S1 Data -

Fig A. Eggshell purification. Fig B. Mass spectrometry data. Fig C. Annexin interspecies alignment. Fig D. Primers. Fig E. Antibody information. Fig F. qPCR data. Fig G. RNAi eggshell measurements. Fig H. Pre-diapause hatching data. Fig I. Circular dichroism. Figure legends for S1 Data. Fig A in S1 Data. A–Eggshell/nematode sample after cysts are crushed with a tissue homogeniser. B–Eggshell lysis after a period in the ultrasonic water bath. C–purified eggshells after sodium-potassium tartrate gradient density centrifugation. Images taken on a moticam microscope camera attached by a 0.5x magnification mount seated above a 40x objective lens. Fig B in S1 Data. Mass spectrometry data of proteins extracted from eggshells. Fig C in S1 data. Alignment of GROS_g03104 against orthologues in other plant parasitic nematodes. Free-living nematodes from the genus Caenorhaditis were included as non-parasitic nematode comparatives. D. melanogastor provides a non-nematode outlier. GROS_g03104 orthologues were identified through parasite.wormbase. Sequence alignment was completed using the MUSCLE server with default settings. CLUSTAL X colours highlighting amino acid profile were added using Jalview (2.11.2.5). Fig D in S1 Data. Primers used in this study. Fig E in S1 Data. Information on region of protein used for antibody production. Fig F in S1 Data. qPCR data from developing G.rostochiensis females reflecting change in expression of GROS_g03104 following host induced gene silencing (HIGS). EV—empty vector, 2,6,7,20 represent plant lines determined to be expressing the RNAi construct. Variation in success of HIGS can be seen between females multiplying on the same plants lines. Fig G in S1 Data. Cyst diameter measurements from G. rostochiensis populations grown on RNAi plant lines (6, 20) or control plant lines not expressing the HIGS construct. Population A2012 represents the initial population used to infect RNAi plant lines. Diameters were measured using imageJ measuring from left to right lateral sides of the cyst. ANOVA including 2nd generation EV and HIGS shows no significant variance between the diameter of these populations. Fig H in S1 Data. Hatching of juveniles from RNAi treated and control eggs. Fig I in S1 Data. Circular dichroism using recombinant GROS_g03104 under various conditions. Recombinant annexin was diluted in 50 mM Tris (pH7.5), 100 mM NaCl, 10% glycerol and either 1 mM CaCl2, 1 mM EDTA, tomato or potato root diffusates. 2 mL sample sizes including 1 mg/mL protein were used for each sample. Samples were loaded into a 10 mm cuvette and readings were taken between 260 nm-320 nm using a Mos-500 circular dichroism spectrophotometer (Biologic) coupled with and ALX-300 lamp unit running above 145 W. Slit size was set to 2 nm and readings were taken stepwise every 0.25 nm. No large variation in CD traces can be seen for the recombinant annexin under any conditions suggesting that neither host RD or the presence/absence of calcium drastically changes the annexin tertiary structure.
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