The Arabidopsis Mei2 homologue AML1 binds AtRaptor1B, the plant homologue of a major regulator of eukaryotic cell growth

Abstract

Background: TOR, the target of the antibiotic rapamycin in both yeast and mammalian cells, is a potent cell growth regulator in all eukaryotes. It acts through the phosphorylation of downstream effectors that are recruited to it by the binding partner Raptor. In Arabidopsis, Raptor activity is essential for postembryonic growth. Though comparative studies suggest potential downstream effectors, no Raptor binding partners have been described in plants.

Results: AtRaptor1B, a plant Raptor homologue, binds the AML1 (Arabidopsis Mei2-like 1) protein in a yeast two-hybrid assay. This interaction is mediated by the N-terminal 219 residues of AML1, and marks AML1 as a candidate AtTOR kinase substrate in plants. The AML1 N-terminus additionally carries transcriptional activation domain activity. Plants homozygous for insertion alleles at the AML1 locus, as well as plants homozygous for insertion alleles at all five loci in the AML gene family, bolt earlier than wild-type plants.

Conclusion: AML1 interacts with AtRaptor1B, homologue of a protein that recruits substrates for phosphorylation by the major cell-growth regulator TOR. Identification of AML1 as a putative downstream effector of TOR gives valuable insights into the plant-specific mode of action of this critical growth regulator.

Figure 1

AML1 interacts with AtRaptor1B in a yeast two-hybrid assay. A. Schematic diagram of AML1 fragments cloned into pGADT7. The AML1 RNA Recognition Motifs (RRMs) are indicated. Fragments interacting with AtRaptor1B are labeled in red. B. Yeast two-hybrid results. Numbers on each plate refer to the pGAD construct in A. For each plate, the set of six cultures on the top half were co-transformed with pGBK:Raptor; the set of six cultures on the bottom half were co-transformed with pGBKT7 vector. The plate at left lacks leucine, tryptophan, histidine and adenine and selects for a protein-protein interaction. The control plate at right lacks leucine and tryptophan, and indicates co-transformation of the yeast cells with both a pGAD and a pGBK plasmid.

Figure 2

The AML1 N-terminus harbors transcriptional activation domain activity. One-hybrid assay on AML1 fragments cloned into pGBK. The plate at left lacks tryptophan and adenine, and growth indicates transcriptional activation domain activity in the tested fragments. The control plate at right lacks adenine, and indicates transformation of the cells with the desired pGBK construct.

Figure 3

AML loci and insertion alleles. The position of the insertion in each locus is indicated by a triangle below the locus picture. The wild-type loci were described previously (17). The thin central line indicates genomic DNA. Solid blue blocks spanning the central line indicate coding exons. Solid blue blocks below the line indicate untranslated regions. T-DNA insertion left borders are indicated with a triangle below the locus. Binding sites for primers used to genotype the wild-type allele of each locus are indicated with arrows. Binding sites for primers used to ascertain the effect of homozygosity at the mutant allele are indicated with triangles tagged by a single hash line if the PCR product spans the insertion site, or a doublet hash line if the PCR product is from a region downstream of the insertion site.

Figure 4

AML mutant allele characterization. i) PCR product to identify the wild-type allele of each AML locus. ii) PCR product to identify the mutant allele of each AML locus. iii) PCR product to assay for accumulation of cDNA from transcripts transcribed across the insertion site. iv) PCR product to assay for accumulation of cDNA from transcripts transcribed from the AML locus downstream of the insertion site. AML4 was excluded from this assay because the mutant allele is disrupted in the center of its coding region. Q6 refers to genomic DNA or cDNA template extracted from quintuple insertion allele homozygotes; Ws refers to genomic DNA or cDNA template extracted from wild-type (Wassilijewskia ecotype) plants.

Figure 5

AML insertion mutants bolt early. A. Wassilijewskia (Ws) and AML1 insertion homozygotes (AML1-/-) grown under long days. B. Bolting time and number of rosette leaves at time of bolting for AML single insertion and higher order insertion mutants. Plants were grown under 16 hour days. Ws and Columbia (Col 0) ecotypes are the two points in the top left of the graph; the cluster of points at the lower right are all AML-/- mutants.