Ectopic Expression of Constitutively Activated RACB
in Barley Enhances Susceptibility to Powdery Mildew
and Abiotic Stress1
Holger Schultheiss2, Götz Hensel2, Jafargholi Imani, Sylvia Broeders3, Uwe Sonnewald,
Karl-Heinz Kogel, Jochen Kumlehn, and Ralph Hückelhoven*
Institute of Phytopathology and Applied Zoology, University of Giessen, D–35392 Giessen, Germany
(H.S., J.I., K.-H.K., R.H.); Institute of Plant Genetics and Crop Plant Research, D–06466 Gatersleben,
Germany (G.H., S.B., J.K.); and Institute of Microbiology, Biochemistry and Genetics, University of
Erlangen-Nürnberg, D–91058 Erlangen, Germany (U.S.)
Small RAC/ROP-family G proteins regulate development and stress responses in plants. Transient overexpression and RNA
interference experiments suggested that the barley (Hordeum vulgare) RAC/ROP protein RACB is involved in susceptibility to
the powdery mildew fungus Blumeria graminis f. sp. hordei. We created transgenic barley plants expressing the constitutively
activated RACB mutant racb-G15V under control of the maize (Zea mays) ubiquitin 1 promoter. Individuals of the T1 generation
expressing racb-G15V were significantly more susceptible to B. graminis when compared to segregating individuals that did not
express racb-G15V. Additionally, racb-G15V-expressing plants showed delayed shoot development from the third leaf stage on,
downward rolled leaves, and stunted roots. Expression of racb-G15V decreased photosynthetic CO2-assimilation rates and
transpiration of nonstressed leaves. In contrast, racb-G15V-expressing barley leaves, when detached from water supply,
showed increased water loss and enhanced transpiration. Water loss was associated with reduced responsiveness to abscisic
acid in regard to transpiration when compared to segregants not expressing racb-G15V. Hence, RACB might be a common
signaling element in response to both biotic and abiotic stress.
Early interaction of plants with the biotic and abiotic
environment requires perception and transduction of
extracellular signals. Communication between extracellular and intracellular compartments in eukaryotes
is mediated by endocytotic pathways and/or plasma
membrane receptor-mediated signaling. The processing of extracellular signals often involves GTP-binding
proteins. The RHO-related subclass of plant small
monomeric GTPases is called RAC or ROP (Rho of
plants). RAC/ROPs act as molecular switches in cell
polarity, hormone signaling, and plant defense. Activation of downstream effectors by RAC/ROP requires
binding of GTP, and, in turn, GTP-mediated RAC/
ROP activity is abolished by intrinsic or stimulated
GTP-hydrolyzing activity. Hence, RAC/ROP proteins
can be constitutively activated by mutation of the
intrinsic GTPase function. In contrast, wild-type
RAC/ROP activity is strictly regulated, in plants
1
This work was supported by the GABI program of the Federal
Ministry of Education and Research (BMBF; grant GABI-AGROTEC
to K.-H.K. and U.S.) and by the German Research Foundation (grant
no. DFG HU886/1 to R.H.).
2
These authors contributed equally to the paper.
3
Present address: European Commission, JRC-IRMM-RM Unit,
Retieseweg 111, 2440 Geel, Belgium.
* Corresponding author; e-mail ralph.hueckelhoven@agrar.
uni-giessen.de; fax 49–641–9937499.
Article, publication date, and citation information can be found at
www.plantphysiol.org/cgi/doi/10.1104/pp.105.066613.
most likely by receptor-like kinases, GTPase-activating
proteins, guanine nucleotide exchange factors, and
guanine nucleotide dissociation inhibitors (Gu et al.,
2004; Berken et al., 2005). Winge et al. (2000) subdivided the 11 Arabidopsis (Arabidopsis thaliana) RAC/
ROP proteins into two major subgroups that can be
distinguished by length due to an additional exon in
group II. Cereals appear to express six to nine RAC/
ROP genes (Christensen et al., 2003; Schultheiss et al.,
2003). Based on the available barley (Hordeum vulgare)
transcriptome data, six barley full-length RAC/ROP
cDNAs have been isolated, and no further family
members could be identified in more than 300,000
expressed sequence tags (Schultheiss et al., 2003).
Similar to Arabidopsis RAC/ROPs, barley family members can be subdivided into group I and group II RAC/
ROPs by length. Likewise, similar to Arabidopsis,
barley RAC/ROPs can be further distinguished into
a total of four clades (Schultheiss et al., 2003).
Plant susceptibility to biotrophic fungi is little understood (Schulze-Lefert and Panstruga, 2003; Hückelhoven,
2005). Successful pathogens target host proteins to
bypass or to suppress basic defense mechanisms. Seemingly, such target proteins are essential to the host
because resistant mutants often show pleiotropic growth
or cell death phenotypes (Vogel and Somerville, 2000).
Barley susceptibility to the biotrophic powdery
mildew fungus Blumeria graminis f. sp. hordei (Bgh) is
dependent on the host receptor-like MLO protein.
MLO is believed to be used by the fungus for host
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353
Schultheiss et al.
defense suppression (for review, see Schulze-Lefert
and Panstruga, 2003; Hückelhoven, 2005). Mutants
that do not express functional MLO are completely
resistant to penetration by Bgh. This type of resistance
is in turn limited in ror1 and ror2 mutants, which are
affected in independent loci required for mlo-mediated
resistance (Freialdenhoven et al., 1996). ROR1 is not
yet identified, but ROR2 is a plasma membrane syntaxin that locally accumulates at the site of attempted
fungal penetration, where it appears to be involved in
exocytosis of defense-related compounds (Collins
et al., 2003; Assaad et al., 2004; Bhat et al., 2005).
