Post-transcriptional gene silencing (PTGS) of transgenes is graft-transmissible and acts systemically throughout plants. A forward genetic screen identified one mutant, rtp1-1, that showed delayed onset of systemic PTGS and a variety of developmental abnormalities. We used map-based cloning to identify rtp1-1, and found it to be a new allele of the previously described PLEIOTROPIC REGULATORY LOCUS 1 (PRL1). Recently, it was reported that PRL1 is an RNA binding protein required for the accumulation of endogenous small regulatory RNAs in Arabidopsis shoot tissues or whole plants. PRL1 is a member of a two-gene family in Arabidopsis, and we also characterized the prl2-1 T-DNA insertion mutant and the prl1(rtp1-1) prl2-1 double mutant for defects in systemic RNA silencing. PRL1 is also a core component of a plant defense pathway required for plant innate immunity, and we have characterized prl1, prl2 and mutants defective in small regulatory RNA (sRNA) biogenesis for an altered response to the root-rot pathogen Fusarium oxysporum (Fo).