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MicroRNAs (miRNAs) play important regulatory roles in plant development and stress responses. Tomato is an economically important vegetable crop in the world with publicly available genomic information database, but only a limited number of tomato miRNAs have been identified. In this study, two independent small RNA libraries from mock and Cucumber mosaic virus (CMV)-infected tomatoes were constructed, respectively, and sequenced with a high-throughput Illumina Solexa system. Based on sequence analysis and hairpin structure prediction, a total of 50 plant miRNAs and 273 potentially candidate miRNAs (PC-miRNAs) were firstly identified in tomato, with 12 plant miRNAs and 82 PC-miRNAs supported by both the 3p and 5p strands. Comparative analysis revealed that 79 miRNAs (including 15 new tomato miRNAs) and 40 PC-miRNAs were differentially expressed between the two libraries, and the expression patterns of some new tomato miRNAs and PC-miRNAs were further validated by qRT-PCR. Moreover, potential targets for some of the known and new tomato miRNAs were identified by the recently developed degradome sequencing approach, and target annotation indicated that they were involved in multiple biological processes, including transcriptional regulation and virus resistance. Gene ontology analysis of these target transcripts demonstrated that defense response- and photosynthesis-related genes were most affected in CMV-Fny-infected tomatoes. Because tomato is not only an important crop but also is a genetic model for basic biology research, our study contributes to the understanding of miRNAs in response to virus infection.

Usually, modifications in normal microRNAs (miRs)-dependent gene expression are induced in transgenic plants expressing viral silencing suppressors or in viruses-inoculated plants. These phenomena are generally interpreted as a side effect of the antiviral silencing suppression. However, it has been recently reported that certain animal miRs prevent viral infection by directly interfering with pathogen replication or by binding to viral transcripts. To investigate whether such mechanisms operate in plants, we performed in silico analysis using tomato miR/miR* sequences and genomes of Cucumber mosaic virus (CMV)-Fny (the severe strain), CMV-Q (the mild strain), the aggravated satellite RNA (satRNA) variant satT1 and the attenuated variant satYn12. Results showed that a total of 38 and 37 miR/miR* sequences could bind to genomic RNAs of CMV-Fny and CMV-Q, respectively. But only one sequence could bind to safT1 and three to satYn12. Interestingly, we found that these miRs/miR*s exhibited significant complementarity within 2a and 3a Open Reading Frames (ORFs) of CMV. And most of the sequences with potential to bind viral genomes were of miR*s, suggesting novel role of miR*s in host defense response. This finding is noteworthy as plant miR* sequences are presently thought very unstable and are not assigned with any function. Finally, we could predict targets in tomato database for the miR/miR* sequences that are highlighted in our study, most of them are involved in plant metabolism. Overall, these results shed light on the possible novel role of plant miR/miR* in antiviral defense mechanisms.

The economic importance of Solanaceae plant species is well documented, and tomato has become a model for fleshy fruit development and ripening studies. Plant microRNAs (miRNAs) are small endogenous RNAs that are involved in a variety of activities including plant development, signal transduction and protein degradation, as well as response to environment stress and pathogen invasion. Here in this study, we aimed at quantifying the expression alterations of nine miRNAs and target mRNAs in tomato flower and fruit development upon Cucumber mosaic virus (CMV) and Tomato aspermy virus infections. Three different CMV strains CMV-Fny, CMV-FnyΔ2b and CMV-Fny-satT1 were used in our investigation, and the miRNA/mRNA expression alterations were analyzed by real-time quantitative RT-PCR. The results shown the levels of several miRNA/mRNA pairs were increased upon virus infections. However, the increased level of individual miRNA differed for different virus strains, reflecting differences in severity of symptom phenotypes. The altered expression patterns of these miRNA/mRNA pairs and their predicted functions indicate the possible roles in flower and fruit development, and provide experimental data for understanding the miRNA-mediated phenotype alterations in tomato fruit.

Viral infections generally cause disease symptoms by interfering with the microRNA (miRNA)-mediated regulation of gene expression of host plants. In tomato leaves, the accumulation levels of eleven miRNAs and ten target mRNAs were enhanced by different degrees upon Cucumber mosaic virus (CMV)-Fny and Tomato aspermy virus (TAV)-Bj infections. The ability of CMV-Fny to interfere with miRNA pathway was dramatically suppressed in the addition of the benign satellite (sat) RNA variant (satYn12), but was slightly affected when CMV-Fny was co-inoculated with the aggressive satRNA variant (satT1). In plants harboring the infection of CMV-FnyΔ2b (a CMV-Fny 2b-deletion mutant), the unaltered miRNAs and target mRNAs levels compared with mock inoculated plants indicated that 2b ORF was essential for perturbation of miRNA metabolism. When the amounts of viral open reading frames (ORFs) in these infections were quantified, we found satYn12 caused a higher reduction of CMV-Fny accumulation levels than satT1. These results indicate the complex mechanism by which satRNAs participate in CMV-tomato interaction, and suggest that the severity of disease symptoms positively correlates to some extent with the perturbation of miRNA pathway in tomato.

