A pectin acetyl-transferase facilitates secondary plasmodesmata formation and RNA silencing movement between plant cells

Some silencing small (s)RNAs, comprising micro (mi)RNAs and small-interfering (si)RNAs, move between plant cells to orchestrate gene expression and defense. Besides possible redundancy or embryo lethality, a prevalent challenge in genetic studies of mobile silencing is to discriminate bona fide alterations to sRNA movement from impaired cell-autonomous sRNA activity within silencing-recipient cells. Without such clarifications, cell-to-cell mobility factors are yet to be unequivocally identified. Consequently, known properties of sRNA movement, including contextuality and directionality, remain poorly explained. Circumstantial evidence and synthetic biology pinpoint plasmodesmata (PDs) – the pores traversing plant cell walls (CWs) – as the likely channels involved. Yet, how plants control the number of primary and secondary PDs developing respectively before and after CW formation remains largely unknown. Here, we address these intertwined issues in Arabidopsis using a forward screen for compromised epidermis-to-mesophyll movement of an artificial (a)miRNA. We identify a pectin acetyl-transferase mutation that, we demonstrate, reduces amiRNA physical trafficking but also impedes siRNA, GFP, and viral movement by decreasing the frequency of leaf secondary PDs. sRNA movement at leaf interfaces involving primary PDs remains unaffected, however, as does miRNA and GFP cell-to-cell mobility in roots, hinting at how movement's contextuality and directionality might be achieved. We also show that reducing de-esterified pectin depolymerization decreases leaves' symplasmic connectivity, whereas defective pectin biogenesis increases PD number. Combining genetics with antibody-based pectin probing and atomic force microscopy helps delineate a mechanistically coherent framework whereby pectin esterification and/or abundance impact CW loosening, a process required for CW extension during which secondary PDs form to enable macromolecular trafficking.