Analysis of Small RNAs of Barley Genotypes Associated with Resistance to Barley Yellow Dwarf Virus

Abstract

Barley yellow dwarf virus (BYDV) causes an often-devastating disease of cereals that is most effectively controlled by using plant genotypes that are resistant or tolerant to the virus. New barley lines Vir8:3 and Vir13:8, with pyramided resistance genes against different pathogens and resistance gene Ryd2 against BYDV, are currently being tested. Because microRNAs (miRNAs) are associated with antiviral plant defense, here we compared the miRNA profiles in these lines and in cultivar Wysor (carrying one resistance gene, Ryd2), with and without BYDV infection and after feeding by virus-free aphids, to determine whether the miRNA profile in the resistant variety bear similarities with the newly developed lines. The BYDV titer for each group was also determined and compared to the titer in sensitive cultivar Graciosa. Among 746 miRNAs identified in barley, 66 were known miRNAs, and 680 were novel. The expression of 73 miRNAs differed significantly after BYDV infection, including the strong, specific upregulation of novel miRNA10778 that was conserved across all the barley genotypes. This miRNA belongs to the H box and ACA box (H/ACA) snoR14 family of RNAs (Rf01280) and is associated with pseudourydilation. The expression of 48 miRNAs also differed depending on the barley genotype. The profile of miRNAs expressed in Vir8:3 and Vir13:8 in response to BYDV was similar and differed from that of Wysor. Insights into the expression patterns of miRNAs in response to BYDV in barley provided here will benefit further studies toward understanding the resistance mechanisms and developing novel strategies against virus infections.

Keywords: BYDV; NGS; Ryd2; miRNA; sRNA; sequencing.

Figure 1

Cultivars used in the present study. Wysor has Ryd2 for moderate resistance to BYDV, whereas line Vir13:8 is a cross between six-rowed, non-malting winter barley cultivar Tramine and the Ryd2-carrying, two-rowed feed barley cultivar Doria.

Figure 2

Relative Barley yellow dwarf virus (BYDV) titer in infected barley (Hordeum vulgare L.) genotypes 7 and 14 days post inoculation (dpi). The standard deviation is expressed as error bars. The whole plants (above-ground biomass) was collected and assayed. Relative titer values were calculated using the 2^−ΔΔCt method.

Figure 3

Length distribution of individual miRNAs. (A) Total calculated reads of all miRNAs from healthy, aphid-infested non-infected, and BYDV-infected plants. (B) Individual miRNAs.

Figure 4

Karyogram showing the chromosomal positions of novel miRNAs on chromosomes 1 to 7H of barley (Hordeum vulgare). The position of each miRNA is shown using a green circle. Centromeres are represented using a red oval. The final karyogram was drawn using the ggplot function in the ggplot package of R and an R script, as described [34].

Figure 5

Number of differentially expressed miRNAs from barley cultivars Wysor, Vir8:3, and Vir13:8 in response to BYDV infection. The differential expression was analyzed using the R bioconductor packages DESEq2 and EdgeR. The miRNAs with a log fold-change (Log₂ FC) more than 1.5 (1.2) or less than Log₂FC—1.5 (1.2) and with p-value less than 0.05 were considered significantly differentially expressed.

Figure 6

Expression of selected miRNAs as influenced by the barley genotype in response to BYDV infection. The miRNA expression in the BYDV infected group is shown. The average number of reads from the biological triplicates was used and transformed to normalized values such that a graphical comparison could be made. The values on the left axis are the fold-change values. In the top graph (A), miRNAs varying in their expression between Vir8:3 and the other two genotypes (Vir13:8 and Wysor) are displayed; in the middle graph (B), miRNAs varying in their expression between Wysor and the other two genotypes (Vir8:3 and Vir13:8) are displayed; and in the bottom graph (C), miRNAs varying in their expression between Vir13:8 and the other two genotypes (Vir8:3 and Wysor) are displayed.

Figure 7

Gene ontology (GO) annotations for known and novel miRNAs from Hordeum vulgare: 39 for molecular function (MF), 79 for biological process (BP), and 16 for cellular component (CC). (A) Venn diagram for miRNAs. (B) Network analysis of individual GO terms: molecular function in red, biological process in black, and cellular component in green. The functional enrichment analysis of miRNAs was performed using g:GOSt implemented in the g:profile online web server (p < 0.05).

Figure 8

siRNA analysis in healthy, aphid-infested, and Barley yellow dwarf virus (BYDV)-infected barley genotypes. (A) Distribution of sense and antisense vsRNAs calculated as a percentage. (B) Length distribution of siRNAs derived from plants (upper) and the BYDV-PAV strain of Barley yellow dwarf virus (lower). X-axis: length of vsRNAs (nucleotides). Y-axis: number of reads. (C) Abundancy of BYDV-derived siRNAs in BYDV-infected samples of individual genotypes. Values are scaled logarithmically (value 0 is graphed as −1). (D) distribution of vsRNAs on the BYDV-PAV genome. X-axis: BYDV genomic position. Y-axis: number of reads.