Figure 1.
Schematic diagram of the cyst nematode feeding process.
The nematode feeds by inserting the stylet through the wall of the feeding cell. A feeding tube is formed within the host cell cytoplasm, and host cell assimilate is withdrawn through the walls of the feeding tube in the direction of the arrows. Inset shows a cross section of a feeding tube induced by Globodera pallida in potato (Solanum tuberosum) and viewed under a transmission electron microscope.
Figure 2.
RotaMol: Varying Pixelskip and its effect on measurement accuracy.
The images represent every pixel analysed at varying Pixelskips for a single viewing angle of the green fluorescent protein PDB file. At a Pixelskip of 1 every pixel is measured, at a Pixelskip of 50, 1 in every 50 pixels is measured. The number of pixels is then multiplied by the Pixelskip for X and Y.
Figure 3.
Optimum values for RotaMol parameters.
(A) RotaMol analysis of mRFP at θ = 30° varying Pixelskip in a stepwise manner from 3 to 100. Average area (black) in Angstroms2 plotted against Pixelskip. Computation time (red) plotted on left axis in seconds, shows exponential increase with decreasing Pixelskip. The optimum value for Pixelskip was defined as 20, as it has the highest accuracy with the shortest computational time (arrow). (B) RotaMol analysis of Luteinising hormone-releasing hormone (LHRH) using a Pixelskip of 20 and varying the number of viewing angles from 1 to 32,400 (θ = 180° to 1°). The optimum value for the number of viewing angles is defined as 32 (θ = 30°) as it has no appreciable difference when compared to 32,400 viewing angles (θ = 1°).
Figure 4.
Comparison between RotaMol predictions and existing prediction methods.
Size predictions shown for MOBCAL Exact Hard Sphere Scattering (EHSS) in grey bars, and Projection Approximation (PA) in white bars, compared to RotaMol, in black bars, for a range of proteins (PDB code given) from published sources. For the majority of predictions (70%) RotaMol lies between that of PA and EHSS (• indicates the cases where RotaMol predictions are not between PA and EHSS) [23], [28], [29].
Figure 5.
ESI-TWIMS-MS drift plots of GFP, mRFP and Dual PI.
In each case, left represents mass spectrum and right represents the corresponding drift plot. (A) GFP drift plot shows both monomeric species of 27334 Da and a dimeric species at 54627 Da only (red). (B) mRFP drift plot shows a monomeric species at 25786 Da and a less intense dimeric species at 51575 Da (red). (C) Dual PI drift plot shows a single weak dimeric species below the measurement threshold and two monomeric species, both at 21690 Da, presumed to be folded and unfolded variants.
Table 1.
Observed uptake of proteins by cyst nematodes correlated with their molecular mass and size.
Figure 6.
Native-PAGE comparison between Dual PI and mRFP.
Dual PI migrates as multiple bands under native conditions indicating multimerisation.
Figure 7.
Absorbance data from analytical ultra-centrifugation for mRFP, GFP and Dual PI.
For all samples, sedimentation coefficient is plotted against concentration distribution, c(s). A single monomeric species was detected for mRFP (red), both monomeric and dimeric species were detected for GFP (green). A monomeric form and large heterogeneous multimers estimated at >150 kDa were detected for Dual PI (black).