Transient transformation-mediated overexpression
and RNA interference are used as test systems to assess
gene function in the interaction of cereals with powdery
mildew fungi (Nielsen et al., 1999; Schweizer et al.,
1999, 2000; Panstruga, 2004). Transient RNA interference-mediated knock down of the barley RAC/ROP
protein RACB renders epidermal cells more resistant to
penetration by Bgh. In contrast, transient overexpression of the constitutively activated RACB mutant racbG15V but not of wild-type RACB supports fungal
penetration (Schultheiss et al., 2002, 2003). Hence, abundance and activity of RACB appear to be crucial for
single-cell accessibility to Bgh. The exact mechanism of
RACB activity is not known. However, RACB effects on
susceptibility are dependent on functional MLO and
ROR1 (Schultheiss et al., 2002, 2003). RACB could thus
be involved in MLO-dependent ROR1 antagonism.
RACB is a group I RAC/ROP falling into clade IV
with maize (Zea mays) ROP2, ROP4, and ROP9, rice
(Oryza sativa) RACB and RACD, and Arabidopsis ROPs
1 to 6 (Christensen et al., 2003; Schultheiss et al., 2003).
RACB is a plasma membrane-associated protein. This
localization is mediated by a CAAX-box prenylation
motif at the C terminus, which is required for RACB
function in susceptibility to Bgh (Schultheiss et al., 2003).
Additionally, single-cell RACB overactivation hampers actin-filament reorganization in Bgh-attacked cells,
whereas RACB knock down promotes polarization
(Opalski et al., 2005). Hence, RACB modulates actin reorganization during attack by Bgh. In contrast to barley
RACB, rice RAC1 is functioning in the oxidative burst
and defensive cell death in response to the hemibiotrophic fungus Magnaporthe grisea (Ono et al., 2001).
Hence, small RAC/ROP G proteins appear to be active
in resistance and susceptibility to plant pathogenic fungi.
In this study, we genetically transformed susceptible
barley to constitutively express racb-G15V. Our results
support the role of RACB in susceptibility to Bgh and
suggest a potential involvement of RACB in plant development as well as in response to abiotic stress.
RESULTS
Generation of Transgenic racb-G15V Barley
and Phenotypes
Transient transformation of barley epidermal cells is
widely used to assess gene function in the interaction
of cereals and powdery mildew fungi (Nielsen et al.,
1999; Schweizer et al., 1999, 2000). However, little
direct evidence exists for transferability of results
from transient single-cell expression to entire plants
(Altpeter et al., 2005). To validate that transient transformation is a suitable tool to assess gene function in
interaction with powdery mildew fungi and to assess
further functions of barley RACB, we produced transgenic barley plants that express the constitutively activated RACB-G15V protein under control of the maize
ubiquitin 1 promoter ZmUbi1 (Christensen and Quail,
1996). To this end, the barley cultivar Golden Promise
was stably transformed with Agrobacterium tumefaciens
strain AGL1 (Lazo et al., 1991) employing a method
described earlier (Hensel and Kumlehn, 2004). Seventyone independent primary transgenic lines were generated from 470 immature embryos infected. We observed
that the T0 generation of candidate racb-G15V barley
was reduced in growth as compared to nontransgenic
donor plants of Golden Promise, and only 43 of these
lines set seeds, indicating an impact of the transferred
expression cassette on plant development.
To test individual plants of the T1 generation for
transgene expression by reverse transcription (RT)PCR, we designed oligo DNA primers that distinguish
endogenous RACB mRNA from racb-G15V mRNA. In
contrast to what was expected, the T1 generation of
nine independent transgenic lines segregated about 1:1
(65:62) instead of 1:2:1 (3:1 for transgene expression;
lines 17-1-11, 17-1-15, 17-1-22, 17-1-23, 18-1-6, 18-1-10,
18-1-16, 18-1-19, and 18-1-21). A x 2 test confirmed a 1:1
segregation (x 2 test for a 1:1 segregation: x 25 0.07; P 5
0.79). One:one segregation was also found for 50
individuals of the T2 generation of line 17-1-23 (x 25
0.08; P 5 0.78). This might indicate male sterility of racbG15V barley. The transgenic status of individuals was
confirmed by genomic DNA blotting. Thereby, DNA
analysis of individual T1 segregants consistently corroborated previous RT-PCR results (data not shown).
Therefore, we considered individuals with negative
results in the racb-G15V RT-PCR as T1 plants that lost
racb-G15V by segregation (segregants not expressing
racb-G15V; synonym: racb-G15V-negative individuals).
Out of seven lines tested in DNA-blot experiments,
six contained single transgene copies (17-1-11, 17-1-23,
18-1-6, 18-1-7, 18-1-10, and 18-1-21) and one contained
two copies (17-1-37; data not shown).
To assess a potential RACB function during plant
development, we judged plant habit over time. Individuals that expressed racb-G15V were macroscopically
indistinguishable from segregants that lost racb-G15V
until they reached the three-leaf stage 3 weeks after
germination. The subsequently developing leaves
showed a downward rolled phenotype and unfolded
about 1 week later when compared to segregants not
expressing racb-G15V in eight out of nine independent
lines (Table I). After leaf unfolding, leaves often remained twisted. This phenotype was associated with
expression of racb-G15V in 100% of cases as checked by
RT-PCR. From 3 to 5 weeks after germination onward,
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RACB-Induced Altered Barley Phenotypes
Table I. Overview of transgenic lines expressing racb-G15V and corresponding phenotypes
Phenotypea
racb-G15VExpressing Line
17-1-3d
17-1-11
17-1-15
17-1-22
17-1-23
18-1-6
18-1-10
18-1-19
Enhanced Fungal
Penetration
(RT-PCR1/Total)b
(4/9)
(6/10)
(3/9)
(4/10)
n.d.
