Figure 1.Morphology of germinating A. algerae spores. (A)Overall organization of organelles in an A. algerae spore. The spore coat consists of 3 layers: a proteinaceous exospore (orange), a chitin-containing endospore (yellow), and a plasma membrane. Within the spore, the polar tube (PT) (blue), which is the infection organelle, surrounds other organelles like a rib cage. The PT is anchored to the apical end of the spore via a structure called the anchoring disc (green). At the apical end, the PT is linear, and then forms a series of coils, which end at the posterior end of the spore. The PT interacts closely with other spore organelles, including the posterior vacuole (red), and a membranous organelle called the polaroplast (purple). The organization of the spore shown here comes from SBF-SEM data (bright colors) and TEM images (nuclei positioning, and plasma membrane, grey). (B-D) Examples of slices from SBF-SEM imaging and the corresponding 3D reconstructions for ungerminated (B), incompletely germinated (C) and germinated (D) A. algerae spores. Colored according to the color key shown in (C). All scale bars are 500 nm. (E) Kymograph of the PT ejection process in A. algerae. The PT ejection process can be divided into 3 phases: PT elongation phase (blue), PT static phase (pink), and emergence of infectious cargo phase (green). This kymograph was generated from data deposited in Jaroenlak et al. (2020).Figure 2.Possible hypotheses for the topological connectivity and morphology of spore organelles. The selection process of the hypotheses for the energetics calculation is shown. We considered 6 critical topological questions regarding the connections between different spaces in the spore that is relevant to the energetics calculation and developed a standard nomenclature to describe the hypotheses. The combinatorics of the 6 questions gave us 64 hypotheses. By evaluating the topological compatibility of these combinations, we are left with 10 hypotheses, and we further narrow this down to 5 hypotheses based on the fact that the posterior vacuole expands during the germination process (see Figure S1). The list of all the hypotheses is summarized in Table S1, and a detailed calculation of each hypothesis is described in Figure S2-S4.Figure 3.PT ring kinematics in the presence of varying external viscosity. (A) Schematic outlining the protocol for hypothesis testing. We experimentally measured the PT ring kinematics of A. algerae spores in buffers with varying viscosity, by varying the methylcellulose (MC) concentrations up to 4%. We next calculated the required total energy, peak pressure and peak power for each experimentally measured data according to our physical framework proposed in Figure S2-S4, and we see if the required energy changes with respect to changes in surrounding viscosity. We assume that changing surrounding viscosity should not change the energy sources of the spores. Thus if the calculated energy requirement changes significantly with respect to changes in surrounding viscosity (p < 0.05), the hypothesis is inconsistent with experimental observations. (B) Experimental measurement of PT ejection kinematics of A. algerae spores in different concentrations of methylcellulose. The kinematics was t to a sigmoid function and then normalized by L. The additional term in the sigmoid function is to ensure the curve passes the origin. (0%: n=12; 0.5%: n=10; 1%: n=10; 2%: n=8; 3%: n=5; 4%: n=9) The inset shows the original data in MC0%. The changes in MC concentration does not cause obvious changes in overall kinematics of PT ring. The complete set of original data can be found in Supplementary Figure S6. (C) The dependence of maximum PT ejection velocity on MC concentration in germination buffer. Increasing MC concentration up to 4% does not change the maximum PT ejection velocity. (p=0.848, Kruskal–Wallis test) (D) Viscosity measurements of germination buffer with various concentrations of methylcellulose, corresponding to the concentrations used in PT extrusion experiments. As the PT ejection process is a high shear rate phenomenon (∼3000 1/sec), we used the measurement at shear rate γ = 1000 sec−1. The maximum tested shear rate was 1000 sec−1 as that reaches the operation limit of the shear rheometer. (n = 5 for 0%, 0.5%, 1%. n = 3 for 2%, 3%, 4%.)Figure 4.Energetic analysis to identify hypotheses that are consistent with experimental results of PT extrusion kinematics in varying external viscosities. Each row (A-E) shows calculations based on the ve different hypotheses, and the three columns show the calculation for total energy requirement (left column), peak pressure difference requirement (middle column), and peak power requirement (right column) for each PT ring event shown in Figure S6. Kruskal–Wallis test was used, and only the p-values which are significant or near-significant are shown. Only