Genomic profiling of exogenous abscisic acid-responsive microRNAs in tomato (Solanum lycopersicum)
The miRNAs that target pathogen defense genes
Biotic stresses, including fungal, bacterial and viral pathogens, are a major constraint to crop production [52]. The phytohormone ABA plays multifaceted and crucial roles in plant pathogen resistance. In this study, we identified miRNAs which target nucleotide-binding site and leucine-rich repeat (NBS-LRR), AP2/EREBP, serine/threonine-protein kinase (STK), jasmonate ZIM-domain protein genes, and globally analyzed the miRNAs and their target genes. In total, we identified 64 miRNAs involved in regulating the expression of disease resistance genes that respond to exogenous ABA (Additional file 1: Table S8). Among them, 14 were known miRNAs with ?2 TPM in one sample, and these were selected for target gene analysis. The results showed that most of these miRNAs were down-regulated by ABA (Table 2).
To date, the majority of known plant disease-resistance proteins contain NBS-LRR. NBS-LRR resistance proteins directly or indirectly recognize pathogen avirulence factors, triggering signal transduction cascades that lead to rapid defense responses, hypersensitive reactions, and programmed cell death [53–55]. Here, we predicted that seven miRNAs act on NBS-LRR gene members, such as miR6024-3p, miR482a, sly-miR482a, sly-miR482c, and miR5083, miR6026-3p and novel_mir_674.
The miR6024-3p was predicted to target 27 members of NBS-LRR resistance genes. Interestingly, these 27 members included all the NBS-LRR targets of miR482a (one NBS-LRR target), sly-miR482a (2), sly-miR482c (6), and miR6026-3p (1). Analysis of miR6024-3p showed a decrease in expression from 132.63 to 0.01 TPM following ABA treatment, although the abundance of other four miRNAs were elevated from 33.5 to 41.77, 3.58 to 7.43, 3.58 to 7.43 and 12.82 to 18.13 TPM. These four miRNAs were predicted to act together with miR6024-3p, and most of their target NBS-LRR showed an increased expression. In the predicted 27 members of NBS-LRR resistance genes, 11 transcripts were elevated and one was decreased after ABA treatment, and 15 exhibited less obvious changes (Table 2). A novel miRNA, novel_mir_674 (3.47 to 0.01 TPM), was predicted to target six members of NBS-LRRs, where two were elevated, three were unaltered and the other was not detected. In addition, the target of miR5083 was not detected in the transcriptomes.
Globally, the results indicated the miRNA that were more abundant had larger influence on target gene expression. Admittedly, the appearance of the expression levels did not completely conform to the negative correlation between a miRNA and the target gene, but the overall trend was in accordance to this hypothesis. This could be explained by the regulatory complexity of miRNAs, which often have one or more targets, and a gene can also be targeted by multiple miRNAs.
Serine/threonine protein kinase (STK) family genes not only play crucial roles in adaption of abiotic stresses but also in pathogen defense in plant [56]. Here, we predicted four differentially expressed miRNAs that target STK genes, including miR5813, miR6024-3p, miR4376a-3p and miR319b (Table 2). A STK gene was predicted to be targeted by miR5813, and was down-regulated from 185.65 to 107.58 TPM. And the target gene had three transcripts in the transcriptome, with two that were increased in expression and one remained unchanged. miR4376a-3p was down-regulated from 48.85 to 10.30 TPM under ABA treatment, and was predicted to target seven genes. One of its targets is a mitogen-activated protein (MAP) kinase gene, which belongs to STK family. We compared the related transcriptome data and found one transcript that showed increased expression. MAP kinase is involved in the sphingolipid elicitor-dependent defense signaling pathway, which acts downstream of the heterotrimeric G protein alpha subunit and small GTPase RAC1, and may regulate the expression of various genes involved in biotic and abiotic stress responses [57].
Moreover, the receptor-like kinase (RLK), which also belongs to STK family, is implicated in plant pathogen interaction and defense responses [58]. The targets of miR6024-3p comprise two RLK genes, and both showed an increase in expression in the transcriptome upon ABA treatment. However, the novel miRNA novel_mir_418 was up-regulated (from 3.36 to 17.73 TPM) upon ABA treatment, and was predicted to target RLK, which showed one transcript with increased expression and one without change in expression level. In addition, miR319b (from 0.01 to 2.14 TPM) also targets a RLK gene, but the target gene transcript was not detected in the transcriptome. Generally, the expressions of the miRNAs described here were reduced by exogenous ABA application, and their targets of STK were elevated (Table 2).
With respect to jasmonic acid signal transduction pathway, a jasmonate ZIM-domain protein 3 encoding gene was identified as a target of miR5658. This protein, a repressor of jasmonate, is negatively regulated by the proteasome in an SCF (COI1) E3 ubiquitin-protein ligase complex-dependent manner [59, 60]. The expression level of miR5658 decreased from 21.23 to 16.55 TPM, but the target transcript was not detected (Table 2).
AP2/EREBP family regulation is usually involved in biotic stress responses, such as pathogen attack and jasmonate and ethylene pathways [61]. Here, we identified two known miRNAs that target AP2/EREBP, miR172a and miR5658, which were predicted to target eight and one AP2/EREBP genes respectively (Table 2). The expressions of these miRNAs and their targets were analyzed as described above.
Taken together, the miRNAs related to disease resistance showed a tendency to be down-regulated by ABA treatment, and the target disease resistance genes were mainly up-regulated in the transcriptome of ABA treated plants. Reportedly, ABA was considered to have multifaceted role in plant resistance to both biotrophic and necrotrophic fungi and bacteria [62]. Our results suggested that ABA improves broad-spectrum pathogen resistance in tomato, as supported by the years-field trials (data not shown). However, the underlying mechanism of pathogen resistance induced by ABA in plants remains to be fully explored.
A miRNA usually targets multiple genes, which possibly exert roles in different development stages and stresses, playing various functions. Likewise, some genes also are targeted by one or more miRNAs. Accordingly, a miRNA probably is in the presence of functional overlap in biotic and abiotic stress, such as miR6024a-3p, miR172a, miR5658, implying that a complex crosstalk between the global regulation of miRNA metabolism and ABA signaling functions enables the fine-tuning of stress response in plants [63].