Fv1200 ix83

The recombinant plasmid 35S::TaBBX-3B-nYFP was generated by inserting the full-length CDS (without the stop codon) of TaBBX-3B into the pCAMBIA2300-VYNE vector, which carried the N-terminal half of YFP. However, the recombinant plasmid 35S::TaHY5-7A-cYFP was generated by inserting the full-length CDS (without the stop codon) of TaHY5-7A into the pCAMBIA2300-VYCE vector, which carried the C-terminal half of YFP. All primers are listed in Table S3. These two recombinant plasmids and empty vectors were independently transformed into A. tumefaciens strain GV3101. A. tumefaciens harboring all possible combinations of plasmids were co-infiltrated into the epidermal cells of N. benthamiana leaves. The specific methods are detailed in the Subcellular Localization Analysis section (see above). The fluorescence signals were observed and imaged with a confocal laser scanning microscope (IX83-FV1200, Olympus, Tokyo, Japan).

The full-length coding sequence of BdGATA13 was amplified by PCR and cloned into the pCambia-1301 vector harboring the CaMV35S promoter. The recombinant vector was transformed into Arabidopsis strain Col-0 using the GV3101-mediated floral dip method (Clough and Bent, 1998 (link)). Transgenic lines were screened using a 0.1% hygromycin B solution and further confirmed by PCR. The full-length BdGATA13 coding sequence without a stop codon was inserted into the pCambia-1301-GFP vector to produce construct 35S: BdGATA13-GFP. For subcellular localization assays this construct and the vector pCAMBIA1301-GFP were co-transformed into tobacco leaves. Subcellular localization in tobacco leaves using GFP and DAPI staining was expedited by confocal microscopy (Olympus IX83-FV1200, Japan).

To identify PpE4 and GFP co‐silencing transformants, transformants with attenuated GFP signal were identified using an Olympus BX‐51TRF fluorescence microscope (Olympus, Tokyo, Japan) with the GFP filter (BP450‐480). For E4FL‐mCherry‐expressing lines, the putative transformants were observed under the mCherry filter (BP520‐550). Images of vegetative mycelia and infection hyphae in N. benthamiana leaves were captured on an Olympus IX83‐FV1200 confocal microscope with 488 nm excitation and a 500–530 nm emission spectrum for GFP. For mCherry, the emission spectrum was acquired between 595 and 625 nm under 559 nm excitation to eliminate potential autofluorescence from P. parasitica hyphae and cell damage. The detached N. benthamiana leaves inoculated with the transformants and the control strain were incubated at 23 °C for 12–48 h to allow the penetration and formation of intercellular hyphae with haustoria. The control strain and transformants were observed under the same conditions.

Arabidopsis seedlings were grown in a growth chamber under a 16 h light (22°C; 08:00–00:00)/8 h darkness (20°C; 00:00–08:00) regime. Root length was measured on the tenth day and counted using the ImageJ software (Rueden et al., 2017 (link)). Photos of the root apical meristem cell on the 4-day-old plants were taken after staining with propidium iodide (PI, 0.01 mg/ml) for 1-2 min using confocal microscopy (Olympus IX83-FV1200, Japan) with a 561-nm laser for PI. Data were analyzed and plotted using the IBM SPSS Statistics software (USA). Values are shown as means ± SD, and significant differences are indicated by different letters or e-values (P <0.05, one-way ANOVA).

The amplified fragments with additional Nde I and EcoR I sites through corresponding primers (Additional file 1: Table S8) were cloned separately into the DNA binding vector pGBKT7 and activation domain vector pGADT7. Recombined vectors were transformed into the yeast strain Y2H using the LiAc transformation method [92 (link)] and coated on synthetic dextrose (SD) -Trp or SD-Trp-Leu for growth tests. Yeast clones were plated on SD-His-Trp-Ade and SD-His-Trp-Leu-Ade medium for 3 days at 30 °C to assay for self-activation and protein interaction.
The coding sequence were amplified using primers listed in Additional file 1: Table S8 and then introduced into vectors p1302-eYFP-N and p1302-eYFP-C using recombination reactions. Recombined plasmids were transformed into the Agrobacterium tumefaciens strain GV3101 and then co-infiltrated with Agrobacterium carrying the p19 silencing plasmid into leaves of 1-month-old Nicotiana benthamiana plants. Two days after infiltration, eYFP signals were observed with a fluorescence microscopy (Olympus IX83-FV1200).
The coding sequence were cloned with primers in Additional file 1: Table S8 and ligated into the over-expression vector pCAMBIA1300 using recombination reactions. Recombined plasmids were introduced into GV3101 and then transformed into Arabidopsis Col-0 via the flowerer-dipping method [93 (link)].