Botrytis cinerea Pers. Fr. is an important pathogen causing stem rot in tomatoes grown indoors for extended periods. MicroRNAs (miRNAs) have been reported as gene expression regulators related to several stress responses and B. cinerea infection in tomato. However, the function of miRNAs in the resistance to B. cinerea remains unclear. The miRNA expression patterns in tomato in response to B. cinerea stress were investigated by high-throughput sequencing. In total, 143 known miRNAs and seven novel miRNAs were identified and their corresponding expression was detected in mock- and B. cinerea-inoculated leaves. Among those, one novel and 57 known miRNAs were differentially expressed in B. cinerea-infected leaves, and 8 of these were further confirmed by quantitative reverse-transcription PCR (qRT-PCR). Moreover, five of these eight differentially expressed miRNAs could hit 10 coding sequences (CDSs) via CleaveLand pipeline and psRNAtarget program. In addition, qRT-PCR revealed that four targets were negatively correlated with their corresponding miRNAs (miR319, miR394, and miRn1). Results of sRNA high-throughput sequencing revealed that the upregulation of miRNAs may be implicated in the mechanism by which tomato respond to B. cinerea stress. Analysis of the expression profiles of B. cinerea-responsive miRNAs and their targets strongly suggested that miR319, miR394, and miRn1 may be involved in the tomato leaves’ response to B. cinerea infection.

Gray mold of tomato is caused by the pathogen Botrytis cinerea. MicroRNAs play a crucial role in the biotic and abiotic stress responses of plants and regulate their targets by gene silencing. miR394 is an ancient and conserved miRNA in plants, and it participates in the regulation of plant development and stress responses. In our previous study, miR394 was found to respond to B. cinerea infection in tomato, but the roles and regulatory mechanisms of miR394 in B. cinerea-infected tomato remain unclear. miR394 was down-regulated in tomato in response to B. cinerea infection, showing an expression pattern opposite to the previous finding that miR394 was up-regulated in tomato cv. Jinpeng 1 infected by B. cinerea. We obtained transgenic Arabidopsis overexpressing miR394, which resulted in low expression levels of its target LEAF CURLING RESPONSIVENESS (LCR). Leaf lesion size and trypan blue staining showed that miR394 overexpression led to increased sensitivity of transgenic Arabidopsis to B. cinerea compared to wild type. We also detected changes in the expression levels of stress-related miRNAs, including miR159, miR156, miR168, and miR172. In the transgenic plants, it indicated potential cross talk between these miRNAs and miR394, except for miR159. miR394 also enhanced the expression of ARGONAUTE 1 (AGO1), DSRNA-BINDING PROTEIN 4 (DRB4) and the RNA-binding protein gene DAWDLE (DDL), which are involved in the pathways of miRNA biosynthesis and regulation, suggesting that miR394 overexpression has a feedback effect on these genes. Our data indicate that overexpression of miR394 in Arabidopsis increased the susceptibility of plants to B. cinerea by affecting the expression of its target gene LCR along with a number of key genes involved in plant miRNA metabolism (AGO1). Thus, miR394 is a negative regulator of Arabidopsis resistance to B. cinerea infection by targeting LCR.

Replanting disease is a major factor limiting the artificial cultivation of the traditional Chinese medicinal herb Salvia miltiorrhiza. At present, little information is available regarding the role of miRNAs in response to replanting disease. In this study, two small RNA libraries obtained from first-year (FPR) and second-year plant (SPR) roots were subjected to a high-throughput sequencing method. Bioinformatics analysis revealed that 110 known and 7 novel miRNAs were annotated in the roots of S. miltiorrhiza. Moreover, 39 known and 2 novel miRNAs were identified and validated for differential expression in FPR compared with SPR. Thirty-one of these miRNAs were further analyzed by qRT-PCR, which revealed that 5 miRNAs negatively regulated the expression levels of 7 target genes involved in root development or stress responses. This study not only provides novel insights into the miRNA content of S. miltiorrhiza in response to replanting disease but also demonstrates that 5 miRNAs may be involved in these responses. Interactions among the differentially expressed miRNAs with their targets may form an important component of the molecular basis of replanting disease in S. miltiorrhiza.