Genome rearrangements during macronuclear development in Paramecium tetraurelia have been proposed to be guided by meiosis-specific small RNAs, called scnRNAs, which are produced from micronuclear transcripts by the Dicer-like proteins Dcl2 and Dcl3. ScnRNAs are likely bound by the Piwi proteins Ptiwi01 and Ptiwi09 and are required to target the excision of a subset of MIC-specific sequences in the developing MAC, including maternally controlled IESs: silencing of both Dicer-like or both Piwi genes during autogamy results in genome rearrangement defects and in cell death. To obtain viable mutants in scnRNA-pathway genes, we designed a mutagenesis screen based on mating type reversal, which relies on the hypersensitivity of the mtA promoter IES to the slightest perturbation of the scnRNA pathway. Mating type O is normally determined during MAC development by the excision of the promoter of the mtA gene as an IES. This excision event is impaired by the silencing of a single gene from the Dcl2/Dcl3 or Ptiwi01/Ptiwi09 pairs, which results in mating type E viable progeny, whereas other IESs are hardly affected. Furthermore, the promoter-retaining version of the mtA gene is ‘dominant’ over the promoter-excised version, since it allows expression of the gene, while retention of intragenic IESs results in non-functional genes. Thus, screening populations of mutagenized O cells for E-expressing progeny should allow us to recover non-lethal mutations that only partially impair the scnRNA pathway, such as null alleles of genes with redundant ohnologs or hypomorphic alleles of essential genes. This scheme allowed us to isolate 2 mutants: one is a null allele of a developmentally regulated zinc finger protein with a WGD1 ohnolog, called mtG, and the other is a mis-sense mutation in Ptiwi09. An mtG-GFP fusion protein was shown to localize in the new MAC, with an initially punctate pattern. As development proceeds, mtG-GFP progressively concentrates at a single spot in the DNA-poor region of the MAC, as does the heterochromatin mark, H3K9me3. RNAi-mediated silencing of mtG and/or its ohnolog during autogamy of wild-type O cells impaired excision of the mtA promoter and caused progeny to switch to mating type E. In addition, resequencing of the MAC genome showed that a very small subset of ~100 IESs are retained in these conditions. These IESs are significantly enriched in a 5-bp motif, GCTAA, and have a specific size range. Preliminary analysis of mtG function in the sibling species P. octaurelia suggested that orthologous IESs also require mtG for excision, but only when the GCTAA motifs are conserved. I will discuss the possible mechanisms and biological significance of mtG-dependent IES excision.