Dark septate endophytes (DSE) are a form-group of root endophytic fungi with elusive functions. Here, the genomes of two common DSE of semiarid areas, Cadophora sp. and Periconia macrospinosa were sequenced and analyzed with another 32 ascomycetes of different lifestyles. Cadophora sp. (Helotiales) and P. macrospinosa (Pleosporales) have genomes of 70.46 Mb and 54.99 Mb with 22,766 and 18,750 gene models, respectively. The majority of DSE-specific protein clusters lack functional annotation with no similarity to characterized proteins, implying that they have evolved unique genetic innovations. Both DSE possess an expanded number of carbohydrate active enzymes (CAZymes), including plant cell wall degrading enzymes (PCWDEs). Those were similar in three other DSE, and contributed a signal for the separation of root endophytes in principal component analyses of CAZymes, indicating shared genomic traits of DSE fungi. Number of secreted proteases and lipases, aquaporins, and genes linked to melanin synthesis were also relatively high in our fungi. In spite of certain similarities between our two DSE, we observed low levels of convergence in their gene family evolution. This suggests that, despite originating from the same habitat, these two fungi evolved along different evolutionary trajectories and display considerable functional differences within the endophytic lifestyle.

Homologous recombination (HR) is crucial for the error-free repair of DNA double-strand breaks (DSBs) and the restart of stalled replication. However, imprecise HR can lead to genome instability, highlighting the importance of HR quality control. After DSB formation, HR proceeds via DNA end resection and recombinase loading, whereas helicase-catalyzed disruption of a subset of subsequently formed DNA invasions is thought to be essential for maintaining HR accuracy via inhibiting illegitimate (non-allelic) recombination. Here we show that in vitro characterized mechanistic aberrations of E. coli RecBCD (resection and recombinase loading) RecQ (multifunctional DNA-restructuring helicase) mutant enzyme variants, on one hand, cumulatively deteriorate cell survival under certain conditions of genomic stress. On the other hand, we find that RecBCD and RecQ defects functionally compensate each other in terms of HR accuracy. The abnormally long resection and unproductive recombinase loading activities of a mutant RecBCD complex (harboring the D1080A substitution in RecB) cause enhanced illegitimate recombination. However, this compromised HR-accuracy phenotype is suppressed in double mutant strains harboring mutant RecQ variants with abnormally enhanced helicase and inefficient invasion disruptase activities. These results frame an in vivo context for the interplay of biochemical activities leading to illegitimate recombination, and underscore its long-range genome instability effects manifest in higher eukaryotes.

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The degradation capacity and utilisation of complex plant substrates are crucial for the functioning of saprobic fungi and different plant symbionts with fundamental functions in ecosystems. Measuring the growth capacity and biomass of fungi on such systems is a challenging task. We established a new micro-scale experimental setup using substrates made of different plant species and organs as media for fungal growth. We adopted and tested a reliable and simple titration-based method for the estimation of total fungal biomass within the substrates using fluorescence-labelled lectin. We found that the relationship between fluorescence intensity and fungal dry weight was strong and linear but differed among fungi. The effect of the plant organ (i.e. root vs. shoot) used as substrate on fungal growth differed among plant species and between root endophytic fungal species. The novel microscale experimental system is useful for screening the utilisation of different substrates, which can provide insight into the ecological roles and functions of fungi. Furthermore, our fungal biomass estimation method has applications in various fields. As the estimation is based on the fungal cell wall, it measures the total cumulative biomass produced in a certain environment.

Cells must protect their genome from harmful alterations, caused directly by DNA damage or indirectly by inappropriate repair, to avoid cell death or cancerous transformation. DNA double-strand (ds) breaks, the most severe type of DNA damage, are repaired in a potentially error-free manner by homologous recombination (HR). During HR, broken dsDNA ends are transformed into 3' single-stranded (ss) overhangs that form nucleoprotein filaments with recombinases. These filaments can search for intact, homologous dsDNA regions that serve as template for repair and can invade into the homologous DNA molecule to form a displacement loop (D-loop). D-loops are thus key HR DNAintermediate structures. D-loops are thought to be processed by multiple pathways with different outcomes and can serve as early decision points in HR regulation. Members of the RecQ helicase family are implicated both in D-loop stabilization and disruption. The human Bloom's syndrome RecQ helicase (BLM) is thought to channel HR into error-free pathways partially by processing D-loops. However, the exact mechanism of this action is poorly understood. Via a novel kinetic assay, here we show that the conserved domain architecture of BLM maintains an almost 1:1 balance in D-loop disruption and stabilization. Interestingly, the activity profile of BLM markedly differs from that of the very effective D-loop disruptor Escherichia coli RecQ. However, our single-molecule magnetic tweezers experiments reveal that the similar domain architecture of BLM and RecQ, while having different roles in D-loop processing, allows both proteins to sense DNA substrate geometry and unwind dsDNA in a repetitive fashion. Our results highlight how functions of the conserved architecture of RecQ helicases evolved to specialize these enzymes to different cellular requirements.