Changes in relative expression levels of viroid-specific small RNAs and microRNAs in tomato plants infected with severe and mild symptom-inducing isolates of Potato spindle tuber viroid

Floral initiation and floral organ development are both regulated by the phytohormone gibberellin (GA). For example, in short-day photoperiods, the Arabidopsis floral transition is strongly promoted by GA-mediated activation of the floral meristem-identity gene LEAFY. In addition, anther development and pollen microsporogenesis depend on GA-mediated opposition of the function of specific members of the DELLA family of GA-response repressors. We describe the role of a microRNA (miR159) in the regulation of short-day photoperiod flowering time and of anther development. MiR159 directs the cleavage of mRNA encoding GAMYB-related proteins. These proteins are transcription factors that are thought to be involved in the GA-promoted activation of LEAFY, and in the regulation of anther development. We show that miR159 levels are regulated by GA via opposition of DELLA function, and that both the sequence of miR159 and the regulation of miR159 levels by DELLA are evolutionarily conserved. Finally, we describe the phenotypic consequences of transgenic over-expression of miR159. Increased levels of miR159 cause a reduction in LEAFY transcript levels, delay flowering in short-day photoperiods, and perturb anther development. We propose that miR159 is a phytohormonally regulated homeostatic modulator of GAMYB activity, and hence of GAMYB-dependent developmental processes.

Currently, there are very few loss-of-function mutations in micro-RNA genes. Here, we characterize two members of the Arabidopsis MIR159 family, miR159a and miR159b, that are predicted to regulate the expression of a family of seven transcription factors that includes the two redundant GAMYB-like genes, MYB33 and MYB65. Using transfer DNA (T-DNA) insertional mutants, we show that a mir159ab double mutant has pleiotropic morphological defects, including altered growth habit, curled leaves, small siliques, and small seeds. Neither mir159a nor mir159b single mutants displayed any of these traits, indicating functional redundancy. By using reporter-gene constructs, it appears that MIR159a and MIR159b are transcribed almost exclusively in the cells in which MYB33 is repressed, as had been previously determined by comparison of MYB33 and mMYB33 (an miR159-resistant allele of MYB33) expression patterns. Consistent with these overlapping transcriptional domains, MYB33 and MYB65 expression levels were elevated throughout mir159ab plants. By contrast, the other five GAMYB-like family members are transcribed predominantly in tissues where miR159a and miR159b are absent, and consequently their expression levels are not markedly elevated in mir159ab. Additionally, mMYB33 transgenic plants can phenocopy the mir159ab phenotype, suggesting that its phenotype is explained by deregulated expression of the redundant gene pair MYB33 and MYB65. This prediction was confirmed; the pleiotropic developmental defects of mir159ab are suppressed through the combined mutations of MYB33 and MYB65, demonstrating the narrow and specific target range of miR159a and miR159b.

Although they induce symptoms in plants similar to those accompanying virus infections, viroids have unique structural, functional, and evolutionary characteristics. They are composed of a small, nonprotein-coding, single-stranded, circular RNA, with autonomous replication. Viroid species are clustered into the families Pospiviroidae and Avsunviroidae, whose members replicate (and accumulate) in the nucleus and chloroplast, respectively. Viroids replicate in three steps through an RNA-based rolling-circle mechanism: synthesis of longer-than-unit strands catalyzed by host RNA polymerases; processing to unit-length, which in the family Avsunviroidae is mediated by hammerhead ribozymes; and circularization. Within the initially infected cells, viroid RNA must move to its replication organelle, with the resulting progeny then invading adjacent cells through plasmodesmata and reaching distal parts via the vasculature. To carry out these movements, viroids must interact with host factors. The mature viroid RNA could be the primary pathogenic effector or, alternatively, viroids could exert their pathogenic effects via RNA silencing.