Cut-and-Paste DNA Insertion with Engineered Type V-K CRISPR-associated Transposases

CRISPR-associated transposases (CASTs) enable recombination-independent, multi-kilobase DNA insertions at RNA-programmed genomic locations. Type V-K CASTs offer distinct technological advantages over type I CASTs given their smaller coding size, fewer components, and unidirectional insertions. However, the utility of type V-K CASTs is hindered by a replicative transposition mechanism that results in a mixture of desired simple cargo insertions and undesired plasmid co-integrate products. Here, we overcome this limitation by engineering new CASTs with dramatically improved product purity. To do so, we compensate for the absence of the TnsA subunit in multiple type V-K CASTs by engineering a Homing Endonuclease-assisted Large-sequence Integrating CAST compleX, or HELIX system. HELIX utilizes a nicking homing endonuclease (nHE) fused to TnsB to restore the 5-prime nicking capability needed for dual-nicking of the DNA donor. By leveraging distinct features of both type V-K and type I systems, HELIX enables cut-and-paste DNA insertion with up to 99.3% simple insertion product purity, while retaining robust integration efficiencies on genomic targets. Furthermore, we demonstrate the versatility of this approach by generating HELIX systems for other CAST orthologs. We also establish the feasibility of creating a minimal, 3-component HELIX, simplifying the number of proteins that must be expressed. Together, HELIX streamlines and improves the application of CRISPR-based transposition technologies, eliminating barriers for efficient and specific RNA-guided DNA insertions.

Mitochondrial Genomics of Six Cacao Pathogens From the Basidiomycete Family Marasmiaceae

Thread blight disease has recently been described as an emerging disease on cacao (Theobroma cacao) in Ghana. In Ghana, thread blight disease is caused by multiple species of the Marasmiaceae family: Marasmius tenuissimus, M. crinis-equi, M. palmivorus, and Marasmiellus scandens. Interestingly, two additional members of the Marasmiaceae; Moniliophthora roreri (frosty pod rot) and Moniliophthora perniciosa (witches’ broom disease), are major pathogens of cacao in the Western hemisphere. It is important to accurately characterize the genetic relationships among these economically important species in support of their disease management. We used data from Illumina NGS-based genome sequencing efforts to study the mitochondrial genomes (mitogenomes) of the four cacao thread blight associated pathogens from Ghana and compared them with published mitogenomes of Mon. roreri and Mon. perniciosa. There is a remarkable interspecies variation in mitogenome size within the six cacao-associated Marasmiaceae species, ranging from 43,121 to 109,103 bp. The differences in genome lengths are primarily due to the number and lengths of introns, differences in intergenic space, and differences in the size and numbers of unidentified ORFs (uORF). Among seven M. tenuissimus mitogenomes sequenced, there is variation in size and sequence pointing to divergent evolution patterns within the species. The intronic regions show a high degree of sequence variation compared to the conserved sequences of the 14 core genes. The intronic ORFs identified, regardless of species, encode GIY-YIG or LAGLIDADG domain-containing homing endonuclease genes. Phylogenetic relationships using the 14 core proteins largely mimic the phylogenetic relationships observed in gene order patterns, grouping M. tenuissimus with M. crinis-equi, and M. palmivorus with Mon. roreri and Mon. perniciosa, leaving Mar. scandens as an outlier. The results from this study provide evidence of independent expansion/contraction events and sequence diversification in each species and establish a foundation for further exploration of the evolutionary trajectory of the fungi in Marasmiaceae family.

Mitochondrial and Plastid Genomes of the Monoraphid Diatom Schizostauron trachyderma

We provide for the first time the complete plastid and mitochondrial genomes of a monoraphid diatom: Schizostauron trachyderma. The mitogenome is 41,957 bp in size and displays two group II introns in the cox1 gene. The 187,029 bp plastid genome features the typical quadripartite architecture of diatom genomes. It contains a group II intron in the petB gene that overlaps the large single-copy and the inverted repeat region. There is also a group IB4 intron encoding a putative LAGLIDADG homing endonuclease in the rnl gene. The multigene phylogenies conducted provide more evidence of the proximity between S. trachyderma and fistula-bearing species of biraphid diatoms.

