The 'Candidatus Phytoplasma ziziphi' effectors SJP1/2 negatively control leaf size by stabilizing ZjTCP2 in jujube.

Phytoplasmas manipulate host plant development to benefit their invasion and insect vector colonization. However, the virulence factors and mechanisms underlying small leaf formation caused by jujube witches' broom (JWB) phytoplasmas remain largely unknown. Here, effectors SJP1 and SJP2 from JWB phytoplasmas were identified to induce small leaf formation in jujube. In vivo interaction and expression assays showed that SJP1 and SJP2 interacted with and stabilized ZjTCP2. Over-expression of SJP1 and SJP2 in jujube induced ZjTCP2 accumulation. In addition, the abundance of miRNA319f-1 was significantly repressed in leaves of SJP1 and SJP2 transgenic jujube plants and showed the opposite pattern with its target ZjTCP2, which was consistent with that in the diseased leaves. Overexpression of ZjTCP2 in Arabidopsis promoted ectopic leaves arising from the adaxial side of cotyledons and reduced the leaf size. Constitutive expression of the miRNA319f-1 precursor in the 35S::ZjTCP2 background reduced the abundance of ZjTCP2 mRNA and reversed the cotyledon and leaf defects in Arabidopsis. Therefore, these observations suggest that effectors SJP1 and SJP2 induced small leaf formation, at least partly, by interacting with and activating ZjTCP2 expression both at the transcriptional and protein levels, providing new knowledge about small leaf formation caused by phytoplasmas in woody plants.