the p-values calculated for total energy requirement were used for ranking. The p-values for peak pressure difference requirement and peak power requirement are just for reference. The data shown here is calculated assuming a cytoplasmic viscosity of 0.05 Pa-sec, and a zero boundary slip. The effect of ambiguity in cytoplasmic viscosity and slip length of the boundaries are discussed in Table S4-S5. Under these assumptions, Model 1 and Model 3 are the two hypotheses that are least likely to be true. Also note that for the other three hypotheses (Model 2, Model 4, and Model 5), the total energy requirement is roughly 10−11J, the peak pressure difference requirement is roughly 60-300 atm, and the peak power requirement is roughly 10−10W.Figure 5.Hypotheses that can potentially explain the two-stage translocation of the cargo. (A) Kymograph of nuclear transport inside the PT. Nuclei were stained with NucBlue prior to germination, and imaged using fluorescence microscopy. Previously deposited data from Jaroenlak et al. (2020) were used in this gure. A two-stage process is observed for nuclear translocation, with a long pause in the middle. The second stage of nuclear movement is overlaid with red, and the asterisk indicates the beginning of the second stage movement, in which the nuclei are expelled out of the PT. (B) Quantification of the nuclear position relative to spore coat over time (n=4). (C) 3D reconstructions of incompletely germinated and germinated spores from SBF-SEM data. 100% of spores in which the nuclei have been expelled are buckled (Table S6). The translocation of nuclei at the nal stage can be explained by spore buckling. (D) Volumes of ungerminated and germinated spores calculated from SBF-SEM 3D reconstructions. Ungerminated: mean = 8.78 µm3, std = 1.41 µm3, n=19; Germinated: mean = 5.52 µm3, std = 1.03 µm3, n = 14; p <0.0001. (E) Schematic model of an A. algerae spore used for calculating the spore wall buckling pressure, the relevant parameters used in the calculation and the formulae. Using the theory of elastic shell buckling (see text for detail), we showed that the pressure built up during the PT ring process is enough to buckle the spore wall, and the predicted buckling volume is enough to push cytoplasmic content in PT forward by 129-261 µm. (F) The predicted time series of pressure from Model 4 and Model 5 (n = 54), overlaid with the critical pressure of spore wall buckling, water cavitation pressure and bubble nucleation. All three phenomena can cause volume displacement at the later stage of the germination process, and provide a driving force to push the cargo/nuclei forward. Model 5 is more compatible with experimental data than Model 4. The downward arrows indicate the mean time when the negative pressure rst reaches the critical pressure. (detailed numbers mentioned in the main text.) (G) Theoretical predictions and experimental measurements from orthogonal approaches are compiled and are in agreement with each other. We obtained the prediction based on spore wall buckling theory and hydrodynamic energy dissipation theory, and we compiled the experimental observations from the SBF-SEM data.Symbols: Rspore: spore radius; ΔV: volume changes of spore after buckling; t: spore wall thickness; E: Young’s modulus of the spore wall; v: Poisson ratio of the spore wall; W: work; Δx: predicted fluid displacement distance; LPT: full length of the ejected PT.Figure 6.Summary and a model for the most likely hypothesis of the PT ring mechanism. We evaluated 64 possible topological connectivities, eliminated those that are incompatible with our knowledge of the process, and further explored 10 viable hypotheses. We retained the 5 hypotheses that assume an expanding posterior vacuole during the germination process, which are consistent with the SBF-SEM data. The hydrodynamic energy dissipation analysis allows us to rank 2 hypotheses over the other 3, and our analysis on the pressure requirement for spore wall buckling suggests Model 5 (E-OE-PTPV-ExP, “Eversion, with PT tip open to external environment, and PT connected to posterior vacuole, with expanding posterior vacuole”) is the most preferred hypothesis. The schematic shows our understanding of the process based on Model 5. After initiation of germination, the PT extrudes via an eversion-based mechanism. Vacuole contents may be connected to the original PT contents. The eversion brings the end of the PT away from the posterior vacuole, which allows the infectious cargo to later enter the PT through fluid entrainment. Tube eversion causes negative pressure to build up within the spore. Eventually this negative pressure either initiates buckling of the spore wall or causes bubble formation in the spore to push the nucleus outward. Key numbers related to the process and the predictions from E-OE-PTPV-ExP hypothesis are summarized in the text box.