Genome-Wide microRNA Profiling Using Oligonucleotide Microarray Reveals Regulatory Networks of microRNAs in Nicotiana benthamiana During Beet Necrotic Yellow Vein Virus Infection

Abstract

Beet necrotic yellow vein virus (BNYVV) infections induce stunting and leaf curling, as well as root and floral developmental defects and leaf senescence in Nicotiana benthamiana. A microarray analysis with probes capable of detecting 1596 candidate microRNAs (miRNAs) was conducted to investigate differentially expressed miRNAs and their targets upon BNYVV infection of N. benthamiana plants. Eight species-specific miRNAs of N. benthamiana were identified. Comprehensive characterization of the N. benthamiana microRNA profile in response to the BNYVV infection revealed that 129 miRNAs were altered, including four species-specific miRNAs. The targets of the differentially expressed miRNAs were predicted accordingly. The expressions of miR164, 160, and 393 were up-regulated by BNYVV infection, and those of their target genes, NAC21/22, ARF17/18, and TIR, were down-regulated. GRF1, which is a target of miR396, was also down-regulated. Further genetic analysis of GRF1, by Tobacco rattle virus-induced gene silencing, assay confirmed the involvement of GRF1 in the symptom development during BNYVV infection. BNYVV infection also induced the up-regulation of miR168 and miR398. The miR398 was predicted to target umecyanin, and silencing of umecyanin could enhance plant resistance against viruses, suggesting the activation of primary defense response to BNYVV infection in N. benthamiana. These results provide a global profile of miRNA changes induced by BNYVV infection and enhance our understanding of the mechanisms underlying BNYVV pathogenesis.

Keywords: Beet necrotic yellow vein virus; Nicotiana benthamiana; defense; hormone signaling; microRNAs; microarray; reactive oxygen intermediates; superoxide free radicals O2.

Figure 1

Stunting, leaf curling, and floral and root development defects in Nicotiana benthamiana, induced by Beet necrotic yellow vein virus (BNYVV), requires the presence of RNA4. BNYVV infections containing RNA4 induced; (a) stunting and leaf curling, (b) leaf area reduction, (c) leaf senescence, (d) floral development defects, and (e) root development defects. (d) We observed more than seven full capsules formed at the base of flowers with normal length in BN12-infected plants; in contrast, there were about four small, shriveled, deformed capsules at the base of abnormally short flowers. Statistical analysis of the stem and root length of different plants, shown in (a) and (e), are indicated in (f), and (g), respectively. Error bars represent standard deviation of three individual plants (n = 3); *** p < 0.01.

Figure 2

The distribution of microRNAs (miRNAs) of N. benthamiana from the phylogenetic perspective of plant miRNAs. The phylogenetic perspective was proposed by Taylor et al. [41]; the graphic mode was generated, according to the model constructed by Yin et al. [42]. Underlined black bold numbers indicate miRNA families in N. benthamiana; italics indicate missing miRNAs; * indicates that miRNAs belong to at least one member of an identified miRNA family; black * represents miRNAs that have been reported previously; red * represents newly identified miRNAs.

Figure 3

Differentially expressed miRNAs and their putative targets in mock- and BN1234-infected N. benthamiana. (a) Validation of the relative expression levels of selected miRNAs in response to BN1234, as determined by stem-loop quantitative real-time PCR (qRT-PCR) (gray), and microarray analysis (black). (b) Correlation of the expression ratio of selected miRNAs measured by stem-loop qRT-PCR and microarray analysis. (c) Validation of the relative expression levels of selected targets in response to BN1234, as determined by qRT-PCR (gray) and RNA-Seq (black) [10]. (d) Correlation of the expression ratio of selected targets measured by qRT-PCR and RNA-Seq [10]. EF1A was used as the internal reference control. The experiments were independently repeated three times; error bars represent the standard error of the mean (n = 3).

Figure 4

Gene ontology (GO) analysis of the predicted miRNA target genes and expression pattern of two signaling pathways that may contribute to the symptom induction by BNYVV. (a) GO analysis of predicted targets of differentially expressed miRNAs; (b) the expression changes of auxin signaling pathway caused by BNYVV infection; (c) the expression pattern of genes belonging to the auxin signaling pathway from transcriptome analysis, and IAA synthetic gene YUCCA from qRT-PCR analysis of mock- and BN1234-infected N. benthamiana. (d) The expression pattern of genes that function in lignin biosynthesis pathway. * represents the differential expression of this gene could significantly change the content of lignin [54,55,56,57,58,59,60].

Figure 5

The phenotypes of silencing of the LAC11, GRF1, and GRF3 genes at 14 days post-inoculation (dpi). (a) Silencing of LAC11 inhibited the formation of new upper leaves; N. benthamiana plants agroinfiltrated with TRV:mCherry showed no obvious abnormality at 14 dpi. In contrast, silencing of LAC11 led to the pronounced inhibition of plant apical meristem growth (see the leaves indicated by the yellow arrowheads). (b) qRT-PCR analysis confirmed the silencing of these target genes as described in (a,c,d). (c) Silencing of T-164-1 interfered with the growth of apical meristems, and there were no obvious phenotypes in TRV:NAM-inoculated plants. NAM and T-164-1 are two different genes of NAC (NAM/ATAF/CUC) gene family. (c) Silencing of GRF1 caused the dwarf symptom and inhibited the growth of leaves; TRV:mCherry- and TRV:PDS (phytoene desaturase gene)-inoculated N. benthamiana served as the negative, and positive controls, respectively.

Figure 6

The changes in ethylene and gibberellin signal pathways caused by BN1234 infection. The expression pattern of genes involved in (a,b) ethylene signal pathway and (c,d) gibberellin signal pathway from transcriptome analysis, except GA20-OX, which was identified from qRT-PCR analysis of mock- and BN1234-inoculated N. benthamiana.

Figure 7

Silencing of miR398′s target gene umecyanin enhances plant resistance against viruses, which is related to the increased level of O₂⁻. (a) The level of O₂⁻ was higher in TRV: umecyanin-treated N. benthamiana than that of TRV:mCherry-treated plants. (b) Silencing of miR398′s target gene umecyanin could enhance plant resistance against BNYVV; (c) qRT-PCR analysis was used to confirm the silencing of umecyanin and the upregulation of PR-1 in TRV:umecyanin-inoculated N. benthamiana plants. (d) The level of O₂⁻ increased in different viruses-infected plants. (e) A proposed model for the miR398- and O₂⁻-mediated plant defense against virus infection.