28
This was carried out under stringency conditions that
did not allow cross-hybridization with AtSTP1 and
-2. These results also show that there is a marked
increase in AtSTP4 expression in infected material
compared with noninfected leaves. Light brushing
without fungal inoculum (equivalent to the brushing
that was carried out to apply the fungus) also caused
an increase in AtSTP4 expression, although this was
far less than the expression induced by infection with
the powdery mildew. In contrast, the actin control
showed little change throughout infection or with
brushing (Fig. 2). Similar results were obtained in a
semiquantitative analysis using reverse transcriptase
(RT)-PCR and gene-specific primers for AtSTP4 (data
not shown). RT-PCR with gene-specific primers for
AtSTP1 and AtSTP2 indicated that the former did not
alter markedly in expression after infection, whereas
the latter was not expressed in noninfected or in-
fected leaf material (J.K. Pittman and L.E. Williams,
unpublished data).
To investigate the induction of AtSTP4 expression
during infection in more detail, we studied the re-
sponse in transgenic plants transformed with an
AtSTP4-promoter- -glucuronidase (GUS) construct.
Initially, the AtSTP4 promoter-GUS construct was in
the Arabidopsis C24 ecotype (Truernit et al., 1996),
which is not susceptible to E. cichoracearum. Therefore
these plants were crossed with the susceptible acces-
sion Col-0, and seed was collected. Crossing was
repeated three times to increase the Col-0 back-
ground. The resulting plants were susceptible to E. Figure 2. Northern analysis of AtSTP4 expression after infection with
cichoracearum. We confirmed that the healthy unin- E. cichoracearum. a, Forty micrograms of total RNA per lane was
fected Col-0 plants containing the AtSTP4-promoter separated on a 1.2% (w/v) agarose gel and transferred to a nylon
GUS construct showed the same pattern of expres- membrane. Hybridization was carried out with a digoxigenin (DIG)-
sion for AtSTP4 as originally observed in the C24 labeled AtSTP4 (top) and actin (bottom) probes. Blots were washed at
ecotype i.e. high expression in the sink organs (roots high stringency. Tissue was treated with fungal inoculum by gently
and anthers) but very low in the mature source brushing with a paintbrush. Controls were brushed without inocu-
lum. Lane 1, Noninfected, non-brushed tissue (d 0); lane 2, nonin-
leaves. When detected in the mature leaves, it was
fected brushed tissue (d 0); lane 3, control tissue (d 3); lane 4,
often found at the hydathodes (M.J. Gilbert, J.L. Hall, infected tissue (d 3); lane 5, control tissue (d 6); and lane 6, infected
and L.E. Williams, unpublished data). Expression of tissue (d 6). A representative blot is shown. The experiment was
AtSTP4, as indicated by GUS staining, was induced performed three times for AtSTP4 and twice for actin. Densitometry
in leaves infected with E. cichoracearum at 1 d postin- data were obtained for each experiment and the mean band intensity
fection and increased with time after inoculation is shown for AtSTP4 (b) and actin (c).
Plant Physiol. Vol. 132, 2003 823
Fotopoulos et al.
To determine the cellular pattern of expression, tigated here (AtSTP1–4), AtSTP4 shows the most
sections were taken from the infected and nonin- marked response with a severalfold increase in ex-
fected leaves of the AtSTP4 promoter-GUS trans- pression after infection with a compatible mildew
formed plants. In the sections from control plants, no strain.
GUS expression was observed, whereas in those ob-
tained from infected material, GUS staining was Effect of Powdery Mildew Infection on Cell
present in many cells of the leaf including the vascu- Wall Invertase
lar tissue (Fig. 3b).
