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First published online August 26, 2005; 10.1104/pp.105.066613 Plant Physiology 139:353-362 (2005) © 2005 American Society of Plant Biologists
Ectopic Expression of Constitutively Activated RACB in Barley Enhances Susceptibility to Powdery Mildew and Abiotic Stress1Institute 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 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
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 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.
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
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 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, 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 (
RACB-G15V-Expressing Individuals Are Photosynthetically Less Active To further test whether photosynthesis is impaired in racb-G15V-expressing plants, we measured CO2 assimilation 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, racb-G15V-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 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 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
Arabidopsis RAC/ROP-like proteins are involved in abiotic stress signaling. Therefore, we tested racb-G15V 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-G15V-negative 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 about 60% of their original weight, whereas racb-G15V-expressing 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 = 0), whereas racb-G15V-negative individuals were able to reduce transpiration to about 20% to 40% of what was measured at t = 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 µM ABA solution. This strongly reduced transpiration in T1 plants not expressing racb-G15V over time to 4% of what was measured at t = 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 = 0 (Fig. 7). Similar results were obtained with 10 µM ABA, except that final transpiration rates were higher (14% of t = 0 in racb-G15V-negative versus 36% of t = 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-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.
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
The function of RACB was linked to the function of MLO (Schultheiss et al., 2002
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
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
Besides constitutively activated RACB-G15V, barley RAC3-G17V similarly induces enhanced accessibility to Bgh in transiently transformed cells (Schultheiss et al., 2003
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 racb-G15V 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 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
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 µmol s–1 m–2 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 µmol s–1 m–2 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 For microscopic evaluation, Bgh was inoculated onto detached primary leaves of 10-d-old barley plants to give a density of 30 conidia mm–2. 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 mm–2 and evaluated 5 to 7 d after inoculation.
Stable genetic transformation of barley with ZmUbi::racb-G15V was performed as described by Hensel and Kumlehn (2004)
Barley plants were grown and transformed with ZmUbi::GFP as described by Tingay et al. (1997)
For constitutive expression of the racb-G15V gene in barley, the BamHI/SalI fragment from plasmid pGY1-RacB-V15 (Schultheiss et al., 2003
For in planta expression, the GFP gene (GFP out of pGY1-GFP; Schweizer et al., 1999
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)
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 µmol s–1 m–2 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 µmol s–1 m–2 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 µmol s–1 m–2 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).
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 µg of total RNA was separated on a 1.2% agarose gel and blotted on Hybond N+ 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. 32P-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 0.1x SSC/0.1% SDS for 5 min each. Signals were detected with a Molecular Imager FX Phosphorimager (Bio-Rad).
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.
Genomic DNA prepared from leaves (Palotta et al., 2000 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.
We thank Henning Tschiersch (Plantalytics GmbH, Gatersleben, Germany) for independent measurement of photosynthesis and ABA effects. We would like to thank Cornelia Marthe and Christiane Börnke for excellent technical assistance. Received June 3, 2005; returned for revision June 24, 2005; accepted July 15, 2005.
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. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.066613. * Corresponding author; e-mail ralph.hueckelhoven{at}agrar.uni-giessen.de; fax 49–641–9937499.
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TOP
ABSTRACT
RESULTS
2 test confirmed a 1:1 segregation (
-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 6x SSC for 5 min followed by a washing step with 2x SSC, 0.1% (w/v) SDS for 30 min and twice with 0.1x SSC, 0.1% (w/v) SDS for 15 min. The radioactivity was detected using Bio-Imaging Analyzer BAS 2000 (Fuji Film).