A red algal polysaccharide influences the multicellular development of the choanoflagellate Salpingoeca rosetta

Abstract

We uncovered an interaction between a choanoflagellate and alga, in which porphyran, a polysaccharide produced by the red alga Porphyra umbilicalis, induces multicellular development in the choanoflagellate Salpingoeca rosetta. We first noticed this possible interaction when we tested the growth of S. rosetta in media that was steeped with P. umbilicalis as a nutritional source. Under those conditions, S. rosetta formed multicellular rosette colonies even in the absence of any bacterial species that can induce rosette development. In biochemical purifications, we identified porphyran, a extracellular polysaccharide produced by red algae, as the rosette inducing factor The response of S. rosetta to porphyran provides a biochemical insight for associations between choanoflagellates and algae that have been observed since the earliest descriptions of choanoflagellates. Moreover, this work provides complementary evidence to ecological and geochemical studies that show the profound impact algae have exerted on eukaryotes and their evolution, including a rise in algal productivity that coincided with the origin of animals, the closest living relatives of choanoflagellates.

Figure 1:. Growth media prepared from Porphyra umbilicalis induces multicellular development of the choanoflagellate Salpingoeca rosetta

(A) S. rosetta senses environmental cues to develop into multicellular rosettes. Upon sensing lipids from the bacterium Algoriphagus machipongonensis, S. rosetta develops into rosettes through serial cell divisions while secreting an adhesive protein, Rosetteless (green), at the basal end of cells. Here we show that the red macroalga P. umbilicalis also induces rosette development. (B) A medium prepared from the red alga P. umbilicalis induces rosette development. We optimized preparation of media enriched with P. umbilicalis at low (25°C) and high (80°C) temperatures (Fig. S1). We compared rosette induction with (+) or without (–) bacterial lipids in media prepared from P. umbilicalis, the green alga Ulva spp., and Cereal Grass. Rosette induction was quantified by counting the number of single swimmers and cells in rosettes for a total of 500 cells. Independent triplicates of those counts (black dots) are shown with their mean and standard deviation (lines). Only media prepared from P. umbilicalis at 80°C induced rosette development in the absence of bacterial lipids delivered in outer membrane vesicles from A. machipongonensis. (C) S. rosetta secretes Rosetteless into the interior of multicellular rosettes induced with bacterial lipids or media enriched with P. umbilicalis. Immunofluorescent microscopy of rosettes induced with bacterial lipids or media enriched with P. umbilicalis visualizes the multicellular architecture of rosettes through an anti-alpha tubulin antibody (DM1A), which highlights, microtubules (grey), and phalloidin, which highlights filamentous actin (magenta). An antibody raised against Rosetteless (refs levin and rutaganira) shows the localization of this protein (green) in the interior of rosettes where the basal ends of cells meet. Scale bars denote 10 µm.

Figure 2:. Porphyran, a sulfated galactan from P. umbilicalis, induces rosette development.

(A and B) The purification of rosette inducing activity from P. umbilicalis extracts. (A) The purification scheme for rosette inducing activity started with the preparation of a soluble extract from P. umbilicalis. A hydrophobic resin (C₁₈-silica) retained hydrophobic molecules while hydrophilic molecules passed through the resin in the ‘flow through,’ to which we added enzymes that degrade nucleic acids, agar, and proteins to produce the ‘digested extract.’ In this sample, we measured rosette inducing activity (as described in Fig. 1B) to confirm that the ‘digest extract’ retained rosette inducing activity (B). Likewise, rosette inducing activity remained after dialyzing the ‘digested extract’ in a membrane with a 40 kD molecular weight cutoff. To this dialyzed sample, the addition of ethanol formed precipitates that were pelleted by centrifugation. The dissolved pellet retained rosette inducing activity. (C) The chemical structure of porphyran. Porphyran consists of linked galactose molecules with either a sulfonate or methyl functional group on the sixth carbon of alternating monosaccharides. (D) Porphyran induces multicellular rosette development. Porphyran purchased from a commercial source induces rosettes to the same extent as the purified rosette inducing activity from P. umbilicalis (labeled purification) and bacterial lipids from A. machipongonensis. Rosette induction was quantified as described in Figure 1B. (E) Rosette induction is directly proportional to the concentration of porphyran. Across a serial dilution, the effective concentration of porphyran that induces 50% of cells to form rosettes (EC50) is 210±50 µg/ml, an average calculated from fitting the hill equation (line) to independent triplicates of the dilution series (dots).