The RIX domain defines a class of polymorphic T6SS effectors and secreted adaptors.

Bacteria use the type VI secretion system (T6SS) to deliver toxic effectors into bacterial or eukaryotic cells during interbacterial competition, host colonization, or when resisting predation. Identifying effectors is a challenging task, as they lack canonical secretion signals or universally conserved domains. Here, we identify a protein domain, RIX, that defines a class of polymorphic T6SS cargo effectors. RIX is widespread in the Vibrionaceae family and is located at N-termini of proteins containing diverse antibacterial and anti-eukaryotic toxic domains. We demonstrate that RIX-containing proteins are delivered via T6SS into neighboring cells and that RIX is necessary and sufficient for T6SS-mediated secretion. In addition, RIX-containing proteins can enable the T6SS-mediated delivery of other cargo effectors by a previously undescribed mechanism. The identification of RIX-containing proteins significantly enlarges the repertoire of known T6SS effectors, especially those with anti-eukaryotic activities. Furthermore, our findings also suggest that T6SSs may play an underappreciated role in the interactions between vibrios and eukaryotes.