Lumar 43 filter set

Cross-sections (70 µm) of haustoria 3 d after FR light induction were prepared using a Leica VT1000E vibratome (Leica Biosystems, Nussloch GmbH, Nussloch, Germany). Free binding sites were blocked for 30min with 5% (w/v) non-fat milk powder in standard phosphate-buffered saline buffer (1× PBS) (blocking buffer). After washing with PBS, 1:20 dilutions of polyclonal anti-XTH rabbit IgGs or IgGs from the pre-immune serum (both provided by Dr E. Labrador and her group at the University of Salamanca, Spain) in blocking buffer were applied to the sections for 2h followed by three 5min washes with PBS. The sections were then incubated for 1h in the dark with Alexa Fluor 555 Goat Anti-Rabbit IgG (Life Technologies, Carlsbad, CA, USA) (1:200 in blocking buffer) and subsequently washed with PBS. Labelled sections were stained with toluidine blue O in order to quench autofluorescence. Micrographs were taken with a SteREO Lumar V12 equipped with an AxioCam MRc5 camera and the Lumar 43 filter set (all from Carl Zeiss, Jena, Germany).

Different stages of Cuscuta infection sites on P. zonale were collected based on morphological characteristics [6 (link)]. Cross-sections (100 μm thick) through the sites were prepared using a Leica VT1000E vibratome (Leica Biosystems, Nussloch GmbH, Nussloch, Germany) and provided confirmation regarding the stage of haustorium development. Localization of XET activity using endogenous xyloglucan as donor substrate was carried out as described by Vissenberg et al. [23 (link)] with minor modifications. Cross-sections were incubated in 5 μM XyGO-SR dissolved in 50 mM Na-acetate (pH 5.5), 300 mM NaCl for 1 h in the dark. Sections incubated in buffer without XyGO-SR served as controls. After 10 min washing in ethanol: formic acid: water (15:1:4), the sections were further de-stained overnight in 5% formic acid. Micrographs were taken with a SteREO Lumar V12 equipped with an AxioCam MRc5 camera and the Lumar 43 filter set (all from Carl Zeiss, Jena, Germany). Exposure times were between 300 and 520 ms. The fluorescence emission spectrum from 560 nm to 690 nm was recorded using a LSM780 confocal laser scanning microscope (Carl Zeiss).