2 (3/7)
1 (11/19)
1? (5/11)
2
1
1
1
Enhanced Bgh
Colony Development
(RT-PCR1/Total)b
2
1
1?
2
1
1
1
(5/10)
(6/10)
(2/10)
(4/10)
(20/39)b
(3/10)
(14/20)
n.d.
Rolled
Leavesc
Dwarfedc
Enhanced
Water Loss
Reduced
Response to ABA
Reduced Photosynthetic
Capacity
–
1
1
1
1
1
1
1
–
1
1
1
1
1
1
1
n.d.
1
n.d.
n.d.
1
1
1
n.d.
n.d.
1
n.d.
n.d.
n.d.
1
n.d.
n.d.
n.d.
1
n.d.
n.d.
n.d.
1
1?
n.d.
a
1 labels lines with a detectable phenotype;2 labels lines without detectable phenotype; 1? labels lines with an altered phenotype that was not
b
statistically significant at P , 0.05.
Numbers of segregants expressing racb-G15V of total n tested in the T1 generation; for line 17-1-23, the T2
c
d
generation was tested.
Phenotypes could be observed from 3 weeks after germination onward.
Line 17-1-3 expressed racb-G15V on a very
low level (Fig. 5).
racb-G15V-positive individuals showed a delayed development and dwarfed growth, shorter internodes,
less tillering, and delayed or missing heading (Fig. 1;
Table I; data not shown). To assess root phenotypes, we
grew T1 segregants in Oildri that makes easy liberation
of roots from the substrate possible. Roots of segregants
expressing racb-G15V were stunted and had fewer
branches when compared to segregants of the same
line not expressing racb-G15V. We measured the root
length of the segregating T1 populations. In average,
root length of 3-week-old racb-G15V-positive segregants
was reduced by about 40%. In contrast, root hairs of
segregants expressing racb-G15V appeared normal.
Together, segregants expressing racb-G15V appeared
to be retarded in organ expansion. However, when
segregants that expressed racb-G15V completed heading, shoot length was indistinguishable from that of
controls.
To confirm that the maize ubiquitin promoter used
to drive racb-G15V expression is active in all barley
tissues where we observed altered phenotypes, we
generated barley Golden Promise expressing the green
fluorescent protein (GFP) under control of ZmUbi1.
GFP expression was observed by confocal laser scanning microscopy in all tissues of the GFP-expressing
T1 individuals analyzed. This confirmed ubiquitous
activity of the ZmUbi1 promoter in barley (Fig. 2). To
confirm a 1:1 segregation for one-copy insertions in
pollen, we counted the frequencies of pollen expressing and not expressing GFP under the fluorescence
microscope. We found a 224:231 segregation (x 2 test
for a 1:1 segregation: x 25 0.11; P 5 0.75), which
underscored the possibility that male sterility of racbG15V-expressing plants might be caused by strong
expression of racb-G15V in pollen.
RACB-G15V-Expressing Individuals Are
Photosynthetically Less Active
To further test whether photosynthesis is impaired
in racb-G15V-expressing plants, we measured CO2 as-
similation rates under different light conditions. When
we compared segregants from three independent
lines, we observed that racb-G15V-expressing segregants had a reduced photosynthetic capacity under
medium to high light conditions (shown for line 18-1-6
in Fig. 3). This effect was of similar strength in three
lines tested (17-1-11, 18-1-6, and 18-1-10).
RACB-G15V-Expressing Individuals Are More
Susceptible to Bgh
We analyzed cellular accessibility to Bgh and mildew
symptom development after artificial inoculation of
the T1 generation. We tested first leaves from 10-d-old
plants because they had no obvious developmental
phenotype. About 10 segregants of seven independent
lines were analyzed for fungal penetration success and
haustorium establishment on the microscopic level
at 48 h after inoculation. In total, on racb-G15V segregants, 20% more fungal germlings succeeded in penetration when compared to segregants not expressing
racb-G15V. In individual lines, racb-G15V enhanced
penetration rates by 10% to 40%. Due to the high
variability of penetration frequencies on individual
leaves and the partially limited numbers of seeds, this
effect was statistically significant for only five of the
seven individual lines tested (Table I). However, racbG15V-mediated susceptibility to penetration was statistically significant at P , 0.01 over all seven lines of
the T1 generation (Student’s t test). The frequency of
hypersensitive cell death reactions of single attacked
cells was not affected by racb-G15V in any line (data
not shown). We scored 200 interaction sites on each of
the segregants and grouped them into categories of
fungal penetration rates. This also indicated that the
population of segregants expressing racb-G15V was
significantly shifted toward higher accessibility to
fungal penetration (Fig. 4A).
Five days after inoculation of third leaves, mildew
colonies were counted on the same lines. Segregants
expressing racb-G15V supported about 40% more
Plant Physiol. Vol. 139, 2005
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Schultheiss et al.
two lines showing strongly altered phenotypes (Fig. 5).
Thus, the lack of a racb-G15V-mediated phenotype in
17-1-3 appeared to be due to low transgene expression.
It was speculated that susceptibility to one fungal pathogen might limit susceptibility to fungi with
different lifestyles (Brown, 2002). Since racb-G15Vexpressing T1 individuals were more susceptible
to the biotrophic fungus Bgh, we tested racb-G15Vtransgenic leaves for their ability to resist inoculation
with the hemibiotrophic fungus Bipolaris sorokiniana
and the necrotrophic fungus Fusarium graminearum. In
both cases, segregants expressing racb-G15V showed
as much necrotic symptoms as segregants not expressing racb-G15V (data not shown).
