Proteomic characterization of the Rph15 barley resistance gene-mediated defence responses to leaf rust

Abstract

Background: Leaf rust, caused by the biotrophic fungal pathogen Puccinia hordei, is one of the most important foliar disease of barley (Hordeum vulgare) and represents a serious threat in many production regions of the world. The leaf rust resistance gene Rph15 is of outstanding interest for resistance breeding because it confers resistance to over 350 Puccinia hordei isolates collected from around the world. Molecular and biochemical mechanisms responsible for the Rph15 effectiveness are currently not investigated. The aim of the present work was to study the Rph15-based defence responses using a proteomic approach.

Results: Protein pattern changes in response to the leaf rust pathogen infection were investigated in two barley near isogenic lines (NILs), Bowman (leaf rust susceptible) and Bowman-Rph15 (leaf rust resistant), differing for the introgression of the leaf rust resistance gene Rph15. Two infection time points, 24 hours and four days post inoculation (dpi), were analysed. No statistically significant differences were identified at the early time point, while at 4 dpi eighteen protein spots were significantly up or down regulated with a fold-change equal or higher than two in response to pathogen infection. Almost all the pathogen-responsive proteins were identified in the Bowman-Rph15 resistant NIL. Protein spots were characterized by LC-MS/MS analysis and found to be involved in photosynthesis and energy metabolism, carbohydrate metabolism, protein degradation and defence. Proteomic data were complemented by transcriptional analysis of the respective genes. The identified proteins can be related to modulation of the photosynthetic apparatus components, re-direction of the metabolism to sustain defence responses and deployment of defence proteins.

Conclusions: The identification of leaf rust infection-modulated defence responses restricted to the resistant NIL support the hypothesis that basal defence responses of Bowman, but not the Rph15 resistance gene-based ones, are suppressed or delayed by pathogen effectors to levels below the detection power of the adopted proteomic approach. Additionally, Rph15-mediated resistance processes identified mainly resides on a modulation of primary metabolism, affecting photosyntesis and carbohydrate pool.

Figure 1

Phenotype and defense genes activation of barley leaves subjected to proteomic analyses. (A) Barley leaves images of the two NILs Bowman and Bowman-Rph15 inoculated with the leaf rust pathogen at the indicated time points and utilized for the proteomic analyses. After sampling, in some plants for each biological replicate the disease was left to proceed until 8 dpi to assess the success of the infection experiments. (B) The same genotypes and time points of inoculation as in (A) were verified for the transcriptional activation of the defense related genes coding for oxalate oxidase and callose synthase using quantitative RT-PCR analysis. Normalization was carried out with the β-actin constitutively expressed gene. Values are expressed as log2 fold changes of transcript levels in the inoculated samples with respect to the transcript levels in un-inoculated barley leaves. Error bars represent SD across all RT-PCR replicates (three from each of two independent inoculations). Statistical significance of differential expression was evaluated with a Wilcoxon two group test (P<0.05, Methods).

Figure 2

2-DE maps. Representative 2-DE maps of soluble protein fractions extracted from Bowman and Bowman-Rph15 leaves at 4 days after mock inoculation (A and C, respectively) or after inoculation with leaf rust spores (B and D, respectively). Proteins (300 μg) were analyzed by IEF at pH 4–7, followed by 12.5% SDS-PAGE and visualized by SYPRO-staining. Numbers, corresponding to those in Table 1 and Figure 3, indicate the spots, identified by LC-ESI-MS/MS, showing significant changes of at least two-fold in their relative volumes (t-test, p < 0.05) after 4 dpi. Proteins that increased or decreased after this treatment are reported in red or in green, respectively. Spot 3142 is highlighted in blue, indicating its absence in the Bowman-Rph15 inoculated sample.

Figure 3

Changes in protein accumulation in the two NILs in response to leaf rust infection. Changes in the relative volumes of the identified proteins whose concentration is increased or decreased in the two NILs Bowman (Bow) and Bowman-Rph15 (Bow-Rph15) at 4 days after mock inoculation (C) or after inoculation with leaf rust spores (I). Values are the means of six 2-DE gels derived from two independent biological replicates analyzed in triplicate (n=6). Error bars represent SD across all replicates. Numbers identify the spots as indicated in Table 1 and Figure 2; proteins were ordered into five functional classes, as indicated in Table 1.

Figure 4

Western blot analysis with α-Rubisco antibody. (A) Western blot of five micrograms of proteins extracted from the two biological replicates of Bowman (Bow) and Bowman-Rph15 (Bow-Rph15) at 4 days after mock inoculation (C) or after inoculation with leaf rust spores (I) were separated on 12% SDS-PAGE and hybridized with a Rubisco antibody. Bands representing the main Rubisco degradation forms are numbered from 1 to 5 on the Western right side. (B) Quantification data of Rubisco degradation forms; evaluation of degradation forms was conducted for the five bands indicated in (A) in Bowman (Bow) and Bowman-Rph15 (Bow-Rph15) under control (C) or after inoculation with leaf rust spores (I) conditions. The data are the mean of three independent experiments. Different letters on the bars indicate significant difference P-values (P < 0.05) of the t-TESTs performed comparing control and inoculated samples.

Figure 5

Gene expression analysis of eight target genes. Quantitative RT-PCR at 1, 2, 3, 4 and 5 dpi for eight genes in leaves of Bowman (open circles) and Bowman-Rph15 (open squares). Values are expressed as log2 fold changes of transcript levels in the inoculated samples with respect to the transcript levels in mock-inoculated barley leaves. Numbers and name abbreviations are corresponding to those in Table 1. Error bars represent SD across all RT-PCR replicates (three from each of two independent inoculations). Statistical significance of differential expression for the tested genes was evaluated with a Wilcoxon two group test (P<0.05, Methods).