UV-B-induced modulation of constitutive heterochromatin content in Arabidopsis thaliana.

Sunlight regulates transcriptional programs and triggers the shaping of the genome throughout plant development. Among the different sunlight wavelengths that reach the surface of the Earth, UV-B (280-315 nm) controls the expression of hundreds of genes for the photomorphogenic responses and also induces the formation of photodamage that interfere with genome integrity and transcriptional programs. The combination of cytogenetics and deep-learning-based analyses allowed determining the location of UV-B-induced photoproducts and quantifying the effects of UV-B irradiation on constitutive heterochromatin content in different Arabidopsis natural variants acclimated to various UV-B regimes. We identified that UV-B-induced photolesions are enriched within chromocenters. Furthermore, we uncovered that UV-B irradiation promotes constitutive heterochromatin dynamics that differs among the Arabidopsis ecotypes having divergent heterochromatin contents. Finally, we identified that the proper restoration of the chromocenter shape, upon DNA repair, relies on the UV-B photoreceptor, UV RESISTANCE LOCUS 8 (UVR8). These findings shed the light on the effect of UV-B exposure and perception in the modulation of constitutive heterochromatin content in Arabidopsis thaliana.

Structural and Functional Implication of Natural Variants of Gαs.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) are among the most important cellular signaling components, especially G protein-coupled receptors (GPCRs). G proteins comprise three subunits, Gα, Gß, and Gγ. Gα is the key subunit, and its structural state regulates the active status of G proteins. Interaction of guanosine diphosphate (GDP) or guanosine triphosphate (GTP) with Gα switches G protein into basal or active states, respectively. Genetic alteration in Gα could be responsible for the development of various diseases due to its critical role in cell signaling. Specifically, loss-of-function mutations of Gαs are associated with parathyroid hormone-resistant syndrome such as inactivating parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) signaling disorders (iPPSDs), whereas gain-of-function mutations of Gαs are associated with McCune-Albright syndrome and tumor development. In the present study, we analyzed the structural and functional implications of natural variants of the Gαs subtype observed in iPPSDs. Although a few tested natural variants did not alter the structure and function of Gαs, others induced drastic conformational changes in Gαs, resulting in improper folding and aggregation of the proteins. Other natural variants induced only mild conformational changes but altered the GDP/GTP exchange kinetics. Therefore, the results shed light on the relationship between natural variants of Gα and iPPSDs.

Diversity of root hydrotropism among natural variants of Arabidopsis thaliana.

Root gravitropism affects root hydrotropism. The interference intensity of root gravitropism with root hydrotropism differs among plant species. However, these differences have not been well compared within a single plant species. In this study, we compared root hydrotropism in various natural variants of Arabidopsis under stationary conditions. As a result, we detected a range of root hydrotropism under stationary conditions among natural Arabidopsis variants. Comparison of root gravitropism and root hydrotropism among several Arabidopsis natural variants classified natural variants that decreased root hydrotropism into two types; namely one type that expresses root gravitropism and root hydrotropism weaker than Col-0, and the other type that expresses weaker root hydrotropism than Col-0 but expresses similar root gravitropism with Col-0. However, root hydrotropism of all examined Arabidopsis natural variants was facilitated by clinorotation. These results suggested that the interference of root gravitropism with root hydrotropism is conserved among Arabidopsis natural variants, although the intensity of root gravitropism interference with root hydrotropism differs.

The mutational landscape of human olfactory G protein-coupled receptors.

BACKGROUND: Olfactory receptors (ORs) constitute a large family of sensory proteins that enable us to recognize a wide range of chemical volatiles in the environment. By contrast to the extensive information about human olfactory thresholds for thousands of odorants, studies of the genetic influence on olfaction are limited to a few examples. To annotate on a broad scale the impact of mutations at the structural level, here we analyzed a compendium of 119,069 natural variants in human ORs collected from the public domain. RESULTS: OR mutations were categorized depending on their genomic and protein contexts, as well as their frequency of occurrence in several human populations. Functional interpretation of the natural changes was estimated from the increasing knowledge of the structure and function of the G protein-coupled receptor (GPCR) family, to which ORs belong. Our analysis reveals an extraordinary diversity of natural variations in the olfactory gene repertoire between individuals and populations, with a significant number of changes occurring at the structurally conserved regions. A particular attention is paid to mutations in positions linked to the conserved GPCR activation mechanism that could imply phenotypic variation in the olfactory perception. An interactive web application (hORMdb, Human Olfactory Receptor Mutation Database) was developed for the management and visualization of this mutational dataset. CONCLUSION: We performed topological annotations and population analysis of natural variants of human olfactory receptors and provide an interactive application to explore human OR mutation data. We envisage that the utility of this information will increase as the amount of available pharmacological data for these receptors grow. This effort, together with ongoing research in the study of genetic changes in other sensory receptors could shape an emerging sensegenomics field of knowledge, which should be considered by food and cosmetic consumer product manufacturers for the benefit of the general population.

