Crane fly semiochemical overrules coordinate cyanobiont differentiation in Azolla ferns
Semiochemicals from insects that target plant-microbe interactions to restrict symbiont dinitrogen fixation had not been known. Here we report on an intertrophic signal mediated by the glycosylated triketide d-lactone from Nephrotoma cornicina crane flies, cornicinine, that causes chlorosis in the floating-fern symbioses from the genus Azolla. Cornicinine was chemically synthesized, as well as its aglycone and diastereoisomer. Only the glycosylated trans-A form was active: 500 nM cornicinine in the growth medium turned the dinitrogen- fixing cyanobacterial filaments from Nostoc azollae inside the host leaf pockets into akinete- like cells. Chlorosis was not alleviated with nitrate supplementation. Cornicinine inhibited akinete germination when Azolla sporelings re-establish the symbiosis. It did not affect the plant Arabidopsis thaliana or several free-living cyanobacteria from the genera Anabaena or Nostoc. Cornicinine, therefore, interfered with Azolla symbiosis crosstalk. Sequence profiling of the polyA-enriched RNA from isolated leaf pockets confirmed high NH 4 -assimilation and proanthocyanidin biosynthesis in this trichome-rich tissue. Leaf pocket transcripts accumulating in ferns grown on cornicinine reflected activation of Cullin-RING ubiquitin- ligase pathways, known to mediate metabolite signaling. Transcripts accumulating when akinetes are formed, in leaf pockets of ferns on cornicinine and in megasporocarps, affected jasmonic acid metabolism consistent with elicitation of the jasmonic-acid immune pathway, sulfate transport and exosome formation.