A novel approach for multi-domain and multi-gene family identification provides insights into evolutionary dynamics of disease resistance genes in core eudicot plants

Background

Recent advances in DNA sequencing techniques resulted in more than forty sequenced plant genomes representing a diverse set of taxa of agricultural, energy, medicinal and ecological importance. However, gene family curation is often only inferred from DNA sequence homology and lacks insights into evolutionary processes contributing to gene family dynamics. In a comparative genomics framework, we integrated multiple lines of evidence provided by gene synteny, sequence homology and protein-based Hidden Markov Modelling to extract homologous super-clusters composed of multi-domain resistance (R)-proteins of the NB-LRR type (for NUCLEOTIDE BINDING/LEUCINE-RICH REPEATS), that are involved in plant innate immunity.

Results

To assess the diversity of R-proteins within and between species, we screened twelve eudicot plant genomes including six major crops and found a total of 2,363 NB-LRR genes. Our curated R-proteins set shows a 50% average for tandem duplicates and a 22% fraction of gene copies retained from ancient polyploidy events (ohnologs). We provide evidence for strong positive selection and show significant differences in molecular evolution rates (Ka/Ks-ratio) among tandem- (mean = 1.59), ohnolog (mean = 1.36) and singleton (mean = 1.22) R-gene duplicates. To foster the process of gene-edited plant breeding, we report species-specific presence/absence of all 140 NB-LRR genes present in the model plant Arabidopsis and describe four distinct clusters of NB-LRR “gatekeeper” loci sharing syntenic orthologs across all analyzed genomes.

Conclusion

By curating a near-complete set of multi-domain R-protein clusters in an eudicot-wide scale, our analysis offers significant insight into evolutionary dynamics underlying diversification of the plant innate immune system. Furthermore, our methods provide a blueprint for future efforts to identify and more rapidly clone functional NB-LRR genes from any plant species.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-966) contains supplementary material, which is available to authorized users.     

Structural plasticity of an invariant hydrophobic triad in the switch regions of Rab GTPases is a determinant of effector recognition

Rab GTPases function as regulatory components of an evolutionarily conserved machinery that mediates docking, priming, and fusion of vesicles with intracellular membranes. We have previously shown that the active conformation of Rab3A is stabilized by a substantial hydrophobic interface between the putative conformational switch regions (Dumas, J. J., Zhu, Z., Connolly, J. L., and Lambright, D. G. (1999) Structure 7, 413-423). A triad of invariant hydrophobic residues at this switch interface (Phe-59, Trp-76, and Tyr-91) represents a major interaction determinant between the switch regions of Rab3A and the Rab3A-specific effector Rabphilin3A (Ostermeier, C., and Brunger, A. T. (1999) Cell 96, 363-374). Here, we report the crystal structure of the active form of Rab5C, a prototypical endocytic Rab GTPase. As is true for Rab3A, the active conformation of Rab5C is stabilized by a hydrophobic interface between the switch regions. However, the conformation of the invariant hydrophobic triad (residues Phe-58, Trp-75, and Tyr-90 in Rab5C) is dramatically altered such that the resulting surface is noncomplementary to the switch interaction epitope of Rabphilin3A. This structural rearrangement reflects a set of nonconservative substitutions in the hydrophobic core between the central beta sheet and the alpha2 helix. These observations demonstrate that structural plasticity involving an invariant hydrophobic triad at the switch interface contributes to the mechanism by which effectors recognize distinct Rab subfamilies. Thus, the active conformation of the switch regions conveys information about the identity of a particular Rab GTPase as well as the state of the bound nucleotide.     

Sepsis progression and outcome: a dynamical model

Background  

Sepsis (bloodstream infection) is the leading cause of death in non-surgical intensive care units. It is diagnosed in 750,000 US patients per annum, and has high mortality. Current understanding of sepsis is predominately observational and correlational, with only a partial and incomplete understanding of the physiological dynamics underlying the syndrome. There exists a need for dynamical models of sepsis progression, based upon basic physiologic principles, which could eventually guide hourly treatment decisions.     

RUTBC2 protein, a Rab9A effector and GTPase-activating protein for Rab36

Rab GTPases regulate vesicle budding, motility, docking, and fusion. In cells, their cycling between active, GTP-bound states and inactive, GDP-bound states is regulated by the action of opposing enzymes called guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). The substrates for most RabGAPs are unknown, and the potential for cross-talk between different membrane trafficking pathways remains uncharted territory. Rab9A and its effectors regulate recycling of mannose 6-phosphate receptors from late endosomes to the trans Golgi network. We show here that RUTBC2 is a TBC domain-containing protein that binds to Rab9A specifically both in vitro and in cultured cells but is not a GAP for Rab9A. Biochemical screening of Rab protein substrates for RUTBC2 revealed highest GAP activity toward Rab34 and Rab36. In cells, membrane-associated RUTBC2 co-localizes with Rab36, and expression of wild type RUTBC2, but not the catalytically inactive, RUTBC2 R829A mutant, decreases the amount of membrane-associated Rab36 protein. These data show that RUTBC2 can act as a Rab36 GAP in cells and suggest that RUTBC2 links Rab9A function to Rab36 function in the endosomal system.     