MicroRNAs (miRNAs) play vital roles in regulating plant growth and development. Recent work has shown that miRNA-mediated regulation of cellular mRNA expression is involved in pathogen-host interactions. However, knowledge about the timing and spatial regulation of plant miRNA expression is still limited. Here, we use stem-loop real-time reverse transcription-polymerase chain reaction to quantify the expression changes of seven miRNAs and their target mRNAs in different tomato tissues during the pathogenic processes. Compared with mock-inoculated plants, the expression levels of investigated miRNAs and mRNAs were enhanced by different degrees upon Cucumber mosaic virus (CMV)-Fny and Tomato aspermy virus-Bj infections, but were almost unchanged in CMV-FnyΔ2b (a CMV-Fny 2b-deletion mutant)-infected tomato seedlings. In addition, the obvious up-regulation of several miRNAs and target mRNAs in some tomato tissues suggested their special roles in these tissues' organogenesis and development. Temporal analyses also revealed that the expressions of these miRNAs and mRNAs were highly regulated by different viral infections. Taken together, the observed spatially and temporally changes in miRNAs and target mRNAs expression levels indicate the abilities of different viruses to interfere with miRNA pathway, and are correlated with their respective functions in phenotype determination in different tomato tissues.

MicroRNAs (miRNAs) are a newly identified class of non-coding small RNAs of about 21-24 nucleotides. They play important roles in multiple biological processes by degrading targeted mRNAs or repressing mRNA translation. To date, a total of 2,043 plant miRNAs are present in the miRNA Registry database (miRBase Release 14.0), and none for tobacco (Nicotiana tabacum). In this research, we used known plant miRNAs against both genomic survey sequence (GSS) and expressed sequence tags (EST) databases to search for potential miRNAs in tobacco. A total of 25 potential miRNAs were identified following a range of strict filtering criteria, and 33 potential targets of miRNAs were predicted by searching the tobacco Unigene database. Most of these miRNA targeting genes were predicted to encode transcription factors which play important roles in tobacco development. Additionally, real-time PCR assays were performed to profile the expression levels of 10 miRNAs after the infection of Cucumber mosaic virus (CMV) and Potato virus X (PVX). The results showed that symptom severity is correlated to the miRNA accumulation, and increased miR168 expression during virus infection is a common, plant- and virus-independent response.

Potato (Solanum tuberosum) is an important crop around the world, and accounts for a significant amount of the food consumed by humans. However, little information is available about potato miRNAs which play important regulatory roles in plant growth and development. In the present study, computational prediction of potential miRNAs from potato revealed 71 miRNAs belonging to 48 families. Amongst these 71 mRNAs, 65 were predicted for the first time. Most potato miRNA families have one to three members, and sequence analysis showed that the candidate pre-miRNA sequences varied from 48 to 224 bp in length. To verify the predicted miRNAs, specific stem-loop RT primers were designed and real-time PCR assays were used to profile the expression levels of seven miRNAs from different tissues of potato. The results showed that all the selected miRNAs were successfully amplified. Most of them had their highest expression levels in leaves, and the lowest levels in the stem, while miR159 and miR164 presented a different expression pattern. The specific expression levels of each miRNAs in the tested tissues may be related to their particular functions in regulating potato vegetative growth and organ development.

MicroRNAs (miRNAs) are RNA molecules consisting of 20-24 nucleotides that play important roles in regulating plant's gene expression for growth and development, cell viability and stress responses. Viral infection often has a noticeable influence on host gene expression, which may result in a range of developmental abnormalities. To investigate the molecular mechanisms underlying viral infection, miRNA pathway and host gene expression, we report herein the application of the novel miRNAs quantification method in tomato, using a stem-loop reverse transcription followed by SYBR Green PCR assay. For the seven tested miRNAs of Solanum lycopersicum, which are related to the regulation of plant development, hormone response, and their own biogenesis, this quantification method showed high sensitivity, specificity, and wide dynamic range. Precise quantification could be achieved with as little as 0.01 ng of total RNAs for most cases. Additionally, their target mRNAs could be quantified from the same RNA sample simultaneously, by the conventional real-time RT-PCR assay. In comparison with mock inoculation, accumulation levels of the tested miRNAs and target mRNAs were found obviously altered in tomato seedlings, indicating that the miRNA pathway was interrupted by Cucumber mosaic virus and Tomato aspermy virus infection.