Barley Expressing racb-G15V Shows Enhanced Water
Loss under Stress Conditions
Arabidopsis RAC/ROP-like proteins are involved
in abiotic stress signaling. Therefore, we tested racbG15V T1 plants for their ability to maintain turgor
under stress conditions. Excised leaves of segregants
that expressed racb-G15V completely lost turgor overnight when kept at room temperature on the bench.
In contrast, segregants not expressing racb-G15V partially retained turgor. Within 48 h racb-G15V-positive
segregants dried out completely, whereas racb-G15Vnegative individuals partially retained water (data not
shown). To quantify water loss, we gravimetrically
analyzed excised leaves of three independent lines
during a period of 24 h at 20°C and 40% relative
humidity. Segregants not expressing racb-G15V lost
Figure 1. Development of T1 individuals expressing racb-G15V. A,
Greenhouse growth habit of racb-G15V expressing (1) and not
expressing (2) T1 individuals 90 d after germination (transgenic line
17-1-23). B, Third leaves of racb-G15V expressing (1) and not
expressing (2) T1 individuals 28 d after germination. C, Roots of
racb-G15V expressing (1) and not expressing (2) T1 individuals 28 d
after germination.
disease development than individuals not expressing
racb-G15V (Fig. 4B). This effect was detected in five of
the seven lines analyzed (Table I). Again, despite the
fact that the individual susceptibility level of single
leaves is generally variable, the racb-G15V effect was
statistically significant over all lines at P , 0.01.
Line 17-1-3 showed neither a growth phenotype nor
a susceptibility phenotype. Hence, we re-evaluated
racb-G15V expression in line 17-1-3 by RNA gel-blot
experiments. This revealed that this line expressed the
transgene at a much lower level when compared to
Figure 2. Ubiquitous expression of GFP under control of the ZmUbi1
promoter. A to I, Confocal laser scanning microscopy of ZmUbi1::GFP
barley. The maize ubiquitin promoter drives GFP expression in the leaf
epidermis and mesophyll (A); pericarp but not kernel hairs (B and C);
roots, root apex, and root hairs (D–F); and stamen and pollen (G–I). The
size bars represent 100 mm.
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RACB-Induced Altered Barley Phenotypes
G15V-expressing segregants were treated with water
instead of ABA, they showed up to 50% less transpiration than racb-G15V-negative segregants before, during, and at the end of the experiments. These results
indicate that racb-G15V-expressing individuals were
not generally more conductive for water but were less
responsive to ABA, and, hence, they could not effectively reduce transpiration.
Figure 3. T1 individuals expressing racb-G15V have limited CO2
assimilation rates. Photosynthesis was measured as CO2 assimilation
rate depending on light intensities. Segregants expressing racb-G15V
according to RT-PCR show less photosynthetic capacity when compared to segregants of the same T1 generation (line 18-1-6) not
expressing racb-G15V. Error bars show SDs of the mean (n 5 4). Lines
18-1-10 and 17-1-11 showed similar results.
about 60% of their original weight, whereas racb-G15Vexpressing individuals lost 80% of their original
weight (shown for line 18-1-10 in Fig. 6A). Expression
of racb-G15V did not influence leaf dry weight (data
not shown).
To distinguish potential transgene-dependent cuticle
defects (Chen et al., 2004) from altered stomatal conductivity, we measured chlorophyll release into 80%
watery ethyl alcohol over time and transpiration rates,
respectively. Transcuticular chlorophyll release was
not different in segregants not expressing racb-G15V
versus those expressing racb-G15V (data not shown).
However, porometry indicated that transpiration rates
remained on a high level in racb-G15V-positive individuals 16 h after leaf detachment (50%–80% of t 5 0),
whereas racb-G15V-negative individuals were able to
reduce transpiration to about 20% to 40% of what was
measured at t 5 0 (shown for line 18-1-10 in Fig. 6B).
Hence, leaves expressing racb-G15V held up high
transpiration rates when detached from water supply.
This was in contrast to nonstressed leaves.
Since transpiration under stress condition is regulated by abscisic acid (ABA), we measured the influence of ABA on stomatal conductivity. After 4 min
of adaptation to the measuring cell, excised leaves
were placed in a 50 mM ABA solution. This strongly
reduced transpiration in T1 plants not expressing racbG15V over time to 4% of what was measured at t 5
0 when ABA was applied. In contrast, following the
same treatment, individuals expressing racb-G15V less
effectively reduced transpiration to 21 % of the value
at t 5 0 (Fig. 7). Similar results were obtained with
10 mM ABA, except that final transpiration rates were
higher (14% of t 5 0 in racb-G15V-negative versus 36%
of t 5 0 in racb-G15V-positive individuals). Absolute
transpiration rates of racb-G15V barley plants remained at a higher level after ABA treatment when
compared to wild-type segregants. In contrast, if racb-
Figure 4. T1 individuals expressing racb-G15V show enhanced susceptibility to powdery mildew. Susceptibility was measured as fungal
penetration success and colony development. A, A total of 15,000
interaction sites on 75 T1 individuals from seven lines were judged for
penetration success by Bgh. Cells were considered as penetrated if
a fungal haustorium was easily visible within the first attacked cell.
