( Ralph Panstruga Brigitt...)Single-Cell Complementation of Barley Mlo Mutants Using a PDS-1000/He Hepta System

Ralph Panstruga, Brigitte Schauf, and Paul Schulze-Lefert, Max-Planck-Institut fr Zchtungsforschung, Carl-von-Linn-Weg 10, 50829 Kln, Germany e-mail: panstrug@mpiz-koeln.mpg.de

Abstract
Transient expression via particle bombardment is widely used as a means of gene transfer to bacteria, yeast, animals, and plants. In this study we describe the use of the Hepta adaptor for the PDS-1000/He biolistic system for transformation and transient expression of Mlo in single epidermal cells of detached barley leaves. Barley Mlo is known to dampen plant defense and its expression in single epidermal cells of mlo resistant mutants restores susceptibility to attack from the powdery mildew fungal pathogen, Blumeria graminis. Consistently high transformation efficiencies were obtained upon delivery of a plasmid carrying both Green Fluorescent Protein (GFP) and Mlo. After bombardment and fungal spore inoculation, we found leaf sectors with numerous green fluorescent epidermal cells supporting growth of the pathogen. Likewise, we observed a high transformation efficiency of Arabidopsis leaf cells upon delivery of a GUS (β-glucuronidase) reporter gene construct. Application of the Hepta adaptor reduces the number of biolistic transfers necessary to obtain sufficient numbers of transformed plant cells for quantitative scoring of single-cell traits.

Introduction
Mutation induced recessive alleles (mlo) of the barley Mlo gene confer broad spectrum resistance against Blumeria graminis f. sp. hordei, the causal agent of powdery mildew. Conversely, the presence of wild-type Mlo leads to susceptibility upon attack from this obligate biotrophic fungal pathogen. Mlo encodes the founder of a novel family of plant-specific integral membrane proteins (Bschges et al. 1997). The 7-transmembrane-domain protein resides in the plasma membrane and is presumed to act as a negative regulator of a basal defense mechanism (Devoto et al. 1999).

We have previously shown that transient single-cell expression of wild-type Mlo in Mlo resistant leaves restores susceptibility to Blumeria graminis (Shirasu et al. 1999). This was based on cobombardment experiments involving 2 plasmids harboring either Mlo or GFP (Figure 1).

Here we describe a modification of the transformation procedure by using the PDS-1000/He Hepta adaptor. This device fits into the shocking chamber of the PDS-1000/He unit and splits the helium shock wave over 7 outlets, permitting a more even dispersal of DNA-coated particles and a greater target area.

Methods
Plant and Fungal Material
Leaves of mlo resistant barley (Hordeum vulgare cv. BC Ingrid mlo-5) and Arabidopsis thaliana (ecotype Ms-0) were used for this study. Barley plants were grown in a controlled environment at 18C (16 hr light/8 hr darkness), whereas Arabidopsis plants were grown in short-day (8 hr light/16 hr darkness) conditions. First leaves of 8-day-old barley plants or rosette leaves of approximately 5-week-old Arabidopsis plants were used for the experiments. Barley leaves (apical 57 cm segments) were harvested and kept on 1% agar containing 10% sucrose 4 hr prior to bombardment.

Blumeria graminis f. sp. hordei K1 was propagated on H. vulgare cv. Golden Promise as previously described (Shirasu et al. 1999).

Plasmids
Plasmid pUGLUM (Zhou et al. 2001) carrying GFP and Mlo coding sequences under the control of the strong constitutive maize ubiquitin 1 promoter was used for the barley experiments. For transformation of A. thaliana, plant binary vector pPam-GUS (T Rademacher and R Panstruga, unpublished) was used. In this plasmid the β-glucuronidase reporter gene is driven by a double constitutive cauliflower mosaic virus 35S promoter.

Preparation of DNA-Coated Gold Microcarriers
The preparation of DNA-coated gold microcarriers (1 M particle size) was carried out according to the manufacturers instructions (Bio-Rad PDS-1000/He manual). Briefly, gold particles were soaked in 70% ethanol (v/v), washed thoroughly with sterile water, and resuspended in 50% glycerol at a concentration of 60 mg/ml. To coat the particles with plasmid DNA, 50 l (3 mg) of microcarriers were mixed with 5 l DNA (1 g/l), 50 l 2.5 M CaCl₂ and 20 l 0.1 M spermidine (free base). After 2 washing steps with ethanol (first 140 l 70%, second 140 l 100%), the coated particles were finally resuspended in 48 l 100% ethanol.

