Is this how complex life evolved? Experiment that put bacteria inside fungi offers clues

Scientists wielding a minute hollow needle — and a bike pump — have managed to implant bacteria into a larger cell, creating a relationship similar to those that sparked the evolution of complex life.
The feat — described1 in Nature on 2 October — could help researchers to understand the origins of pairings that gave rise to specialized organelles called mitochondria and chloroplasts more than one billion years ago.

Fungi borrowed bacterial gene again and again

Endosymbiotic relationships — in which a microbial partner lives harmoniously within the cells of another organism — are found in numerous life forms, including insects and fungi. Scientists think that mitochondria, the organelles that are responsible for cells’ energy production, evolved when a bacterium took up residence inside an ancestor of eukaryotic cells. Chloroplasts emerged when an ancestor of plants swallowed a photosynthetic microorganism.
Determining the factors that formed and sustained these couplings is difficult because they occurred so long ago. To get around this problem, a team led by microbiologist Julia Vorholt, at the Swiss Federal Institute of Technology in Zurich (ETH Zurich), has spent the past few years engineering endosymbioses in the laboratory. Their approach uses a 500–1,000-nanometre-wide needle to puncture host cells and then deliver bacterial cells one at a time.
Even with this technical wizardry, initial pairings tended to fail; for instance, because the would-be symbiont divided too fast and killed its host2. The team’s luck changed when the researchers recreated a natural symbiosis that occurs between some strains of a fungal plant pathogen, Rhizopus microsporus, and the bacterium Mycetohabitans rhizoxinica, which produces a toxin that protects the fungus from predation.
Yet delivering bacterial cells into the fungi, which have thick cell walls that maintain a high internal pressure, was a challenge. After piercing the wall with the needle, the researchers used a bicycle pump — and later an air compressor — to maintain enough pressure to deliver the bacteria.
After overcoming the initial shock of surgery, the fungi continued their life cycles and produced spores, a fraction of which contained bacteria. When these spores germinated, bacteria were also present in the cells of the next generation of fungi. This showed that the new endosymbiosis could be passed onto offspring — a key finding.
But the germination success of the bacteria-containing spores was low. In a mixed population of spores (some with bacteria and some without), those with bacteria vanished after two generations. To see whether relations could be improved, the researchers used a fluorescent cell sorter to select spores containing bacteria — which had been labelled with a glowing protein — and propagated only these spores in future rounds of reproduction. By ten generations, the bacteria-containing spores germinated nearly as efficiently as did those without bacteria.
The basis of this adaptation isn’t clear. Genome sequencing identified a handful of mutations associated with improved germination success in the fungus — which was a strain of R. microsporus not known to carry endosymbionts naturally — and found no changes in the bacteria.

The line that germinated most efficiently tended to limit the number of bacteria in each spore, says study co-author Gabriel Giger, a microbiologist at ETH Zurich. “There are ways for these two partners to make a better, easier living with each other. That’s something that’s really important for us to understand.”
Researchers don’t know much about the genetics of R. microsporus. But Thomas Richards, an evolutionary biologist at the University of Oxford, UK, wonders whether a fungal immune system is preventing symbiosis — and whether mutations to this system could be easing relations. “I’m a big fan of this work,” he adds.
Eva Nowack, a microbiologist at Heinrich Heine University Düsseldorf in Germany, was surprised at how quickly adaptations to symbiotic life seemed to evolve. In the future, she would love to see what happens after even longer time periods; for example, more than 1,000 generations.
Engineering such symbioses could lead to the development of novel organisms with useful traits, such as the ability to consume carbon dioxide or atmospheric nitrogen, says Vorholt. “That’s the idea: to bring in new traits that an organism doesn’t have, and that would be difficult to implement otherwise.”