Acetosyringone

Before agroinfiltration, glycerol stocks of agrobacteria were inoculated into 10 mL of LB containing 50 mg/L of rifampicin and 200 mg/L of carbenicillin, as well as 100 μM of acetosyringone (Merck, Saint Loius, MO, USA, D134406). The cultures were grown in the dark overnight at 28 °C with 220 rpm shaking. The cultures were then centrifuged at 2900 g, suspended in MMA buffer (10 mM MES, Formedium, Norfolk, England, MES03; 10 mM MgCl₂, Molecula, Krasnodar, Russia, 29218779; 200 μM acetosyringone Merck, Saint Loius, MO, USA, D134406), and incubated at 28 °C, 100 rpm for 3–4 h. Next, optical density at 600 nm was measured and used to dilute each culture to the optical density of 0.6. In addition, suspension of agrobacteria containing a plasmid encoding pNOS–P19–tOCS was added at the optical density of 0.2. The final optical density at 600 nm of agrobacterial suspension used for infiltration was 0.8. We then used these cultures to infiltrate leaves of 4–6-week-old N. benthamiana, using a 1 mL medical syringe without needle. At least four leaves of at least three different plants were infiltrated for each experiment. The exact numbers of leaves are included in the figure’s legends.

A. tumefaciens GV3101 carrying either pJL TRBO-G or pCBNoX P19, were grown in 4 ml LB medium for 24 h at 28 °C and shaking at 250 rpm. The cultures were then transferred into 100 ml LB medium having 200 μM of acetosyringone (Sigma-Aldrich) grown overnight at 28 °C and shaking at 250 rpm. Cells were harvested by centrifugation at 3000 g for 10 min and re-suspended in infiltration buffer (pH 5.6, 10 mM MES, 10 mM MgCl₂ and 200 μM acetosyringone) to achieve an OD₆₀₀ of 0.4. The pJL TRBO-G expression vector was mixed in a 1:1 volume ratio with the gene-silencing suppressor (pCBNoX P19). The mixed Agrobacterium suspensions were incubated in the dark at room temperature for 2–3 h before infiltration. The incubated Agrobacterium suspensions were infiltrated into the abaxial surface of leaves using a 1-ml syringe without needle. The agroinfiltrated plants were incubated in the growth chamber for 4–8 days after which the leaves were harvested.

Maize cp1a (GRMZM2G166281) without the sequence encoding for the granulin domain and cp2 (GRMZM2G038636) genes were amplified by PCR from maize (EGB) cDNA. PCR products were directly cloned with BsaI overhangs into the level 1 binary vector (pICH47732) (Addgene) containing 2x35S promotor and a C-terminal Streptwin tag using the golden gate procedure^{43 (link)}. Binary plasmids pL1M-F1-CP2-Streptwin::2x35S and pL1M-F1-CP1A_nogran-Streptwin::2x35S were generated and transformed into Agrobacterium tumefaciens GV3101 competent cells for overexpression in N. benthamiana. GV3101 strains containing the desired plasmids were grown in liquid media overnight and diluted to OD = 2 in 10 mM magnesium chloride with 200 µM acetosyringone final concentration (Sigma-Aldrich, Taufkirchen, Germany). After 1 h incubation cultures were mixed with GV3101 strains containing the p19 construct⁴⁴ to a final OD = 1 and cultures were syringe infiltrated in leaves of 5−6-week-old N. benthamiana plants. Three days post infiltration leaves were harvested and apoplastic fluid was isolated.

Four-week-old N. benthamiana plants grown at 22–24°C in culture rooms were used for Agrobacterium tumefaciens-mediated transient expression as described previously (Sparkes et al., 2006 (link)). The β1- and β6-conglutin coding sequence were cloned into the vector pMDC83 to generate C-terminal GFP fusion proteins and transformed into A. tumefaciens AGL1. Transformed A. tumefaciens AGL1 were cultured at 28°C until stationary phase (∼24 h), washed and resuspended in infiltration medium (50 mM MES, 0.5% (w/v) glucose, 100 μM acetosyringone (Sigma-Aldrich⁷ pH 5.6). The bacterial suspension was inoculated using a 1-mL syringe without a needle by gentle pressure through a <1 mm hole punched on the lower epidermal surface of the upper leaves of N. benthamiana plants. Following infiltration, plants were incubated under normal growth conditions at 22–24°C. This protocol was used for in vivo fungal growth inhibition assays, oxyblot assays, and subcellular localization studies.

The genes encoding Ghlac WT and variants were ordered from Genscript. ABTS, DMP, guaiacol, SGZ, 1-hydroxybenzotriazole (HBT), acetosyringone (ASG), and Sypro Orange were obtained from Sigma-Aldrich (Saint Louis, MO, USA). Methyl syringate (MeS) and violuric acid (VA) were purchased from BioRuler (Danbury, CT, USA). Malachite Green chloride (>98%) was purchased from Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China). All other chemicals and reagents were of analytical grade.

Agrobacterium strains EHA105 harboring the binary constructs were grown on AB minimal medium with 50 mg/L kanamycin for 16 h at 28°C. Cultures (OD₆₀₀ = 0.1) were resuspended in liquid co-cultivation medium supplemented with 100 µM acetosyringone (Sigma, Aldrich, India). Calli (45 day old) were incubated with bacterial (carrying mtlD or UidA gene construct) suspension medium for 10 min. They were then blotted dry on sterile filter paper to remove excess Agrobacterium and co-cultivated for 48 h at 25°C in dark. Subsequently, they were washed using sterile water containing 300 mg/L cefotaxime for four to five times, blotted on sterile filter paper and inoculated on antibiotic selection medium (MS+0.5 mg/L BA+3 mg/L 2, 4-D+30 mg/L hygromycin and 300 mg/L cefotaxime). This step was repeated if Agrobacterium growth occurred.

Bisphenol A, vanillin, acetosyringone, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 1-hydroxy-benzotriazole (HBT) were purchased from Sigma-Aldrich (USA). All other chemicals were of analytical grade.
Corn straw, corncob, rice straw, sawdust, bagasse, and wheat bran, which were obtained from local farms, were air dried and milled to pass through a 20-mesh screen.