Characterization of AIR9 - a novel plant microtubule-associated protein that marks the division plane

Lead Research Organisation: John Innes Centre
Department Name: Contracts Office


The preprophase band of microtubules (PPB) predicts where the cell plate will fuse with the parental wall yet disappears before mitosis. The nature of this 'memorization' is unknown but we have found a microtubule-associated protein (AIR9) that labels the PPB then reappears during cytokinesis as a cortical ring. AIR9 is the first molecular marker for this key process.
1. By gene truncation, we will identify the domain responsible for locating AIR9 to the cortical ring during cytokinesis. The cell cycle regulation of AIR9 cortical binding will then be analysed: (a) by site-directed mutagenesis of potential phosphorylation sites, (b) by western blotting synchronized cells.
2. A yeast two-hybrid screen has indicated that AIR9 interacts with a cellulose synthase, CESA1 and with a microtubule motor, ZWICHEL. We will raise antibodies to AIR9 to check if it co-immunoprecipitates ZWI and CESA. We will use cell homogenates and bacterially-expressed protein fragments, also testing if AIR9 helps its putative interactors bind microtubules.
3. We will confirm that the genomic AIR9 gene complements two insertional alleles.
4.(A). Embryo-lethal AIR9 mutants will be examined: the pollen/tube phenotype using cytoskeletal marker lines; the early female gametophyte by confocal microscopy.
(B). To test depletion of AIR9 in development of Arabidopsis plants, we will express RNAi constructs under an inducible promoter. In synchronized cell cultures, we will see if changes to MT arrays affect cross wall formation and alignment. (Successful RNAi would also allow us to study early development in plants).
5. We will characterize 3 TILLING alleles with defects in axial growth. After back-crossing with wildtype we will see if these alleles co-segregate with observed growth phenotypes. Immunofluorescence will test if mutants have abnormal MT arrays. Field emission scanning EM will be used to examine alignment of cellulose microfibrils in mutants vs wildtype.


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