Article Figures & Data
Figures
- Fig. 1.
Characterization of two splicing-defectivemlo mutant alleles. RNA-blot analysis (mRNA panels) and schematic representation of transcript splice products inmlo-16 (A) and mlo-30 genotypes (B). Northern-blot analysis was performed as described in “Materials and Methods” using wild-type Mlo plants as a control. Arrows indicate the Mlo wild-type transcript. Exonic sequences in the schematic representations are in uppercase and intron sequences in lowercase. Underlined dinucleotides in lowercase denotes 5′-splice donor and acceptor sites, and the underlined dinucleotides in uppercase represent the cryptic 3′-splice acceptor sites utilized in themlo-16 and mlo-30 alleles. The arrows highlight the point mutations in mlo-16 (G→A) and mlo-30 (A→T) genotypes. Major splicing events in mutant cDNAs and corresponding effects on Mlo coding sequences are indicated by black lines.
- Fig. 2.
Bgh-triggered cell death in a partially resistantmlo mutant. Macroscopic phenotypes of wild-typeMlo, partially and fully resistant mlo mutants 7 d after fungal challenge. In the Mlo genotype (compatible interaction), no sign of cell death is visible beneath the sporulating colony. In the mlo-28 mutant (partially resistant), necrotic mesophyll cells are visible beneath a small fungal colony, and in the mlo-5 null mutant (fully resistant), a cluster of necrotic mesophyll cells can be seen beneath the failed penetration attempt. Bar = 50 μm.
- Fig. 3.
Inactivation of the MLO protein leads to enhanced H2O2 accumulation at epidermal cells and to cell death in the mesophyll. A, In situ detection of H2O2 by DAB precipitation at 24 h after inoculation. For each genotype, three sites are shown to illustrate the range of DAB staining (area and intensity) observed at sites of attempted fungal penetration (white arrow). Bar = 10 μm. B, Quantitative analysis of DAB-stained areas beneath fungal appressorial germ tubes (A, white arrows). C, DAB staining in mesophyll cells beneath sites of attempted penetration (top) at 36 h following inoculation; retention of trypan blue 60 h after fungal challenge (bottom). In the fully compatible interaction (Mlo Ror1), no DAB staining or trypan blue retention was observed. DAB staining and trypan blue retention were found to be significantly reduced in the double mutant mlo-5ror1-2. Bar in top = 10 μm; bar in bottom = 50 μm.
- Fig. 4.
Accelerated leaf senescence in mlomutants. A, Time course analysis of chlorophyll and carotenoid pigment content in Mlo wild-type and mlo-5 genotypes. First foliage leaves were collected at 2-d intervals from 9 to 21 d after sowing, and pigment analysis was carried out as described in “Materials and Methods.” Each time point in the graphs shows the mean (±sd) of six leaf samples. B, Time course analysis of MLO transcript abundance throughout first foliage leaf development in Mlo wild-type and mlo-5 genotypes. The same blot was probed sequentially with the MLO-, GAPDH-, and ubiquitin (UBI)-labeled cDNAs. The fold changes in MLO transcript abundance were calculated using the GAPDH signal as a control. C, Mid-portions of first foliage leaves primary leaves of 24-d-old seedlings of the Mlo (top) and mlo-5 (bottom) genotypes. Chlorosis (yellowing) and, at later time points, necrotic lesions are noticeable in the mlo-5 genotype.
- Fig. 5.
Time course analysis of MLO transcript and MLO protein upon powdery mildew inoculation. A and B, Accumulation of MLO transcript following Bgh challenge in Mlowild-type (A) and mlo-5 (B) leaves. The same blot was sequentially probed with the MLO, GST, and GAPDH cDNA probes. The fold changes in MLO transcript abundance were calculated using the GAPDH signal as a control. The ×1 level was defined as ratio MLO:GAPDH ratio as at time 0. Figures showing MLO transcript levels were calculated using three independent experiments. C, Accumulation of MLO transcript following Bgh challenge in epidermal peels (see “Materials and Methods”) of mlo-5 mutant (left) and Mlowild-type (right) leaves. Abaxial leaf epidermal tissue was harvested during the indicated time intervals after spore inoculation, and the same blot was sequentially probed with the MLO and GAPDH cDNA probes. The fold changes in MLO transcript abundance were calculated using the GAPDH signal as a control. The ×1 level was defined as ratio MLO:GAPDH ratio as at time 0. D, Accumulation of MLO protein upon powdery mildew inoculation. Western blots of plasma membrane vesicle preparations from inoculated and uninoculated leaves were probed with the barley MLO-specific and the plasma membrane ATPase (PM-ATPase) antibodies. Positive signals were analyzed using a phosphor imager, and the fold MLO protein induction was calculated relative to the PM-ATPase signal. The ×1 level was defined as ratio MLO:PM-ATPase as at time 0. Figures showing MLO protein induction were calculated using two independent experiments.
- Fig. 6.
Time course analysis of MLO transcript accumulation upon M. grisea challenge and abiotic stresses. A, Accumulation of MLO transcript upon challenge of barleyMlo wild-type (top) and mlo-5 (bottom) leaves with M. grisea. The same blot was sequentially probed with the MLO and GAPDH probes. Signals were analyzed using a phosphorimager, and fold Mlo transcript accumulation was calculated using the GAPDH signal as control. Figures showing MLO transcript accumulation were calculated using two independent experiments. B, Accumulation of MLO mRNA upon a carbohydrate elicitor derived from the wheat (Tritcum aestivum) powdery mildew fungus (Egt elicitor). The same blot was probed sequentially with the barley MLO and GAPDH cDNAs. C, Accumulation of MLO mRNA upon wounding. First foliage barley leaves were harvested from 8-d-old seedlings and were mechanically wounded. Representative data from two independent experiments are shown. D, Accumulation of MLO mRNA upon paraquat treatment. Paraquat was sprayed onto 8-d-old seedlings to generate an oxidative burst in chloroplasts. After spraying, plants were kept for 2 h in the dark and were then transferred to light to ensure homogenous distribution of the chemical in the seedlings. The same blot was probed sequentially with the barley MLO and GAPDH cDNAs.
Tables
Allele Mutant IDs1-a Mutational Event at Mlo Effect on MLO Protein Protein Domain Affected Bgh Resistance Mutagen mlo-12 Do 4122 C1396 → A F240 → L Second intracellular loop Partial Nitrosomethylurea mlo-16 Do 2376 G1917 → A1-b Not applicable Not applicable Full Ethymethane sulfonate (EMS) mlo-27 Do 20211-c G2004 → A G318 → E Third intracellular loop Full EMS mlo-28 Do 4228 C1224 → T T222→ I Second intracellular loop Partial NaN3 mlo-29 This study C2051 → T P334→ L Third intracellular loop Full NaN3 mlo-30 Do 2234, 2235 A2242 → T1-d Δ 6 amino acids C terminus (intracellular) Full EMS Nucleotide nos. based on the genomic Mlo DNA sequence starting from the translational start site (ATG).
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↵F1-a According to Habekuss and Hentrich (1988).
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↵F1-b Nucleotide substitution in the conserved 3′ splice site of intron 9.
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↵F1-c Also Do 2002, 2003, 2004, 2005, 2014, 2015, 2016, and 2019.
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↵F1-d Nucleotide substitution in the conserved 3′ splice site of intron 11.
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