Subsequently, each individual was categorized for fungal success from
less than 20% successful fungal germlings to more than 45% of
successful germlings. Each individual was tested for racb-G15V
expression by RT-PCR, and the T1 generation was subdivided into
two subpopulations: white columns represent segregants expressing
racb-G15V, and black columns represent segregants not expressing
racb-G15V. Finally, percentages of individuals of each subpopulation
falling into a category of accessibility to Bgh were calculated and
displayed. x 2 test confirmed that subpopulations were statistically
different (x 25 54.7; P , 0.001). B, About 10 individuals each of seven
independent lines were judged for early symptom development 5 d after
powdery mildew inoculation. Growing colonies of heavily branched
surface hyphae and developing conidiophores of Bgh were counted.
Error bars show SDs of the mean.
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Schultheiss et al.
Figure 5. Expression of racb-G15V in T1 individuals of barley Golden
Promise transformed with ZmUbi1::racb-G15V. RNA gel-blot analysis
of racb-G15V expression in arbitrarily selected individuals of three
transgenic lines (18-1-10 [individuals 1, 5, and 8], 17-1-3 [individuals
4, 6, and 8], 17-1-11 [individuals 9, 10, and 11]) and Golden Promise
(GP). All individuals that express racb-G15V according to RT-PCR also
show a racb-G15V RNA-hybridization signal. However, the expression
level in 17-1-3 is very low. Accordingly, RT-PCR-positive individuals of
17-1-3 did not show racb-G15V-mediated phenotypes like observed in
18-1-10 and 17-1-11. In gel-stained rRNA serves as a measure for RNA
loading.
ceptibility at the macroscopic level 5 d after inoculation
(Fig. 4B). Bgh developed more colonies on racb-G15Vpositive segregants than on racb-G15V-negative segregants. This supports that single-cell accessibility limits
background resistance. The fact that early fungal
colony growth is supported by RACB activity in third
leaves is astonishing considering the observation that
racb-G15V barley had limited photosynthetic capacity
(Fig. 3) because Bgh, as an obligate biotroph, is dependent on host carbohydrate supply.
The function of RACB was linked to the function of
MLO (Schultheiss et al., 2002, 2003), and Bgh-resistant
barley mlo mutants show enhanced susceptibility to
the hemibiotrophic fungus M. grisea and to the toxic
culture filtrate of B. sorokiniana (Jarosch et al., 1999;
Kumar et al., 2001). Additionally, RAC/ROP functions
have been linked to effective defense responses in
several plant pathogen interactions (Ono et al., 2001;
Agrawal et al., 2003; Moeder et al., 2005). Therefore,
we speculated that RACB might have a role in background resistance to other pathogens. However, when
DISCUSSION
We have generated transgenic barley lines expressing the constitutively active mutant RAC/ROP protein
RACB-G15V. Individuals expressing racb-G15V show
multiple altered phenotypes when compared to individuals not expressing racb-G15V from the same
generation. Among them, organ expansion defects, enhanced transpiration rates under stress conditions, and
enhanced susceptibility to Bgh are most obvious. This
suggests that small G proteins are involved in barley
development and response to both biotic and abiotic
stress.
Biolistic transformation of single cereal cells with
subsequent challenge by B. graminis has been widely
used to assess gene function in defense and susceptibility (Panstruga, 2004). However, direct evidence for
gene function on entire plant level assessed by singlecell transformation is rare (Altpeter et al., 2005). Here,
we report that a similar degree of enhanced susceptibility to penetration by Bgh was obtained when we
compared racb-G15V barley plants with single-cell
transformation (Fig. 4; Schultheiss et al., 2003). RACBG15Venhanced susceptibility of cultivars Ingrid, Pallas,
and Golden Promise in transient or stable overexpression experiments (Schultheiss et al., 2003; this study;
H. Schultheiss, unpublished data). Together, this supports a function of active RACB in background susceptibility to Bgh and further validates the transient
transformation assay as a suitable tool for gene function assessment in the interaction with B. graminis.
Enhanced susceptibility to penetration by Bgh was
observed on the first leaf, not having an obvious
racb-G15V-dependent developmental phenotype. This,
together with evidence from transient single-cell transformation in fully developed leaves and dependency
of RACB effects on MLO and/or Ror-1, indicates
that RACB activity may directly support entry by Bgh.
T1 plants expressing racb-G15V showed enhanced sus-
Figure 6. T1 individuals expressing racb-G15V have reduced water
retention capacities and enhanced transpiration rates when detached
from water supply. A, Average weight loss of 10 detached leaves during
the first 24 h after detachment (line 18-1-10). B, Average transpiration
rates of 10 T1 individuals 16 h after leaf detachment. Each individual
was tested for racb-G15V expression by RT-PCR, and the population
subdivided in two groups (segregants expressing racb-G15V and segregants not expressing racb-G15V ). Error bars show SDs of the mean.
Lines 18-1-6 and 17-1-11 showed very similar results.
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RACB-Induced Altered Barley Phenotypes
Figure 7. T1 individuals expressing racb-G15V are less responsive to
ABA treatment. Transpiration was measured after placing excised
leaves in a solution of 50 mM ABA or aqua destillata. (control). Data
are shown for line 18-1-6 examined for racb-G15V expression by RTPCR. Very similar results were obtained in experiments with line 17-111 and in independent reproductions. Error bars represent SEs of four
independent plants.
we inoculated racb-G15V-positive leaf segments with
either F. graminearum or B. sorokiniana, we did not
detect a significant difference in background resistance
when compared to racb-G15V-negative segregants
(data not shown). Although this does not exclude
altered responses of racb-G15V barley to other pathogens or in other tissues, data indicate that there is no
general link between barley susceptibility to Bgh and
resistance to necrotizing pathogens.