More recently another monosaccharide transporter Cell wall invertase activity increased markedly 3
gene, AtSTP3, has been reported, which is also ex- and 6 d postinfection (Fig. 5). The response was also
pressed in leaves (Buttner et al., 2000), and therefore
¨ investigated at the RNA level using RT-PCR. Degen-
we investigated whether this transporter had any erate oligonucleotide primers were designed based
role in the host response to infection. Little change in on highly conserved amino acid regions identified in
expression over the first 3 d and only a small induc- cell wall invertases from carrot (Daucus carota), mung
tion at 6-d postinfection was observed in northern bean (Vigna radiata), tomato (Lycopersicon esculentum),
analysis (Fig. 4) and RT-PCR (V. Fotopoulos, unpub- and Arabidopsis. RT-PCR was carried out using
lished data). The response was minor compared with RNA prepared from noninfected and infected mate-
that seen for AtSTP4. No marked response of AtSTP3 rial, and greater amplification was observed in in-
was observed in the incompatible interaction as de- fected samples. To investigate this in more detail, we
termined by RT-PCR (V. Fotopoulos, unpublished cloned the RT-PCR products produced with 3-d
data). Therefore, of the four sugar transporters inves- postinfected material. Five clones were selected and
Figure 3. Histochemical analysis of mature
transgenic Arabidopsis leaves expressing the
GUS gene under the control of the AtSTP4 pro-
moter after inoculation with compatible and in-
compatible mildew strains. a, To visualize the
location of GUS activity (indicated by blue col-
or), leaves were incubated at 22°C for 15 h in
the presence of 5-bromo-4-chloro-3-indolyl- -
D-glucuronide-sodium salt trihydrate. Results
are shown for 1, 3, and 6 d postinoculation for
(i), healthy, uninfected leaf; (ii), leaf with the
incompatible B. graminis; and (iii), leaf with the
compatible E. cichoracearum. b, Tissue was
stained for GUS then fixed, embedded, and sec-
tioned. Cell walls are stained pink with Schiff
reagent. (i), Control healthy leaf; (ii), 6 d post-
inoculation with E. cichoracearum. UE, Upper
epidermis; LE, lower epidermis; VB, vascular
bundle. Representative leaves for each condi-
tion are shown.
824 Plant Physiol. Vol. 132, 2003
Sugar Transport and Erysiphe cichoracearum Infection
sequenced, and in all cases they represented partial
cDNAs of At fruct1, a cell wall invertase (Schwebel-
Dugue et al., 1994). We therefore monitored expres-
sion of this particular gene during infection. For com-
parison, At fruct2 was included in the analysis. RT-
PCR was carried out on RNA samples obtained from
noninfected and infected material (1, 3, and 6 d
postinfection). In these particular experiments, fun-
gal inoculum was supplied by shaking spores onto
Figure 5. Effect of infection on cell wall invertase activity in Arabi-
dopsis source leaves. Invertase activity was assayed at pH 4.5 in
extracts isolated from leaves treated with (black bars) or without
(white bars) fungal inoculum. Noninfected control tissue was gently
brushed without fungal inoculum. Values are the means of four
replicated experiments. Asterisk indicates significant difference from
control plants (P 0.001).
leaves to avoid any effects of brushing. The products
were separated by gel electrophoresis, probed, and
blotted with probes specific for At fruct1 and
At fruct2 under conditions where minimal cross-
reactivity occurred. At fruct1 expression was de-
tected in all samples but showed a marked increase
in expression 1, 3, and 6 d postinfection (Fig. 6). In
contrast, actin did not change markedly on infection.
With similar exposure times, At fruct2 expression
could not be detected in any of the samples, and this
is consistent with previous reports showing that this
gene is only expressed in flowers (Tymowska-
Lalanne and Kreis, 1998). After prolonged exposure,
we observed some binding of At fruct2, which also
occurred to the ladder, indicating nonspecific bind-
ing (V. Fotopoulos, unpublished data). Northern
analysis was also carried out with At fruct1, and
similar results were observed to the RT-PCR experi-
ments with an increase in expression 1, 3, and 6 d
postinfection compared with controls (V. Fotopoulos,
unpublished data). In contrast, in the incompatible
interaction, RT-PCR showed no marked increase in
the expression of At fruct1 up to 6 d postinoculation
(V. Fotopoulos, unpublished data).
Figure 4. Northern analysis of AtSTP3 expression after infection with
E. cichoracearum. a, Twenty-five micrograms of total RNA per lane The nature of transporters involved in powdery
was separated on a 1.5% (w/v) agarose gel and transferred to a nylon mildew infection in the host has never been resolved
membrane. Hybridization was carried out with a 32P-labeled AtSTP3 at the molecular level. The Arabidopsis/E. cichoracea-
probe (top). Methylene blue staining of the 25s rRNA band is also rum interaction provides us with a useful system for
shown (bottom). Blots were washed at moderate stringency. Tissue investigation because Arabidopsis has been used
was treated with fungal inoculum by gently brushing with a paint- extensively for studying the role of sugar carriers
brush. Controls were brushed without inoculum. Lane 1, Nonin-
in source/sink interactions (Williams et al., 2000).
