The miR159-GAMYB pathway: silencing and function of GAMYB homologues amongst diverse plant species
Creators: name: Zheng, Zihui
Publisher: name: The Australian National University
Year: 2018
Subjects: miRNA
miR159
GAMYB
RNA secondary structure
Arabidopsis
tabacoo
plant defence
rice

description: MicroRNAs (miRNAs) are a class of small RNAs that regulate gene expression in eukaryotes. In plants, many miRNA families mediate silencing of target genes, which are involved in plant development and plant defence. For my thesis, I have been investigating the miR159-GAMYB pathway, which appears conserved from basal vascular plants to angiosperms. GAMYB transcription factors have been demonstrated to have conserved roles of programmed cell death (PCD) in both the seed aleurone and the anther tapetum in a number of different plant species. However, what the functional role of GAMYB is in vegetative tissues remains unknown. In Arabidopsis, miR159 is critical for proper growth, as its inhibition has a strong negative impact on vegetative growth, due to deregulated GAMYB expression. However, gamyb loss-of-functional mutants display a wild-type phenotype, as their expression is silenced to phenotypically inconsequential levels by miR159 in vegetative tissues. This raises two questions: (1) how is GAMYB so strongly silenced; (2) why is GAMYB strongly and widely transcribed in vegetative tissues for it to be then completely repressed by miR159? These two questions were the focus of my thesis. Firstly, how the Arabidopsis MYB33 and MYB65 are so strongly silenced in the model plant Arabidopsis was investigated. Both genes were predicted to contain a distinctive RNA secondary structure abutting the miR159 binding site, composed of two stem-loop (SL) structures; whereas such SL structures were not predicted to form in other GAMYB-like genes that are targeted less efficiently by miR159 for expression regulation. Functional analysis found that the RNA structure in MYB33 correlated with strong silencing efficacy; introducing mutations to disrupt either SL attenuated miR159 efficacy, while introducing mutations to form an artificial stem-loop structure adjacent to a miRNA-binding site restored strong miR159-mediated silencing. Although how these predicted structures promote miR159-mediated silencing are not determined, we speculate that the stem-loop structures in the vicinity of the miR159 binding site promotes accessibility of the binding site, where if adjacent sequences form strong stem structures, they are less likely to base-pair with binding site nucleotides, maintaining high accessibility of the binding site. Interestingly, the RNA SL structures are predicted to reside in GAMYB-like homologues of numerous angiosperm and gymnosperm plant species, arguing that these structures have been integral in the miR159-GAMYB regulatory relationship over a long period of time. In addition, these structures are not present in the Arabidopsis GAMYB-like homologues that are not transcribed in vegetative tissues, suggesting that selection for such structures only occurs for homologues transcribed in vegetative tissues as to prevent their expression and demarcating them as sensitive targets of miR159. Secondly, to investigate the functional role of the miR159-GAMYB pathway, target MIMIC159 (MIM159) transgenes, which can inhibit endogenous miR159 activity, were expressed in a number of Arabidopsis ecotypes, as well as in tobacco and rice. Inhibiting miR159 in all three plant species resulted in similar phenotypic outcomes, predominantly stunted growth and irregular leaf shape. This implies that the function and expression of the miR159-GAMYB pathway is strongly conserved in distant plant species. This raises several questions: why is GAMYB widely transcribed if its expression is strongly silenced by miR159 throughout the plant to result in little to no phenotypic impact; and why has this been strongly conserved across multiple plant species. When miR159 activity is inhibited in MIM159 tobacco leaves, pathways related to plant defence response are most up-regulated. This included PATHOGENESIS-RELATED PROTEIN (PR) mRNA levels that were 100-1000s fold up-regulated compared to wild type, and correlated with deregulated GAMYB expression. This finding suggests that the miR159-GAMYB pathway is involved in the plant defence response to biotic stress. However, PR expression is not up-regulated in Arabidopsis or rice when miR159 is inhibited, suggesting that despite the conserved nature of the miR159-GAMYB pathway, there are species-specific differences in its function.