Microarray-based identification of tomato microRNAs and time course analysis of their response to Cucumber mosaic virus infection

Abstract

A large number of plant microRNAs (miRNAs) are now documented in the miRBase, among which only 30 are for Solanum lycopersicum (tomato). Clearly, there is a far-reaching need to identify and profile the expression of miRNAs in this important crop under various physiological and pathological conditions. In this study, we used an in situ synthesized custom microarray of plant miRNAs to examine the expression and temporal presence of miRNAs in the leaves of tomato plants infected with Cucumber mosaic virus (CMV). Following computational sequence homology search and hairpin structure prediction, we identified three novel tomato miRNA precursor genes. Our results also show that, in accordance with the phenotype of the developing leaves, the tomato miRNAs are differentially expressed at different stages of plant development and that CMV infection can induce or suppress the expression of miRNAs as well as up-regulate some star miRNAs (miRNA*s) which are normally present at much lower levels. The results indicate that developmental anomalies elicited by virus infection may be caused by more complex biological processes.

Fig. 1

Comparison of the expression patterns of miRNAs in mock (a) and CMV-infected (b) tomato leaves The color scale is based on the Z-value of the log2 detected signal, from green relatively-low to red relatively-high expression of miRNAs in samples. The heat maps presented here summarize four distinct white line expression patterns in (a), over the time course in healthy plants. The expression patterns of miRNA’s response to the CMV infection [yellow lines in (b)] were also surveyed and characterized by the regulation blue arrow, as compared with their expression in healthy plants

Fig. 2

Microarray image of miRNA*s accumulation in CMV-infected plants at 20 dpi Image in a region containing probes for miRNA*s for (a) CMV-inoculated samples at 20 dpi with Cy3 labeling and (b) mock samples at 20 dpi with Cy5 labeling; (c) Probe layout for the image regions shown in (a) and (b). For example, ath-miR399f annotates the probes complementary to the mature miRNAs; S-ath-miR158a annotates the probes complementary to miRNA*s; ath-miR403-1 and S-ath-miR165a-1 annotate the mismatch probes with one different mismatched nucleotide. The red boxes annotate the miRNA* probes only appeared during CMV-inoculated samples. The hybridization images were obtained using the Array-Pro image analysis software Media Cybernetics

Fig. 3

Northern blots of small RNAs extracted from mock and CMV-inoculated tomato plants at 20 dpi 5S rRNA stained with ethidium bromide was used as loading control. The graph shows the calculated absolute fold changes between mock and CMV-inoculated samples at 20 dpi deduced from Northern blotting after quantification by a Phosphor-Imager and microarray data analysis

Fig. 4

Homologous analysis of miR156/miR157 family sequences and their expression patterns A total of eight different miR156/157 analog probes on the array, and eight probes of the two expression groups (shaded in yellow) had detectable hybridization signals. They may be homologous to the sequence we predicted (sly-miR156d, shaded in blue) or the sequence which was reported in miRBase (sly-miR156a–c shaded in gray). Red letters are the mismatched nucleotides