An Update on Trichoderma Mitogenomes: Complete De Novo Mitochondrial Genome of the Fungal Biocontrol Agent Trichoderma harzianum (Hypocreales, Sordariomycetes), an Ex-Neotype Strain CBS 226.95, and Tracing the Evolutionary Divergences of Mitogenomes in Trichoderma

Members of the genus Trichoderma (Hypocreales), widely used as biofungicides, biofertilizers, and as model fungi for the industrial production of CAZymes, have actively been studied for the applications of their biological functions. Recently, the study of the nuclear genomes of Trichoderma has expanded in the directions of adaptation and evolution to gain a better understanding of their ecological traits. However, Trichoderma’s mitochondria have received much less attention despite mitochondria being the most necessary element for sustaining cell life. In this study, a mitogenome of the fungus Trichoderma harzianum CBS 226.95 was assembled de novo. A 27,632 bp circular DNA molecule was revealed with specific features, such as the intronless of all core PCGs, one homing endonuclease, and a putative overlapping tRNA, on a closer phylogenetic relationship with T. reesei among hypocrealean fungi. Interestingly, the mitogenome of T. harzianum CBS 226.95 was predicted to have evolved earlier than those of other Trichoderma species and also assumed with a selection pressure in the cox3. Considering the bioavailability, both for the ex-neotype strain of the T. harzianum species complex and the most globally representative commercial fungal biocontrol agent, our results on the T. harzianum CBS 226.95 mitogenome provide crucial information which will be helpful criteria in future studies on Trichoderma.

A Phylogenetic Approach to Structural Variation in Organization of Nuclear Group I Introns and Their Ribozymes

Nuclear group I introns are restricted to the ribosomal DNA locus where they interrupt genes for small subunit and large subunit ribosomal RNAs at conserved sites in some eukaryotic microorganisms. Here, the myxomycete protists are a frequent source of nuclear group I introns due to their unique life strategy and a billion years of separate evolution. The ribosomal DNA of the myxomycete Mucilago crustacea was investigated and found to contain seven group I introns, including a direct repeat-containing intron at insertion site S1389 in the small subunit ribosomal RNA gene. We collected, analyzed, and compared 72 S1389 group IC1 introns representing diverse myxomycete taxa. The consensus secondary structure revealed a conserved ribozyme core, but with surprising sequence variations in the guanosine binding site in segment P7. Some S1389 introns harbored large extension sequences in the peripheral region of segment P9 containing direct repeat arrays. These repeats contained up to 52 copies of a putative internal guide sequence motif. Other S1389 introns harbored homing endonuclease genes in segment P1 encoding His-Cys proteins. Homing endonuclease genes were further interrupted by small spliceosomal introns that have to be removed in order to generate the open reading frames. Phylogenetic analyses of S1389 intron and host gene indicated both vertical and horizontal intron transfer during evolution, and revealed sporadic appearances of direct repeats, homing endonuclease genes, and guanosine binding site variants among the myxomycete taxa.

An alternative domain-swapped structure of the Pyrococcus horikoshii PolII mini-intein

Protein splicing is a post-translational process by which an intein catalyzes its own excision from flanking polypeptides, or exteins, concomitant with extein ligation. Many inteins have nested homing endonuclease domains that facilitate their propagation into intein-less alleles, whereas other inteins lack the homing endonuclease (HEN) and are called mini-inteins. The mini-intein that interrupts the DNA PolII of Pyrococcus horikoshii has a linker region in place of the HEN domain that is shorter than the linker in a closely related intein from Pyrococcus abyssi. The P. horikoshii PolII intein requires a higher temperature for catalytic activity and is more stable to digestion by the thermostable protease thermolysin, suggesting that it is more rigid than the P. abyssi intein. We solved a crystal structure of the intein precursor that revealed a domain-swapped dimer. Inteins found as domain swapped dimers have been shown to promote intein-mediated protein alternative splicing, but the solved P. horikoshii PolII intein structure has an active site unlikely to be catalytically competent.

