Transcriptional dynamic in response to environmental and developmental cues are fundamental to biology, yet many mechanistic aspects are poorly understood. One such example is fungal plant pathogens, which use secreted proteins and small molecules, termed effectors, to suppress host immunity and promote colonization. Effectors are highly expressedin plantabut remain transcriptionally repressedex planta, but our mechanistic understanding of these transcriptional dynamics remains limited. We tested the hypothesis that repressive histone modification at H3-Lys27 underlies transcriptional silencingex planta, and that exchange for an active chemical modification contributes to transcription ofin plantainduced genes. Using genetics, chromatin immunoprecipitation and sequencing and RNA-sequencing, we determined that H3K27me3 provides significant local transcriptional repression. We detail how regions that lose H3K27me3 gain H3K27ac, and these changes are associated with increased transcription. Importantly, we observed that manyin plantainduced genes were marked by H3K27me3 during axenic growth, and detail how altered H3K27 modification influences transcription. ChIP-qPCR duringin plantagrowth suggests that H3K27 modifications are generally stable, but can undergo dynamics at specific genomic locations. Our results support the hypothesis that dynamic histone modifications at H3K27 contributes to fungal genome regulation and specifically contributes to regulation of genes important during host infection.

One major threat to global food security that requires immediate attention, is the increasing incidence of host shift and host expansion in growing number of pathogenic fungi and emergence of new pathogens. The threat is more alarming because, yield quality and quantity improvement efforts are encouraging the cultivation of uniform plants with low genetic diversity that are increasingly susceptible to emerging pathogens. However, the influence of host genome differentiation on pathogen genome differentiation and its contribution to emergence and adaptability is still obscure. Here, we compared genome sequence of 6 isolates of Magnaporthe species obtained from three different host plants. We demonstrated the evolutionary relationship between Magnaporthe species and the influence of host differentiation on pathogens. Phylogenetic analysis showed that evolution of pathogen directly corresponds with host divergence, suggesting that host-pathogen interaction has led to co-evolution. Furthermore, we identified an asymmetric selection pressure on Magnaporthe species. Oryza sativa-infecting isolates showed higher directional selection from host and subsequently tends to lower the genetic diversity in its genome. We concluded that, frequent gene loss or gain, new transposon acquisition and sequence divergence are host adaptability mechanisms for Magnaporthe species and this coevolution processes is greatly driven by directional selection from host plants.

DNA double-strand breaks require repair or risk corrupting the language of life. To ensure genome integrity and viability, multiple DNA double-strand break repair pathways function in eukaryotes. Two such repair pathways, canonical non-homologous end joining and homologous recombination, have been extensively studied, while other pathways such as microhomology-mediated end joint and single-strand annealing, once thought to serve as back-ups, now appear to play a fundamental role in DNA repair. Here, we review the molecular details and hierarchy of these four DNA repair pathways, and where possible, a comparison for what is known between animal and fungal models. We address the factors contributing to break repair pathway choice, and aim to explore our understanding and knowledge gaps regarding mechanisms and regulation in filamentous pathogens. We additionally discuss how DNA double-strand break repair pathways influence genome engineering results, including unexpected mutation outcomes. Finally, we review the concept of biased genome evolution in filamentous pathogens, and provide a model, termed Biased Variation, that links DNA double-strand break repair pathways with properties of genome evolution. Despite our extensive knowledge for this universal process, there remain many unanswered questions, for which the answers may improve genome engineering and our understanding of genome evolution.

CRISPR-Cas mediated genome engineering has revolutionized functional genomics. However, understanding of DNA repair following Cas-mediated DNA cleavage remains incomplete. Using Cas12a ribonucleoprotein genome editing in the fungal pathogen, Magnaporthe oryzae, we detail non-canonical DNA repair outcomes from hundreds of transformants. Sanger and nanopore sequencing analysis reveals significant variation in DNA repair profiles, ranging from small INDELs to kilobase size deletions and insertions. Furthermore, we find the frequency of DNA repair outcomes varies between loci. The results are not specific to the Cas-nuclease or selection procedure. Through Ku80 deletion analysis, a key protein required for canonical non-homologous end joining, we demonstrate activity of an alternative end joining mechanism that creates larger DNA deletions, and uses longer microhomology compared to C-NHEJ. Together, our results suggest preferential DNA repair pathway activity in the genome that can create different mutation profiles following repair, which could create biased genome variation and impact genome engineering and genome evolution.

While increased mutation rates typically have negative consequences in multicellular organisms, hypermutation can be advantageous for microbes adapting to the environment. Previously, we identified two hypermutator Cryptococcus neoformans clinical isolates that rapidly develop drug resistance due to transposition of a retrotransposon, Cnl1. Cnl1-mediated hypermutation is caused by a nonsense mutation in a gene encoding an RNA interference (RNAi) component, ZNF3 , combined with a tremendous transposon burden. To elucidate adaptive mechanisms following RNAi loss, two bioinformatic pipelines were developed to identify RNAi loss-of-function (LOF) mutations in a collection of 387 sequenced C. neoformans isolates. Remarkably, several RNAi-loss isolates were identified that are not hypermutators and have not accumulated transposons. To test whether these RNAi LOF mutations can cause hypermutation, the mutations were introduced into a nonhypermutator strain with a high transposon burden, which resulted in a hypermutator phenotype. To further investigate whether RNAi-loss isolates can become hypermutators, in vitro passaging was performed. Although no hypermutators were found in two C. neoformans RNAi-loss strains after short-term passage, hypermutation was observed in a passaged Cryptococcus deneoformans strain with an increased transposon burden. Consistent with a two-step evolution, when an RNAi-loss isolate was crossed with an isolate containing a high Cnl1 burden, F1 hypermutator progeny inheriting a high transposon burden were identified. In addition to Cnl1 transpositions, insertions of a gigantic DNA transposon KDZ1 (~11 kb) contributed to hypermutation in the progeny. Our results suggest that RNAi loss is relatively common (7/387, ~1.8%) and enables distinct evolutionary trajectories: hypermutation following transposon accumulation or survival without hypermutation.