A Selective Bottleneck During Host Entry Drives the Evolution of New Legume Symbionts.

During the emergence of new host-microbe symbioses, microbial fitness results from the ability to complete the different steps of symbiotic life cycles, where each step imposes specific selective pressures. However, the relative contribution of these different selective pressures to the adaptive trajectories of microbial symbionts is still poorly known. Here, we characterized the dynamics of phenotypic adaptation to a simplified symbiotic life cycle during the experimental evolution of a plant pathogenic bacterium into a legume symbiont. We observed that fast adaptation was predominantly explained by improved competitiveness for host entry, which outweighed adaptation to within-host proliferation. Whole-population sequencing of bacteria at regular time intervals along this evolution experiment revealed the continuous accumulation of new mutations (fuelled by a transient hypermutagenesis phase occurring at each cycle before host entry, a phenomenon described in previous work) and sequential sweeps of cohorts of mutations with similar temporal trajectories. The identification of adaptive mutations within the fixed mutational cohorts showed that several adaptive mutations can co-occur in the same cohort. Moreover, all adaptive mutations improved competitiveness for host entry, while only a subset of those also improved within-host proliferation. Computer simulations predict that this effect emerges from the presence of a strong selective bottleneck at host entry occurring before within-host proliferation and just after the hypermutagenesis phase in the rhizosphere. Together, these results show how selective bottlenecks can alter the relative influence of selective pressures acting during bacterial adaptation to multistep infection processes.

The G-patch activators Pfa1 and PINX1 exhibit different modes of interaction with the Prp43 RNA helicase.

Prp43 is a DEAH-box RNA helicase involved in both splicing and ribosome biogenesis. Its activities are directly stimulated by several co-activators that share a G-patch domain. The substrates of Prp43, its mechanism of action and the modes of interaction with and activation by G-patch proteins have been only partially characterized. We investigated how Pfa1 and PINX1, two G-patch proteins involved in ribosome biogenesis, interact with Prp43. We demonstrate that a protruding loop connecting the ß4 and ß5 strands of Prp43 OB fold is crucial for the binding of the G-patch domain of Pfa1. However, neither this loop nor the entire OB fold of Prp43 is essential for PINX1 binding. We conclude that the binding modes of Pfa1 and PINX1 G-patches to Prp43 are different. Nevertheless, stimulation of the ATPase and helicase activities of Prp43 by both full-length Pfa1 and PINX1 requires the ß4-ß5 loop. Moreover, we show that disruption of this loop completely abrogates Prp43 activity during yeast ribosome biogenesis but does not prevent its integration within pre-ribosomal particles. We propose that the ß4-ß5 loop plays a crucial role in the transmission of conformational changes induced by binding of the G-patch to Prp43 active site and substrate RNA.

The telomerase inhibitor Gno1p/PINX1 activates the helicase Prp43p during ribosome biogenesis.

We provide evidence that a central player in ribosome synthesis, the ribonucleic acid helicase Prp43p, can be activated by yeast Gno1p and its human ortholog, the telomerase inhibitor PINX1. Gno1p and PINX1 expressed in yeast interact with Prp43p and the integrity of their G-patch domain is required for this interaction. Moreover, PINX1 interacts with human PRP43 (DHX15) in HeLa cells. PINX1 directly binds to yeast Prp43p and stimulates its adenosine triphosphatase activity, while alterations of the G patch abolish formation of the PINX1/Prp43p complex and the stimulation of Prp43p. In yeast, lack of Gno1p leads to a decrease in the levels of pre-40S and intermediate pre-60S pre-ribosomal particles, defects that can be corrected by PINX1 expression. We show that Gno1p associates with 90S and early pre-60S pre-ribosomal particles and is released from intermediate pre-60S particles. G-patch alterations in Gno1p or PINX1 that inhibit their interactions with Prp43p completely abolish their function in yeast ribosome biogenesis. Altogether, our results suggest that activation of Prp43p by Gno1p/PINX1 within early pre-ribosomal particles is crucial for their subsequent maturation.

Prp43p contains a processive helicase structural architecture with a specific regulatory domain.

The DEAH/RNA helicase A (RHA) helicase family comprises proteins involved in splicing, ribosome biogenesis and transcription regulation. We report the structure of yeast Prp43p, a DEAH/RHA helicase remarkable in that it functions in both splicing and ribosome biogenesis. Prp43p displays a novel structural architecture with an unforeseen homology with the Ski2-like Hel308 DNA helicase. Together with the presence of a beta-hairpin in the second RecA-like domain, Prp43p contains all the structural elements of a processive helicase. Moreover, our structure reveals that the C-terminal domain contains an oligonucleotide/oligosaccharide-binding (OB)-fold placed at the entrance of the putative nucleic acid cavity. Deletion or mutations of this domain decrease the affinity of Prp43p for RNA and severely reduce Prp43p ATPase activity in the presence of RNA. We also show that this domain constitutes the binding site for the G-patch-containing domain of Pfa1p. We propose that the C-terminal domain, specific to DEAH/RHA helicases, is a central player in the regulation of helicase activity by binding both RNA and G-patch domain proteins.

The ex planta signal activity of a Medicago ribosomal uL2 protein suggests a moonlighting role in controlling secondary rhizobial infection.

We recently described a regulatory loop, which we termed autoregulation of infection (AOI), by which Sinorhizobium meliloti, a Medicago endosymbiont, downregulates the root susceptibility to secondary infection events via ethylene. AOI is initially triggered by so-far unidentified Medicago nodule signals named signal 1 and signal 1' whose transduction in bacteroids requires the S. meliloti outer-membrane-associated NsrA receptor protein and the cognate inner-membrane-associated adenylate cyclases, CyaK and CyaD1/D2, respectively. Here, we report on advances in signal 1 identification. Signal 1 activity is widespread as we robustly detected it in Medicago nodule extracts as well as in yeast and bacteria cell extracts. Biochemical analyses indicated a peptidic nature for signal 1 and, together with proteomic analyses, a universally conserved Medicago ribosomal protein of the uL2 family was identified as a candidate signal 1. Specifically, MtRPuL2A (MtrunA17Chr7g0247311) displays a strong signal activity that requires S. meliloti NsrA and CyaK, as endogenous signal 1. We have shown that MtRPuL2A is active in signaling only in a non-ribosomal form. A Medicago truncatula mutant in the major symbiotic transcriptional regulator MtNF-YA1 lacked most signal 1 activity, suggesting that signal 1 is under developmental control. Altogether, our results point to the MtRPuL2A ribosomal protein as the candidate for signal 1. Based on the Mtnf-ya1 mutant, we suggest a link between root infectiveness and nodule development. We discuss our findings in the context of ribosomal protein moonlighting.