Changes of Gut Microbiota by Natural mtDNA Variant Differences Augment Susceptibility to Metabolic Disease and Ageing.

We recently reported on two mouse strains carrying different single nucleotide variations in the mitochondrial complex I gene, i.e., B6-mtBPL mice carrying m.11902T>C and B6-mtALR carrying m.4738C>A. B6-mtBPL mice exhibited a longer lifespan and a lower metabolic disease susceptibility despite mild mitochondrial functional differences in steady-state. As natural polymorphisms in the mitochondrial DNA (mtDNA) are known to be associated with distinct patterns of gut microbial composition, we further investigated the gut microbiota composition in these mice strains. In line with mouse phenotypes, we found a significantly lower abundance of Proteobacteria, which is positively associated with pathological conditions, in B6-mtBPL compared to B6-mtALR mice. A prediction of functional profile of significantly differential bacterial genera between these strains revealed an involvement of glucose metabolism pathways. Whole transcriptome analysis of liver samples from B6-mtBPL and B6-mtALR mice confirmed these findings. Thus, both host gene expression and gut microbial changes caused by the mtDNA variant differences may contribute to the ageing and metabolic phenotypes observed in these mice strains. Since gut microbiota are easier to modulate, compared with mtDNA variants, identification of such mtDNA variants, specific gut bacterial species and bacterial metabolites may be a potential intervention to modulate common diseases, which are differentially susceptible to individuals with different mtDNA variants.

Learning about quantitative genetics from Marla Sokolowski.

Marla Sokolowski is a true pioneer in behavioral genetics, having made the first molecular delineation of a naturally occurring behavioral polymorphism in her work on the foraging locus in Drosophila melanogaster. The gene was subsequently found to be responsible for behavioral variants and types in many other species, both invertebrate and mammal (human). The path to get there is a paradigmatic example of how to use the power of genetic analysis, including some rather esoteric techniques, to zero in on a gene and delineate its molecular identity and its pleiotropic roles.

Cellular expression and function of naturally occurring variants of the human ABCG2 multidrug transporter.

The human ABCG2 multidrug transporter plays a crucial role in the absorption and excretion of xeno- and endobiotics; thus the relatively frequent polymorphic and mutant ABCG2 variants in the population may significantly alter disease conditions and pharmacological effects. Low-level or non-functional ABCG2 expression may increase individual drug toxicity, reduce cancer drug resistance, and result in hyperuricemia and gout. In the present work we have studied the cellular expression, trafficking, and function of nine naturally occurring polymorphic and mutant variants of ABCG2. A comprehensive analysis of the membrane localization, transport, and ATPase activity, as well as retention and degradation in intracellular compartments was performed. Among the examined variants, R147W and R383C showed expression and/or protein folding defects, indicating that they could indeed contribute to ABCG2 functional deficiency. These studies and the applied methods should significantly promote the exploration of the medical effects of these personal variants, promote potential therapies, and help to elucidate the specific role of the affected regions in the folding and function of the ABCG2 protein.

Molecular and Functional Evolution at the Odorant Receptor Or22 Locus in Drosophila melanogaster.

Insect odorant receptor (Or) genes determine the responses of sensory neurons that mediate critical behaviors. The Drosophila melanogaster Or22 locus represents an interesting example of molecular evolution, with high levels of sequence divergence and copy number variation between D. melanogaster and other Drosophila species, and a corresponding high level of variability in the responses of the neuron it controls, ab3A. However, the link between Or22 molecular and functional diversity has not been established. Here, we show that several naturally occurring Or22 variants generate major shifts in neuronal response properties. We determine the molecular changes that underpin these response shifts, one of which represents a chimeric gene variant previously suggested to be under natural selection. In addition, we show that several alternative molecular genetic mechanisms have evolved for ensuring that where there is more than one gene copy at this locus, only one functional receptor is generated. Our data thus provide a causal link between the striking levels of phenotypic neuronal response variation found in natural populations of D. melanogaster and genetic variation at the Or22 locus. Since neuronal responses govern animal behavior, we predict that Or22 may be a key player in underlying one or more olfactory-driven behaviors of significant adaptive importance.

