Silwet L-77

The Agrobacterium strain GV3101 containing TRV-VIGS vectors was grown at 28 °C in Luria–Bertani medium supplemented with 10mM MES, 20mM acetosyringone, 50mg l^–1 gentamicin, and 50mg l^–1 kanamycin for ~24h. Agrobacterium cells were harvested and suspended in the infiltration buffer (10mM MgCl₂, 200mM acetosyringone, and 10mM MES, pH 5.6). A mixture of Agrobacterium cultures containing pTRV1 and pTRV2 and its derivatives, in a ratio of 1:1 (v/v), were placed in the dark at room temperature for 4h before inoculation. For vacuum infiltration, Silwet L-77 was added to a final concentration of 0.01% (v/v).
Nicotiana benthamiana infiltration was performed as described by Liu et al. (2002a). Agrobacterium cultures containing pTRV1 and pTRV2 or its derivatives (1:1, OD₆₀₀=1.0) were injected into the lower leaf of four-leaf stage plants by using a 1ml needleless syringe. For axil injection, 20 μl of bacterial suspension (1:1, OD₆₀₀=4.0) were injected per plant into the leaf axil using a 1ml syringe with a needle at the four-leaf stage period.
Since rose leaves and axils are hard to inject, the vacuum infiltration method was used for rose cuttings, and seedlings were infiltrated as described previously (Ma et al., 2008 (link); Yan et al., 2012 (link)). Four-week-old rose cuttings were removed from the soil, the roots were rinsed with distilled water, and whole plants were submerged in infiltration mixture containing pTRV1 and pTRV2 or its derivatives (OD₆₀₀=1.0) and subjected to a vacuum at –25 kPa twice, each for 60 s. This was repeated once. Treated plants were briefly washed with distilled water and then planted in pots. For seedlings, the seeds generated were submerged in infiltration mixture containing pTRV1 and pTRV2 or its derivatives (OD₆₀₀=1.0) and subjected to a vacuum at –25 kPa twice, each for 60 s. The infiltrated seeds were briefly washed with distilled water and placed on wet filter paper for several days, and then the seedlings were planted in pots.
Strawberry was vacuum infiltrated in the same way as rose cuttings and seedlings. After the release of the vacuum, the plants were briefly washed with distilled and planted in pots.
Arabidopsis and chrysanthemum infiltration was performed as described by Burch-Smith et al. (2006) (link). A mixture of Agrobacterium culture containing pTRV1 and pTRV2-GFP (OD₆₀₀=1.5) was infiltrated into the two leaves of 2-week-old Arabidopsis plants and the lower leaves of six- to eight-leaf-stage chrysanthemum plants using a needleless syringe. The infected chrysanthemum plants were transferred into pots.

The day before cocultivation, liquid cultures of A. tumefaciens were inoculated from colonies on agar plates or frozen glycerol stock. After growth at 28°C in 2 mL LB medium with appropriate antibiotics (25 μg/mL streptomycin plus 50 μg/mL kanamycin or 100 μg/mL spectinomycin, or both) for 18–24 hr, 1.6 mL saturated culture was diluted the next day into 10 mL fresh YEB medium (5 g/L beef extract, 1 g/L yeast extract, 5 g/L peptone, 5 g/L sucrose, 0.5 g/L MgCl₂) to OD600 = 0.3 and was grown until the OD₆₀₀ reached more than 1.5. Bacteria cells were harvested by centrifugation at 6,000 g for 5 min and washed once with 10 mL washing solution containing 10 mM MgCl₂ and 100 μM acetosyringone. After centrifugation at 6,000 g for another 5 min, the pellet of bacteria cells was resuspended in 1 mL washing solution. We also tested a simplified protocol where A. tumefaciens cells were directly scraped from agar plates and resuspended into wash solution at a final OD600 = 0.3. This simplified procedure resulted in similar transformation efficiency as the protocol described above.
In a clean Petri dish (100 × 20 mm), 30–40 4-day-old Arabidopsis (or tobacco) seedlings, 10–15 4-day-old tomato seedlings or 5-day-old rice (or swichgrass) seedlings were soaked with 20 mL cocultivation medium containing 0.25 × MS (pH 6.0, Caisson Laboratories), 1% sucrose, 100 μM acetosyringone, 0.005% (v/v; i.e. 50 μL/L) Silwet L-77 and A. tumefaciens cells at final density of OD600 = 0.5 (6 × 10⁸ cfu/mL). Cocultivation was carried out in darkness at the same temperature as seedling growth for 36–40 hr for Arabidopsis seedlings, 36–60 hr for tobacco (or tomato) seedlings or 6 days for rice (or switchgrass) seedlings before microscopic observation or other analysis was performed. For cocultivation assay in 96-well plate, 2–3 4-day-old Arabidopsis seedlings geminated directly on 30 μL MS-agar (0.8%) plus 1% sucrose in each well were cocultivated with A. tumefaciens cells in 100 μL cocultivation medium.

dsRNAs of ~300 bp in size that were complementary to MaATG8F and MaATG4B were generated with the T7 RNAi Transcription Kit following the manufacturer’s protocol (Vazyme Biotech, Nanjing, China). The PCR technique was used to add the T7 promoter sequence to both ends of the RNA interference (RNAi) target fragments. The primers used for PCR are listed in Table S2. Sequences including the T7 promoters were purified and used as the templates for dsRNA amplification. Infection assays were then performed to establish the effects of knocking out target genes performed as in a previous study with some modifications [32 (link)]. Specifically, banana leaves of the same age were detached and pressed with the tip of a pipette head to make even wounds, then treated with 10 μL dsRNA at a concentration of 500 ng/μL with 0.02% Silwet L-77 (Solarbio Science & Technology, Beijing, China). The sites in the same leaves treated with 10 μL water with 0.02% Silwet L-77 were used as a control. The Silwet L-77 was a surfactant used for facilitating the adsorption and spread of dsRNA or water on the banana leaves. The dsRNA solution and water were allowed to dry for about 1 h, then mycelial plugs of 5 mm in diameter were taken from plates containing 6-day-old Foc TR4 strain II5. Plugs were taken only from the growing edges and were placed onto the portion of banana leaves treated with dsRNA. The mycelium was facing down, touching the banana leaves. A total of 10 banana leaves were used for each treatment. Each experiment was repeated three times independently. All leaves were then moved to plastic trays, each of which was lined with two layers of moistened paper towel. To maintain sufficiently high humidity, each tray was covered with plastic film. Trays containing inoculated leaves were incubated at 28 °C in the dark for 10 days. Leaves were then photographed, and the fungal lesion size was quantified in ImageJ V1.8.0 software (https://imagej.nih.gov/ij/, access on 20 November 2023). Leaves were also harvested for qRT-PCR to verify gene silencing.

Aboveground plant parts were detached from belowground parts using sterilized equipment and placed individually into preweighed 1.5 mL tubes. After determining the plant weight, 1 mL PBS with 0.02% Silwet L‐77 (Helena Chemical Company) was added to each tube. Bacteria were dislodged from the sample by shaking twice at 2.6 m s⁻¹ for 5 min (Omni Bead Ruptor 24) and sonicated for 5 min. Bacterial CFU were enumerated using plate counting on R2A media plates.