Characterization of Phytoplasmal Effector Protein Interaction with Proteinaceous Plant Host Targets Using Bimolecular Fluorescence Complementation (BiFC)

A Novel Effector Protein of Apple Proliferation Phytoplasma Disrupts Cell Integrity of Nicotiana spp. Protoplasts

“…The correct assembly of the plant transformation constructs was confirmed by sequencing. Plasmid-DNA for protoplast transformation was obtained as described elsewhere [60], using the NucleoSnap ® Plasmid Midi preparation kit (Macherey-Nagel, Düren, Germany) and PEG precipitation.…”

“…Protoplasts of N. benthamiana and N. occidentalis were isolated from four- to five-week-old plants, cultivated under long photoperiod conditions (16 h/8 h, 24 °C/22 °C, 70% rH) and transformed as described in [60] using 10 µg plasmid-DNA per 20,000 protoplasts. After 18 h, at least 100 protoplasts of each transformation were checked for the occurrence of GFP or mCherry-fluorescence using a confocal laser scanning microscope (LSM800, Zeiss, Oberkochen, Germany) with an excitation wavelength of 488 nm for GFP and 561 nm for mCherry.…”

The ‘Candidatus Phytoplasma mali’ effector protein SAP11CaPm interacts with MdTCP16, a class II CYC/TB1 transcription factor that is highly expressed during phytoplasma infection

“…Nicotiana benthamiana leaf mesophyll protoplasts were isolated for bimolecular fluorescence complementation analysis (BiFC) from leaves of four-week-old plants and protoplasts were transformed with the pBiFC vectors as described in Janik et al (2017) [37]. Transformed protoplasts were analyzed 16h after transformation using a confocal laser scanning microscope (Zeiss LSM800, Carl Zeiss Microscopy, Oberkochen, Germany).…”

“…For further confirmation of the interaction between SAP11 CaPm and MdTCP16, the sequences of SAP11 CaPm and MdTCP16 were subcloned into different pBiFC-2in1 vectors [36] by Gateway-cloning [40]. The pBiFC-2in1 vectors were transformed into N. benthamiana mesophyll protoplasts [37]…”

“…For further confirmation of the interaction between SAP11 CaPm and MdTCP16, the sequences of SAP11 CaPm and MdTCP16 were subcloned into different pBiFC-2in1 vectors [36] by Gateway-cloning [40]. The pBiFC-2in1 vectors were transformed into N. benthamiana mesophyll protoplasts [37] and interaction was analyzed by confocal microscopy 16 h after transformation. Up to 92.5% of the transformed protoplasts showed a YFP signal, resulting from the interaction between SAP11 CaPm and MdTCP16 (Fig 2).…”