Toxin-producing endosymbionts shield pathogenic fungus against micropredators: Supplementary Information Videos

ABSTRACT

The fungus Rhizopus microsporus harbours a bacterial endosymbiont (Mycetohabitans rhizoxinica) for the production of the antimitotic toxin rhizoxin. Although rhizoxin is the causative agent of rice seedling blight, the toxinogenic bacterial-fungal alliance is, however, not restricted to the plant disease. It has been detected in numerous environmental isolates from geographically distinct sites covering all five continents, thus raising the question on the ecological role of rhizoxin beyond rice seedling blight.

Here we show that rhizoxin serves the fungal host in fending off protozoan and metazoan predators. Fluorescence microscopy and co-culture experiments with the fungivorous amoeba Protostelium aurantium revealed that ingestion of R. microsporus spores is toxic to P. aurantium. This amoebicidal effect is caused by the dominant bacterial rhizoxin congener rhizoxin S2, which is also lethal towards the model nematode Caenorhabditis elegans. By combining stereomicroscopy, automated image analyses, and quantification of nematode movement we show that the fungivorous nematode Aphelenchus avenae actively feeds on R. microsporus that is lacking endosymbionts, while worms co-incubated with symbiotic R. microsporus are significantly less lively.

This work uncovers an unexpected ecological role of rhizoxin as shield against micropredators. This finding suggests that predators may function as an evolutionary driving force to maintain toxin-producing endosymbionts in non-pathogenic fungi.

Legends for the Supplementary Information Videos

Video S1. Predation of Protostelium aurantium on swollen spores from Rhizopus microsporus. Timelapse
movie showing ingestion of a swollen R. microsporus spore (stained with FITC) by P. aurantium. Scale
bar: 5 μm.

Video S2. Aphelenchus avenae co-incubated with symbiotic Rhizopus microsporus. R. microsporus
ATCC62417 was co-incubated with A. avenae for 24 hrs in a micro-channel slide (Ibidi). Time-lapse movie,
recorded on a spinning disc microscope, showing dead/unhealthy nematodes. Scale bar: 100 μm.

Video S3. Aphelenchus avenae movement after incubation with solvent control (DMSO). Time-lapse
movie, recorded on a spinning disc microscope, showing healthy nematodes. Scale bar: 200 μm.

Video S4. Aphelenchus avenae movement after incubation with 100 μM rhizoxin S2. Time-lapse
movie, recorded on a spinning disc microscope, showing unhealthy nematodes. Scale bar: 200 μm.

Video S5. Aphelenchus avenae movement after incubation with 250 μM rhizoxin S2. Time-lapse
movie, recorded on a spinning disc microscope, showing dead/unhealthy nematodes. Scale bar: 200 μm.

Video S6. Aphelenchus avenae movement after incubation with 500 μM rhizoxin S2. Time-lapse
movie, recorded on a spinning disc microscope, showing dead/unhealthy nematodes. Scale bar: 200 μm.

Video S7. Aphelenchus avenae movement after incubation with 114 μM ivermectin (positive
control). Time-lapse movie, recorded on a spinning disc microscope, showing dead/unhealthy
nematodes. Scale bar: 200 μm.

Video S8. Segmented worms and their summarized tracks. The segmented worms are shown in white,
whereas the worm outlines at each time point are shown in yellow. The time series shows the individual
worms per time point, whereas the yellow outlines are superimposed over the entire time series and shown
at each time point of the video.

Video S9. The segmented worms and their tracks of a time series experiment. The worms and the
tracks are shown here as provided by the automated tracking algorithm applied to a time series experiment.
The worms are coloured randomly, whereas the tracks (thin lines) are coloured from blue to red for each
track, blue corresponding to time zero and red to the final time point. When worms merge, they become of
the same colour until they separate again.

Video S10. The X component of the per-worm and per time-point velocity vector of each worm as a
function of the Y component of the velocity vector. The time series shows the velocity vector
components at individual time points, playing from time zero to the final time point. Line colours correspond
to the time, whereas the worm colours indicate the area of the worm, see colour scale bars at the bottom of
the window.

Video S11. A segmented worm and its footprint for LR = 11.5. The orange objects shows the segmented
worm at each time point per movie frame, whereas the red area shows the worm's footprint calculated for
the entire time series.
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Video S12. A segmented worm and its footprint for LR = 4.0. The green object corresponds to the
segmented worm shown at each time point, the orange area indicates the footprint of this worm, calculated
for the entire time series.