The role of small RNAs in plant and insect interactions
Nicotiana attenuata is a native plant which regularly attacked by number of insect herbivore species. N. attenuata recognizes herbivores by the content of herbivore oral secretions (OS). Elicitors in OS can rapid induce jasmonate response and large-scale transcriptional and metabolic changes. This is required to maximize plant fitness in the face of herbivory. Small RNAs are also regulators of plant defense responses. To deepen our understanding of the role of smRNAs, we characterized four Dicer-like (DCL) genes, a key processor of smRNAs in the ecological model plant system N. attenuata. Time-course expression analysis of DCL transcripts with wounding and OS-elicitation compared to control treatments demonstrated different transcriptional responses and accumulated diurnally. We used reverse genetics approach to describe them in term of ecological relevance. DCLs independently regulate or co-regulate jasmonates and defense responses to Manduca sexta herbivory. Herbivores grew larger on ir-dcl3 and ir-dcl4 plants suggesting that these plants defense is impaired. Ir-dcl3 reduced nicotine and protease inhibitor (TPI) whereas ir-dcl4 has lower nicotine and altered phenylpropanoid-polyamine conjugates. In contrast, DCL2 has antagonistic effect on DCL3 or DCL4 in term of nicotine production. In addition I showed independent transcriptional regulation of DCLs by using microarray expression analysis. Moreover, to understand the role of microRNAs and endogenous tasiRNAs, I identified 59 miRNAs belonging to 36 MIR families and two miRNA-regulated TAS transcripts, and predicted their stem and loop structures and their targets. The average minimum free energy for stem and loop folding was comparable to Arapidopsis. We found TAS members using BLAST search by their miRNA-binding site. Results revealed that tasiRNAs are conserved with one in Arabidopsis. Quantitative PCR analysis showed that smRNAs had different and specific transcriptional responses to wounding and oral secretions treatment. Moreover, I also found that some oral secretions-induced miRNAs were specifically regulated in a jasmonate-dependent manner. Microarray expression analysis showed miRNA-target abundance which was correlated to miRNA abundance after treatments.