Abstract <p>Some yeast isolates secrete toxic proteins called yeast killer toxins, which kill other yeast and filamentous fungi through toxin-specific modes of action. These toxins hold potential as food preservatives, as therapeutics, and for biotechnology. Toxin production typically necessitates self-protection during and after secretion, as several toxins also act against their own producer species. Self-protection can occur through genomic mutations or through dedicated immunity factors. Here, we focus on dedicated immunity factors that are encoded on the same extrachromosomal element as their cognate toxin. These co-encoded toxin-immunity pairs reside on cytoplasmic elements that rely on autonomous replication for their maintenance and expression: either as double-stranded RNA (dsRNA) mycovirus satellites or double-stranded DNA virus-like elements (VLEs). On these systems, the immunity factors are either encoded within the same open reading frame (ORF) as their cognate toxin or encoded on a separate neighboring ORF. While the mode of action of several toxins has been studied, relatively little is known about the mode of protection of their immunity factors. Here, we consolidate current molecular knowledge of exemplary toxin-immunity pairs and the methods used to clone, study, and engineer them, with the goal of advancing fundamental understanding and enabling their use in synthetic biology and biotechnology, analogous to intracellular bacterial toxin-antitoxin systems that have historically been used in synthetic biology.</p> Key points <p>• <i>Yeast extrachromosomal elements encoding toxins often also encode immunity factors.</i></p> <p>• <i>Systematic studies have increased molecular understanding of toxin-immunity pairs.</i></p> <p>• <i>When understood further, toxin-immunity pairs could be used in synthetic biology.</i></p>