Integrating mRNA and Protein Sequencing Enables the Detection and Quantitative Profiling of Natural Protein Sequence Variants of Populus trichocarpa.

Next-generation sequencing has transformed the ability to link genotypes to phenotypes and facilitates the dissection of genetic contribution to complex traits. However, it is challenging to link genetic variants with the perturbed functional effects on proteins encoded by such genes. Here we show how RNA sequencing can be exploited to construct genotype-specific protein sequence databases to assess natural variation in proteins, providing information about the molecular toolbox driving cellular processes. For this study, we used two natural genotypes selected from a recent genome-wide association study of Populus trichocarpa, an obligate outcrosser with tremendous phenotypic variation across the natural population. This strategy allowed us to comprehensively catalogue proteins containing single amino acid polymorphisms (SAAPs), as well as insertions and deletions. We profiled the frequency of 128 types of naturally occurring amino acid substitutions, including both expected (neutral) and unexpected (non-neutral) SAAPs, with a subset occurring in regions of the genome having strong polymorphism patterns consistent with recent positive and/or divergent selection. By zeroing in on the molecular signatures of these important regions that might have previously been uncharacterized, we now provide a high-resolution molecular inventory that should improve accessibility and subsequent identification of natural protein variants in future genotype-to-phenotype studies.

Comparative genomic analysis of Bradyrhizobium strains with natural variability in the efficiency of nitrogen fixation, competitiveness, and adaptation to stressful edaphoclimatic conditions.

Bradyrhizobium is known for fixing atmospheric nitrogen in symbiosis with agronomically important crops. This study focused on two groups of strains, each containing eight natural variants of the parental strains, Bradyrhizobium japonicum SEMIA 586 (=CNPSo 17) or Bradyrhizobium diazoefficiens SEMIA 566 (=CNPSo 10). CNPSo 17 and CNPSo 10 were used as commercial inoculants for soybean crops in Brazil at the beginning of the crop expansion in the southern region in the 1960s-1970s. Variants derived from these parental strains were obtained in the late 1980s through a strain selection program aimed at identifying elite strains adapted to a new cropping frontier in the central-western Cerrado region, with a higher capacity of biological nitrogen fixation (BNF) and competitiveness. Here, we aimed to detect genetic variations possibly related to BNF, competitiveness for nodule occupancy, and adaptation to the stressful conditions of the Brazilian Cerrado soils. High-quality genome assemblies were produced for all strains. The core genome phylogeny revealed that strains of each group are closely related, as confirmed by high average nucleotide identity values. However, variants accumulated divergences resulting from horizontal gene transfer, genomic rearrangements, and nucleotide polymorphisms. The B. japonicum group presented a larger pangenome and a higher number of nucleotide polymorphisms than the B. diazoefficiens group, possibly due to its longer adaptation time to the Cerrado soil. Interestingly, five strains of the B. japonicum group carry two plasmids. The genetic variability found in both groups is discussed considering the observed differences in their BNF capacity, competitiveness for nodule occupancy, and environmental adaptation.IMPORTANCEToday, Brazil is a global leader in the study and use of biological nitrogen fixation with soybean crops. As Brazilian soils are naturally void of soybean-compatible bradyrhizobia, strain selection programs were established, starting with foreign isolates. Selection searched for adaptation to the local edaphoclimatic conditions, higher efficiency of nitrogen fixation, and strong competitiveness for nodule occupancy. We analyzed the genomes of two parental strains of Bradyrhizobium japonicum and Bradyrhizobium diazoefficiens and eight variant strains derived from each parental strain. We detected two plasmids in five strains and several genetic differences that might be related to adaptation to the stressful conditions of the soils of the Brazilian Cerrado biome. We also detected genetic variations in specific regions that may impact symbiotic nitrogen fixation. Our analysis contributes to new insights into the evolution of Bradyrhizobium, and some of the identified differences may be applied as genetic markers to assist strain selection programs.

Dynamics of TUBB protein with five majorly occurring natural variants: a risk of cortical dysplasia.

