Molecular characterization reveals no functional evidence for naturally occurring cross-kingdom RNA interference in the early stages of Botrytis cinerea-tomato interaction.

Plant immune responses are triggered during the interaction with pathogens. The fungus Botrytis cinerea has previously been reported to use small RNAs (sRNAs) as effector molecules capable of interfering with the host immune response. Conversely, a host plant produces sRNAs that may interfere with the infection mechanism of an intruder. We used high-throughput sequencing to identify sRNAs produced by B. cinerea and Solanum lycopersicum (tomato) during early phases of interaction and to examine the expression of their predicted mRNA targets in the other organism. A total of 7042 B. cinerea sRNAs were predicted to target 3185 mRNAs in tomato. Of the predicted tomato target genes, 163 were indeed transcriptionally down-regulated during the early phase of infection. Several experiments were performed to study a causal relation between the production of B. cinerea sRNAs and the down-regulation of predicted target genes in tomato. We generated B. cinerea mutants in which a transposon region was deleted that is the source of c.10% of the fungal sRNAs. Furthermore, mutants were generated in which both Dicer-like genes (Bcdcl1 and Bcdcl2) were deleted and these displayed a >99% reduction of transposon-derived sRNA production. Neither of these mutants was significantly reduced in virulence on any plant species tested. Our results reveal no evidence for any detectable role of B. cinerea sRNAs in the virulence of the fungus.

Considerations and practical implications of performing a phenotypic CRISPR/Cas survival screen.

Genome-wide screens that have viability as a readout have been instrumental to identify essential genes. The development of gene knockout screens with the use of CRISPR-Cas has provided a more sensitive method to identify these genes. Here, we performed an exhaustive genome-wide CRISPR/Cas9 phenotypic rescue screen to identify modulators of cytotoxicity induced by the pioneer transcription factor, DUX4. Misexpression of DUX4 due to a failure in epigenetic repressive mechanisms underlies facioscapulohumeral muscular dystrophy (FHSD), a complex muscle disorder that thus far remains untreatable. As the name implies, FSHD generally starts in the muscles of the face and shoulder girdle. Our CRISPR/Cas9 screen revealed no key effectors other than DUX4 itself that could modulate DUX4 cytotoxicity, suggesting that treatment efforts in FSHD should be directed towards direct modulation of DUX4 itself. Our screen did however reveal some rare and unexpected genomic events, that had an important impact on the interpretation of our data. Our findings may provide important considerations for planning future CRISPR/Cas9 phenotypic survival screens.

Ontogeny and Anatomy of the Dimorphic Pitchers of Nepenthes rafflesiana Jack.

An enigmatic feature of tropical pitcher plants belonging to the genus Nepenthes is their dimorphic prey-capturing pitfall traps. In many species, the conspicuously shaped upper and lower pitchers grow from a swollen leaf tendril tip until finally opening as insect-alluring devices. Few have studied the ontogeny of these traps from an anatomical and quantitative morphological perspective. We investigated whether the anatomy and development of lower and upper type pitchers of N. rafflesiana differ or overlap in terms of 3D geometric morphology and microstructure progression and presence. We hypothesized that there is an overlap in the initial, but not all, developmental stages of the two pitcher types and that one pitcher type is suspended in development. We identified four important morphological changes of pitcher ontogeny and defined these as curvation, elongation, inflation and maturation phases. Pitcher length indicated progress through developmental phases, and we propose to use it as a tool for indication of developmental stage. Microstructure development coincided with the developmental phases defined. Additionally, we discovered a new anatomical feature of extrafloral nectariferous peristomal glands between the inner peristome ridges of upper and lower pitchers being hollow and analyze the chemistry of the sugars on the outside of these glands. Ontogenetic shape analysis indicated that upper and lower pitcher types develop with similar phase progression but have no directly overlapping morphology. This means that upper pitchers are not a derived state from lower pitchers. Independent developmental programs evolved to produce distinctly shaped upper and lower pitchers in Nepenthes, likely to exploit different food sources.

Recently Evolved Enhancers Emerge with High Interindividual Variability and Less Frequently Associate with Disease.

Mutations in non-coding regulatory DNA such as enhancers underlie a wide variety of diseases including developmental disorders and cancer. As enhancers rapidly evolve, understanding their function and configuration in non-human disease models can have important clinical applications. Here, we analyze enhancer configurations in tissues isolated from the common marmoset, a widely used primate model for human disease. Integrating these data with human and mouse data, we find that enhancers containing trait-associated variants are preferentially conserved. In contrast, most human-specific enhancers are highly variable between individuals, with a subset failing to contact promoters. These are located further away from genes and more often reside in inactive B-compartments. Our data show that enhancers typically emerge as instable elements with minimal biological impact prior to their integration in a transcriptional program. Furthermore, our data provide insight into which trait variations in enhancers can be faithfully modeled using the common marmoset.

Hominin-specific regulatory elements selectively emerged in oligodendrocytes and are disrupted in autism patients.

Speciation is associated with substantial rewiring of the regulatory circuitry underlying the expression of genes. Determining which changes are relevant and underlie the emergence of the human brain or its unique susceptibility to neural disease has been challenging. Here we annotate changes to gene regulatory elements (GREs) at cell type resolution in the brains of multiple primate species spanning most of primate evolution. We identify a unique set of regulatory elements that emerged in hominins prior to the separation of humans and chimpanzees. We demonstrate that these hominin gains perferentially affect oligodendrocyte function postnatally and are preferentially affected in the brains of autism patients. This preference is also observed for human-specific GREs suggesting this system is under continued selective pressure. Our data provide a roadmap of regulatory rewiring across primate evolution providing insight into the genomic changes that underlie the emergence of the brain and its susceptibility to neural disease.

AML Subtype Is a Major Determinant of the Association between Prognostic Gene Expression Signatures and Their Clinical Significance.

Relapse in acute myeloid leukemia (AML) may result from variable genetic origins or convergence on common biological processes. Exploiting the specificity and sensitivity of regulatory DNA, we analyze patient samples of multiple clinical outcomes covering various AML molecular subtypes. We uncover regulatory variation among patients translating into a transcriptional signature that predicts relapse risk. In addition, we find clusters of coexpressed genes within this signature selectively link to relapse risk in distinct patient subgroups defined by molecular subtype or AML maturation. Analyzing these gene clusters and the AML subtypes separately enhances their prognostic value substantially and provides insight in the mechanisms underlying relapse risk across the distinct patient subgroups. We propose that prognostic gene expression signatures in AML are valid only within patient subgroups and do not transcend these subgroups.