F-box proteins promote hermaphrodite development in two different species of Caenorhabditis. In C. elegans, the F-box protein FOG-2 interacts with GLD-1 to repress the translation of
tra-2 mRNAs, and the decrease in TRA-2 activity allows XX hermaphrodites to make sperm. By contrast, in C. briggsae the F-box protein SHE-1 down-regulates TRA-2, but does so without help from GLD-1. We want to learn how SHE-1 controls sex-determination.Because a missense mutation in the F-box inactivates SHE-1, it might act as a classical F-box protein, bringing a target to the E3 ubiquitin ligase complex to be marked for degradation. To identify potential SHE-1 targets, we used the yeast two-hybrid system. From a screen of about 340,000 cDNAs, we identified three genes that that were represented by multiple, independent clones. The
pqn-94 gene passed two further tests. First, PQN-94 also interacts with SHE-1 when used as bait. Second,
pqn-94(RNAi) partially suppresses
she-1. At 25deg C,
she-1(
v49); control(RNAi) mothers never produced hermaphrodites, but
she-1(
v49);
pqn-94(RNAi) mothers produced 7% hermaphrodites. Tests with
she-1(
v35) showed the same pattern of suppression, whereas knocking down
pqn-94 on its own had no phenotype. To confirm that SHE-1 controls germ cell fates by interacting with PQN-94, we used genome editing to make a
pqn-94 null mutant. These
pqn-94(
v203) animals are healthy, but
she-1(
v35);
pqn-94(
v203) mothers produced 12% hermaphrodites at 25degC, whereas
she-1(
v35) controls were all female. Since these experiments with the null allele resembled those done with RNAi, we conclude that the absence of PQN-94 partially compensates for a loss of SHE-1.This result supports models in which SHE-1 targets PQN-94 for degradation. However, PQN-94 cannot be its sole target, since
pqn-94(
v203) is not completely epistatic to
she-1. To study their interactions in living animals, we used genome editing to produce a
pqn-94::FLAG strain. Preliminary results show that PQN-94 is expressed in the cytoplasm of germ cells, as suggested by its phenotype. We are now characterizing its interaction with SHE-1.Finally, phylogenetic studies revealed that the family of genes most closely related to
she-1 underwent a large expansion before the divergence of C. briggsae and C. nigoni. As a result, the closest relatives of SHE-1 are all C. nigoni proteins, even though C. nigoni XX animals develop as females. This result implies that
she-1 underwent an important change in either expression or activity during C. briggsae evolution, or that the context in which it acts changed dramatically.