Beta-tubulin (TUBB) protein is one of the components of the microtubule cytoskeleton that plays a critical role in the central nervous system. Genetic variants of TUBB cause cortical dysplasia, a developmental brain defect implicated in axonal guidance and the neuron migration. In this study, we assess pathogenic variants (Q15K, Y222F, M299V, V353I, and E401K) of TUBB protein and compared with non-pathogenic variant G235S to determine their impact on protein dynamic to cause cortical dysplasia. Among the analyzed variants, Q15K, Y222F, M299V, and E401K were noticed to have deleterious effect. Then, variant structures were modeled and their affinity with their known cofactor Guanosine-5'-triphosphate (GTP) was assessed which showed diverse binding energies ranged between (-7.436 to -6.950 kcal/mol) for the variants compared to wild-type (-7.428 kcal/mol). Finally, the molecular dynamics simulation of each variant was investigated which showed difference in trajectory between the pathogenic and non-pathogenic variant. Our analysis suggests change in amino acid residue of TUBB structure has notably affects the protein flexibility and their interactions with known cofactor. Overall, our findings provide insight on the relationship between TUBB variants and their structural dynamics that may cause diverse effects leading to cortical dysplasia.

Assessing Genomic Mutations in SARS-CoV-2: Potential Resistance to Antiviral Drugs in Viral Populations from Untreated COVID-19 Patients.

Naturally occurring SARS-CoV-2 variants mutated in genomic regions targeted by antiviral drugs have not been extensively studied. This study investigated the potential of the RNA-dependent RNA polymerase (RdRp) complex subunits and non-structural protein (Nsp)5 of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) to accumulate natural mutations that could affect the efficacy of antiviral drugs. To this aim, SARS-CoV-2 genomic sequences isolated from 4155 drug-naive individuals from southern Italy were analyzed using the Illumina MiSeq platform. Sequencing of the 4155 samples showed the following viral variant distribution: 71.2% Delta, 22.2% Omicron, and 6.4% Alpha. In the Nsp12 sequences, we found 84 amino acid substitutions. The most common one was P323L, detected in 3777/4155 (91%) samples, with 2906/3777 (69.9%) also showing the G671S substitution in combination. Additionally, we identified 28, 14, and 24 different amino acid substitutions in the Nsp5, Nsp7, and Nsp8 genomic regions, respectively. Of note, the V186F and A191V substitutions, affecting residues adjacent to the active site of Nsp5 (the target of the antiviral drug Paxlovid), were found in 157/4155 (3.8%) and 3/4155 (0.07%) samples, respectively. In conclusion, the RdRp complex subunits and the Nsp5 genomic region exhibit susceptibility to accumulating natural mutations. This susceptibility poses a potential risk to the efficacy of antiviral drugs, as these mutations may compromise the drug ability to inhibit viral replication.

MUG: A mutation overview of GPCR subfamily A17 receptors.

G protein-coupled receptors (GPCRs) mediate several signaling pathways through a general mechanism that involves their activation, upholding a chain of events that lead to the release of molecules responsible for cytoplasmic action and further regulation. These physiological functions can be severely altered by mutations in GPCR genes. GPCRs subfamily A17 (dopamine, serotonin, adrenergic and trace amine receptors) are directly related with neurodegenerative diseases, and as such it is crucial to explore known mutations on these systems and their impact in structure and function. A comprehensive and detailed computational framework - MUG (Mutations Understanding GPCRs) - was constructed, illustrating key reported mutations and their effect on receptors of the subfamily A17 of GPCRs. We explored the type of mutations occurring overall and in the different families of subfamily A17, as well their localization within the receptor and potential effects on receptor functionality. The mutated residues were further analyzed considering their pathogenicity. The results reveal a high diversity of mutations in the GPCR subfamily A17 structures, drawing attention to the considerable number of mutations in conserved residues and domains. Mutated residues were typically hydrophobic residues enriched at the ligand binding pocket and known activating microdomains, which may lead to disruption of receptor function. MUG as an interactive web application is available for the management and visualization of this dataset. We expect that this interactive database helps the exploration of GPCR mutations, their influence, and their familywise and receptor-specific effects, constituting the first step in elucidating their structures and molecules at the atomic level.

VarSite: Disease variants and protein structure.

