Deciphering the transcriptional regulation and spatiotemporal distribution of immunity response in barley to Pyrenophora graminea fungal invasion

Abstract

Background: Barley leaf stripe disease, caused by the fungus Pyrenophora graminea (Pg), is a worldwide crop disease that results in significant loss of barley yield. The purpose of the present work was to use transcriptomic profiling to highlight barley genes and metabolic pathways affected or altered in response to Pg infection and consequently elucidate their involvement and contribution in resistance to leaf stripe.

Results: Our study examined and compared the transcriptomes of two barley genotypes using an established differential display reverse-transcription polymerase chain reaction (DDRT-PCR) strategy at 14 and 20 days post-inoculation (dpi). A total of 54 significantly modulated expressed sequence tags (ESTs) were identified. The analysis of gene expression changes during the course of infection with Pg suggested the involvement of 15 upregulated genes during the immunity response. By using network-based analyses, we could establish a significant correlation between genes expressed in response to Pg invasion. Microscopic analysis and quantitative PCR (qPCR) profiling of callose synthase and cellulose synthases revealed a direct involvement of cell wall reinforcement and callose deposition in the Pg-resistant phenotype.

Conclusions: We have identified a number of candidate genes possibly involved in the host-pathogen interactions between barley and Pg fungus, 15 of which are specifically expressed in Pg-resistant plants. Collectively, our results suggest that the resistance to leaf stripe in barley proceeds through callose deposition and different oxidation processes.

Keywords: Callose deposition; Differential display; Expressed sequence tags; Hordeum vulgare; Leaf stripe; Pyrenophora graminea; Transcriptional gene networking.

Fig. 1

Analysis of infection on barley genotypes. a Seeds of Banteng, Fourat-1 and Thibaut cultivars were P. graminea-inoculated with isolate Sy3 and disease symptoms were monitored at 18 and 26 dpi. b The upper panel represents the Rdg2a gene expression by RT-PCR. The middle panel represents the barley HvEf1-α that was used as an internal control and the Pg-1 marker was used for estimating the fungal DNA content (Pg) by RT-PCR using specific primers

Fig. 2

Monitoring of barley phenotypic and molecular changes. a temporal kinetic of infection in susceptible and resistant genotypes between 6 and 24 dpi. b Relative gene expression using qPCR analysis of PAL and PR2 transcripts and quantification of fungal Pg DNA present in R and S barley seedlings at selected time-points. Student t test was applied on gene expression data. Asterisks designate a statistical difference at P < 0.001 on each sample mean. The error bars correspond to the 95 % confidence interval calculated from the Student t test

Fig. 3

Temporal expression of ESTs (genes) selected by RT-PCR differential screening. After extraction of total RNA from plant tissues at 14 and 20 dpi, reverse-transcripts were produced for carrying-out qPCR analysis. Control to normalize amplification was run with the Ef1-α specific primers. Transcripts were analyzed using comparative Ct method. Data are presented in color scales: five shades of red from dark red to light red for upregulated genes and five shades of green for the downregulated genes. Non-differential expression is mentioned in black. The value of relative quantification (RQ) from qPCR was represented by the ladder of both colors (top panel)

Fig. 4

Spatial expression of ESTs (genes) selected by RT-PCR differential screening Barley seedlings were inoculated for 14 and 20 dpi. Roots or shoots of all inoculated plants mixed together in pools representing14 and 20 dpi together. qPCR runs were performed and illustrated like presented in Fig. 3

Fig. 5

Numbers of differentially expressed genes (DEGs) in different barley genotypes tested in roots and shoots. The Venn diagram shows the number of genes up- or downregulated in root and shoot tissues in response to Pg inoculation at levels of 2 folds or more and a P value < 0.05

Fig. 6

Classification of Gene Ontology (GO) analysis of selected DEGs after inoculation with Pg. A total of 54 genes were categorized in three groups after GO enrichment: Molecular function, Cell component and Biological process

Fig. 7

Functional network of enriched gene set in the resistant genotype. The 15 genes selected by qPCR analysis for their resistance-specific pattern of expression, were annotated using Blast2GO and the functional GO terms were manually selected. Network map of GO terms interactions was generated using Cytoscape. The network-interacting genes annotated with a particular term are represented by nodes. Nodes are sized according to annotated/enriched gene number. The connections between nodes means at least one gene is shared between enriched genes. The thinness of links represents the significance of linking between nodes. Gene expression analysis using qPCR of each annotated gene was incorporated in the network to represent the behavior of each of them in both susceptible and resistant genotypes

Fig. 8

Elevated callose deposition in resistant genotype prevents Pg fungal growth but not susceptible genotype. Tests were performed at 14 dpi. a Leaves were stained with aniline blue to visualize callose deposition by blue fluorescence using florescence microscopy (upper panel). Relative fluorescence intensity emitted by aniline blue-stained callose depositions (CD) was calculated (lower panel) on photographs taken under UV filter. The average of 10 tissue samples of each category is presented as a percentage. Scale bar = 20 μm. Samples with mycelium and second hyphae (or control tissues) were also washed off, stained by trypan blue and passed on optical microscope to visualize the presence of fungus. b Quantification of fungal Pg DNA presence in roots and shoots of resistant and susceptible plants inoculated in liquid culture for 10 days was performed using qPCR. Student’s t test was applied and asterisks indicate a statistical difference at P < 0.001 on each sample mean. The error bars correspond to the 95 % confidence interval calculated from the test

Fig. 9

Expression levels of callose deposition related gene families after inoculation with Pg fungus. Phylogenetic relatedness of the barley callose synthase, cellulose synthase and related gene families to callose formation: HvGSLs, HvCSLAs, HvGTs, HvCesAs and HvCsIFs including Hv-Pg54. Phylogenetic tree was generated with the clustalX program and is based on nucleic acid sequences. The relative gene expression of transcripts of all genes from the five gene families was analyzed using comparative Ct method in roots and shoots of resistant and susceptible inoculated plants at 10 dpi. The error bars correspond to the calculated 95 % confidence interval