Conditions for Particle Bombardment and Fungal Inoculation
Each of the 7 macrocarriers of the Hepta adaptor was loaded with 6 l of the coated microcarriers corresponding to 0.62 g plasmid DNA. Hence, a total of approximately 4.3 g plasmid DNA was delivered per shot. The target shelf carrying the petri dish with detached leaf segments was placed 6 cm from the Hepta adaptor. Usually 810 leaf sections (barley) or 1020 rosette leaves (A. thaliana) were placed side by side in a single 9 cm petri dish for the biolistic transfer. After evacuating the shocking chamber to 27" Hg, specimens were bombarded with a helium pressure of 900 or 1,100 psi. Immediately after bombardment, the vacuum was released and leaf segments were kept on the plates 24 hr for recovery. The leaves were transferred to fresh plates (1% agar, 0.002 g/L benzimidazole) and inoculated with a high density of conidiospores of B. graminis f. sp. hordei K1. Petri dishes carrying the detached leaf sections were incubated in a growth cabinet under conditions described above.

Staining for β-glucuronidase (GUS) Activity
Arabidopsis rosette leaves were stained for GUS activity 34 days after bombardment by vacuum infiltration (1 hr) in 100 mM Na₂HPO₄/NaH₂PO₄, pH 7.0, 0.1% Triton X-100, 2 mM K₃Fe(CN)₆ containing 0.5 mg/ml 5-bromo-4-chloro-3- indoxyl-β-D-glucuronic acid and incubated overnight at 37C. The stained leaves were cleared thereafter in ethanol (96%) for several hours.

Results and Discussion
Detached leaves of mlo resistant barley cultivar Ingrid containing the null mutant allele mlo-5 were bombarded with plasmid DNA of pUGLUM as described in Methods (Figure 1). The plasmid harbors the Mlo wild type and the GFP reporter genes whose expression is driven by a ubiquitin promoter. Approximately 24 hr after the biolistic transfer, we observed leaf sectors with numerous green fluorescent epidermal cells and occasionally single fluorescent cells outside of the clusters (Figure 2). Optimal transformation efficiencies were obtained with He pressures of 900 psi and 1,100 psi. Higher He pressures (1,350 psi) resulted in disruption of foliar tissue, whereas lower pressures (450 or 650 psi) led to a reduction in the number of detectable green fluorescent cells. GFP expression was observed in different cell types of the leaf epidermis, e.g., guard cells and interstomatal short and long cells (Figure 2).

Detached leaf sections were heavily inoculated with spores of B. graminis f. sp. hordei isolate K1 approximately 24 hr after bombardment. Microscopic fungal growth was visible 34 days after inoculation as the formation of aerial hyphae and conidiophores. In the vast majority of sections, formation of fungal colonies originated from single infected cells expressing GFP (Figure 3). Because plasmid pUGLUM contains both GFP and Mlo, our data suggest that transient expression of wild-type Mlo complements mlo resistance at the single cell level. This is consistent with results obtained after cobombardment of 2 plasmids containing GFP or Mlo (Shirasu et al. 1999).

A particle inflow gun with a single outlet and a much smaller target area was used in a previous study (Shirasu et al. 1999). Under those conditions, leaf sections of 3 cm length could be bombarded in a single shot. About 20 leaf segments and several bombardments were necessary to obtain 100300 GFP expressing cells (Shirasu et al. 1999).

Modification of the experimental setup described here generated a comparable number of cells expressing the GFP reporter gene after a single bombardment. Application of the Hepta adaptor resulted in consistently high numbers of transfected cells in several independent experiments. Generally, we found multiple clusters of transfected epidermal cells in bombarded leaf segments as shown in Figure 2. Thus, the Hepta adaptor is useful for experiments in which several independent constructs need to be tested or in which high numbers of transformed cells are desired.

Next we tested the efficiency of the Hepta system transformation procedure in the dicot plant A. thaliana by delivering a plasmid expressing GUS to detached leaf tissue. In this case we used plasmid pPam-GUS driving expression of the reporter gene from the doubled 35S promoter. Experimental conditions were identical as described above for the transformation of barley leaves. We obtained a high density of GUS-stained cells that were clustered in multiple leaf areas (Figure 4) 34 days following bombardment.

Our findings demonstrate the usefulness of the Hepta particle delivery system in obtaining high transformation efficiencies in leaves of monocot and dicot plant species.

References
Bschges R et al., The barley Mlo gene: A novel control element of plant pathogen resistance, Cell 88, 695705 (1997)

Devoto A et al., Topology, subcellular localization, and sequence diversity of the Mlo family in plants, J Biol Chem 274, 3499335004 (1999)

Shirasu K et al., Cell-autonomous complementation of mlo resistance using a biolistic transient expression system, Plant J 17, 293299 (1999)

Zhou F et al., Molecular isolation and functional analysis of the barley Mlo1 powdery mildew resistance gene by using a transient single-cell expression assay, Plant Cell 13, 337350 (2001)

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