SUMMARYThe genusTrichodermacontains fungi with high relevance for humans, with applications in enzyme production for plant cell wall degradation and use in biocontrol. Here, we provide a broad, comprehensive overview of the genomic content of these species for “hot topic” research aspects, including CAZymes, transport, transcription factors, and development, along with a detailed analysis and annotation of less-studied topics, such as signal transduction, genome integrity, chromatin, photobiology, or lipid, sulfur, and nitrogen metabolism inT. reesei,T. atroviride, andT. virens, and we open up new perspectives to those topics discussed previously. In total, we covered more than 2,000 of the predicted 9,000 to 11,000 genes of eachTrichodermaspecies discussed, which is >20% of the respective gene content. Additionally, we considered available transcriptome data for the annotated genes. Highlights of our analyses include overall carbohydrate cleavage preferences due to the different genomic contents and regulation of the respective genes. We found light regulation of many sulfur metabolic genes. Additionally, a new Golgi 1,2-mannosidase likely involved inN-linked glycosylation was detected, as were indications for the ability ofTrichodermaspp. to generate hybrid galactose-containingN-linked glycans. The genomic inventory of effector proteins revealed numerous compounds unique toTrichoderma, and these warrant further investigation. We found interesting expansions in theTrichodermagenus in several signaling pathways, such as G-protein-coupled receptors, RAS GTPases, and casein kinases. A particularly interesting feature absolutely unique toT. atrovirideis the duplication of the alternative sulfur amino acid synthesis pathway.

Genetic deletion of the essential GTPase Gpn1 or replacement of the endogenous gene by partial loss of function mutants in yeast is associated with multiple cellular phenotypes, including in all cases a marked cytoplasmic retention of RNA polymerase II (RNAPII). Global inhibition of RNAPII-mediated transcription due to malfunction of Gpn1 precludes the identification and study of other cellular function(s) for this GTPase. In contrast to the single Gpn protein present in Archaea, eukaryotic Gpn1 possesses an extension of approximately 100 amino acids at the C-terminal end of the GTPase domain. To determine the importance of this C-terminal extension in Saccharomyces cerevisiae Gpn1, we generated yeast strains expressing either C-terminal truncated (gpn1ΔC) or full-length ScGpn1. We found that ScGpn1ΔC was retained in the cell nucleus, an event physiologically relevant as gpn1ΔC cells contained a higher nuclear fraction of the RNAPII CTD phosphatase Rtr1. gpn1ΔC cells displayed an increased size, a delay in mitosis exit, and an increased sensitivity to the microtubule polymerization inhibitor benomyl at the cell proliferation level and two cellular events that depend on microtubule function: RNAPII nuclear targeting and vacuole integrity. These phenotypes were not caused by inhibition of RNAPII, as in gpn1ΔC cells RNAPII nuclear targeting and transcriptional activity were unaffected. These data, combined with our description here of a genetic interaction between GPN1 and BIK1, a microtubule plus-end tracking protein with a mitotic function, strongly suggest that the ScGpn1 C-terminal tail plays a critical role in microtubule dynamics and mitotic progression in an RNAPII-independent manner.

Fungal blue-light photoreceptors have been proposed as integrators of light and oxidative stress. However, additional elements participating in the integrative pathway remain to be identified. In Trichoderma atroviride, the blue-light regulator (BLR) proteins BLR-1 and -2 are known to regulate gene transcription, mycelial growth, and asexual development upon illumination, and recent global transcriptional analysis revealed that the histone deacetylase-encoding gene hda-2 is induced by light. Here, by assessing responses to stimuli in wild-type and Δhda-2 backgrounds, we evaluate the role of HDA-2 in the regulation of genes responsive to light and oxidative stress. Δhda-2 strains present reduced growth, misregulation of the con-1 gene, and absence of conidia in response to light and mechanical injury. We found that the expression of hda-2 is BLR-1 dependent and HDA-2 in turn is essential for the transcription of early and late light-responsive genes that include blr-1, indicating a regulatory feedback loop. When subjected to reactive oxygen species (ROS), Δhda-2 mutants display high sensitivity whereas Δblr strains exhibit the opposite phenotype. Consistently, in the presence of ROS, ROS-related genes show high transcription levels in wild-type and Δblr strains but misregulation in Δhda-2 mutants. Finally, chromatin immunoprecipitations of histone H3 acetylated at Lys9/ Lys14 on cat-3 and gst-1 promoters display low accumulation of H3K9K14ac in Δblr and Δhda-2 strains, suggesting indirect regulation of ROS-related genes by HDA-2. Our results point to a mutual dependence between HDA-2 and BLR proteins and reveal the role of these proteins in an intricate gene regulation landscape in response to blue light and ROS.

GPN‐loop GTPases 1 and 3 are required for RNA polymerase II (RNAPII) nuclear import. Gpn1 and Gpn3 display some sequence similarity, physically associate, and their protein expression levels are mutually dependent in human cells. We performed here Fluorescence Resonance Energy Transfer (FRET), molecular modeling, and cell biology experiments to understand, and eventually disrupt, human Gpn1–Gpn3 interaction in live HEK293‐AD cells. Transiently expressed EYFP‐Gpn1 and Gpn3‐CFP generated a strong FRET signal, indicative of a very close proximity, in the cytoplasm of HEK293‐AD cells. Molecular modeling of the human Gpn1–Gpn3 heterodimer based on the crystallographic structure of Npa3, the Saccharomyces cerevisiae Gpn1 ortholog, revealed that human Gpn1 and Gpn3 associate through a large interaction surface formed by internal α‐helix 7, insertion 2, and the GPN‐loop from each protein. In site‐directed mutagenesis experiments of interface residues, we identified the W132D and M227D EYFP‐Gpn1 mutants as defective to produce a FRET signal when coexpressed with Gpn3‐CFP. Simultaneous but not individual expression of Gpn1 and Gpn3, with either or both proteins fused to EYFP, retained RNAPII in the cytoplasm and markedly inhibited global transcription in HEK293‐AD cells. Interestingly, the W132D and M227D Gpn1 mutants that showed an impaired ability to interact with Gpn3 by FRET were also unable to delocalize RNAPII in this assay, indicating that an intact Gpn1–Gpn3 interaction is required to display the dominant‐negative effect on endogenous Gpn1/Gpn3 function we described here. Altogether, our results suggest that a Gpn1–Gpn3 strong interaction is critical for their cellular function.

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