Structural requirements of the phytoplasma effector protein SAP54 for causing homeotic transformation of floral organs

Abstract

Phytoplasmas are intracellular bacterial plant pathogens that cause devastating diseases in crops and ornamental plants by the secretion of effector proteins. One of these effector proteins, termed SECRETED ASTER YELLOWS-WITCHES BROOM PROTEIN 54 (SAP54), leads to the degradation of a specific subset of floral homeotic proteins of the MIKC-type MADS-domain family via the ubiquitin-proteasome pathway. In consequence, the developing flowers show the homeotic transformation of floral organs into vegetative leaf-like structures. The molecular mechanism of SAP54 action involves physical binding to the keratin-like K-domain of MIKC-type proteins, and to some RAD23 proteins, which translocate ubiquitylated substrates to the proteasome. The structural requirements and specificity of SAP54 function are poorly understood, however. Here we report, based on biophysical and molecular biological analyses, that SAP54 folds into -helical structures. We also show that the insertion of helix-breaking mutations disrupts correct folding of SAP54, which interferes with the ability of SAP54 to bind to its target proteins and to cause disease phenotypes in vivo. Surprisingly, dynamic light scattering data together with electrophoretic mobility shift assays suggest that SAP54 preferentially binds to multimeric complexes of MIKC-type proteins rather than to dimers or monomers of these proteins. Together with literature data this finding suggests that MIKC-type proteins and SAP54 constitute multimeric -helical coiled-coils, possibly also involving other partners such as RAD23 proteins. Our investigations clarify the structure-function relationship of an important phytoplasma effector protein and thus may ultimately help to develop treatments against some devastating plant diseases.\n\nSIGNIFICANCE STATEMENTPhytoplasmas are bacterial plant pathogens that cause devastating diseases in crops and ornamental plants by the secretion of effector proteins such as SAP54, which leads to the degradation of some floral homeotic proteins. Our study clarifies the structural requirements of SAP54 function and illuminates the molecular mode of interaction and thus may ultimately help to develop treatments against some devastating plant diseases.