Diverse set of microRNAs are responsive to powdery mildew infection and heat stress in wheat (Triticum aestivum L.)

Abstract

Background: MicroRNAs (miRNAs) are a class of small non-coding regulatory RNAs that regulate gene expression by guiding target mRNA cleavage or translational inhibition. MiRNAs can have large-scale regulatory effects on development and stress response in plants.

Results: To test whether miRNAs play roles in regulating response to powdery mildew infection and heat stress in wheat, by using Solexa high-throughput sequencing we cloned the small RNA from wheat leaves infected by preponderant physiological strain Erysiphe graminis f. sp. tritici (Egt) or by heat stress treatment. A total of 153 miRNAs were identified, which belong to 51 known and 81 novel miRNA families. We found that 24 and 12 miRNAs were responsive to powdery mildew infection and heat stress, respectively. We further predicted that 149 target genes were potentially regulated by the novel wheat miRNA.

Conclusions: Our results indicated that diverse set of wheat miRNAs were responsive to powdery mildew infection and heat stress and could function in wheat responses to both biotic and abiotic stresses.

Figure 1

small RNA size distribution with Solexa's high-throughput sequencing. a) Total abundance of sequences vs. size. b) Number of distinct signatures vs. size.

Figure 2

Expression patterns of novel miRNAs in wheat. RNA gel blots of low molecular weight RNA from different tissues, including spikes, stems, leaves and roots, were probed with labeled oligonucleotides. The tRNA and 5 S RNA bands were visualized by ethidium bromide staining of polyacrylamide gels and served as loading controls.

Figure 3

Expression profiling of MiRNA families using high-throughput sequencing. Blue and red bars represented the JD8 and JD8-Pm30 at 12 hr post-inoculation with Egt relative to uninoculated leaves. Green bar represented the TAM107 after the 40°C for 1 h relative to un-inoculated leaves.

Figure 4

Powdery mildew infection responsive expression of wheat miRNAs by Northern blot. The tRNA and 5 S RNA bands were visualized by ethidium bromide staining of polyacrylamide gels and served as loading controls. Disease susceptible wheat cultivar Jingdong8 (JD8) and its near-isogenic line Jingdong8-Pm30 (JD8-Pm30) having a resistant gene Pm30 were used. "0 h" means the uninoculated leaves and "12 h" means the leaves were inoculated by the Erysiphe graminis f. sp. tritici (Egt) (Isolate E09).

Figure 5

heat stress responsive expression of wheat miRNAs by Northern blot. The tRNA and 5 S RNA bands were visualized by ethidium bromide staining of polyacrylamide gels and served as loading controls. Heat tolerant cultivar TAM107 and heat susceptible cultivar CS were treated by heat treatment for 0.5, 1, 2 hours and were returned to normal growth condition (R).

Figure 6

Different members in miRNA family miR166 represented the different expression patterns.

Figure 7

Semi-quantitative RT-PCR analysis of putative target genes of miR156 in response to powdery mildew infection. Disease susceptible wheat cultivar Jingdong8 (JD8) and its near-isogenic line Jingdong8-Pm30 (JD8-Pm30) having a resistant gene Pm30 were used. "0 h" means the uninoculated leaves and "12 h" refers to the leaves were inoculated by the Erysiphe graminis f. sp. tritici (Egt) (Isolate E09).

Figure 8

Realtime-PCR analysis of TaGAMYB1 and TaGAMYB2 response to heat stress. Heat tolerant cultivar TAM107 and heat susceptible cultivar CS were treated by heat treatment for 0.5, 1, 2 hours and were returned to normal growth condition (R).