Constitutive RACB activity in transgenic barley
apparently provoked developmental phenotypes in
roots and shoots (Fig. 1). To confirm that RACB-G15V
might be responsible for these phenotypes, we analyzed racb-G15V expression by northern blots and by
use of a reporter plant expressing GFP under control of
the same promoter that was used to drive racb-G15V
expression. Northern blots showed that strong leaf
expression of racb-G15V is associated with leaf phenotypes in regard to leaf folding and susceptibility to
Bgh. The GFP-expressing reporter lines showed GFP
expression in all tissues analyzed, including those
showing racb-G15V-mediated phenotypes. This is in
accordance with observations in wheat (Triticum aestivum), in which the maize ubiquitin promoter drives
ubiquitous reporter gene expression, in particular in
young metabolically active tissue and pollen grains
(Rooke et al., 2000). Hence, RACB-G15V might be
directly responsible for shoot and root defects. Additionally, our analyses of expression of RACB from its
endogenous promoter and data mining in public
barley databases (http://www.tigr.org/tigr-scripts/tgi/
T_index.cgi?species5barley; http://pgrc.ipk-gatersleben.
de/b-est/; http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db5unigene) for expressed barley sequence tag
abundance (e.g. Zhang et al., 2004) indicate that RACB
is expressed in leaf epidermis, leaf mesophyll, earlets,
male and female inflorescences, caryopses, pericarps,
embryos, coleoptiles, stems, and roots (Schultheiss
et al., 2002; data not shown). Since we observed a 1:1
segregation in T1 and T2 populations, we speculate
that racb-G15V may cause male sterility at high expression levels. RACB-like proteins have already been
implicated in pollen tube growth and male gametophyte development in Arabidopsis, rice, and maize.
Overexpression of constitutively activated RAC/ROPs
induces loss of polarity in pollen tube growth (Kost
et al., 1999; Arthur et al., 2003; Xu et al., 2004; Gu et al.,
2005).
RACB-G15V provoked obvious organ expansion
phenotypes in roots and shoots. Leaves showed a
rolled downward phenotype rather than a leaf expansion phenotype. Heading was delayed or inhibited.
Individuals that expressed racb-G15V and produced
ears finally reached the height of individuals not expressing racb-G15V. Some of the racb-G15V-induced
phenotypes are reminiscent of hormone signaling defects such as those observed in auxin or ABA mutants.
Since RACB function was recently linked to cell polarization and racb-G15V inhibits actin reorganization in
host defense (Opalski et al., 2005), racb-G15V barley may
be decelerated in cell polarization required for organ
expansion. Because double-stranded-RNA-mediated
knock down of RACB induces resistance to Bgh, we
also speculated that RACB participates in the polar
membrane growth process that is involved in plasmalemma invagination during haustorium establishment
(Schultheiss et al., 2002, 2003).
Besides constitutively activated RACB-G15V, barley
RAC3-G17V similarly induces enhanced accessibility
to Bgh in transiently transformed cells (Schultheiss
et al., 2003). Arabidopsis RAC10, a relative of barley
RAC3, was recently ectopically overexpressed in Arabidopsis to study potential protein functions (Bloch et al.,
2005). This revealed a role for RAC10 in actin organization and endocytosis. Similar to racb-G15V barley,
rac10-G15V Arabidopsis had downward rolled leaves.
However, in contrast to racb-G15V barley but similar to
other constitutively activated Arabidopsis RAC/ROP
mutants (Molendijk et al., 2001; Jones et al., 2002; Fu
et al., 2005), rac10-G15V Arabidopsis had an altered
root hair phenotype. Together, one might speculate
that constitutively activated RAC/ROPs partially cross
activate downstream events of individual RAC/ROPs
when ectopically expressed.
Constitutive RACB activity in transgenic barley
limited leaf water retention capacity. Barley excised
leaves expressing racb-G15V completely lost turgor
within 24 h. This can be explained by a failure of racbG15V barley to reduce transpiration when cut off from
water supply (Fig. 6). The reduced ABA responsiveness of racb-G15V barley in regard to reduction of
transpiration argues for a defect in ABA-responsive
stomata closure. We observed neither a cuticula defect
nor a morphological stomata defect in racb-G15V
Plant Physiol. Vol. 139, 2005
359
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Copyright © 2005 American Society of Plant Biologists. All rights reserved.
Schultheiss et al.
barley. Hence, constitutive RACB activity appears to
antagonize ABA function in regulation of leaf water
conductivity. In contrast, transpiration was lower in
racb-G15V-expressing T1 plants that were not treated
with ABA than in segregants not expressing racb-G15V
under high light conditions. Reduced transpiration
rates under optimal light conditions might also indicate a reduced CO2 exchange capacity, and this might
explain limited photosynthesis. Arabidopsis ROP10,
another relative of barley RAC3, is specifically involved in negative regulation of ABA effects (Zheng
et al., 2002). The close RACB relative Arabidopsis
RAC1 negatively regulates ABA-induced stomata closure and ABA-induced actin disassembly in stomata
(Lemichez et al., 2001). We compared patterns of
assembled filamentous actin in stomata of racb-G15Vpositive and racb-G15V-negative segregants by confocal laser scanning microscopy after ABA treatment
and phalloidin staining. We observed that individuals
not expressing racb-G15V disassembled a part of the
filamentous actin in stomata after ABA treatment and
that barley expressing racb-G15V had a tendency to
hold up a greater portion of actin in a filamentous form
(data not shown). Transient expression of racb-G15V
inhibits actin dynamics and polarization processes in
the interaction with Bgh (Opalski et al., 2005). Therefore, the function of RACB in actin reorganization may
not only explain enhanced susceptibility, but also
reduced ABA responsiveness and delayed organ expansion of racb-G15V barley. The overlap of phenotypes
induced in dicots and the monocot barley by activated
RAC/ROP supports the view that small RHO-like plant
GTPases regulate similar processes in many higher
plants. Eventually, barley RACB appears to be a susceptibility factor in the interaction with Bgh and to have
a role in plant development and adaptation to leaf
water potentials. This further supports the idea that
biotrophic fungi corrupt essential plant signaling pathways for reprogramming of the attacked host.
