Molecular characterization of the oxalate oxidase involved in the response of barley to the powdery mildew fungus

Abstract

Previously we reported that oxalate oxidase activity increases in extracts of barley (Hordeum vulgare) leaves in response to the powdery mildew fungus (Blumeria [syn. Erysiphe] graminis f.sp. hordei) and proposed this as a source of H2O2 during plant-pathogen interactions. In this paper we show that the N terminus of the major pathogen-response oxalate oxidase has a high degree of sequence identity to previously characterized germin-like oxalate oxidases. Two cDNAs were isolated, pHvOxOa, which represents this major enzyme, and pHvOxOb', representing a closely related enzyme. Our data suggest the presence of only two oxalate oxidase genes in the barley genome, i.e. a gene encoding HvOxOa, which possibly exists in several copies, and a single-copy gene encoding HvOxOb. The use of 3' end gene-specific probes has allowed us to demonstrate that the HvOxOa transcript accumulates to 6 times the level of the HvOxOb transcript in response to the powdery mildew fungus. The transcripts were detected in both compatible and incompatible interactions with a similar accumulation pattern. The oxalate oxidase is found exclusively in the leaf mesophyll, where it is cell wall located. A model for a signal transduction pathway in which oxalate oxidase plays a central role is proposed for the regulation of the hypersensitive response.

Figure 7

Tissue localization of the pathogen-response oxalate oxidase in barley leaves (P-01, 24 h after inoculation with Bgh [C15]) demonstrated on 12% SDS-PAGE gels using samples of the whole leaf (lanes 1), epidermal tissue (lanes 2), and mesophyll tissue (lanes 3). Twenty microliters of protein extract (representing equal amounts of fresh weight tissue) was loaded in each lane as unboiled samples using a loading buffer lacking reducing agent. A, Silver-stained gel. B, Immunoblot incubated with purified oxalate oxidase antibody. C, In-gel oxalate oxidase activity assay.

Figure 1

N-terminal amino acid sequence of the monomer of the 100-kD oligomeric oxalate oxidase HvOxOa. Alignment to the deduced amino acid sequences of the cDNA clones pHvOxO-Lane (Lane et al., 1993) and pHvOxOb (Hurkman et al., 1994) and to the N-terminal amino acid sequence of HvOxO-Dumas (Dumas et al., 1993) are shown. Dots indicate amino acids also found in HvOxOa; X, amino acid not determined.

Figure 2

Accumulation of oxalate oxidase transcripts in barley leaves inoculated with the powdery mildew fungus. Total RNA was extracted from P-01 and P-02 barley leaves following inoculation with isolate C15 of Bgh (+) and from noninoculated control leaves (−). The transcripts were detected on northern blots by hybridization with the pHvOxO-Lane cDNA (Lane et al., 1993). Low- to medium-stringency wash was with 2× SSC at 68°C.

Figure 3

Nucleotide sequence of pHvOxOa. Alignment to the oxalate oxidase encoding cDNA clones pHvOxO-Lane (Lane et al., 1993) and pHvOxOb (Hurkman et al., 1994) are shown. Dashes indicate introduced gaps; dots indicate nucleotides also found in pHvOxOa. Start and stop codons are in bold. Underlined 3′ sequences represent segments used as gene-specific probes.

Figure 4

Amino acid sequence encoded by the pHvOxOa cDNA. Alignment to the deduced amino acid sequences of the cDNA clones pHvOxO-Lane (Lane et al., 1993) and pHvOxOb (Hurkman et al., 1994) and to the amino acid sequences of HvOxO-Dumas (Dumas et al., 1993) and of Gs1 and Gs2 (Hurkman et al., 1991). Lowercase letters indicate leader sequences; dots represent amino acids also found in HvOxOa. Identity (%), Amino acid identity between a polypeptide and the mature HvOxOa.

Figure 5

HvOxOa and HvOxOb transcripts accumulate in barley leaves inoculated with the powdery mildew fungus. Total RNA samples of Figure 2 were applied. The transcripts were detected on northern blots by parallel hybridization with the gene-specific 3′ UTR probes pHvOxOa-3′-UTR (A) and pHvOxOb'-3′-UTR (B). The degree of cross-hybridization determined on plasmid DNA was dotted onto the northern blot. High-stringency washing was with 0.1× SSC at 68°C. Lanes +, Inoculated; lanes −, noninoculated.

Figure 6

Detailed expression pattern of oxalate oxidase transcripts demonstrated on northern blots of total RNA extracted from P-01 and P-02 barley leaves following inoculation with isolates A6 and C15 of Bgh. The transcripts were detected by hybridization with the pHvOxOa cDNA. High-stringency washing was with 0.1× SSC at 68°C.

Figure 8

Cross-sections of oxalate oxidase activity-stained barley leaves (P-02, 24 h after inoculation with Bgh [C15]). The staining reaction was performed without (A) and with (B and C) oxalate. Images are of epidermis (top)/mesophyll section (A and B) and vascular bundle (C). e, Boundary between epidermal cells; m, boundary between mesophyll cells. Bar represents 10 μm on all images.

Figure 9

Southern blot of barley (P-01) genomic DNA hybridized with full-length cDNA clones (A and B) and with gene-specific 3′ UTRs (C and D). Low-/medium-stringency washes with 2× SSC at 68°C (A and B), and medium-stringency washes with 1× SSC at 68°C (C and D).