Phospholipase D (PLD) is a key enzyme in signal transduction – mediating plant responses to various environmental stresses including drought and salinity. Isotype PLDd interacts with the microtubule cytoskeleton, although it is unclear if, or how, each of the 12 PLD isotypes in Arabidopsis may be involved mechanistically. We employed RNA interference in epidermal cells of Allium porrum L. (leek) leaves, in which the developmental reorientation of cortical microtubule arrays to a longitudinal direction is highly sensitive to experimental manipulation. Using particle bombardment and transient transformation with synthetic siRNAs targeting AtPLDa, ß, ?, d, ? and ?, we examined the effect of ‘cross-target’ silencing orthologous A. porrum genes on microtubule reorientation dynamics during cell elongation. Co-transformation of individual siRNAs together with a GFP-MBD microtubule-reporter gene revealed that siRNAs targeting AtPLDd promoted, whereas siRNAs targeting AtPLDß and ? reduced, longitudinal microtubule orientation in A. porrum. These PLD isotypes, therefore, interact, directly or indirectly, with the cytoskeleton and the microtubule-plasma membrane interface. The unique response of PLDd to silencing, along with its exclusive localisation to the plasma membrane, indicates that this isotype is specifically involved in promoting microtubule-membrane anchorage.