Miracloth filter

To ensure comparable growth phases for all substrates tested, cell harvest for RNA isolation was performed at different time points of the cultivation. While sampling for glucose and pure acetate was performed after 24 h, the biomass of the PAC cultures was harvested after 48 h. For the biomass harvest, the content of the entire shake flask was poured through a Miracloth filter (Merck, Darmstadt, Germany) and then rinsed with ultrapure water. The washed biomass was then snap-frozen with liquid nitrogen and stored at −80 °C until further use. Total RNA isolation, the poly A enrichment of mRNA and the construction of Illumina-stranded TruSeq RNA libraries as well as mRNA sequencing (Illumina NextSeq, 75 bp, paired-end), were all performed by Microsynth (Balgach, Switzerland).

Agrobacteria containing the appropriate constructs were grown overnight at 28°C in lysogeny broth containing 25 μg/mL rifampicin and 50 μg/mL kanamycin. For the expression of IgG or monomeric IgA1 and IgA2 variants, the overnight cultures containing the respective constructs for the heavy and light chains were diluted in infiltration buffer (10 mM morpholinoethanesulfonic acid, 10 mM MgSO₄, and 0.1 mM acetosyringone) to an optical density 600 (OD₆₀₀) of 0.1. For secretory IgA variants, heavy- and light-chain constructs were diluted to an OD₆₀₀ of 0.05 and mixed with the JC construct at an OD₆₀₀ of 0.2 and the SC construct at an OD₆₀₀ of 0.1. Agrobacteria solutions were then introduced into 6- to 8-week-old glycoengineered N. benthamiana ΔXT/FT plants by vacuum infiltration.³⁴ (link)^,67 (link) Plants were grown in a controlled environment room at 25°C with an 16/8-h light/dark cycle. After 5 days, infiltrated leaf material was harvested, and crude leaf extract was prepared by adding 3 volumes of ice-cold PBS pH 7.4 containing 0.1% (v/v) Tween 20 in a blender. Homogenized leaf material was passed through a Miracloth filter (Merck Millipore, Germany) and centrifuged at 20,000 × g for 1 h, followed by filtration through 0.45-μm pore size filters (Durapore membrane filter, Merck Millipore).

Gibberellin A3 was purchased from Sigma-Aldrich (St. Louis, MO). The National Research Council Hormone Profiling Facility provided (+)-ABA, while 3′-hexasulfanyl-(+)-ABA was synthesized as described in Takeuchi et al., [20 (link)] and provided by Kenneth Nelson and Suzanne Abrams at the University of Saskatchewan. Phytohormone stocks were solubilized in deionized water as sodium salts by 1.0 N NaOH titration and stored at − 20 °C in amber vials. Working solutions were made in deionized water and pH was adjusted to 7.0 ± 0.05. Fg GZ3639 [66 (link)] was propagated on potato dextrose agar (PDA; Sigma-Aldrich) at 25 °C for 5 days. To obtain spores, carboxymethylcellulose (CMC) liquid media (1.5% CMC (Sigma), 0.1% NH₄NO₃, 0.1% KH₂PO₄, 0.05% MgSO₄·7H₂O, and 0.1% yeast extract) was inoculated with a marginal 5 mm square PDA plug and grown for 5 days at 27 °C, shaking at 170 rpm. Macroconidia were isolated by filtering through one layer of cheese cloth and 25 μm Miracloth filter (EMD Millipore; Billerica, MA), washed three times with sterile water, and quantified using a haemocytometer and light microscopy.