Self-assembly of MPG1, a hydrophobin protein from the rice blast fungus that forms functional amyloid coatings, occurs by a surface-driven mechanism

Abstract

Rice blast is a devastating disease of rice caused by the fungus Magnaporthe oryzae and can result in loss of a third of the annual global rice harvest. Two hydrophobin proteins, MPG1 and MHP1, are highly expressed during rice blast infections. These hydrophobins have been suggested to facilitate fungal spore adhesion and to direct the action of the enzyme cutinase 2, resulting in penetration of the plant host. Therefore a mechanistic understanding of the self-assembly properties of these hydrophobins and their interaction with cutinase 2 is crucial for the development of novel antifungals. Here we report details of a study of the structure, assembly and interactions of these proteins. We demonstrate that, in vitro, MPG1 assembles spontaneously into amyloid structures while MHP1 forms a non-fibrillar film. The assembly of MPG1 only occurs at a hydrophobic:hydrophilic interface and can be modulated by MHP1 and other factors. We further show that MPG1 assemblies can much more effectively retain cutinase 2 activity on a surface after co-incubation and extensive washing compared with other protein coatings. The assembly and interactions of MPG1 and MHP1 at hydrophobic surfaces thereby provide the basis for a possible mechanism by which the fungus can develop appropriately at the infection interface.

Figure 1. Structure and dynamics of MPG1 in solution.

(A) SEC-MALLS elution profile of purified MPG1. (B) Overlay of the 20 lowest energy conformers of MPG1, with the four disulphide bonds shown in yellow. (C) Ribbon representation of secondary structure of MPG1. (D) Surface-charge distribution of MPG1 showing the amphipathic nature of MPG1, with charges coloured in red and blue and hydrophobic regions in white. (E) Distribution of secondary structure elements along the primary sequence of MPG1. (F) Plots of ¹⁵N T₁, T₂, Heteronuclear NOE values and angle order parameter vs. residue number.

Figure 2. Hydrophobins adopt an open β-barrel structure in solution.

(A) Solution structures of MPG1 and other Class I hydrophobins (EAS and DewA) and a Class II hydrophobin (NC2). (B) An overlay of hydrophobin structures based on the core β-strands illustrates the common half β-barrel structure. (C) Schematic representation of the general sequence features of hydrophobins, in particular the unique pattern of eight cysteine residues and four disulphide bonds.

Figure 3. Morphology of self-assembled MPG1 and MHP1.

(A) Image of a droplet of MHP1 on a hydrophobic OTS-coated silicon surface. (B) AFM image of MPG1 on HOPG with the inset showing a height profile across MPG1 rodlets as indicated. (C) Negatively stained TEM image of MPG1 rodlets assembled on TEM grid. (D) AFM image of MHP1 on HOPG.

Figure 4. MPG1 but not MHP1, assembles into amyloid-like rodlets.

(A) In vitro agitation of MPG1 solutions at pH 7.2 over protein concentration range of 0–80 μg/mL. (B) No increase in ThT fluorescence is observed when MHP1 solutions up to 100 μg/mL are incubated with agitation.

Figure 5. Recombinant expression and purification of Cut2 yield a fully folded and functional enzyme.

(A) Far-UV CD spectrum from purified recombinant Cut2 indicates a predominantly α-helical structure. (B) Structural homology model of M. oryzae Cut2, based on the structure of Cut1 from F. solani. (C) Recombinant Cut2 is active as indicated by the increase in esterase activity upon addition of increasing enzyme concentrations.

Figure 6. Activation of Cut2 enzymatic activity by proteins and detergents.

(A,B) Effect of MPG1 and lysozyme in molar excess on the esterase activity of Cut2. (C) Addition of mixtures composed of single amino acids has no effect on the activity of Cut2. (D) Effect of the detergent Triton X-100 on the activity of Cut2. (E) Recruitment and activation of Cut2 by proteins dried onto a HOPG surface.

Figure 7. MPG1 rodlet formation is modulated by the presence of other proteins and surface active agents.

(A) Dose-dependent introduction of a lag phase in MPG1 assembly when the Class II hydrophobin MHP1 is co-incubated with MPG1. (B) Assembly of MPG1 in the presence of the non-surface-active protein lysozyme and the surface active protein BSA at stoichiometric concentrations. (C,D) Reductions in the surface tension by addition of alcohols such as isopropanol (panel C) or a detergent such as Triton X-100 (panel D) result in introduction of a lag phase, followed by a rapid elongation.

Figure 8. The hydrophobic:hydrophilic interface is essential for rodlet formation by MPG1.

(A) Addition of pre-formed rodlets or seeds has no effect on rodlet formation by MPG1. (B) Fit of “dock-lock” kinetic mechanism to the normalised aggregation data presented in Fig. 4A. (C) In the absence of an interface, no assembly is observed (open circles) while upon introduction of an interface (arrow), assembly is initiated and parallels that observed when an interface is present from the start of assay (closed circles). (D) Rate of elongation increases with increase in surface area. (E) The rate of self-assembly is positively correlated with the rate of agitation of the solutions.

Figure 9. Schematic model of MPG1 assembly and the potential role played by the Cys7-Cys8 loop.

(A) Conformational change to an assembly-competent form of MPG1 is limited to the interface. Elongation occurs via a two-step process that involves docking of monomer to the growing rodlet and locking into a rodlet form. (B) Ribbon diagram of MPG1 is overlaid with a sausage-style cartoon representation of the MPG1 structure with the diameter of the tubular spline corresponding to the flexibility experienced by the backbone atoms. (C) Overlay showing the position of the Cys7-Cys8 loop in the MPG1 structure (blue) in solution and that of EAS in solution (green) and in the EAS rodlet model (red).