fected tissue (d 0); lane 2, infected tissue (d 0); lane 3, control tissue
(d 1); lane 4, infected tissue (d 1); lane 5, control tissue (d 3); lane 6,
cDNAs encoding monosaccharide transporters have
infected tissue (d 3); lane 7, control tissue (d 6); and lane 8, infected been cloned from several species, and there is now
tissue (d 6). A representative blot is shown. The experiment was evidence for the existence of multigene families. Ara-
performed three times with similar results. Densitometry data were bidopsis contains a family of STP genes coding for
obtained for two experiments, and the mean band intensity is shown monosaccharide transporters that appear to be tissue
for AtSTP3 (b) and 25S rRNA (c). specific or switched on in response to particular stim-
Plant Physiol. Vol. 132, 2003 825
Fotopoulos et al.
fungal mycelium is removed before measuring up-
take. This indicates that there is a change in nutrient
demand of the host in this plant/fungal interaction
that is independent of a direct requirement of the
pathogen for assimilates. The host response could be
related to an increased requirement by leaf cells for
carbon to support repair or defense processes. Our
objective was to examine which transporters may
have a role in this response. Truernit et al. (1996)
have suggested that the hexose-transport response to
stress may be linked to a specific member of the
monosaccharide gene family, AtSTP4. We have
shown, using a variety of molecular techniques
(northern analysis, RT-PCR, and reporter gene tech-
nology), that AtSTP4 expression is induced after in-
fection with E. cichoracearum, a compatible mildew
strain. A significant increase in AtSTP4 expression
was observed in leaf tissue 1, 3, and 6 d postinfection
compared with the noninfected controls. Leaves in-
oculated with the incompatible B. graminis spores
showed some increase in GUS staining at d 1 posti-
noculation but, unlike the compatible reaction, no
further increase in expression was observed at d 3
and 6 postinfection. This difference presumably re-
lates to the progression, or not (in the case of the
incompatible interaction), of the disease and may
reflect the change in the leaf from source to sink
metabolism in the compatible reaction.
In tissue sections of E. cichoracearum-infected leaves
Figure 6. Effect of infection on invertase expression in Arabidopsis (prepared from AtSTP4 promoter-GUS transformed
source leaf using RT-PCR. a, RT-PCR was carried out with primers plants), GUS appeared to be present in many cells of
designed to generate partial-length invertase (top) or actin cDNAs the leaf, whereas the fungal haustoria are restricted
(bottom). Products were run on a 1.2% (w/v) agarose gel, hybridized to the epidermis in this infection (Adam and Somer-
with DIG-labeled At fruct1 and actin cDNA probes, and washed at ville, 1996). In contrast, in some biotrophic rust in-
high stringency. Lane 1, Noninfected (d 0); lane 2, infected (d 0); lane 3 fections, gene up-regulation is restricted to the infec-
noninfected (d 1); lane 4, infected (d 1); lane 5, noninfected (d 3); lane
tion sites (Chou et al., 2000; Ayliffe et al., 2002). The
6, infected (d 3); lane 7, noninfected (d 6); and lane 8, infected (d 6). b
and c, Densitometry data were obtained for each amplified product and
initial penetration events during the early stages of
expressed relative to the products at d 0 (healthy), which were normal- powdery mildew infection could be expected to in-
ized to a value of 1. The data are from a representative experiment duce AtSTP4, because it is a wound-inducible carrier
repeated twice. (Truernit et al., 1996). However, the findings that
AtSTP4 expression can also be induced by pathogen
elicitors (Truernit et al., 1996) and the fact that we
uli (Buttner et al., 2000; Williams et al., 2000). Hex-
¨ observed AtSTP4 induction in cells other than those
oses, as well as Suc, may be important signaling penetrated by the fungus in the epidermis suggest
molecules in source/sink regulation including re- this is not the case. Therefore it is possible that a
sponses to biotic stress (Roitsch, 1999), and the trans- general retrieval response may be induced in all leaf
porters that regulate their distribution in stressed cells to retain carbon for defense purposes and may
tissues may be an important component of this reg- be linked to the increased invertase activity that also
ulatory and defense activity. accompanies infection (see below).
