Molecular mechanism of modulating miR482b level in tomato with botrytis cinerea infection

Abstract

Background: Plant miRNAs are involved in the response to biotic and abiotic stresses by altering their expression levels, and they play an important role in the regulation of plant resistance to stress. However, the molecular mechanism that regulates the expression levels of miRNAs in plants with biotic and abiotic stress still needs to be explored. Previously, we found that the expression of the miR482 family was changed in tomato infected by Botrytis cinerea. In this study, we investigated and uncovered the mechanism underlying the response of miR482 to B. cinerea infection in tomato.

Results: First, RT-qPCR was employed to detect the expression patterns of miR482b in tomato infected by B. cinerea, and results showed that miR482b primary transcripts (pri-miR482b) were up-regulated in B. cinerea-infected leaves, but the mature miR482b was down-regulated. Subsequently, we used rapid amplification cDNA end method to amplify the full-length of pri-miR482b. Result showed that the pri-miR482b had two isoforms, with the longer one (consisting 300 bp) having an extra fragment of 53 bp in the 3'-end compared with the shorter one. In vitro Dicer assay indicated that the longer isoform pri-miR482b-x1 had higher efficiency in the post-transcriptional splicing of miRNA than the shorter isoform pri-miR482b-x2. In addition, the transcription level of mature miR482b was much higher in transgenic Arabidopsis overexpressing pri-miR482b-x1 than that in OE pri-miR482b-x2 Arabidopsis. These results confirmed that this extra 53 bp in pri-miR482b-x1 might play a key role in the miR482b biogenesis of post-transcription processing.

Conclusions: Extra 53 bp in pri-miR482b-x1 enhanced miR482b biogenesis, which elevated the transcription level of miR482b. This study clarified the response of miR482 to B. cinerea infection in tomato, thereby helping us further understand the molecular mechanisms that regulate the expression levels of other miRNAs.

Keywords: Botrytis cinerea; pathogen response; posttranscriptional processing; pri-miR482b isoforms; sly-miR482b.

Fig. 1

Expression patterns of miR482b and its target genes in mock- and B. cinerea-infected leaves at 0, 12, 24 and 72 hpi. A-B) The expressions of pri-miR482b (A) and miR482b (B). SlyU6 were used as the internal control. Results are expressed as means ± SD of three biological replicates. Asterisks indicate a significant difference (P < 0.05) compared with the corresponding 0 hpi leaves. C-F) The expressions of four NBS-LRR genes (Solyc02g036270.2, Solyc04g009070.1, Solyc12g016220.2, and Solyc05g008070.2) targeted by miR482b. SlyUbq3 were used as the internal control. Results are expressed as means ± SD of three biological replicates. Asterisks indicate a significant difference (P < 0.05) compared with the corresponding 0 hpi leaves

Fig. 2

Cloning and sequence analysis of full-length pri-miR482b. A) RACE Amplification of pri-miR482b. Lane M: DL 2000 DNA marker; Lane 1: 5’ end of pri-miR482b; Lane2: 3’ end of pri-miR482b. B) Schematic of two isoforms of pri-miR482b. C) Alignment of two isoforms of pri-miR482b. D) Secondary structures of two pri-miR482b isoforms

Fig. 3

Expression patterns of two pri-miR482b isoforms in mock- and B. cinerea-infected leaves at 0, 12, 24 and 72 hpi. SlyUbq3 was used as the internal control. Results are expressed as means ± SD of three biological replicates. Various letters indicate a significant difference among samples (P < 0.05)

Fig. 4

Dicer splicing activities of two pri-miR482b isoforms. (A) Splicing efficiencies of two pri-miR482b isoforms in vitro. Land 1: pri-miR482b-x1; Lane 2: pri-miR482b-x2; Lane 3: pri-miR482b-x1 with 2 mg/mL protein extraction; Lane 4: pri-miR482b-x2 with 2 mg/mL protein extraction. (B) Expression levels of pri-miR482b-x1 in pri-miR482b-x1 OE plants and the expression level of pri-miR482b-x2 in pri-miR482b-x2 OE plants via semi-quantification RT-PCR. Lanes 1–3: three OE lines of pri-miR482b-x1; Lanes 4–6: three OE lines of pri-miR482b-x2. C-D) Expression levels of pre-miR482b (C) and miR482b (D) in pri-miR482b-x1 and pri-miR482b-x2 OE plants via quantification RT-PCR. AtUBQ10 was used as the internal control. Results are expressed as means ± SD of three biological replicates. Asterisks indicates a significant difference (P < 0.05) compared with the corresponding OE_pri-miR482b-x1-1

Fig. 5

Resistance analysis of transgenic Arabidopsis overexpressing pri-miR482b-x1 and pri-miR482b-x2 against B. cinerea. (A) Disease symptoms on the B. cinerea-infected leaves of WT and transgenic Arabidopsis overexpressing pri-miR482b-x1and pri-miR482b-x2 for 48 h. (B) Trypan blue staining confirmed the pathological spots. (C) Statistical analysis of the pathogenic spot diameters. Asterisks indicate a significant difference (P < 0.01) compared with the corresponding WT

Fig. 6

Expression levels of two genes (PDF1.2 and PR4) of JA-signal pathway in WT and transgenic Arabidopsis. SlyUbq3 was used as the internal control. Results are expressed as means ± SD of three biological replicates. Various letters indicate a significant difference among samples (P < 0.05)