MATERIALS AND METHODS
Plant and Pathogen Material
Transgenic plants and the parent cultivar Golden Promise were grown in
a growth chamber at 22°C, 60% relative humidity, and a photoperiod of 16 h
(150 mmol s21 m22 photon flux density) up to E.C. 30. From the fifth week after
germination onward, plants were grown in a greenhouse at 20°C (16-h day;
60 mmol s21 m22 photon flux density) and 16°C (8-h night), each with 60%
relative humidity.
The barley (Hordeum vulgare) powdery mildew fungus Blumeria graminis
(DC) Speer f. sp. hordei Em. Marchal, race A6 (Bgh; Wiberg, 1974), was
maintained on barley cultivar Siri under the same conditions.
For microscopic evaluation, Bgh was inoculated onto detached primary
leaves of 10-d-old barley plants to give a density of 30 conidia mm22. The
outcome of the interaction was evaluated after 48 h using light and UV
microscopy. For macroscopical analysis, detached third leaves were inoculated with a density of 5 conidia mm22 and evaluated 5 to 7 d after inoculation.
Barley Transformation by Agrobacterium tumefaciens
Stable genetic transformation of barley with ZmUbi::racb-G15V was performed as described by Hensel and Kumlehn (2004). In brief, immature
embryos of barley Golden Promise were excised 10 to 12 d after anthesis and
subsequently infected with Agrobacterium tumefaciens strain AGL1 (Lazo et al.,
1991) harboring the appropriate binary vector. After infection, callus development was induced on medium containing 50 mg/L hygromycin B
(Roche) to ensure preferential growth of transformed plant cells. Established
calli were then subcultured on regeneration medium supplemented with
25 mg/L hygromycin B until rooted plantlets could be transferred to soil.
Barley plants were grown and transformed with ZmUbi::GFP as described
by Tingay et al. (1997) and Matthews et al. (2001). After surface sterilization for
3 min with 70% ethanol and 20 min with a sodium hypochlorite solution
containing 3% active chlorine and rinsing three times with sterile distilled
water, the infection of immature embryos with Agrobacterium was performed
directly after isolation. We induced callus growth on Murashige and Skoog
medium (Murashige and Skoog, 1962) containing 50 mg/L hygromycin B
(Roche) as a selective agent for transformed plant cells. Regeneration medium
contained 50 mg/L hygromycin B and rooting medium 25 mg/L hygromycin
B. Timentin (150 mg/L; Duchefa Biochemie) was applied as long as tests for
the presence of bacteria were negative.
DNA Cloning
For constitutive expression of the racb-G15V gene in barley, the BamHI/SalI
fragment from plasmid pGY1-RacB-V15 (Schultheiss et al., 2003) was subcloned into appropriate sites of the binary vector pSB181 (S. Broeders,
unpublished data). Plasmid pSB181 was designed by integration of the SfiI
fragment of plasmid pUbi-AB (maize [Zea mays] ubiquitin 1 promoter [ZmUbi-1] containing the first intron in front of the start codon and nos terminator;
DNA Cloning Service) into the pLH6000 vector (GenBank accession no.
AY234328; DNA Cloning Service). Vector pLH6000 contains a T-DNA with the
hygromycin-resistance gene hpt driven by the cauliflower mosaic virus 35S
promoter and terminator, and a multiple cloning site for integration of
additional expression cassettes.
For in planta expression, the GFP gene (GFP out of pGY1-GFP; Schweizer
et al., 1999) was subcloned with restriction enzymes into pUbi-AB. The
transfer of the complete expression cassette into the binary vector pLH6000
was then performed as described above using SfiI sites. All binary plasmids
were then introduced into the A. tumefaciens strain AGL1 (Lazo et al., 1991) by
electroporation (Gene Pulser; Bio-Rad) according to manufacturer’s instructions. Analysis of stably transformed material using the gfp reporter was
performed with either a standard fluorescence microscope or a confocal laser
scanning microscope TCS SP2 AOBS (excitation: laser line 488 nm; emission:
500 to 540 nm; Leica).
Measurement of Photosynthetic Capacities
Photosynthetic capacities were measured in each of nine to 13 T1
individuals of three transgenic lines. Segregants were exposed to increasing
light intensities at 52% relative humidity. Net CO2 uptake rates, transpiration
rates, stomatal conductance, and intercellular CO2 concentrations were determined according to Hajirezaei et al. (2002), using a portable photosynthesis
system LI-6400 (LI-COR). The CO2 concentration of the air entering the leaf
chamber and the leaf temperature were adjusted to 400 mmol mol21 and 20°C,
respectively. Photon flux density was varied between 20 and 2000 mmol
quanta m22 s21.
Water Loss Assay and Porometry
For water loss measurements, third leaves were cut and kept on filter paper
at 20°C (40% relative humidity and 16-h-light period with 60 mmol s21 m22
photon flux). To quantify water loss, we gravimetrically analyzed the leaves
during a period of 24 h. To determine the dry weight, leaves were kept for 48 h
in an incubator at 70°C.
