Identifying Putative Resistance Genes for Barley Yellow Dwarf Virus-PAV in Wheat and Barley

Abstract

Barley yellow dwarf viruses (BYDVs) are one of the most widespread and economically important plant viruses affecting many cereal crops. Growing resistant varieties remains the most promising approach to reduce the impact of BYDVs. A Recent RNA sequencing analysis has revealed potential genes that respond to BYDV infection in resistant barley genotypes. Together with a comprehensive review of the current knowledge on disease resistance in plants, we selected nine putative barley and wheat genes to investigate their involvement in resistance to BYDV-PAV infection. The target classes of genes were (i) nucleotide binding site (NBS) leucine-rich repeat (LRR), (ii) coiled-coil nucleotide-binding leucine-rich repeat (CC-NB-LRR), (iii) LRR receptor-like kinase (RLK), (iv) casein kinase, (v) protein kinase, (vi) protein phosphatase subunits and the transcription factors (TF) (vii) MYB TF, (viii) GRAS (gibberellic acid-insensitive (GAI), repressor of GAI (RGA) and scarecrow (SCR)), and (ix) the MADS-box TF family. Expression of genes was analysed for six genotypes with different levels of resistance. As in previous reports, the highest BYDV-PAV titre was found in the susceptible genotypes Graciosa in barley and Semper and SGS 27-02 in wheat, which contrast with the resistant genotypes PRS-3628 and Wysor of wheat and barley, respectively. Statistically significant changes in wheat show up-regulation of NBS-LRR, CC-NBS-LRR and RLK in the susceptible genotypes and down-regulation in the resistant genotypes in response to BYDV-PAV. Similar up-regulation of NBS-LRR, CC-NBS-LRR, RLK and MYB TF in response to BYDV-PAV was also observed in the susceptible barley genotypes. However, no significant changes in the expression of these genes were generally observed in the resistant barley genotypes, except for the down-regulation of RLK. Casein kinase and Protein phosphatase were up-regulated early, 10 days after inoculation (dai) in the susceptible wheat genotypes, while the latter was down-regulated at 30 dai in resistant genotypes. Protein kinase was down-regulated both earlier (10 dai) and later (30 dai) in the susceptible wheat genotypes, but only in the later dai in the resistant genotypes. In contrast, GRAS TF and MYB TF were up-regulated in the susceptible wheat genotypes while no significant differences in MADS TF expression was observed. Protein kinase, Casein kinase (30 dai), MYB TF and GRAS TF (10 dai) were all up-regulated in the susceptible barley genotypes. However, no significant differences were found between the resistant and susceptible barley genotypes for the Protein phosphatase and MADS FT genes. Overall, our results showed a clear differentiation of gene expression patterns in both resistant and susceptible genotypes of wheat and barley. Therefore, further research on RLK, NBS-LRR, CC-NBS-LRR, GRAS TF and MYB TF can lead to BYDV-PAV resistance in cereals.

Keywords: BYDV-PAV; RT-qPCR; barley; gene expression; resistance; wheat.

Figure 1

BYDV-PAV titre (virus copy number) in barley genotypes (A) and wheat genotypes (B). Significant differences are shown after one-way ANOVA and Tukey’s multiple comparison test, (* = p < 0.05 ** = p < 0.01 and *** = p < 0.001). Bars represent the means and standard errors of three biological replicates.

Figure 2

Heat map showing expression profile of genes associated with resistance to BYDV-PAV infection in barley genotypes (A) and wheat genotypes (B). The blue colour stands for upregulation, the green for downregulation and the grey for control values (1). Up-regulation is mainly observed in susceptible genotypes (left), while down-regulation is more common in resistant genotypes (right). These patterns are particularly consistent for NBS, CC-NBS-LRR and Rec Kin in the inoculated samples of barley and wheat. Slight differences between 10 dai and 30 dai are also observed. The maps were generated with Graphpad Prism software using expression fold change values obtained from qPCR, followed by statistical analysis using two-way ANOVA.

Figure 3

Box plots showing fold change in NBS-LRR resistance genes expression in different barley and wheat genotypes at 10 and 30 dai. The horizontal line in each box represents the mean expression fold change, and the lower and upper box limits the minimum and maximum values, respectively. Two-way ANOVA and Tukey’s multiple comparison test revealed significant differences * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. The straight arrow from Semper to PSR 3628 indicates the samepvalue when Semper is compared to all genotypes.

Figure 4

Box plots showing the expression fold change of CC-NBS-LRR in different barley wheat genotypes at 10 and 30 dai. The horizontal line in each box represents the mean expression fold change, and the lower and upper box limits the minimum and maximum values, respectively. Two-way ANOVA and Tukey’s multiple comparison test revealed significant differences * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001.

Figure 5

Box plots showing the expression fold change of LRR receptor-like kinase in different barley and wheat genotypes at 10 and 30 dai. The horizontal line in each box represents the mean expression fold change, and the lower and upper box limits the minimum and maximum values, respectively. Two-way ANOVA and Tukey’s multiple comparison test revealed significant differences * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. The straight line at the top indicates the samepvalue when that genotype is compared to the rest in line.

Figure 6

Box plots showing the expression fold change of Casein kinase in different barley and wheat genotypes at 10 and 30 dai. The horizontal line in each box represents the mean expression fold change, and the lower and upper box limits the minimum and maximum values, respectively. Two-way ANOVA and Tukey’s multiple comparison test revealed significant differences * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001.

Figure 7

Box plots showing the expression fold change of Protein kinase in different barley and wheat genotypes at 10 and 30 dai. The horizontal line in each box represents the mean expression fold change, and the lower and upper box limits the minimum and maximum values, respectively. Two-way ANOVA and Tukey’s multiple comparison test revealed significant differences * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001.

Figure 8

Box plots showing the expression fold change of Protein phosphatase in different barley and wheat genotypes at 10 and 30 dai. The horizontal line in each box represents the mean expression fold change, and the lower and upper limits the minimum and maximum values, respectively. Two-way ANOVA and Tukey’s multiple comparison test revealed significant differences * = p < 0.05 ** = p < 0.01 and **** = p < 0.0001.

Figure 9

Box plots showing the expression fold change of MYB TF in different barley wheat genotypes at 10 and 30 dai. The horizontal line in each box represents the mean expression fold change, and the lower and upper box limits the minimum and maximum values, respectively. Two-way ANOVA and Tukey’s multiple comparison test revealed significant differences * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001.

Figure 10

Box plots showing the expression fold change of GRAS TF in different barley and wheat genotypes at 10 and 30 dai. The horizontal line in each box represents the mean expression fold change, and the lower and upper box limits the minimum and maximum values, respectively. Two-way ANOVA and Tukey’s multiple comparison test revealed significant differences * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001.

Figure 11

Box plots showing the expression fold change of MADS box transcription factor in different barley and wheat genotypes at 10 and 30 dai. The horizontal line in each box represents the mean expression fold change, and the lower and upper box limits the minimum and maximum values, respectively. Two-way ANOVA and Tukey’s multiple comparison test did not revealed any significant differences.