Genetic encoding of 3-nitro-tyrosine reveals the impacts of 14-3-3 nitration on client binding and dephosphorylation.

14-3-3 proteins are central hub regulators of hundreds of phosphorylated "client" proteins. They are subject to over 60 post-translational modifications (PTMs), yet little is known how these PTMs alter 14-3-3 function and its ability to regulate downstream signaling pathways. An often neglected, but well-documented 14-3-3 PTM found under physiological and immune-stimulatory conditions is the conversion of tyrosine to 3-nitro-tyrosine at several Tyr sites, two of which are located at sites considered important for 14-3-3 function: Y130 (ß-isoform numbering) is located in the primary phospho-client peptide-binding groove, while Y213 is found on a secondary binding site that engages with clients for full 14-3-3/client complex formation and client regulation. By genetically encoding 3-nitro-tyrosine, we sought to understand if nitration at Y130 and Y213 effectively modulated 14-3-3 structure, function, and client complexation. The 1.5 Å resolution crystal structure of 14-3-3 nitrated at Y130 showed the nitro group altered the conformation of key residues in the primary binding site, while functional studies confirmed client proteins failed to bind this variant of 14-3-3. But, in contrast to other client-binding deficient variants, it did not localize to the nucleus. The 1.9 Å resolution structure of 14-3-3 nitrated at Y213 revealed unusual flexibility of its C-terminal α-helix resulting in domain swapping, suggesting additional structural plasticity though its relevance is not clear as this nitrated form retained its ability to bind clients. Collectively, our data suggest that nitration of 14-3-3 will alter downstream signaling systems, and if uncontrolled could result in global dysregulation of the 14-3-3 interactome.

Negotiating mutualism: A locus for exploitation by rhizobia has a broad effect size distribution and context-dependent effects on legume hosts.

In mutualisms, variation at genes determining partner fitness provides the raw material upon which coevolutionary selection acts, setting the dynamics and pace of coevolution. However, we know little about variation in the effects of genes that underlie symbiotic fitness in natural mutualist populations. In some species of legumes that form root nodule symbioses with nitrogen-fixing rhizobial bacteria, hosts secrete nodule-specific cysteine-rich (NCR) peptides that cause rhizobia to differentiate in the nodule environment. However, rhizobia can cleave NCR peptides through the expression of genes like the plasmid-borne Host range restriction peptidase (hrrP), whose product degrades specific NCR peptides. Although hrrP activity can confer host exploitation by depressing host fitness and enhancing symbiont fitness, the effects of hrrP on symbiosis phenotypes depend strongly on the genotypes of the interacting partners. However, the effects of hrrP have yet to be characterised in a natural population context, so its contribution to variation in wild mutualist populations is unknown. To understand the distribution of effects of hrrP in wild rhizobia, we measured mutualism phenotypes conferred by hrrP in 12 wild Ensifer medicae strains. To evaluate context dependency of hrrP effects, we compared hrrP effects across two Medicago polymorpha host genotypes and across two experimental years for five E. medicae strains. We show for the first time in a natural population context that hrrP has a wide distribution of effect sizes for many mutualism traits, ranging from strongly positive to strongly negative. Furthermore, we show that hrrP effect size varies across host genotypes and experiment years, suggesting that researchers should be cautious about extrapolating the role of genes in natural populations from controlled laboratory studies of single genetic variants.

Decreased coevolutionary potential and increased symbiont fecundity during the biological invasion of a legume-rhizobium mutualism.

Although most invasive species engage in mutualism, we know little about how mutualism evolves as partners colonize novel environments. Selection on cooperation and standing genetic variation for mutualism traits may differ between a mutualism's invaded and native ranges, which could alter cooperation and coevolutionary dynamics. To test for such differences, we compare mutualism traits between invaded- and native-range host-symbiont genotype combinations of the weedy legume, Medicago polymorpha, and its nitrogen-fixing rhizobium symbiont, Ensifer medicae, which have coinvaded North America. We find that mutualism benefits for plants are indistinguishable between invaded- and native-range symbioses. However, rhizobia gain greater fitness from invaded-range mutualisms than from native-range mutualisms, and this enhancement of symbiont fecundity could increase the mutualism's spread by increasing symbiont availability during plant colonization. Furthermore, mutualism traits in invaded-range symbioses show lower genetic variance and a simpler partitioning of genetic variance between host and symbiont sources, compared to native-range symbioses. This suggests that biological invasion has reduced mutualists' potential to respond to coevolutionary selection. Additionally, rhizobia bearing a locus (hrrP) that can enhance symbiotic fitness have more exploitative phenotypes in invaded-range than in native-range symbioses. These findings highlight the impacts of biological invasion on the evolution of mutualistic interactions.