Emergent RNA-RNA interactions can promote stability in a nascent phototrophic endosymbiosis

Benjamin H. Jenkins, Finlay Maguire, Guy Leonard, Joshua D. Eaton, Steve West, Benjamin E. Housden, S. Milner David, Thomas A. Richards

Posted on: 17 May 2021

Preprint posted on 13 May 2021

Article now published in Proceedings of the National Academy of Sciences at http://dx.doi.org/10.1073/pnas.2108874118

Unexpected host-endosymbiont RNA-RNA interactions discourage Paramecium from digesting its symbiotic partners.

Selected by Olivia Tidswell

Categories: cell biology, ecology, evolutionary biology, microbiology, molecular biology

Background

Endosymbiosis has played a fundamental role in eukaryote evolution, giving rise to vitally important organelles such as mitochondria and chloroplasts. However, the interests of a host cell and its endosymbionts do not always align, and these relationships are frequently fraught with conflict. What is stopping a host cell from simply digesting its tenants for a quick meal in a pinch?

In this preprint, Jenkins and colleagues investigate this question in the context of a eukaryotic-eukaryotic endosymbiosis, specifically the nascent endosymbiosis between the ciliate protist Paramecium bursaria and the single-celled algae Chlorella. A single Paramecium may contain hundreds of Chlorella cells, housed inside modified phagosomes. Here, Chlorella can evade potential predators and utilise metabolic products from its host (such as amino acids and CO₂). In return, Chlorella provides its host with the products of photosynthesis. This symbiosis is still relatively new, and both partners are able to survive independently of each other.

There is no doubt that P. bursaria wields much of the power in this arrangement. At any time, it could unleash digestive lysosomes to break down its endosymbionts and recycle all of that valuable biomatter. Here, Jenkins and colleagues describe an emergent mechanism that punishes this kind of over-exploitation, and therefore promotes co-operation between host and endosymbiont.

Key findings

Induction of endosymbiont digestion reduces the growth of P. bursaria

One of the benefits of the P. bursaria–Chlorella symbiosis as a study system is that it can be readily manipulated using the protein synthesis inhibitor cycloheximide. This chemical conveniently switches off protein synthesis in Chlorella, but not Paramecium, cells, which induces P. bursaria to digest its endosymbionts (see Kodama and Fujishima, 2008 for more information). The authors show that extended cycloheximide exposure dramatically reduces the growth of symbiotic P. bursaria cultures, an effect that is not observed in the non-symbiotic species P. tetraurelia. These observations suggest that digestion of Chlorella (not exposure to cycloheximide) limits the growth of P. bursaria.

Figure 1C from the preprint. Cycloheximide exposure reduces culture growth of P. bursaria, but not of non-symbiotic P. tetraurelia.

**Reduced growth of P. bursaria after endosymbiont digestion is dependent on endogenous RNAi machinery**

One possible explanation for this reduction in growth is that destroying its endosymbionts renders P. bursaria unable to benefit from photosynthesis, effectively exchanging a valuable ongoing source of sustenance for a temporary feast. However, the authors show that this is unlikely to be the primary downside to Chlorella digestion. Reduced growth of P. bursaria after cycloheximide treatment can be almost completely rescued by knocking down RNA interference (RNAi) machinery, such as Dicer (Dcr1), PiwiA1 andPds1. This suggests that the reduction in growth rate observed after Chlorella digestion is dependent on the endogenous RNAi pathway of Paramecium.

Figure 1D from the preprint. Knockdown of Dicer1, a mediator of the RNAi pathway, can partially rescue growth inhibition after cycloheximide exposure compared to controls.

**Endosymbiont digestion results in Dicer-dependent accumulation of short, endosymbiont-derived RNAs**

The authors propose that digested Chlorella cells release RNAs that are processed into short RNAs (sRNAs) by the host’s Dicer pathway, and that these sRNAs are ultimately responsible for the effects on host fitness. In agreement with this hypothesis, they find that cycloheximide exposure increases the abundance of endosymbiont-derived sRNAs in P. bursaria cells. sRNA abundance is reduced if host Dicer is knocked down by RNAi. This suggests that host Dicer is able to process RNA released from digested Chlorella cells into sRNAs. Furthermore, the authors find that many of these endosymbiont-derived sRNAs have homology to host mRNA transcripts, and therefore could potentially interact with them.

Endosymbiont-derived sRNA sequences can be used by host RNAi machinery to downregulate the expression of homologous host genes

The authors investigated whether endosymbiont-derived sRNAs can interact with host mRNA using an elegant series of experiments. Firstly, they chose ten sRNAs with high homology to host mRNA transcripts, cloned them into a plasmid, and delivered the plasmid into P. bursaria cells by allowing them to feed on transformed E. coli. Exposure to this chimeric plasmid retarded the growth of P. bursaria cultures in a Dicer-dependent manner. Next, the authors narrowed down the cause of this retardation to three of the ten endosymbiont-derived sRNAs, with homology to host elongation factor 1α, heat shock protein 90 and tubulin-ß mRNAs. The expression of at least two of these genes was reduced in P. bursaria cells exposed to the chimeric plasmid, and could be rescued by knockdown of Dicer. Together, these experiments suggest that sRNAs derived from digested Chlorella cells can be utilized by the host’s endogenous RNAi pathway to knock down homologous host genes, and to reduce host fitness.

Figures 3B and E from the preprint. B| P. bursaria culture growth is inhibited after exposure to a vector encoding ten different endosymbiont-derived sRNAs (“chimera”). This effect can be partially rescued by simultaneous knockdown of Dicer1. E| Exposure of P. bursaria to the chimeric vector described in B results in the downregulation of homologous host transcripts.

