The multigene family encoding germin-like proteins of barley. Regulation and function in Basal host resistance

Abstract

Germin-like proteins (GLPs) have been shown to be encoded by multigene families in several plant species and a role of some subfamily members in defense against pathogen attack has been proposed based on gene regulation studies and transgenic approaches. We studied the function of six GLP subfamilies of barley (Hordeum vulgare) by selecting single mRNAs for gene expression studies as well as overexpression and gene-silencing experiments in barley and Arabidopsis (Arabidopsis thaliana). Expression of all six subfamilies was high in very young seedlings, including roots. The expression pattern gradually changed from developmental to conditional with increasing plant age, whereby pathogen attack and exogenous hydrogen peroxide application were found to be the strongest signals for induction of several GLP subfamilies. Transcripts of four of five GLP subfamilies that are expressed in shoots were predominantly accumulating in the leaf epidermis. Transient overexpression of HvGER4 or HvGER5 as well as transient silencing by RNA interference of HvGER3 or HvGER5 protected barley epidermal cells from attack by the appropriate powdery mildew fungus Blumeria graminis f. sp. hordei. Silencing of HvGER4 induced hypersusceptibility. Transient and stable expression of subfamily members revealed HvGER5 as a new extracellular superoxide dismutase, and protection by overexpression could be demonstrated to be dependent on superoxide dismutase activity of the encoded protein. Data suggest a complex interplay of HvGER proteins in fine regulation of basal resistance against B. graminis.

Figure 1.

Phylogenetic tree of GLP proteins from barley and Arabidopsis. Multiple sequence alignment was carried out by using ClustalW with default parameter settings, including the Gonnet series of protein weight matrix (MegAlign software package; DNASTAR). Scale bar indicates amino acid substitutions. Boxed HvGER proteins correspond to genes selected for this study. TC numbers of Barley Gene Index at TIGR (release 9.0) corresponding to HvGER proteins are indicated, too. Only HvGER proteins represented by full CDS sequences are included in the multiple sequence alignment. Three partial cDNAs encoding additional putative HvGER4 paralogs were excluded from this analysis (TIGR TC139506, TC139507, and TC139508).

Figure 2.

Developmental regulation of the HvGER multigene family of barley. Transcript abundance detected on RNA blots was normalized to 26S rRNA signals after rehybridization of blots. Data are expressed as percentage of maximum signal intensity observed per cDNA probe over all RNA samples (for primary data, see Supplemental Fig. 2). RNA from mature plants was extracted three times during a period of 3 weeks (starting approximately 1 week after pollination and covering the yellow-to-white pollen stage). Mean values of all three time points are shown. Numbers (1–6) inside boxes correspond to HvGER1 to HvGER6.

Figure 3.

Stress-regulated expression of the HvGER multigene family of barley. A, HvGER transcript abundance detected by RNA-blot analysis was normalized to 26S rRNA signals after rehybridization of blots. Mean signal intensities ± range of two biological replicates are shown. Data of both biological replicates were normalized to each other by using control values of root samples, except for HvGER2 (leaf sample, controls for salt treatments). Black bars, Treatment; striped bars, corresponding control. B, HvGER transcript abundance detected by reverse RNA-blot analysis was normalized by median centering as described in “Materials and Methods.” Mean signal intensities ± range of two biological replicates are shown. Black bars, Treatment; striped bars, corresponding control. A and B, Units of signal intensities were defined by the different software packages used for RNA-blot or reverse RNA-blot analysis and are therefore not directly comparable. Numbers below bars correspond to duration of treatments (h). Ctr, Control; Epid, epidermis; SBR, soybean rust fungus.

Figure 4.

Localization of HvGER transcripts in leaf epidermis versus inner leaf tissues. Equal amounts of RNA extracted from stripped abaxial epidermis (E) or from the remaining leaf (L) were loaded per lane. RNA was extracted simultaneously 24 h postinoculation with Bgh (+) or from noninoculated control plants (−).

Figure 5.

Accumulation of recombinant HvGER proteins in transgenic Arabidopsis plants. Acid-soluble proteins were extracted from leaves of 10 T₀ plants (nos. 1–10) and used for western blotting. I, Extract from Bgh-inoculated wheat (Gf-2.8 blot) or barley (all other blots) leaves; C, extract from noninoculated wheat (Gf-2.8 blot) or barley (all other blots) leaves; wt, extract from Arabidopsis wild-type plants; H, high molecular mass complex (approximately 100-kD apparent molecular mass); M, monomeric protein band (approximately 22-kD apparent molecular mass). HvGER5a was detected by using a polyclonal serum directed against HvGER1a. Five micrograms of protein were loaded per lane.

Figure 6.

HvGER4 and HvGER5 possess SOD activity that is destroyed by mutation of one conserved His residue. A, SOD in-gel assay with protein extracts from one selected Arabidopsis T₀ line expressing either wild-type (wt) or mutant (M) GLP. H, Protein extract from Bgh-attacked barley leaves; A, protein extract from Arabidopsis wild-type leaves; SOD, commercial SOD from horseradish (Sigma-Aldrich). B, Same protein extracts as shown in A were subjected to western blotting.

Figure 7.

Model of fine regulation of basal resistance by HvGER protein expression in barley. The model is based on TIGS (x axis low) and overexpression (x axis high) data of this study. The y axis shows the relative susceptibility compared to the empty-vector controls (set to 1), which are assumed to correspond to median expression levels. For calculation of the hypothetical additive effect of all three HvGER proteins, the differences between relative susceptibility values and control value 1 were added to value 1. Please note that all three HvGER subfamilies in the model are predominantly expressed in leaf epidermis (Fig. 4).