VarSite is a web server mapping known disease-associated variants from UniProt and ClinVar, together with natural variants from gnomAD, onto protein 3D structures in the Protein Data Bank. The analyses are primarily image-based and provide both an overview for each human protein, as well as a report for any specific variant of interest. The information can be useful in assessing whether a given variant might be pathogenic or benign. The structural annotations for each position in the protein include protein secondary structure, interactions with ligand, metal, DNA/RNA, or other protein, and various measures of a given variant's possible impact on the protein's function. The 3D locations of the disease-associated variants can be viewed interactively via the 3dmol.js JavaScript viewer, as well as in RasMol and PyMOL. Users can search for specific variants, or sets of variants, by providing the DNA coordinates of the base change(s) of interest. Additionally, various agglomerative analyses are given, such as the mapping of disease and natural variants onto specific Pfam or CATH domains. The server is freely accessible to all at: https://www.ebi.ac.uk/thornton-srv/databases/VarSite.

The Genetic Basis of Mutation Rate Variation in Yeast.

Mutations are the root source of genetic variation and underlie the process of evolution. Although the rates at which mutations occur vary considerably between species, little is known about differences within species, or the genetic and molecular basis of these differences. Here, we leveraged the power of the yeast Saccharomyces cerevisiae as a model system to uncover natural genetic variants that underlie variation in mutation rate. We developed a high-throughput fluctuation assay and used it to quantify mutation rates in seven natural yeast isolates and in 1040 segregant progeny from a cross between BY, a laboratory strain, and RM, a wine strain. We observed that mutation rate varies among yeast strains and is heritable (H2 = 0.49). We performed linkage mapping in the segregants and identified four quantitative trait loci underlying mutation rate variation in the cross. We fine-mapped two quantitative trait loci to the underlying causal genes, RAD5 and MKT1, that contribute to mutation rate variation. These genes also underlie sensitivity to the DNA-damaging agents 4NQO and MMS, suggesting a connection between spontaneous mutation rate and mutagen sensitivity.

Multiple levers for overcoming the recalcitrance of lignocellulosic biomass.

Background: The recalcitrance of cellulosic biomass is widely recognized as a key barrier to cost-effective biological processing to fuels and chemicals, but the relative impacts of physical, chemical and genetic interventions to improve biomass processing singly and in combination have yet to be evaluated systematically. Solubilization of plant cell walls can be enhanced by non-biological augmentation including physical cotreatment and thermochemical pretreatment, the choice of biocatalyst, the choice of plant feedstock, genetic engineering of plants, and choosing feedstocks that are less recalcitrant natural variants. A two-tiered combinatoric investigation of lignocellulosic biomass deconstruction was undertaken with three biocatalysts (Clostridium thermocellum, Caldicellulosiruptor bescii, Novozymes Cellic® Ctec2 and Htec2), three transgenic switchgrass plant lines (COMT, MYB4, GAUT4) and their respective nontransgenic controls, two Populus natural variants, and augmentation of biological attack using either mechanical cotreatment or cosolvent-enhanced lignocellulosic fractionation (CELF) pretreatment. Results: In the absence of augmentation and under the conditions tested, increased total carbohydrate solubilization (TCS) was observed for 8 of the 9 combinations of switchgrass modifications and biocatalysts tested, and statistically significant for five of the combinations. Our results indicate that recalcitrance is not a trait determined by the feedstock only, but instead is coequally determined by the choice of biocatalyst. TCS with C. thermocellum was significantly higher than with the other two biocatalysts. Both CELF pretreatment and cotreatment via continuous ball milling enabled TCS in excess of 90%. Conclusion: Based on our results as well as literature studies, it appears that some form of non-biological augmentation will likely be necessary for the foreseeable future to achieve high TCS for most cellulosic feedstocks. However, our results show that this need not necessarily involve thermochemical processing, and need not necessarily occur prior to biological conversion. Under the conditions tested, the relative magnitude of TCS increase was augmentation > biocatalyst choice > plant choice > plant modification > plant natural variants. In the presence of augmentation, plant modification, plant natural variation, and plant choice exhibited a small, statistically non-significant impact on TCS.

Understanding the influences of different pretreatments on recalcitrance of Populus natural variants.

