Cyst nematodes are plant-parasitic roundworms that are of significance in many cropping systems around the world. Cyst nematode infection is facilitated by effector proteins secreted from the nematode into the plant host. The cDNAs of the 25A01-like effector family are novel sequences that were isolated from the esophageal gland cells of the soybean cyst nematode (Heterodera glycines). To aid functional characterization we identified an orthologous member of this protein family (Hs25A01) from the closely related sugar beet cyst nematode H. schachtii, which infects Arabidopsis. Constitutive expression of the Hs25A01 CDS in Arabidopsis plants caused a small increase in root length accompanied by up to 22% increase in susceptibility to H. schachtii. A plant-expressed RNAi construct targeting Hs25A01 transcripts in invading nematodes significantly reduced host susceptibility to H. schachtii. These data document that Hs25A01 has physiological functions in planta and a role in cyst nematode parasitism. In vivo as well as in vitro binding assays confirmed specific interactions of Hs25A01 with an Arabidopsis F-box containing protein, a chalcone synthase, and the translation initiation factor eIF- 2 beta subunit (eIF-2bs), making these proteins likely candidates for being involved in the observed changes in plant growth and parasitism. A role of eIF- 2bs in mediating the Hs25A01 virulence function is further supported by the observation that two independent eIF-2bs Arabidopsis knock-out lines were significantly more susceptible to H. schachtii.