For transpiration measurements, third leaves were cut and incubated
on 0.5% water-agar at 20°C, 60% relative humidity, and a light intensity of
60 mmol s21 m22 photon flux. Transpiration rates of the leaves were measured
under the same conditions 16 h after detachment using a LICOR 1600
porometer.
For measurement of ABA effects, the excised third leaves were kept under
1,500 mmol s21 m22 photon flux at 52% relative humidity and 25°C until they
reached maximal leaf conductivity. Measurement was started 4 min before
ABA application. Subsequently, leaves were transferred into aqua destillata or
ABA solutions, respectively, and water conductivity was measured over time
for 21 min (system LI-6400; LI-COR).
360
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Copyright © 2005 American Society of Plant Biologists. All rights reserved.
RACB-Induced Altered Barley Phenotypes
RNA-Gel Blots
would like to thank Cornelia Marthe and Christiane Börnke for excellent
technical assistance.
Total RNA was extracted from three leaves (fourth and higher) of T1
individuals using RNA extraction buffer (PEQLAB) according to the manufacturer’s instructions. For RNA gel-blot analysis, 15 mg of total RNA was
separated on a 1.2% agarose gel and blotted on Hybond N1 nylon membranes
(Amersham Biosciences Europe GmbH). The RNA content of the extracts was
measured by UV photometry and was adjusted after checking in ethidium
bromide-stained gels taking rRNA bands as a standard.
32
P-probe labeling of PCR-amplified RACB cDNA (GenBank accession no.
AJ344223; primers, 5#-GGATCCGATGAGCGCGTCCAGGTT-3# and 5#-GTCGACCTTCGCCCTTGTTCTTTGTC-3#) was carried out using the random
prime HexaLabel DNA labeling kit (Fermentas GmbH) following the manufacturer’s instructions. The RNA-gel blots were hybridized overnight at 62°C
in 0.5 M sodium phosphate buffer, pH 7.2, containing 0.5% (w/v) bovine
serum albumin, 3.5% (w/v) SDS, and 0.5 mM sodium EDTA and stringently
washed three times at 62°C in 0.13 SSC/0.1% SDS for 5 min each. Signals
were detected with a Molecular Imager FX Phosphorimager (Bio-Rad).
RT-PCR Analysis
Total RNA was extracted from one sary leaf using RNA extraction buffer
(PEQLAB) according to the manufacturer’s instructions. The OneStep RT-PCR
kit (Qiagen) was used for semiquantitative RT-PCR following the manufacturer’s instructions. To demonstrate the expression of transformed racb-G15V,
500 ng of total RNA was used for RT-PCR (prePCR: 50°C 30 min, 95°C 15 min;
PCR: 95°C 30 s, 55°C 30 s, 72°C 60 s, 35 cycles). Primers were designed in a way
to allow amplification of expressed transgenic racb-G15V but not endogenous
RACB (5#-AACCAGATCTCCCCCAAATC-3#and 5#-GTCGACCTTCGCCCTTGTTCTTTGTC-3#). Primers target a nontranslated 5# part of the mRNA
originating from maize ubiquitin and flank a 5# maize ubiquitin 1 intron.
Hence, primers allowed us to test T1 segregating individuals for racb-G15V
mRNA expression by one-step RT-PCR (data not shown). The ubiquitin
coding gene (GenBank accession no. M60175; primers, 5#-ACCCTCGCCGACTACAACAT-3# and 5#-CAGTAGTGGCGGTCGAAGTG-3#) was used to verify quality of used RNA and as a measure for equal RNA amounts. PCR
products were separated in agarose gels.
DNA Blotting
Genomic DNA prepared from leaves (Palotta et al., 2000) was digested
with HindIII, separated on a 0.8% (w/v) agarose gel (30 mg per lane), and
blotted onto a Hybond N1 membrane (Amersham Biosciences Europe GmbH)
by capillary transfer under alkaline conditions as essentially described by
Sambrook et al. (1989). The membrane was hybridized with the PCRamplified hyg probe using the primers 5#-GATCGGACGATTGCGTCGCA3# and 5#-TATCGGCACTTTGCATCGGC-3# as well as plasmid pYF133 (Fang
et al., 2002) as template DNA. Labeling was performed with [a-32P]dCTP
using Ready-To-Go DNA labeling beads (Amersham Biosciences Europe
GmbH), and purification was carried out in a ProbeQuant G-50 microcolumn
(Amersham Biosciences Europe GmbH). The membrane was hybridized with
the labeled probe in ULTRAhyb Ultrasensitive hybridization buffer (Ambion)
at 65°C overnight. After hybridization, the blot was washed first with 63 SSC
for 5 min followed by a washing step with 23 SSC, 0.1% (w/v) SDS for 30 min
and twice with 0.13 SSC, 0.1% (w/v) SDS for 15 min. The radioactivity was
detected using Bio-Imaging Analyzer BAS 2000 (Fuji Film).
Upon request, all novel materials described in this publication will be
made available in a timely manner for noncommercial research purposes,
subject to the requisite permission from any third-party owner of all or parts of
the material. Obtaining any permission will be the responsibility of the
requestor. No restrictions or conditions will be placed on the use of any novel
materials described in this paper that would limit their use in noncommercial
research purposes.
Sequence data from this article can be found in the GenBank/EMBL data
libraries under accession numbers AJ344223, M60175, and AY234328.
ACKNOWLEDGMENTS
We thank Henning Tschiersch (Plantalytics GmbH, Gatersleben, Germany) for independent measurement of photosynthesis and ABA effects. We
Received June 3, 2005; revised June 24, 2005; accepted July 15, 2005; published
August 26, 2005.
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