Nematode rearing, collection and extraction

Heterodera glycines was raised on soybean (Glycine max ‘Kent’), and M. incognita was raised on pepper (Capsicum annuum ‘PA-136’) using a constant moisture system (Sardanelli & Kenworthy, Reference Sardanelli and Kenworthy1997), at 27°C and 16 h light:8 h dark photoperiod. Plants were inoculated near the rhizosphere. Soybean was inoculated with ~1500–2000 H. glycines eggs per plant, and harvested after 5 weeks. Cysts were collected from infected G. max roots, broken open in water to release eggs, and the freed eggs placed into hatching plates. Pepper was inoculated with ~3000 M. incognita eggs per plant, and harvested after 5–6 weeks. Egg masses were collected from infected C. annuum roots under a dissecting microscope, rinsed with tap water, and placed into hatching chambers.

Hatching chambers were six-well assay plates (Falcon B-D, Franklin Lakes, New Jersey, USA) fitted with 12-mm-diameter sieves. Sieves were prepared with Netwell plate inserts (Corning Costar, Corning Incorporated, Corning, New York, USA) consisting of a c. 1-mm-diameter plastic collar supporting a 500 μm pore polyester mesh. A circle of 30 μm pore nylon mesh (Spectrum Laboratories, Rancho Dominguez, California, USA) was placed on top of the 500 μm mesh and secured in place with a tight-fitting ring cut from a polypropylene tube.

Assembled sieves were suspended in each plate well in 2 ml sterile tap water and eggs were added. As infective stage juveniles (J2) hatched from eggs they passed through the mesh into the plate well and were collected daily. Freshly hatched J2 were either used immediately in bioassays, or were collected by centrifugation (15 s, 16,000 × g) to remove water, and the worm pellets stored at − 20°C for later extraction. Panagrellus redivivus were reared in sterile liquid culture at 22°C (Chitwood et al., Reference Chitwood, Lusby, Thompson, Kochansky and Howarth1995) and harvested after 1 week as a mixed-age population. Worms were washed with sterile distilled and de-ionized (SDD) water to remove culture medium, water was removed as above and the worm pellets were stored at − 20°C for later extraction.

Worms were thawed and suspended in ice-cold SDD water (~10 volumes of water/worm volume) and mechanically disrupted in 1.5 ml polypropylene tubes containing 0.5 mm zirconia-silica beads shaken in a Bead Beater (BioSpec Products, Bartlesville, Oklahoma, USA). Processing consisted of six rounds of shaking (30 s per round) alternated with six periods of cooling on ice (3 min each). The processed homogenates were centrifuged at 16,000 × g for 30 s at room temperature and the supernatant (S1) was transferred to a glass high-speed centrifuge tube on ice.

Approximately 1 ml of SDD water was added to the extraction tube and a second set of six rounds of shaking and centrifugation was done as above. The resulting supernatant (S2) was pooled with S1 and an aliquot of the 16,000 × g pellet was examined at 40 × and 100 ×  to assure that all worms were disrupted. The remaining pellet was washed by shaking in 1 ml SDD water followed by 16,000 × g centrifugation and the supernatant added to the pool. This was repeated 2–3 times until the supernatant was clear. All 16,000 × g supernatants were pooled, centrifuged at 40,000 × g for 20 min at 5°C, and aliquots of the resulting supernatant were dried by vacuum centrifugation and stored at − 20°C. Total protein was determined using the microBCA assay (Pierce Chemical Co., Rockford, Illinois, USA).

Preparation of cyst contents and catechin analogues

Heterodera glycines cysts were collected by gently rubbing infected G. max roots under a stream of tap water over an 80-mesh sieve. The collected cysts were rinsed into a sterile 50 ml tube containing 40 ml of SDD water and shaken vigorously to wash the outer surfaces of the cysts. Cysts were allowed to settle and the supernatant wash was decanted. Washing was repeated three additional times with SDD water, and the exhaustively washed cysts transferred to a sterile glass Petri dish for processing. Cysts (c. 5000 cysts in 100 ml SDD) were gently crushed with a sterile rubber stopper to release eggs and contents. Total number of eggs was estimated by direct count of eggs in multiple 0.1 ml aliquots of the crush mixture followed by extrapolation to total mixture volume.

The crushed cyst mixture was filtered through a series of nested sieves (80-, 120-, 635-mesh) and filtrate 1 was collected on ice. The sieve stack with retained cyst materials and eggs was rinsed with c. 400 ml of SDD water. Filtrate 2 was collected, pooled with filtrate 1, and the volume of the pooled filtrates was measured. The filtrate pool was centrifuged at 40,000 × g for 20 min at 5°C, and the resulting supernatant (cyst–egg wash, CE) was dried as aliquots by vacuum centrifugation and stored at − 20°C. Total cysts were estimated by using a ratio of 200 eggs/cyst, and CE equivalents for each preparation were calculated as: total estimated cysts/total filtrate volume = CE equivalent/ml filtrate.

