Biology is full of stories that read like a modern day zombie apocalypse. For instance, the parasite Toxoplasma gondii has been in the news for its ability to infect the brains of rats, and reprogram their normal behavioral responses such that they lose their innate fear of cats. Previously, we reviewed the research about the parasitic fungus that infects ants, causing drastic changes in typical ant behavior to aid in distribution of the fungal spores.
In April of this year, MacLean and colleagues published research in PLOS Biology describing interactions between a phytoplasma parasite and Arabidopsis thaliana. What is nice about this particular “zombie” biology story is that the researchers present the beginnings of the genetics that underlie the plant-parasite-insect relationship, moving beyond a description of the phenotypic changes that occur to describing an actual mechanism for those changes.
Phytoplasmas are bacterial plant parasites with life cycles that require two distinct hosts, the plant and insects that feed on the vegetative structures of the plants. Phytoplasmas can effect dramatic developmental changes on plants that include converting flowers to leaf-like structures or causing a proliferation of stems known as a witches’ broom. In the study authored by Maclean, the researchers looked at the interactions among “Aster Yellows phytoplasma strain Witches’ Broom” (AY-WB), its insect vector (leafhoppers) and the wild brassica plant, Arabidopsis thaliana.
In Arabidopsis plants infected with AY-WB, flowers are transformed into leaf-like structures. The plant loses its ability to reproduce as a result, and exists solely to provide food for its phytoplasma parasite—essential becoming a “zombie” plant.
The patterning and development of the floral meristem involves an intricate sequence of gene regulation turning “on” or “off” the correct genes at the precise time in the precise place to produce the concentric whorls of flower structure: sepals, petals, stamens and carpels. In previous work, this team had identified a protein from AY-WB, SAP54, which is required for this transformation and showed that Arabidopsis plants transformed with this protein develop the same leaf-like structures seen in the infected plants.
In this paper, the researchers began by asking what Arabidopsis proteins interact with SAP54. A yeast two-hybrid screen identified Type II MADS-domain containing transcription factors (MTFs). Members of this class of proteins include AGAMOUS-LIKE 12, MADS AFFECTING FLOWERING1 and several other transcription factors that specifically regulate genes that are responsible for the conversion of vegetative to flowering meristem and the production of the flower structure. Further investigation revealed that the interaction involved the keratin-like domain of these proteins, which is unique to flowering plants and not found in the MTFs of other organisms such as the leaf hopper.
They next looked at the interactions between SAP54 and Arabidopsis proteins in infected plants using GFP tagged SAP54 transgenic plants. Immunoprecipitation and mass spectrometry analysis revealed that many of the MTFs identified in the yeast two-hybrid screen do indeed interact with SAP54 in plants.
The researchers hypothesized that SAP54 may be interfering with MTF function. Analysis of Western blots from healthy and infected plants expressing tagged MTFs indicated that MTF proteins are less abundant in the leaf-like structures of the infected plants. These and other experiments led the team to propose that SAP54 is affecting the stability of the MTF proteins either directly or by upregulating their degradation using a pathway such as the ubiquitin system. Because the treatment of samples with epoxomicin, an inhibitor of the ubiquitin degradation pathway, allowed the Type II MTFs to accumulate, the researchers concluded that SAP54 was promoting the destabilization and degradation of the Type II MTFs using the hosts normal ubiquitin tagging and protein degradation pathways.
The two-hybrid screen used to identify the interaction of Type II MTFs with SAP54, also revealed interaction between SAP54 and RAD23C and RAD23D. These proteins are believed to be proteins responsible for shuttling ubiquitin-tagged proteins to the proteasome for degradation. Thus SAP54 appears to create a “short-circuit” between two cellular pathways, flower development and protein degradation, by directly interacting with key proteins in both pathways.
But that’s not all.
The production of leaf-like structures instead of flowers has the advantage of making the plants more “attractive” to the leaf hopper insect vectors. These insects feed on the vegetative tissues of the plants, and the phytoplasma are spread from plant to plant when a leaf hopper feeds on an infected plant and then moves to another plant. The researchers performed leaf hopper “choice assays” and found that the insects were more likely to colonize both the infected plants and transgenic plants expressing SAP54, than wildtype plants or plants that produced green flowers as a result of an unrelated mutation. Furthermore this preference appears to be RAD23 dependent, although the exact nature of the RAD23 role is not understood.
This research showed how a small, bacterial parasite uses one protein to change the developmental program of a plant, converting the plant into a vegetative food-producing system on which that parasite’s vectors are more likely to lay eggs and feed. Amazing. Zombie Plant Genetics—Just another reason I love science.
MacLean, A.M. et al. (2014) Phytoplasma effector SAP54 hijacks plant reproduction by degrading MADS-box proteins and promotes insect colonization in RAD23-dependent manner. PLOS Biology 12, e1001835.
John Innes Centre News. How plants become zombies. [Internet: accessed 4/28/2014 http://news.jic.ac.uk/2014/04/how-plants-become-zombies/]
Irish, V.F. (2010) The flowering of Arabidopsis flower development. The Plant Journal 61, 1014.