The role of miRNAs in regulating the expression of flavonol pathway genes and its possible impact on the crosstalk between UV-B and flg22 signal cascades in Arabidopsis thaliana
In cell culture, we have shown that flavonol metabolite accumulation depends on expression of the Arabidopsis flavonol pathway genes (FPGs), which are upregulated by UV-B irradiation but repressed by the bacterial elicitor flg22 during MAMP Triggered Immunity (MTI). The suppression of flavonoid production during MTI is believed to allow the plant focusing its metabolism on the pathogen defense by directing phenylalanine resources from UV-B protective flavonol production towards production of phytoalexins and cell wall fortification by lignin incorporation during MTI. Here we extend our observations made initially in cell cultures by describing that this kind of signal crosstalk between UV-B and flg22 is also functional in planta. We demonstrate that such signal crosstalk is fully functional in Arabidopsis in planta. However, we observed some differences in the expression patterns of MYB transcriptions factors (TFs) as compared to data from the cell culture system. Our data suggest that in planta the TF MYB111 might play a more dominant role than the TF MYB12, which was strongly regulated in cell cultures. Thus we can present an updated working model how this crosstalk might function. We believe that this system based on seedlings of the model plant Arabidopsis thaliana constitutes a valuable platform for further dissection of the underlying molecular mechanism, e.g. by deploying gain/loss-of-function of candidate genes. Plants are confronted with various abiotic and biotic stress factors in their natural habitats, and have evolved sophisticated and multifaced mechanisms to defense themselves. In our previous works, we have demonstrated the crosstalk between flg22 and UV-B-induced signal cascades in plants, in which the expression of Arabidopsis flavonol pathway genes (FPGs) are upregulated by UV-B irradiation but simultaneously repressed by the bacterial elicitor flg22 during MAMP Triggered Immunity (PTI). Although several transcription factors proved to be involved in the crosstalk, the underlying regulatory mechanism remains largely unsolved. By deep sequencing we identified 217 miRNAs representing 204 conserved and 13 novel miRNAs involved in the crosstalk. Among them, e.g. 106 miRNAs were upregulated by flg22 and downregulated by UV-B. Furthermore, a set of specific interactions between miRNAs and their targets were confirmed showing reciprocal changes in their expression levels, simultaneously. As revealed by GO and KEGG analysis in silico, predicted miRNA target genes participate in a series of plant biological and molecular processes as well secondary metabolism pathways. Two modulations of miRNA-target interactions were obtained in the crosstalk. The first one consisting of miR158, miR165, miR166, miR167, miR168, miR172, miR391, miR393, miR447, miR824, miR828, miR846 and miR858, which were all repressed by UV-B irradiation, while upregulated by flg22, and the second one constitute miR159, miR164, miR171 and miR822, being repressed by flg22 and upregulated by UV-B. Both miRNA sets display converse regulation with the change in transcript levels of their targets. Furthermore, we demonstrated that knockdown of miR858 (Group I) in Arabidopsis goes along with increased the chalcone synthase (CHS) expression while its overexpression results in its depression. Vice versa, knockout of miR164b (Group II) depresses the CHS gene expression while its overexpression strongly elevated the CHS gene expression, providing the first genetic evidence that miRNAs identified in this study constitute an additional layer in regulating the crosstalk between flg22 and UV-B induced signaling cascades. In nature plants are often simultaneously challenged by different stress factors. The abiotic stress UV-B irradiation induces the production of UV-protective flavonols, but their accumulation is attenuated by biotic stress, e.g. by treatment with pathogen elicitors (flg22). This suppression has been shown to occur via suppression of flavonol pathway genes (FPGs) enabling the plant to direct its secondary metabolism to a more efficient pathogen defense response. Identification of two highly conserved miRNAs (miR858 and miR828) being involved in the crosstalk and their targets MYB111 and MYB75 that proved to play an important role in regulation of FPGs and the flavonoid accumulation provoked us to assume that miR858-MYB111 and miR828-MYB75 interactions play an important role in the crosstalk. Here, we demonstrate that both miR858 and miR828 are regulated by UV-B and flg22 in a UVR8 and FLS2 receptors-dependent manner and relying on the respective signaling pathways. Comparison between miR858/MYB111-promoter-GUS and their transcript levels evidences that the MYB111 is regulated not only at the transcriptional level, but also suffered from post-transcriptional modification in response to the flg22 and UV-B challenges, in which miR858 acts as a determinant regulator. Following this, we conclude that the post- transcriptional regulation mediated by plant-derived miRNAs constitutes the crosstalk between the flg22 and UV-B induced signal cascades in Arabidopsis. This allows an extension of the crosstalk model between plant responses to biotic (flg22) and abiotic (UV-B) stresses in Arabidopsis.
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