AGO1 and HSP90 buffer different genetic variants in Arabidopsis thaliana.

Argonaute 1 (AGO1), the principal protein component of microRNA-mediated regulation, plays a key role in plant growth and development. AGO1 physically interacts with the chaperone HSP90, which buffers cryptic genetic variation in plants and animals. We sought to determine whether genetic perturbation of AGO1 in Arabidopsis thaliana would also reveal cryptic genetic variation, and if so, whether AGO1-dependent loci overlap with those dependent on HSP90. To address these questions, we introgressed a hypomorphic mutant allele of AGO1 into a set of mapping lines derived from the commonly used Arabidopsis strains Col-0 and Ler. Although we identified several cases in which AGO1 buffered genetic variation, none of the AGO1-dependent loci overlapped with those buffered by HSP90 for the traits assayed. We focused on 1 buffered locus where AGO1 perturbation uncoupled the traits days to flowering and rosette leaf number, which are otherwise closely correlated. Using a bulk segregant approach, we identified a nonfunctional Ler hua2 mutant allele as the causal AGO1-buffered polymorphism. Introduction of a nonfunctional hua2 allele into a Col-0 ago1 mutant background recapitulated the Ler-dependent ago1 phenotype, implying that coupling of these traits involves different molecular players in these closely related strains. Taken together, our findings demonstrate that even though AGO1 and HSP90 buffer genetic variation in the same traits, these robustness regulators interact epistatically with different genetic loci, suggesting that higher-order epistasis is uncommon. Plain Language Summary Argonaute 1 (AGO1), a key player in plant development, interacts with the chaperone HSP90, which buffers environmental and genetic variation. We found that AGO1 buffers environmental and genetic variation in the same traits; however, AGO1-dependent and HSP90-dependent loci do not overlap. Detailed analysis of a buffered locus found that a nonfunctional HUA2 allele decouples days to flowering and rosette leaf number in an AGO1-dependent manner, suggesting that the AGO1-dependent buffering acts at the network level.

The Mechanistic Underpinnings of an ago1-Mediated, Environmentally Dependent, and Stochastic Phenotype.

The crucial role of microRNAs in plant development is exceedingly well supported; their importance in environmental robustness is studied in less detail. Here, we describe a novel, environmentally dependent phenotype in hypomorphic argonaute1 (ago1) mutants and uncover its mechanistic underpinnings in Arabidopsis (Arabidopsis thaliana). AGO1 is a key player in microRNA-mediated gene regulation. We observed transparent lesions on embryonic leaves of ago1 mutant seedlings. These lesions increased in frequency in full-spectrum light. Notably, the lesion phenotype was most environmentally responsive in ago1-27 mutants. This allele is thought to primarily affect translational repression, which has been linked with the response to environmental perturbation. Using several lines of evidence, we found that these lesions represent dead and dying tissues due to an aberrant hypersensitive response. Although all three canonical defense hormone pathways (salicylic acid, jasmonate, and jasmonate/ethylene pathways) were up-regulated in ago1 mutants, we demonstrate that jasmonate perception drives the lesion phenotype. Double mutants of ago1 and coronatine insensitive1, the jasmonate receptor, showed greatly decreased frequency of affected seedlings. The chaperone HEAT SHOCK PROTEIN 90 (HSP90), which maintains phenotypic robustness in the face of environmental perturbations, is known to facilitate AGO1 function. HSP90 perturbation has been shown previously to up-regulate jasmonate signaling and to increase plant resistance to herbivory. Although single HSP90 mutants showed subtly elevated levels of lesions, double mutant analysis disagreed with a simple epistatic model for HSP90 and AGO1 interaction; rather, both appeared to act nonadditively in producing lesions. In summary, our study identifies AGO1 as a major, largely HSP90-independent, factor in providing environmental robustness to plants.

Hsp90 promotes kinase evolution.

