Short prokaryotic Argonautes provide defence against incoming mobile genetic elements through NAD⁺ depletion.

Argonaute (Ago) proteins are found in all three domains of life. The so-called long Agos are composed of four major domains (N, PAZ, MID and PIWI) and contribute to RNA silencing in eukaryotes (eAgos) or defence against invading mobile genetic elements in prokaryotes (pAgos). The majority (~60%) of pAgos identified bioinformatically are shorter (comprising only MID and PIWI domains) and are typically associated with Sir2, Mrr or TIR domain-containing proteins. The cellular function and mechanism of short pAgos remain enigmatic. Here we show that Geobacter sulfurreducens short pAgo and the NAD⁺-bound Sir2 protein form a stable heterodimeric complex. The GsSir2/Ago complex presumably recognizes invading plasmid or phage DNA and activates the Sir2 subunit, which triggers endogenous NAD⁺ depletion and cell death, and prevents the propagation of invading DNA. We reconstituted NAD⁺ depletion activity in vitro and showed that activated GsSir2/Ago complex functions as a NADase that hydrolyses NAD⁺ to ADPR. Thus, short Sir2-associated pAgos provide defence against phages and plasmids, underscoring the diversity of mechanisms of prokaryotic Agos.