Transcriptome Analysis and Functional Characterization of the HvLRR_8-1 Gene Involved in Barley Resistance to Pyrenophora graminea

Abstract

Barley leaf stripe, caused by Pyrenophora graminea (Pg), significantly reduces yields across various regions globally. Understanding the resistance mechanisms of barley to Pg is crucial for advancing disease resistance breeding efforts. In this study, two barley genotypes-highly susceptible Alexis and immune Ganpi2-were inoculated with the highly pathogenic Pg isolate QWC for 7, 14, and 18 days. The number of differentially expressed genes (DEGs) in Alexis was 1350, 1898, and 2055 at 7, 14, and 18 days, respectively, while Ganpi2 exhibited 1195, 1682, and 2225 DEGs at the same time points. Gene expression pattern analysis revealed that Alexis responded more slowly to Pg infection compared to Ganpi2. A comparative analysis identified 457 DEGs associated with Ganpi2's immunity to Pg. Functional enrichment of these DEGs highlighted the involvement of genes related to plant-pathogen interactions and kinase activity in Pg immunity. Additionally, 20 resistance genes and 24 transcription factor genes were predicted from the 457 DEGs. Twelve candidate genes were selected for qRT-PCR verification, and the results showed that the transcriptomic data was reliable. We conducted cloning of the candidate Pg resistance gene HvLRR_8-1 by the barley cultivar Ganpi2, and the sequence analysis confirmed that the HvLRR_8-1 gene contains seven leucine-rich repeat (LRR) domains and an S_TKc domain. Subcellular localization in tobacco indicates that the HvLRR_8-1 is localized on the cell membrane. Through the functional analysis using virus-induced gene silencing, it was demonstrated that HvLRR_8-1 plays a critical role in regulating barley resistance to Pg. This study represents the first comparative transcriptome analysis of barley varieties with differing responses to Pg infection, providing that HvLRR_8-1 represents a promising candidate gene for improving durable resistance against Pg in cultivated barley.

Keywords: BSMV-VIGS; Hordeum vulgare L.; HvLRR_8-1; Pyrenophora graminea; barley leaf stripe; transcriptome.

Figure 1

The symptom observation of barley leaf stripe. (A): Barley leaf stripe symptoms of a natural infection at boot stage in the field. (B): Barley leaf stripe symptoms of a natural infection at filling stage in the field. (C): Morphological observation of conidia from leaf infected with barley leaf stripe. (D): The symptoms observed in a scanning microscope for magnification at ×100. (E): The symptoms observed in a scanning microscope for magnification at ×400. (F): The symptoms were observed in a scanning microscope for magnification at ×800. (G): The symptoms observed in a scanning microscope for magnification at ×1500. (H): Barley leaf stripe symptoms of Alexis after an artificial inoculation at seedling stage in the phytotron.

Figure 2

Differentially expressed gene selection related to immunity of Ganpi2 to Pyrenophora graminea. (A): Differentially expressed genes between inoculated and uninoculated barley at the same point in time. (B): Expression pattern differences between Alexis and Ganpi2 after Pyrenophora graminea infection. (C): Differentially expressed genes have special existence in G_7d_T-G_14d_T and G_14d_CK-G_14d_T, but not in A_7d_T-A_14d_T and A_14d_CK-A_14d_T. (D): Differentially expressed genes have a special existence in 700 DEGs exclusively in Ganpi2 and A_14d_T-G_14d_T, but not in A_14d_CK-G_14d_CK. A represents the embryos taken from Alexis, G represents the embryos taken from Ganpi2, T represents the treatment group infected by Pg, and CK represents the control group uninfected; 7d, 14d, and 18d respectively represent the 7th day, 14th day, and 18th day of Pg infecting the embryos.

Figure 3

Annotation and qRT-PCR validation of differentially expressed genes related to barley immunity to Pyrenophora graminea. (A): Bubble chart of KEGG enrichment of 457 DEGs related to immunity of Ganpi2 to Pyrenophora graminea; (B): Bubble chart of GO enrichment of 457 DEGs related to immunity of Ganpi2 to Pyrenophora graminea. (C): Heatmap of 27 DEGs belonging to Plant-Pathogen interaction. Red represents up-regulation, and blue represents down-regulation of gene expression. Expression values were FPKM. (D): Quantitative real-time PCR validation of 12 differentially expressed genes and the corresponding expression data of RNA-Seq during various stages of barley infected by Pyrenophora graminea. The columns represent relative expression levels obtained by qRT-PCR, and the broken line represents relative expression obtained by RNA-Seq. Error bars represent standard errors.

Figure 4

Expression analysis and bioinformatics analysis of the barley receptor protein kinase gene HvLRR_8-1. (A): Prediction of the transmembrane structure of the HvLRR_8-1 protein. (B): Schematic representation of the functional structural domains of the protein encoded by the HvLRR_8-1 gene. Leucine-rich repeat (LRR), transmembrane region, and S_TKc kinase structural domains are represented by green squares, blue squares, and pentagons, respectively. (C): Prediction of HvLRR_8-1 protein tertiary structure. (D): Phylogenetic tree of barley HvLRR_8-1 and other related LRR proteins by adopting maximum livelihood model with bootstrap value n = 1000 using MEGA 11.0. The red dot shows the position of HvLRR_8-1. (E): The expression pattern of HvLRR_8-1 gene response to Pyrenophora graminea. 7 dpi, 14 dpi, and 18 dpi represent 7th, 14th, and 18th days post-infection, A denotes the Pg-susceptible barley cultivar Alexis, and G denotes the Pg-immune barley cultivar Ganpi2. CK represents the uninoculated control group, and QWC represents the inoculated with the highly pathogenic Pyrenophora graminea strain QWC. ns: No significant difference, **: p < 0.05, ***: p < 0.001.

Figure 5

Subcellular localization of the barley HvLRR_8-1 protein in N. benthamiana. Green fluorescence protein (GFP), chlorophyll autofluorescence (Auto), bright-field, and merged images are shown. Scale bar represents 20 μm. pRI101-EGFP: HvLRR_8-1 fusion protein was detected in the cell membrane, and pRI101-EGFP fusion protein was used as a positive control.

Figure 6

Phenotype of the HvLRR_8-1-silenced barley leaves and the identification of Pyrenophora graminea resistance. (A): Comparison leaves phenotype and staining symptoms of the wild-type Ganpi2 barley leaves (Mock), the vector control barley leaves (BSMV: γb), and the HvLRR_8-1-silenced barley leaves (BSMV: HvLRR_8-1) inoculated with Pyrenophora graminea. Scale bar represents 1 cm. (B): Relative expression level of HvLRR_8-1 in BSMV: γb, and BSMV: HvLRR_8-1 after inoculating with Pyrenophora graminea. (C): Lesion areas of HvLRR_8-1 in Mock, BSMV: γb, and BSMV: HvLRR_8-1 after inoculating with Pyrenophora graminea. (D): Relative chlorophyll content (SPAD) of HvLRR_8-1 in Mock, BSMV: γb, and BSMV: HvLRR_8-1 after inoculating with Pyrenophora graminea. **: p < 0.01, ***: p < 0.001.