Heat-shock protein 90 (Hsp90) promotes the maturation and stability of its client proteins, including many kinases. In doing so, Hsp90 may allow its clients to accumulate mutations as previously proposed by the capacitor hypothesis. If true, Hsp90 clients should show increased evolutionary rate compared with nonclients; however, other factors, such as gene expression and protein connectivity, may confound or obscure the chaperone's putative contribution. Here, we compared the evolutionary rates of many Hsp90 clients and nonclients in the human protein kinase superfamily. We show that Hsp90 client status promotes evolutionary rate independently of, but in a small magnitude similar to that of gene expression and protein connectivity. Hsp90's effect on kinase evolutionary rate was detected across mammals, specifically relaxing purifying selection. Hsp90 clients also showed increased nucleotide diversity and harbored more damaging variation than nonclient kinases across humans. These results are consistent with the central argument of the capacitor hypothesis that interaction with the chaperone allows its clients to harbor genetic variation. Hsp90 client status is thought to be highly dynamic with as few as one amino acid change rendering a protein dependent on the chaperone. Contrary to this expectation, we found that across protein kinase phylogeny Hsp90 client status tends to be gained, maintained, and shared among closely related kinases. We also infer that the ancestral protein kinase was not an Hsp90 client. Taken together, our results suggest that Hsp90 played an important role in shaping the kinase superfamily.

The protein chaperone HSP90 can facilitate the divergence of gene duplicates.

The heat-shock protein 90 (HSP90) acts as a chaperone by ensuring proper maturation and folding of its client proteins. The HSP90 capacitor hypothesis holds that interactions with HSP90 allow proteins to accumulate mutations while maintaining function. Following this logic, HSP90 clients would be predicted to show relaxed selection compared with nonclients. In this study, we identify a new HSP90 client in the plant steroid hormone pathway: the transcription factor BES1. Its closest paralog, BZR1, is not an HSP90 client. This difference in HSP90 client status in two highly similar proteins enabled a direct test of the capacitor hypothesis. We find that BES1 shows relaxed selection compared to BZR1, hallmarks of neo- and subfunctionalization, and dynamic HSP90 client status across independent evolutionary paths. These results suggested that HSP90's influence on gene evolution may be detectable if we compare gene duplicates because duplicates share most other properties influencing evolutionary rate that might otherwise conceal the chaperone's effect. We test this hypothesis using systematically identified HSP90 clients in yeast and observe a significant trend of HSP90 clients evolving faster than their nonclient paralogs. This trend was not detected when yeast clients and nonclients were compared without considering paralog status. Our data provide evidence that HSP90 influences selection on genes encoding its clients and facilitates divergence between gene duplicates.

Identical repeated backbone of the human genome.

BACKGROUND: Identical sequences with a minimal length of about 300 base pairs (bp) have been involved in the generation of various meiotic/mitotic genomic rearrangements through non-allelic homologous recombination (NAHR) events. Genomic disorders and structural variation, together with gene remodelling processes have been associated with many of these rearrangements. Based on these observations, we identified and integrated all the 100% identical repeats of at least 300 bp in the NCBI version 36.2 human genome reference assembly into non-overlapping regions, thus defining the Identical Repeated Backbone (IRB) of the reference human genome. RESULTS: The IRB sequences are distributed all over the genome in 66,600 regions, which correspond to approximately 2% of the total NCBI human genome reference assembly. Important structural and functional elements such as common repeats, segmental duplications, and genes are contained in the IRB. About 80% of the IRB bp overlap with known copy-number variants (CNVs). By analyzing the genes embedded in the IRB, we were able to detect some identical genes not previously included in the Ensembl release 50 annotation of human genes. In addition, we found evidence of IRB gene copy-number polymorphisms in raw sequence reads of two diploid sequenced genomes. CONCLUSIONS: In general, the IRB offers new insight into the complex organization of the identical repeated sequences of the human genome. It provides an accurate map of potential NAHR sites which could be used in targeting the study of novel CNVs, predicting DNA copy-number variation in newly sequenced genomes, and improve genome annotation.

Recurrent DNA inversion rearrangements in the human genome.

Several lines of evidence suggest that reiterated sequences in the human genome are targets for nonallelic homologous recombination (NAHR), which facilitates genomic rearrangements. We have used a PCR-based approach to identify breakpoint regions of rearranged structures in the human genome. In particular, we have identified intrachromosomal identical repeats that are located in reverse orientation, which may lead to chromosomal inversions. A bioinformatic workflow pathway to select appropriate regions for analysis was developed. Three such regions overlapping with known human genes, located on chromosomes 3, 15, and 19, were analyzed. The relative proportion of wild-type to rearranged structures was determined in DNA samples from blood obtained from different, unrelated individuals. The results obtained indicate that recurrent genomic rearrangements occur at relatively high frequency in somatic cells. Interestingly, the rearrangements studied were significantly more abundant in adults than in newborn individuals, suggesting that such DNA rearrangements might start to appear during embryogenesis or fetal life and continue to accumulate after birth. The relevance of our results in regard to human genomic variation is discussed.