Why is this work important, and what did I like about it?

This work characterizes a novel phenomenon that punishes host cells for overexploitation of their endosymbionts – namely, RNAi-mediated interactions between endosymbiont-derived short RNAs and host mRNAs, which the authors describe as “RNA collisions”. Importantly, RNA collisions do not rely on a long history of co-evolution between host and endosymbiont, and could act to enforce co-operation even in nascent endosymbioses. This phenomenon is therefore a promising explanation for how endosymbioses might be stabilised before more specific dependencies or manipulations evolve.

I chose to highlight this preprint not just because I think that the results are surprising and exciting, but also because I appreciated how clearly and openly the authors discussed the strengths and limitations of their experiments. They go to great lengths to test alternative explanations for their observations, and to justify and caveat their experimental approaches, making their eventual conclusions all the more convincing. I am excited to see how future work tests the broader relevance of RNA collisions in stabilizing emergent endosymbioses.

Additional references:

Kodama, Y. and Fujishima, M. (2008). Cycloheximide Induces Synchronous Swelling of Perialgal Vacuoles Enclosing Symbiotic Chlorella vulgaris and Digestion of the Algae in the Ciliate Paramecium bursaria. Protist 159, 483–494.

Tags: chlorella, conflict, endosymbiosis, paramecium, rnai

doi: https://doi.org/10.1242/prelights.28985

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Author's response

Benjamin H. Jenkins shared

Ben Jenkins has kindly provided the following answers to my questions about the preprint and its implications.

Paramecium regularly engulfs and digests other single-celled eukaryotes. What mechanisms exist to prevent RNAs from these organisms from being processed by Paramecium’s RNAi machinery, and how are these mechanisms circumvented in the case of endosymbiont digestion?

This is a great question. We haven’t yet encountered any mechanisms that would prevent this processing, and it seems likely that Paramecium will indiscriminately process RNA from any organism that enters the cell via phagocytosis (see Carradec et al., 2015). However, this raises a really interesting point. Does relatedness of the ingested microbe increase the potential harmful RNA-RNA interactions that could occur? Our eDicer analysis – comparing the number of putative 23-nt overlaps between the host, algal and bacterial food transcripts – would suggest that this is the case. If so, one could envisage there being a greater cost associated with eating a more closely related organism (i.e. another eukaryote), or in the extreme, the same species (i.e. cannibalism), due to the increased propensity for harmful ‘RNAi collisions’ arising from the higher level of shared sequence identity between transcripts, many of which represent conditionally essential genes. We are excited to explore this avenue of investigation further.

RNA collisions result from Paramecium’s ability to process exogenously provided ssRNAs into sRNAs through the RNAi pathway. How widespread is this ability, and do you have any thoughts on its purpose?

The RNAi factors involved in this response (Dicer, Argonaute, RdRP) are widespread in eukaryotes, and were likely present in the last eukaryotic common ancestor (LECA; see Shabalina and Koonin, 2008). Originally, these mechanisms are believed to have evolved to protect against exogenous sources of RNA such as viruses, transposons and transgenes. However, these RNAi pathways have since undergone significant diversification in function and form. We believe that this mechanism of ssRNA processing is widespread in ciliates, which represent a hugely diverse and ecologically important group in a range of microbial ecosystems. Across the broader diversity of eukaryotes, however, we cannot say. Nonetheless, by demonstrating this RNAi processing as a viable enforcement mechanism, we indicate that this could have been a factor in many of the secondary endosymbiotic events that have occurred across the eukaryotic tree of life.

Do you think that this mechanism of enforcement might also be relevant (to a lesser extent) in the case of eukaryote-prokaryote endosymbioses?

Another great question, and this follows on very closely from the first. Our eDicer analysis suggests that the potential for harmful ‘RNAi collisions’ arising from digestion of a bacterial endosymbiont would be less so than a eukaryotic endosymbiont – yet the possibility remains. Even a very low cost associated with harmful RNA-RNA interactions could act to stabilise an emergent endosymbiotic interaction over evolutionary time. However, this may indicate that in a eukaryote-prokaryote endosymbiosis, the host is capable of exploiting the interaction to a greater extent if the repercussions for doing so (i.e. the number of harmful ‘RNAi collisions’) are less severe than in a eukaryote-eukaryote endosymbiosis. As above, this is something we are keen to explore further.

References:

Carradec Q, Götz U, Arnaiz O, Pouch J, Simon M, Meyer E, et al. Primary and secondary siRNA synthesis triggered by RNAs from food bacteria in the ciliate Paramecium tetraurelia. Nucleic Acids Res. 2015 Feb;43(3):1818–33.
Shabalina SA, Koonin EV. Origins and evolution of eukaryotic RNA interference. Trends Ecol Evol (Amst). 2008 Oct;23(10):578–87.

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Emergent RNA-RNA interactions can promote stability in a nascent phototrophic endosymbiosis

Background

Key findings

Induction of endosymbiont digestion reduces the growth of P. bursaria

Reduced growth of P. bursaria after endosymbiont digestion is dependent on endogenous RNAi machinery

Endosymbiont digestion results in Dicer-dependent accumulation of short, endosymbiont-derived RNAs

Endosymbiont-derived sRNA sequences can be used by host RNAi machinery to downregulate the expression of homologous host genes

Why is this work important, and what did I like about it?

Additional references:

Share this:

Have your say Cancel reply

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**Reduced growth of P. bursaria after endosymbiont digestion is dependent on endogenous RNAi machinery**

**Endosymbiont digestion results in Dicer-dependent accumulation of short, endosymbiont-derived RNAs**