Characterisation of HvVIP1 and expression profile analysis of stress response regulators in barley under Agrobacterium and Fusarium infections

Abstract

Arabidopsis thaliana's VirE2-Interacting Protein 1 (VIP1) interacts with Agrobacterium tumefaciens VirE2 protein and regulates stress responses and plant immunity signaling occurring downstream of the Mitogen-Activated Protein Kinase (MPK3) signal transduction pathway. In this study, a full-length cDNA of 972bp encoding HvVIP1 was obtained from barley (Hordeum vulgare L.) leaves. A corresponding 323 amino acid poly-peptide was shown to carry the conserved bZIP (Basic Leucine Zipper) domain within its 157th and 223rd amino acid residue. 13 non-synonymous SNPs were spotted within the HvVIP1 bZIP domain sequence when compared with AtVIP1. Moreover, minor differences in the bZIP domain locations and lengths were noted when comparing Arabidopsis thaliana and Hordeum vulgare VIP1 proteins through the 3D models, structural domain predictions and disorder prediction profiling. The expression of HvVIP1 was stable in barley tissues infected by pathogen (whether Agrobacterium tumefaciens or Fusarium culmorum), but was induced at specific time points. We found a strong correlation between the transcript accumulation of HvVIP1 and barley PR- genes HvPR1, HvPR4 and HvPR10, but not with HvPR3 and HvPR5, probably due to low induction of those particular genes. In addition, a gene encoding for a member of the barley MAPK family, HvMPK1, showed significantly higher expression after pathogenic infection of barley cells. Collectively, our results might suggest that early expression of PR genes upon infection in barley cells play a pivotal role in the Agrobacterium-resistance of this plant.

Fig 1

(A) Alignment of the VIP1 bZIP domains of various homologous plant proteins identified by Blast search. The bZIP domain of VIP1 (DDBJ/EMBL/GenBank accession No. NP_564486.1) was aligned (using the clustalW algorithm- EMBL-EBI/MUSCLE) with similar motifs of its homologues from Oryza sativa (OsVIP1), Brachypodium distachyon (BdVIP1), Hordeum vulgare (Both, predicted clone HvVIP1, accession No. BAJ96285.1 and our predicted protein sequence HvVIP1_Martı), Triticum aestivum (TaVIP1), Oryza brachyantha (ObVIP1), Sorghum bicolor (SbVIP1), Setaria italica (SiVIP1), Zea mays (ZmVIP1). Regions of identity are indicated as follows: (i) The basic region is highlighted in violet, (ii) the bipartite NLS within the basic region is denoted by a horizontal bar in grey below its sequence, (iii)the invariant N-X7-K/R motif location is indicated within the basic region, (iv) the Leucine Zipper region (seven Leucine heptad) is highlighted in yellow/green, (v) SNPs within the bZIP domains are marked individually and were identified according to AtVIP1. (*) indicates fully conserved residues, (:) and (.) indicate less conserved residues. (B) Predicted gene model of HvVIP1. The bZIP domain Basic region stretches over Exons 1 and 2 (E-1 and E-2). Within the basic region: bipartite NLS is marked in stars, and the invariant DNA binding motif (N-X7-R/K). The Leucine Zipper region is located on exons 2 and 3 (E-2 and E-3). I 1–3: Introns; (N): Asparagine; (R): Arginine; (K): Lysine; (L): Leucine; ATG: Start codon; TGA: Stop codon; X: any nucleotide.

Fig 2. Phylogeny of the VIP1-like proteins.

The multiple alignment was generated using MUSCLE software and the phylogenetic tree was built with the MEGA5 software using the JTT matrix-based model (the numbers at the nodes indicate the bootstrap scores). The protein accession numbers are indicated in parentheses. Oryza sativa (OsVIP1), Brachypodium distachyon (BdVIP1), Hordeum vulgare (Both, predicted clone HvVIP1, and our predicted protein sequence HvVIP1_Martı), Triticum aestivum (TaVIP1), Oryza brachyantha (ObVIP1), Sorghum bicolor (SbVIP1), Setaria italica (SiVIP1), Zea mays (ZmVIP1), and Arabidopsis thaliana (AtVIP1). Blue nodes are the putative ancestors; the red node represents the historical ultimate ancestor. The Bar (0.050) is a scale for the amount of genetic change (nucleotide substitutions per site).

Fig 3. Time-dependent expression of VIP1 in barley cells.

(A) HvVIP1 expression profile in stress-free barley leaves (L), roots (R), immature embryos (IM), calli (C), and coleoptiles (Col). (B) HvVIP1 significantly overexpressed (12^th hours) in cv. Golden promise calli after Agrobacterium (AGL-1 strain) infection. (C) HvVIP1 induced significantly at the 48^th hour in cv. Martı calli treated with AGL-1 strain. (D) HvVIP1 significantly overexpressed in Fusarium-inoculated barley roots 24 hours after treatment. (*) and (***) designate statistical significance. (*) for P < 0,05; and (***) for P < 0,001.