Structural determinants of phosphoinositide selectivity in splice variants of Grp1 family PH domains

The pleckstrin homology (PH) domains of the homologous proteins Grp1 (general receptor for phosphoinositides), ARNO (Arf nucleotide binding site opener), and Cytohesin-1 bind phosphatidylinositol (PtdIns) 3,4,5-trisphosphate with unusually high selectivity. Remarkably, splice variants that differ only by the insertion of a single glycine residue in the beta1/beta2 loop exhibit dual specificity for PtdIns(3,4,5)P(3) and PtdIns(4,5)P(2). The structural basis for this dramatic specificity switch is not apparent from the known modes of phosphoinositide recognition. Here, we report crystal structures for dual specificity variants of the Grp1 and ARNO PH domains in either the unliganded form or in complex with the head groups of PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3). Loss of contacts with the beta1/beta2 loop with no significant change in head group orientation accounts for the significant decrease in PtdIns(3,4,5)P(3) affinity observed for the dual specificity variants. Conversely, a small increase rather than decrease in affinity for PtdIns(4,5)P(2) is explained by a novel binding mode, in which the glycine insertion alleviates unfavorable interactions with the beta1/beta2 loop. These observations are supported by a systematic mutational analysis of the determinants of phosphoinositide recognition.     

An endocytic pathway as a target of tubby for regulation of fat storage

The tubby loci provide a unique opportunity to study adult-onset obesity. Mutation in either mammalian tubby or its homologue in Caenorhabditis elegans, tub-1, results in increased fat storage. Previously, we have shown that TUB-1 interacts with a new Rab GTPase-activating protein, RBG-3, for the regulation of fat storage. To understand further the molecular mechanism of TUB-1, we identified the Rab GTPase downstream of RBG-3. We found that RBG-3 preferentially stimulates the intrinsic GTPase activity of RAB-7 in both human and C. elegans. Importantly, either mutation or RNA interference knockdown in rab-7 reduces stored fat in wild type and tub-1 mutants. In addition, the small GTPase rab-5 and genes that regulate Rab membrane localization and nucleotide recycling are required for the regulation of fat storage, thereby defining a role for endocytic recycling in this process. We propose that TUB-1 controls receptor or sensory molecule degradation in neurons by regulating a RAB-7-mediated endocytic pathway.     

A helical turn motif in Mss4 is a critical determinant of Rab binding and nucleotide release

Monomeric Rab GTPases function as ubiquitous regulators of intracellular membrane trafficking. Mss4, an evolutionarily conserved Rab accessory factor, promotes nucleotide release from exocytic but not endocytic Rab GTPases. Here we describe the results of a high-resolution crystallographic and mutational analysis of Mss4. The 1.65 A crystal structure of Mss4 reveals a network of direct and water-mediated interactions that stabilize a partially exposed structural subdomain derived from four highly conserved but nonconsecutive sequence elements. The conserved subdomain contains the invariant cysteine residues required for Zn2+ binding as well as the residues implicated in the interaction with Rab GTPases. A strictly conserved DPhiPhi motif, consisting of an invariant aspartic acid residue (Asp 73) followed by two bulky hydrophobic residues (Met 74 and Phe 75), encodes a prominently exposed 3(10) helical turn in which the backbone is well-ordered but the side chains of the conserved residues are highly exposed and do not engage in intramolecular interactions. Substitution of any of these residues with alanine dramatically impairs nucleotide release activity toward Rab3A, indicating that the DPhiPhi motif is a critical element of the Rab interaction epitope. In particular, mutation of Phe 75 results in a defect as severe as that observed for mutation of Asp 96, which is located near the zinc binding site at the opposite end of the conserved subdomain. Despite severe defects, however, none of the mutant proteins is catalytically dead. Taken together, the results suggest a concerted mechanism in which distal elements of the conserved Rab interaction epitope cooperatively facilitate nucleotide release.     

Lactose synthase: effect of alpha-lactalbumin on substrate activity of N-acylglucosamines

N-Acetyl-, N-propionyl-, N-butyryl- and N-valerylglucosamines were synthesized as topographical probes to localize further the interaction site of alpha-lactalbumin on galactosyltransferase. All these compounds were found to be substrates for galactosyltransferase with Km values in the millimolar range. In the presence of alpha-lactalbumin, the Michaelis-Menten constants were diminished. However, the effect on the initial rates of these reactions varied. Thus, at low N-acylglucosamine concentrations, alpha-lactalbumin activated the enzyme activity, but at high concentrations, alpha-lactalbumin became inhibitory. This mixed-type inhibition kinetics indicated that a quaternary complex between galactosyltransferase, alpha-lactalbumin, Mn2+-UDPgalactose and N-acylglucosamine existed during the catalytic process. The ability of these N-acylglucosamine substrates to bind to lactose synthase complex was further substantiated by the physical association of galactosyltransferase onto the solid-bound alpha-lactalbumin in the presence of any one of these compounds. The data revealed that the presence of the N-acyl group up to five carbons in length did not interfere with the interaction between alpha-lactalbumin and galactosyltransferase, suggesting that alpha-lactalbumin was not bound in the vicinity of the C-2 region of the monosaccharide site. The inhibitory effect of alpha-lactalbumin on N-acyllactosamine formation is probably a consequence of conformational changes of galactosyltransferase.