Loss of INCREASED SIZE EXCLUSION LIMIT (ISE)1 or ISE2 Increases the Formation of Secondary Plasmodesmata

Plasmodesmata (PD) transport developmentally important nucleic acids and proteins between plant cells [1-3]. Primary PD form during cell division and are simple, linear channels [4, 5]. Secondary PD form in existing cell walls after cell division and are simple, twinned, or branched [4]. PD function undergoes a marked reduction at the mid-torpedo stage of Arabidopsis embryogenesis [6]. Two mutants, increased size exclusion limit (ise)1 and ise2, fail to undergo this transition, and their null mutations are embryonically lethal [7, 8]. We investigated the ultrastructure of PD in early-, mid-, and late-torpedo-stage embryos and in young leaves. Wild-type (WT) embryos contain twinned and branched (T/B) PD at all stages, but ise1 and ise2 embryos contain significantly higher proportions of T/B PD than WT embryos. WT T/B PD formation occurs in a stage- and tissue-specific pattern that is reversed in ise1 embryos. Silencing ISE1 in Nicotiana benthamiana leaves increases the frequency of secondary PD in existing cell walls. Silencing ISE2 increases the proportion of T/B secondary PD formed. Silenced tissues exhibit increased PD-mediated movement of green fluorescent protein tracers. Thus, silencing of ISE1 and ISE2 phenocopies ise1 and ise2 mutant embryos: when wild-type ISE1 and ISE2 functions are lost, de novo production of PD occurs, leading to increased intercellular transport.