(+)-Catechin, ( − )-epicatechin (EC), ( − )-gallocatechin (GC), ( − )-epigallocatechin (EGC), ( − )-catechin gallate (CG), ( − )-gallocatechin gallate (GCG), ( − )-epicatechin gallate (ECG) and ( − )-epigallocatechin gallate (EGCG) (Sigma Chemical, St. Louis, Missouri, USA) were each dissolved in SDD water and dried as aliquots by vacuum centrifugation. For protease inhibitor assays, each catechin analogue was prepared as a 5 mm stock solution in 100 mm Tris, pH 7.8 protease assay buffer. Aliquots of the stocks were added to reaction mixtures at 1 mm final concentration. For bioassays, analogues were prepared as 2 mm stock solutions in sterile tap water and used at 1 mm final assay concentration.

Protease substrates, inhibitors and assays

FRET-modified peptide substrates QXL520-KSAYMRF-K(5-FAM)a (A2, AnaSpec, Fremont, California USA; used as a general protease substrate) and succinyl-LLVY-AMC (P3, EMD Chemicals, San Diego, California, USA; used as a selective protease substrate to detect proteasome chymotrypsin-like protease, CTL) were prepared as stock solutions in dimethyl sulphoxide (DMSO). The CTL proteasome protease inhibitor MG-132 (EMD) was supplied as a stock solution in DMSO. All were stored at − 30°C until use. Dried CE aliquots were dissolved in TRIS protease assay buffer, heated for 5 min at 95°C, chilled on ice for 5 min, and centrifuged at 16,000 × g for 1 min. The resulting supernatant was used immediately in protease assays or bioassays.

Reactions were performed at 27°C in 384-well assay plates (flat bottom, non-treated, Corning, Inc.) in 100 mm Tris, pH 7.8 assay buffer with 25 μl reaction volume. Nematode extracts were dissolved in assay buffer. Reactions contained 0.04–0.16 μg/μl extract protein for use with QXL520-KSAYMRF-K(5-FAM)a or 0.2–0.8 μg/μl for use with succinyl-LLVY-AMC. Reactions were started by the addition of substrate. Final concentrations of substrates were 1 μm QXL520-KSAYMRF-K(5-FAM)a or 100 μm succinyl-LLVY-AMC with a final concentration of DMSO of 2% in reactions. Final concentrations of inhibitors were 100 μm MG-132, 1 mm catechin analogue, or 0.6 CE eq/μl heated CE wash. Inhibitor was added to the reaction mixture and incubated for 15 min at 27°C prior to addition of the substrate.

Reactions were monitored for 60 min for increasing fluorescence (Ex = 490 nm, Em = 520 nm for substrate QXL520-KSAYMRF-K(5-FAM)a or Ex = 380 nm, Em = 460 nm for succinyl-LLVY-AMC) using a SpectraMAX EM fluorescent plate reader (Molecular Devices, Sunnyvale, California, USA). Substrate controls (no extract) were used to correct for background. Reaction rates were quantified using SoftMax Pro instrument control and data collection software (Molecular Devices), and increase in relative fluorescence units (RFU) over time was recorded and reduced to V_max/s. Data were then converted to V_max/min/total μg reaction protein. Inhibitor data are presented as the per cent reduction in V_max/min/μg in test versus control reactions: ((V_max/min/μg_control− V_max/min/μg_inhibitor)/V_max/min/μg_control) × 100.

Bioassays

Eggs from H. glycines or M. incognita were collected as above and transferred to 96-well microtitre plates (Falcon-BD) using a micropipette, with 20–40 eggs per well, in 200 μl of tap water (controls) or 200 μl test solution in tap water. Test solutions used were either (+)-catechin or analogues (1 mm final concentration), or heated CE wash (0.6 CE eq/μl final concentration). These solutions were prepared just prior to use.

All wells were inspected at 40 ×  magnification for both eggs and hatched J2, and totals for each well were recorded. Plates were covered and incubated for 7 days in a humid chamber at 26°C. On day 7, all eggs and J2 in each well were counted as above, and per cent hatch calculated using the formula: ((J2_D7 – J2_D0)/Eggs_D0) × 100. For each hatch experiment, 6–16 treatment and control replicates were used and experiments were repeated 2–8 times.

Freshly collected H. glycines or M. incognita J2 were transferred to chambers of an Ibidi μ-Slide (Ibidi μ-Slide Angiogenesis, uncoated chamber for cell microscopy, www.ibidi.com) at 5–15 J2/well in 50 μl tap water (controls), 50 μl 1 mm EGCG, or 50 μl of 0.6 CE eq/μl heated CE wash. Slide chambers were covered and slides incubated in a humid chamber at 26°C. After 20–22 h incubation, individual J2 were observed continuously for 1 min at 40 × for signs of movement.

J2 movement was scored using a subjective five-step activity scale with values from 0 (no movement of any kind) through 3 (very highly active, whole body flexing, rapid and consistent anterior waving). Scores were assigned by four individuals to minimize bias, experiments were repeated 2–4 times using separate J2 collections, and data are summarized across all J2. Data are expressed as both mean activity score and mean per cent activity of treated J2 relative to controls.

Data analysis

Individual data means were compared using Student's t-test and relationships among data means were analysed using one-way analysis of variance (ANOVA) with Tukey's multiple comparison. P is indicated for each comparison. Statistical analyses were done using GraphPad Prism (GraphPad Software, La Jolla, California, USA) and JMP10 (SAS Institute, Cary, North Carolina, USA).