Recent and Ongoing Horizontal Transfer of Mitochondrial Introns Between Two Fungal Tree Pathogens

Two recently introduced fungal plant pathogens (Ceratocystis lukuohia and Ceratocystis huliohia) are responsible for Rapid ‘ōhi‘a Death (ROD) in Hawai‘i. Despite being sexually incompatible, the two pathogens often co-occur in diseased ‘ōhi‘a sapwood, where genetic interaction is possible. We sequenced and annotated 33 mitochondrial genomes of the two pathogens and related species, and investigated 35 total Ceratocystis mitogenomes. Ten mtDNA regions [one group I intron, seven group II introns, and two autonomous homing endonuclease (HE) genes] were heterogeneously present in C. lukuohia mitogenomes, which were otherwise identical. Molecular surveys with specific primers showed that the 10 regions had uneven geographic distribution amongst populations of C. lukuohia. Conversely, identical orthologs of each region were present in every studied isolate of C. huliohia regardless of geographical origin. Close relatives of C. lukuohia lacked or, rarely, had few and dissimilar orthologs of the 10 regions, whereas most relatives of C. huliohia had identical or nearly identical orthologs. Each region included or worked in tandem with HE genes or reverse transcriptase/maturases that could facilitate interspecific horizontal transfers from intron-minus to intron-plus alleles. These results suggest that the 10 regions originated in C. huliohia and are actively moving to populations of C. lukuohia, perhaps through transient cytoplasmic contact of hyphal tips (anastomosis) in the wound surface of ‘ōhi‘a trees. Such contact would allow for the transfer of mitochondria followed by mitochondrial fusion or cytoplasmic exchange of intron intermediaries, which suggests that further genomic interaction may also exist between the two pathogens.

Insights into genomic evolution from the chromosomal and mitochondrial genomes of Ustilaginoidea virens

Ustilaginoidea virens, the causal agent of rice false smut, is an economically important filamentous fungal pathogen. A high-quality reference genome of U. virens promotes understanding of molecular mechanisms underlying its virulence and pathogenicity. Here, we report the first chromosome-level assembly of U. virens genome consisting of seven chromosomes ranging from 2.4 to 7.5 Mb. The assembly has dramatic improvements over previous assemblies, including considerably longer contigs, higher proportion of repetitive elements and more functionally annotated genes. Phylogenetic analyses revealed an extremely low intraspecific sequence divergence in U. virens. By contrast, intraspecific genome comparisons uncovered dynamic genomic alterations including massive structural variations and widespread lineage-specific regions (LSRs) among U. virens strains, which were mainly generated by recent burst of retrotransposons. Genomic plasticity created by structural variations and LSRs might drive rapid evolution of U. virens. High-quality mitochondrial genomes of eight U. virens strains exhibit size variations from 94 to 102 kb. Consistently, U. virens contains conserved lengths of exons and highly dynamic mobile introns, which contribute to intraspecific size variations due to gain/loss of homing endonuclease genes. This study highlights unique characteristics in nuclear and mitochondrial genomic divergence and provides new insights into genomic and mitochondrial evolution of U. virens.

The Mitochondrial Genome of the Sea Anemone Stichodactyla haddoni Reveals Catalytic Introns, Insertion-Like Element, and Unexpected Phylogeny

A hallmark of sea anemone mitochondrial genomes (mitogenomes) is the presence of complex catalytic group I introns. Here, we report the complete mitogenome and corresponding transcriptome of the carpet sea anemone Stichodactyla haddoni (family Stichodactylidae). The mitogenome is vertebrate-like in size, organization, and gene content. Two mitochondrial genes encoding NADH dehydrogenase subunit 5 (ND5) and cytochrome c oxidase subunit I (COI) are interrupted with complex group I introns, and one of the introns (ND5-717) harbors two conventional mitochondrial genes (ND1 and ND3) within its sequence. All the mitochondrial genes, including the group I introns, are expressed at the RNA level. Nonconventional and optional mitochondrial genes are present in the mitogenome of S. haddoni. One of these gene codes for a COI-884 intron homing endonuclease and is organized in-frame with the upstream COI exon. The insertion-like orfA is expressed as RNA and translocated in the mitogenome as compared with other sea anemones. Phylogenetic analyses based on complete nucleotide and derived protein sequences indicate that S. haddoni is embedded within the family Actiniidae, a finding that challenges current taxonomy.