Increased uptake of sugars into leaf tissues in re- Although AtSTP4 showed a marked increase in
sponse to abiotic and biotic stress has been observed expression, there was a less marked effect on Glc
after ozone treatment (Sutton and Ting, 1977) and uptake. However, in the latter experiments, the net
pathogen infection (Sutton, 1997; Clark and Hall, uptake is affected by a number of parameters includ-
1998). In the present study, an increase in Glc uptake ing both influx and efflux. The predominant role of
was also observed in the response of Arabidopsis leaf AtSTP4 in this response was supported by analysis of
tissue to powdery mildew infection. This increased other monosaccharide transporters. AtSTP1 expres-
capacity for Glc transport in infected tissues may be sion did not change, whereas AtSTP2 was not ex-
due to the pathogen acting as an additional sink, pressed. AtSTP3, a sugar transporter expressed in
although a similar increase is observed when the green leaves (Buttner et al., 2000), showed some in-
826 Plant Physiol. Vol. 132, 2003
Sugar Transport and Erysiphe cichoracearum Infection
crease in expression with infection but this was small leaves increases the susceptibility to the disease, al-
and not apparent until 6 d postinfection. We cannot though this reverses at still higher levels of sugar
rule out that some of the other AtSTPs may also be (Vanderplank, 1984). More information is required in
involved in this response, but further study is re- this area to investigate the delicate balance between
quired to determine this. sugar content, pathogen invasion, and defense re-
In addition to investigating changes in host sponses in fungal biotrophs.
monosaccharide transporters, our work has focused The finding that infection by powdery mildew in-
on the role of the enzyme invertase. Increased inver- duces AtSTP4 expression in a wide range of host cells
tase activity has been recorded for a number of despite the fungus being confined to the epidermis
biotrophic infections, although the precise form, lo- implicates the role of signaling mechanisms in this
cation, and role of the activity are not clear (for response. There is evidence that defense response
reviews, see Ayres et al., 1996; Hall and Williams, genes are induced by elevated sugar levels (Ehness et
2000). Wright et al. (1995) observed that acid inver- al., 1997; Herbers et al., 2000), and hexose transport
tase activity increased in wheat (Triticum aestivum) into the host cells may be enhanced to cope with this
leaves infected with powdery mildew even when increased energy demand and/or to reduce availabil-
treated with a fungicide to reduce the size of the ity of sugars to the pathogen. Enhanced cell wall
fungal sink; this indicated that a signal from the invertase reduces Suc loading (Stitt et al., 1990; von
fungus caused an increase in invertase activity that Schaewen et al., 1990) and an increase in hexose
was not directly related to the size of the fungal sink. availability as a consequence could increase the con-
The present study has focused on cell wall invertase centration gradient of hexoses to the fungus. The
and shows that there is a marked increase in activity transfer of carbon to the fungus, primarily as Glc, is
of invertase upon infection. This coincides with an thought to occur down a concentration gradient cre-
increase in the expression of the At fruct1 isoform. ated by fungal solute uptake and use. The nature of
Although At frut2 does not seem to be involved in the sugar transporters at the haustorial membrane in
this response, we cannot rule out the possibility that the powdery mildew fungi is unknown, but by anal-
other invertase isoforms may also have a role. ogy with rust, carriers may exist at this membrane to
An induction of cell wall invertase has been re- facilitate transport into the mycelium. A major goal
ported for tobacco (Nicotiana tabacum) leaves infected will be to dissect the signaling pathway in a compat-
with potato (Solanum tuberosum) virus Y (Herbers et ible interaction that results in the diversion of host
resources to the fungus.
al., 2000) and for At fruct1 in Arabidopsis leaves
infected with white blister rust (Chou et al., 2000). In
the latter report, increased invertase activity was con- MATERIALS AND METHODS
fined to the parts of the leaves invaded by the fungal Plant and Fungal Material
mycelium. Such increases in cell wall invertase dur-
ing infection could have a profound effect on the Seeds of Arabidopsis cv Columbia (ecotype Col-0) were sown in Irish
moss peat, John Innes No. 2, and medium graded vermiculite (1:1:1). After
source-sink balance within the plant. For example, sowing, the seeds were vernalized for 24 h at 4°C and then grown at 22°C,
transgenic tobacco plants expressing yeast-derived 75% humidity on a 16-h photoperiod. Arabidopsis GUS transformants were
invertase in the apoplast showed an accumulation of grown on Murashige and Skoog media (Murashige and Sk

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