Four different pretreatment technologies were applied to two Populus natural variants and the effects of each pretreatment on glucose release were compared. Physicochemical properties of pretreated biomass were analyzed by attenuated total reflection Fourier transform infrared spectroscopy, gel permeation chromatography, and cross polarization/magic angle spinning carbon-13 nuclear magnetic resonance techniques. The results revealed that hemicellulose and lignin were removed to different extents during various pretreatments. The degree of polymerization of cellulose was decreased in the order of alkali > hydrothermal > organosolv > dilute acid pretreatment. Cellulose crystallinity index was slightly increased after each pretreatment. The results also demonstrated that organosolv pretreatment resulted in the highest glucose yield. Among the tested properties of Populus, degree of polymerization of cellulose was negatively correlated with glucose release, whereas hemicellulose and lignin removal, and cellulose accessibility were positively associated with glucose release from the two Populus natural variants.

Comparative evaluation of Populus variants total sugar release and structural features following pretreatment and digestion by two distinct biological systems.

BACKGROUND: Populus natural variants have been shown to realize a broad range of sugar yields during saccharification, however, the structural features responsible for higher sugar release from natural variants are not clear. In addition, the sugar release patterns resulting from digestion with two distinct biological systems, fungal enzymes and Clostridium thermocellum, have yet to be evaluated and compared. This study evaluates the effect of structural features of three natural variant Populus lines, which includes the line BESC standard, with respect to the overall process of sugar release for two different biological systems. RESULTS: Populus natural variants, SKWE 24-2 and BESC 876, showed higher sugar release from hydrothermal pretreatment combined with either enzymatic hydrolysis or Clostridium thermocellum fermentation compared to the Populus natural variant, BESC standard. However, C. thermocellum outperformed the fungal cellulases yielding 96.0, 95.5, and 85.9% glucan plus xylan release from SKWE 24-2, BESC 876, and BESC standard, respectively. Among the feedstock properties evaluated, cellulose accessibility and glycome profiling provided insights into factors that govern differences in sugar release between the low recalcitrant lines and the BESC standard line. However, because this distinction was more apparent in the solids after pretreatment than in the untreated biomass, pretreatment was necessary to differentiate recalcitrance among Populus lines. Glycome profiling analysis showed that SKWE 24-2 contained the most loosely bound cell wall glycans, followed by BESC 876, and BESC standard. Additionally, lower molecular weight lignin may be favorable for effective hydrolysis, since C. thermocellum reduced lignin molecular weight more than fungal enzymes across all Populus lines. CONCLUSIONS: Low recalcitrant Populus natural variants, SKWE 24-2 and BESC 876, showed higher sugar yields than BESC standard when hydrothermal pretreatment was combined with biological digestion. However, C. thermocellum was determined to be a more robust and effective biological catalyst than a commercial fungal cellulase cocktail. As anticipated, recalcitrance was not readily predicted through analytical methods that determined structural properties alone. However, combining structural analysis with pretreatment enabled the identification of attributes that govern recalcitrance, namely cellulose accessibility, xylan content in the pretreated solids, and non-cellulosic glycan extractability.

Identification of Mob2, a novel regulator of larval neuromuscular junction morphology, in natural populations of Drosophila melanogaster.

Although evolutionary changes must take place in neural connectivity and synaptic architecture as nervous systems become more complex, we lack understanding of the general principles and specific mechanisms by which these changes occur. Previously, we found that morphology of the larval neuromuscular junction (NMJ) varies extensively among different species of Drosophila but is relatively conserved within a species. To identify specific genes as candidates that might underlie phenotypic differences in NMJ morphology among Drosophila species, we performed a genetic analysis on one of two phenotypic variants we found among 20 natural isolates of Drosophila melanogaster. We discovered genetic polymorphisms for both positive and negative regulators of NMJ growth segregating within the variant line. Focusing on one subline, that displayed NMJ overgrowth, we mapped the phenotype to Mob2 [Monopolar spindle (Mps) one binding protein 2)], a gene encoding a Nuclear Dbf2 (Dumbbell formation 2)-Related (NDR) kinase activator. We confirmed this identification by transformation rescue experiments and showed that presynaptic expression of Mob2 is necessary and sufficient to regulate NMJ growth. Mob2 interacts in a dominant, dose-dependent manner with tricornered but not with warts, to cause NMJ overgrowth, suggesting that Mob2 specifically functions in combination with the former NDR kinase to regulate NMJ development. These results demonstrate the feasibility and utility of identifying genetic variants affecting NMJ morphology in natural populations of Drosophila. These variants can lead to discovery of new genes and molecular mechanisms that regulate NMJ development while also providing new information that can advance our understanding of mechanisms that underlie nervous system evolution.