SAP11 is an effector protein that has been identified in various phytoplasma species. It localizes in the plant nucleus and can bind and destabilize TEOSINE BRANCHES/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factors. Although SAP11 of different phytoplasma species share similar activities, their protein sequences differ greatly. Here, we demonstrate that the SAP11-like protein of ‘Candidatus Phytoplasma mali’ (‘Ca. P. mali’) strain PM19 localizes into the plant nucleus without requiring the anticipated nuclear localization sequence (NLS). We show that the protein induces crinkled leaves and siliques, and witches’ broom symptoms, in transgenic Arabidopsis thaliana (A. thaliana) plants and binds to six members of class I and all members of class II TCP transcription factors of A. thaliana in yeast two-hybrid assays. We also identified a 17 amino acid stretch previously predicted to be a nuclear localization sequence that is important for the binding of some of the TCPs, which results in a crinkled leaf and silique phenotype in transgenic A. thaliana. Moreover, we provide evidence that the SAP11-like protein has a destabilizing effect on some TCPs in vivo.

‘Candidatus Phytoplasma mali’ (‘Ca. P. mali’) has only one major membrane protein, the immunodominant membrane protein (Imp), which is regarded as being close to the ancestor of all phytoplasma immunodominant membrane proteins. Imp binds to actin and possibly facilitates its movement in the plant or insect host cells. However, protein sequences of Imp are quite diverse among phytoplasma species, thus resulting in difficulties in identifying conserved domains across species. In this work, we compare Imp protein sequences of ‘Ca. P. mali’ strain PM19 (Imp-PM19) with Imp of different strains of ‘Ca. P. mali’ and identify its actin-binding domain. Moreover, we show that Imp binds to the actin of apple (Malus x domestica), which is the host plant of ‘Ca. P. mali’. Using molecular and scanning force spectroscopy analysis, we find that the actin-binding domain of Imp-PM19 contains a highly positively charged amino acid cluster. Our result could allow investigating a possible correlation between Imp variants and the infectivity of the corresponding ‘Ca. P. mali’ isolates.

It was shown that the SAP11 effector of different Candidatus Phytoplasma can destabilize some TEOSINE BRANCHES/CYCLOIDEA/PROLIFERATING CELL FACTORs (TCPs), resulting in plant phenotypes such as witches’ broom and crinkled leaves. Some SAP11 exclusively localize in the nucleus, while the others localize in the cytoplasm and the nucleus. The SAP11-like effector of Candidatus Phytoplasma mali strain PM19 (SAP11_PM19) localizes in both compartments of plant cells. We show here that SAP11_PM19 can destabilize TCPs in both the nucleus and the cytoplasm. However, expression of SAP11_PM19 exclusively in the nucleus resulted in the disappearance of leaf phenotypes while still showing the witches’ broom phenotype. Moreover, we show that SAP11_PM19 can not only destabilize TCPs but also relocalizes these proteins in the nucleus. Interestingly, three different transgenic Nicotiana species expressing SAP11_PM19 show all the same witches’ broom phenotype but different leaf phenotypes. A possible mechanism of SAP11-TCP interaction is discussed.

‘Candidatus Phytoplasma mali’ is associated with apple proliferation, a devastating disease in fruit production. Using genome analysis, a gene encoding a hemolysin-like protein was identified. It was postulated that this protein could be an effector. However, the function of this protein is unknown. It is shown that the hemolysin-like protein binds to a GTP binding protein, Toc33, of Arabidopsis thaliana in yeast two-hybrid analysis and that the Toc33-binding domain is located in the C-terminus of the domain of unknown function (DUF21) of the protein. The biochemical studies reveal that the protein can hydrolyze phosphate of purine and pyrimidine nucleotides. Transgenic Nicotiana benthamiana plants expressing the protein show no discernible change in phenotype. Phytoplasma have a much-reduced genome, lacking important genes for catabolic pathways or nucleotide production; therefore, the hemolysin-like protein plays a role in the uptake of plant nucleotides from their host and hydrolyzes these nucleotides for energy and their own biosynthesis.

A bstract: The plant pathogen ‘ Candidatus Phytoplasma mali’ (‘ Ca . P. mali’) is the causing agent of apple proliferation that leads to heavy damage in apple production all over Europe. To identify and analyze effector proteins of plant pathogens is an important strategy in plant disease research. Here, we report that the SAP11-like protein of ‘Ca . P. mali’ induces crinkled leaves and siliques and witches’ broom symptoms in transgenic Arabidopsis thaliana ( A. thaliana ) plants and binds to 6 members of class I and all members of class II TCP (TEOSINE BRANCHES/ CYCLOIDEA/PROLIFERATING CELL FACTOR) transcription factors of A. thaliana in yeast two-hybrid assays. Moreover, we demonstrate that the protein localizes actively into the plant nucleus without requiring the nuclear leader sequence (NLS). We also identified a 17 amino acid stretch previously predicted to be a nuclear leader sequence that is important for the binding of some of the TCPs and also responsible for the crinkled leaf and silique phenotype in transgenic A. thaliana .