Identification and Validation of miRNAs Associated with the Resistance of Maize (<i>Zea mays L</i>.) to <i>Exserohilum turcicum</i>

<div><p>Northern leaf blight, caused by the fungus <i>Exserohilum turcicum</i> (Pass.), is a major disease of maize (<i>Zea mays</i> L.). MicroRNAs (miRNAs) have been recently reported as gene expression regulators related to several stress responses; however, evidence of the role of miRNAs in plant response to biotic stresses is limited. In this study, the miRNA expression patterns in maize in response to <i>E. turcicum</i> stress were investigated using a plant miRNA microarray platform. A total of 118 miRNAs were detected in mock- and <i>E. turcicum</i>-inoculated leaves. Among these miRNAs, miR530, miR811, miR829, and miR845 were identified as new miRNAs in maize through a homology-based approach. The secondary structures and putative targets of these miRNAs were also predicted. In addition, four miRNAs were differentially regulated in response to <i>E. turcicum</i>: miR811, miR829, miR845, and miR408. The functional annotation of the predicted targets indicated that these stress-responsive miRNAs regulate metabolic, morphologic, and physiologic adaptations in maize seedlings at the post-transcriptional level. Four targets were negatively correlated with their corresponding miRNAs (miR811, miR829, and miR408). Furthermore, we have demonstrated for the first time that miR811 and miR829 confers a high degree of resistance to <i>E. turcicum</i>, which can be used in maize breeding programs.</p></div

Overexpression and disease-resistance analysis of <i>E. turcicum</i>-responsive miRNAs.

<p>A) The expression levels of pre-miR811, pre-miR829, and pre-miR408 were detected in <i>Agrobacterium</i>-transformed leaves and control leaves; B) The expression of targets were analyzed in miRNA-overexpressing and control leaves; C) miRNA-overexpressing and control leaves were inoculated with <i>E. turcicum</i> agar discs (4 mm in diameter). Then, the maize leaves infected with <i>E. turcicum</i> were observed at 3 dpi. D) Analysis of <i>E. turcicum</i> amount in maize leaves. The EtTubulin was quantified using quantitative PCR, and the ZmTubulin was used as the internal control. Error bars indicate SD obtained from three biological repeats.</p

Quantitative analysis of miR811 levels by stem-loop real-time RT-PCR at 0, 3, 6, 9 and 12 h using 2^−ΔΔCt method.

<p>U6 RNA was used as the internal control<b>.</b> Error bars indicate SD obtained from three biological repeats.</p

Quantitative analysis of miRNAs levels by stem-loop real-time RT-PCR.

<p>A) Expression profiles of miR829 and miR408 at 0, 1, 3, 5, 7 and 9 d; B) Expression of miR811 and miR845 at <i>E. turcicum</i>-infected leaves of 0, 1, 3, 5, 7 and 9 d using 2^−ΔΔCt method. U6 RNA was used as the internal control. Error bars indicate SD obtained from three biological repeats.</p

Detection of novel and differentially expressed miRNAs by northern blotting.

<p>A) Detection of novel miRNAs RNA; B) Detection of differentially expressed miRNAs. Northern blots of total RNA isolated from leaves of mock (M) and infected (I) leaves were probed with labeled oligonucleotides. The U6 RNA was used as internal control.</p

The expression of predicted targets were detectived using RT-PCR at 0 d, 1 d and 9 d.

<p>ZmTubulin was used as the inner control.</p

Amino acid sequence analysis of DAP3.

<p>(<b>a</b>) The conserved SOD domain of DAP3 revealed by BLASTp analysis online (<a href="http://www.ncbi.nlm.nih.gov/BLASTp" target="_blank">http://www.ncbi.nlm.nih.gov/BLASTp</a>). (<b>b</b>) Specific motifs of DAP3 protein. NIT in the box is the glycosylation site, the shading characters are SOD domain and the asterisks indicate amino acids of oxidative modification sites. Cu = copper binding, Zn = zinc binding, Cu, Zn = copper and zinc binding, S-S = disulfide bond.</p