Identification of miRNAs Involved in Maize-Induced Systemic Resistance Primed by Trichoderma harzianum T28 against Cochliobolus heterostrophus

Abstract

microRNAs (miRNAs) are known to play important roles in the immune response to pathogen infection in different plants. Further, Trichoderma strains are able to activate plant defense responses against pathogen attacks. However, little is known about the involvement of miRNAs in the defense response primed by Trichoderma strains. To explore the miRNAs sensitive to priming by Trichoderma, we studied the small RNAs and transcriptome changes in maize leaves that were systemically induced by seed treatment with Trichoderma harzianum (strain T28) against Cochliobolus heterostrophus (C. heterostrophus) infection in leaves. Through analysis of the sequencing data, 38 differentially expressed miRNAs (DEMs) and 824 differentially expressed genes (DEGs) were identified. GO and KEGG analyses of DEGs demonstrated that genes involved in the plant hormone signal transduction pathway and oxidation-reduction process were significantly enriched. In addition, 15 miRNA-mRNA interaction pairs were identified through the combined analysis of DEMs and DEGs. These pairs were supposed to play roles in the maize resistance primed by T. harzianum T28 to C. heterostrophus, in which miR390, miR169j, miR408b, miR395a/p, and novel miRNA (miRn5231) were more involved in the induction of maize resistance. This study provided valuable information for understanding the regulatory role of miRNA in the T. harzianum primed defense response.

Keywords: C. heterostrophus; Trichoderma harzianum; maize; miRNA; primed defense response.

Figure 1

Trichoderma harzianum T28 primed induced systemic resistance (ISR) in maize against Cochliobolus heterostrophus. (A) The growth of maize with or without T28 coated seeds at the 6–8 leaf stage. Maize B73 seeds were coated with T. harzianum T28 (B73 + T28) or without T28 (B73, as a control) and grew to the 6–8 leaf stage at field conditions. (B) Disease severity against C. heterostrophus in maize B73 and B73 + T28. Maize leaves of B73 and B73 + T28, at the 6–8 leaf stage, were inoculated with C. heterostrophus for 3 days; the lesion spots were photographed, and the lesion areas of the spots were measured. Asterisks indicate statistical differences between treatments (Student’s t-tests; p < 0.05). (C) The expression levels of the defense-related genes (B73 + T28/B73) in maize leaves after inoculation with C. heterostrophus. PR4 (pathogenesis-related protein), LOX (lipoxygenases), and ACO (1-aminocyclopropane-1-carboxylate oxidase) are the marker genes of the salicylic acid-, jasmonic acid-, and ethylene-dependent signaling pathways, respectively. The expression levels of the defense-related genes were analyzed using qRT-PCR and normalized to 18s rRNA. Each sample contained 6 plants, and each treatment was repeated three times. Values represented means ± SD of three replicates.

Figure 2

Differentially expressed maize miRNAs responsive to ISR primed by T. harzianum T28 (A) Statistics and heatmap of differentially expressed miRNAs (DEMs) in maize (B73 vs. B73 + T28). The expression levels of the differentially expressed miRNAs in B73 and B73 + T28 replicates were used to create the heatmap by employing the R package. The different color codes represented the different expression levels, ranging from −2 (blue) to 2 (red). Blue and red show low and high expression levels, respectively. (B) GO and (C) KEGG pathway analyses of the predicted target genes of DEMs.

Figure 3

Differentially expressed genes in maize responsive to ISR primed by T. harzianum T28 (A) Statistics and heatmap of differentially expressed genes (DEGs) in maize (B73 vs. B73 + T28). (B) GO and (C) KEGG pathway analyses of the predicted target genes of DEMs. (D) Heatmap of DEGs in the biological process of the oxidation-reduction category and (E) the plant hormone signal transduction category. The expression levels of the differentially expressed genes in B73 and B73 + T28 replicates were used to create the heatmaps by employing the R package. The different color codes represent the different expression levels; blue and red show low and high expression levels, respectively.

Figure 4

The expression levels of the miRNAs and target genes associated with ISR primed by T. harzianum T28. (A) qRT-PCR analysis of candidate ISR-associated miRNAs and (B) target genes of these candidate ISR-associated miRNAs in maize (B73 + T28/B73). Values represent the means ± SDs of the three replicates. *, **, *** and **** indicate significant differences.

Figure 5

Potential regulatory roles of candidate ISR-associated miRNAs and their targets in maize primed by T. harzianum T28.