MicroR408 regulates defense response upon wounding in sweet potato

Abstract

MiRNAs play diverse roles in plant development and defense responses by binding to their mRNA targets based on sequence complementarity. Here, we investigated a wound-related miR408 and its target genes in sweet potato (Ipomoea batatas) by small RNA deep sequencing and transcriptome analysis. The expression patterns of miR408 and the miR408 precursor were significantly repressed by wounding and jasmonate (JA). In contrast, expression of the putative target genes IbKCS (3-ketoacyl-CoA synthase 4), IbPCL (plantacyanin), and IbGAUT (galacturonosyltransferase 7-like) of miR408 was increased following wounding, whereas only IbKCS was increased after JA treatment. Target cleavage site mapping and Agrobacterium-mediated transient assay demonstrated that IbKCS, IbPCL, and IbGAUT were the targets of miR408. The expression of miR408 target genes was repressed in transgenic sweet potatoes overexpressing miR408. These data indicated a relationship between miR408 and its target genes. Notably, miR408-overexpressing plants showed a semi-dwarf phenotype and attenuated resistance to insect feeding, while transgenic plants overexpressing IbKCS exhibited more insect resistance than plants overexpressing only the empty vector. Collectively, sweet potato reduces the abundance of miR408 upon wounding to elevate the expression of IbKCS, IbPCL, and IbGAUT. The expression of IbKCS enhances the defense system against herbivore wounding.

Fig. 1.

Expression patterns of miR408 precursor (pre408) and miR408 in response to wounding. (A) The secondary structure of pre408 in sweet potato. (B) Expression patterns of miR408 in sweet potato upon wounding were analyzed by northern blotting. Total RNAs were extracted from the leaves wounded for 0 (W–), 15, 30, 45, 60, and 120 min. Northern blotting was used to determine the expression levels of miR408. The expression levels of miR408 were normalized by those of 5.8S rRNA. The expression patterns of pre408 (C) and miR408 (D) in the leaves of sweet potato wounded by mechanical wounding or insect feeding for 0, 15, 30, 45, 60, and 120 min were analyzed by qRT-PCR. The qRT-PCR results were normalized by the expression levels of IbActin. Data are presented as means ±SD (n=4). The asterisks represent a significant difference from unwounded treatment by Student’s t-test (*P<0.05).

Fig. 2.

Expression patterns of miR408 target genes upon wounding and insect feeding. The expression of miR408 targets, IbKCS (A), IbPCL (B), and IbGAUT (C) in the leaves of sweet potato wounded by mechanical wounding or insect feeding for 0, 15, 30, 45, 60, and 120 min were analyzed by qRT-PCR. The qRT-PCR results were normalized by the expression levels of IbActin. Data are presented as means ±SD (n=4). The asterisks represent a significant difference from unwounded treatment by Student’s t-test (*P<0.05).

Fig. 3.

Validations of miR408 target genes by RACE and Agrobacterium-mediated transient assays. The potential cleavage sites of miR408 on three targets, IbKCS (A), IbPCL (B), and IbGAUT (C), in sweet potato were analyzed by 5'-RLM-RACE and 3'-PPM-RACE. The arrows indicate the positions of the cleavage sites, and clone frequencies are indicated by the numbers. In addition, Agrobacterium-mediated transient expression assays of miR408 and its targets, IbKCS, IbPCL, and IbGAUT, in tobacco leaves. Tobacco leaves were infiltrated with agrobacteria with a vector containing the miR408 targets, IbKCS (35S:IbKCS), IbPCL (35S:IbPCL), or IbGAUT (35S:IbGAUT), and with a vector containing the empty vector (EV), the miR408 precursor (Pre408), the short tandem target mimic of miR408 (STTM), or miR408 plus STTM. The expression of IbKCS (D), IbPCL (E), and IbGAUT (F) in tobacco leaves was then analyzed by qRT-PCR, and normalized to the levels of NPTII expression. The ratios relative to the plants with empty vector are shown as relative expression levels. Data are presented as means ±SD (n=4). The asterisks represent a significant difference by Student’s t-test (*P<0.05).

Fig. 4.

Gene expression and insect resistances of sweet potato overexpressing miR408. Expression levels of pre408 (A), miR408 (B), IbKCS (C), IbPCL (D), and IbGAUT (E) in wild-type (WT) and miR408-overexpressing (Pre408-ox1 and Pre408-ox2) sweet potato were analyzed by qRT-PCR. Sweet potato leaves were wounded by tweezers for 120 mins. The expression level of 5.8S rRNA was used as an internal control for miR408 expression. IbACT expression levels were used as internal controls for pre408 and miR408 target expression. Data are presented as means ±SD (n=4). The asterisks represent a significant difference by Student’s t-test (*P<0.05). (F) Representative leaves of WT, Pre408-ox1, and Pre408-ox2 sweet potato before (CK) and after feeding with S. litura. (G) Body weights of the second-instar larvae fed WT or transgenic plants were measured at 5, 7, and 9 d after feeding (DAF). Data are presented as means ±SD (n≥10). The asterisks represent a significant difference between WT and miR408-ox plants by Student’s t-test, *P<0.05; **P<0.01). (H) Representative images of the larvae fed WT or miR408-ox plants after 9 d.

Fig. 5.

Insect resistance of IbKCS-overexpressing, IbPCL-overexpressing, and IbGAUT-overexpressing tobacco plants. (A) Body weights of the second-instar larvae were measured at the indicated time after larvae were fed the leaves from transgenic tobacco with empty vector (EV), and overexpressing IbKCS (KCS-ox4 and KCS-ox11), overexpressing IbPCL (PCL-ox1 and PCL-ox4), and overexpressing IbGAUT (GAUT-ox1 and GAUT-ox5). (B) Representative images of the larvae fed EV, KCS-ox, PCL-ox, and GAUT-ox tobacco leaves after 9 d. Data are presented as means ±SD (n≥8). The asterisks represent a significant difference between EV and overexpressing tobacco plants by Student’s t-test (*P<0.05).

Fig. 6.

Expression patterns of pre408, miR408, and its targets after JA treatment. Total RNAs were extracted from sweet potato leaves sprayed with 50 µM MeJA for 0, 30, 60, and 120 min. The expression patterns of pre408 (A), miR408 (B), IbKCS (C), IbPCL (D), and IbGAUT (E) were determined by qRT-PCR. The expression of 5.8S rRNA was used as an internal control for normalization of miR408 expression. The expression of IbACT was used as an internal control for normalization of pre408 and its target genes. Data are indicated as means ±SD (n=4). The asterisks represent a significant difference by Student’s t-test (*P<0.05).

Fig. 7.

The phenotypes of miR408-overexpressing and wild-type (WT) sweet potato. (A) Water loss rates of WT and miR408-overexpressing (Pre408-ox1 and Pre408-ox2) plants. The water loss rates of the detached sweet potato leaves are shown. (B) The phenotypes of WT and miR408-overexpressing transgenic plants. Plants were grown for 6 weeks under a 16 h light/8 h dark photoperiod before photography. The stem lengths (C), root lengths (D), total chlorophyll contents (E), and PSII efficiencies (F) of WT and miR408-overexpressing sweet potato were measured. Data are presented as means ±SD (n≥6). The asterisk represents a significant difference between WT and miR408-ox plants by Student’s t-test (*P<0.05; **P<0.01).