The chloroplast trans\it trans-splicing RNA–protein supercomplex from the green alga Chlamydomonas reinhardtii\textit {Chlamydomonas reinhardtii}

In eukaryotes, RNA $\it trans$-splicing is a significant RNA modification process for the end-to-end ligation of exons from separately transcribed primary transcripts to generate mature mRNA. So far, three different categories of RNA $\it trans$-splicing have been found in organisms within a diverse range. Here, we review $\it trans$-splicing of discontinuous group II introns, which occurs in chloroplasts and mitochondria of lower eukaryotes and plants. We discuss the origin of intronic sequences and the evolutionary relationship between chloroplast ribonucleoprotein complexes and the nuclear spliceosome. Finally, we focus on the ribonucleoprotein supercomplex involved in $\it trans$-splicing of chloroplast group II introns from the green alga $\textit {Chlamydomonas reinhardtii}$. This complex has been well characterized genetically and biochemically, resulting in a detailed picture of the chloroplast ribonucleoprotein supercomplex. This information contributes substantially to our understanding of the function of RNA-processing machineries and might provide a blueprint for other splicing complexes involved in $\it trans$- as well as $\it cis$-splicing of organellar intron RNAs

Dicer-dependent biogenesis of small RNAs and evidence for microRNA-like RNAs in the penicillin producing fungus Penicillium chrysogenum\textit {Penicillium chrysogenum}

MicroRNAs (miRNAs) are non-coding small RNAs (sRNAs) that regulate gene expression in a wide range of eukaryotes. In this study, we analyzed regulatory sRNAs in $\textit {Penicillium chrysogenum}$, the industrial producer of the $\beta$-lactam antibiotic penicillin. To identify sRNAs and microRNA-like RNAs (milRNAs) on a global approach, two sRNA sequencing libraries were constructed. One library was created with pooled total RNA, obtained from twelve differently grown cultures (RNA Mix), and the other with total RNA from a single submerged cultivation ($\Delta$$\it ku70$FRT2). Illumina sequencing of both RNA libraries produced 84,322,825 mapped reads. To distinguish between Dicer-dependent and independent sRNA formation, we further constructed two single $\it dicer$ gene mutants ($\Delta$$\it dcl2$ and $\Delta$$\it dcl1$) and a $\it dicer$ double mutant ($\Delta$$\it dcl2$$\Delta$$\it dcl1$) and analyzed an sRNA library from the Dicer-deficient double-mutant. We identified 661 Dicer-dependent loci and $\textit {in silico}$ prediction revealed 34 milRNAs. Northern blot hybridization of two milRNAs provided evidence for mature milRNAs that are processed either in a complete or partial Dicer-dependent manner from an RNA precursor. Identified milRNAs share typical characteristics of previously discovered fungal milRNAs, like a strong preference for a 5' uracil and the typical length distribution. The detection of potential milRNA target sites in the genome suggests that milRNAs might play a role in posttranscriptional gene regulation. Our data will further increase our knowledge of sRNA dependent gene regulation processes, which is an important prerequisite to develop more effective strategies for improving industrial fermentations with $\textit {P. chrysogenum}$

The Penicillium chrysogenum tom1\textit {Penicillium chrysogenum tom1} gene a major target of transcription factor MAT1-1-1 encodes a nuclear protein involved in sporulation

Fungal mating-type loci ($\it MAT$) encode transcription factors (TFs) MAT1-1-1 and MAT1-2-1, which govern sexual reproduction as well as other developmental processes. In $\textit {Penicillium chrysogenum}$, the major producer of the beta-lactam antibiotic penicillin, a recent chromatin immunoprecipitation followed by sequencing (ChIP-seq) analysis identified 254 genes as direct targets of MAT1-1-1, many of which encode thus far uncharacterized proteins. Here, we characterized one of the major targets of MAT1-1-1, the $\it tom1$ gene, which encodes a protein highly conserved within the group of Eurotiomycetes fungi. Using fluorescence microscopy, we demonstrated binding of MAT1-1-1 to the $\it tom1$ promoter by reporter gene analysis. Extensive electrophoretic mobility shift assays (EMSAs) further showed that the promoter sequence of $\it tom1$ is bound $\textit {in vitro}$ by both MAT1-1-1 and MAT1-2-1. This indicated an interaction between the two TFs, which was verified by yeast two-hybrid analysis. The sequence of $\it tom1$ carries a nuclear localization sequence, and indeed its nuclear localization was verified by fluorescence microscopy. The $\textit {in vivo}$ function of $\it tom1$ was investigated using $\it tom1$ deletion strains, as well as a complementing strain where the wild-type $\it tom1$ gene was reintroduced. We found a clear sporulation defect in the deletion strain, which became more evident when the fungi were grown at an elevated temperature of 31°C

STRIPAK, a key regulator of fungal development, operates as a multifunctional signaling hub

The striatin-interacting phosphatases and kinases (STRIPAK) multi subunit complex is a highly conserved signaling complex that controls diverse developmental processes in higher and lower eukaryotes. In this perspective article, we summarize how STRIPAK controls diverse developmental processes in euascomycetes, such as fruiting body formation, cell fusion, sexual and vegetative development, pathogenicity, symbiosis, as well as secondary metabolism. Recent structural investigations revealed information about the assembly and stoichiometry of the complex enabling it to act as a signaling hub. Multiple organellar targeting of STRIPAK subunits suggests how this complex connects several signaling transduction pathways involved in diverse cellular developmental processes. Furthermore, recent phosphoproteomic analysis shows that STRIPAK controls the dephosphorylation of subunits from several signaling complexes. We also refer to recent findings in yeast, where the STRIPAK homologue connects conserved signaling pathways, and based on this we suggest how so far non-characterized proteins may functions as receptors connecting mitophagy with the STRIPAK signaling complex. Such lines of investigation should contribute to the overall mechanistic understanding of how STRIPAK controls development in euascomycetes and beyond

Transcriptome analysis of the two unrelated fungal β\beta-lactam producers Acremonium chrysogenum\textit {Acremonium chrysogenum} and Penicillium chrysogenum\textit {Penicillium chrysogenum}

$\textbf {Background:}$ Cephalosporins and penicillins are the most frequently used $\beta$-lactam antibiotics for the treatment of human infections worldwide. The main industrial producers of these antibiotics are $\textit {Acremonium chrysogenum}$ and $\textit {Penicillium chrysogenum}$, two taxonomically unrelated fungi. Both were subjects of long-term strain development programs to reach economically relevant antibiotic titers. It is so far unknown, whether equivalent changes in gene expression lead to elevated antibiotic titers in production strains. $\textbf {Results:}$ Using the sequence of PcbC, a key enzyme of $\beta$-lactam antibiotic biosynthesis, from eighteen different pro- and eukaryotic microorganisms, we have constructed a phylogenetic tree to demonstrate the distant relationship of both fungal producers. To address the question whether both fungi have undergone similar genetic adaptions, we have performed a comparative gene expression analysis of wild-type and production strains. We found that strain improvement is associated with the remodeling of the transcriptional landscape in both fungi. In $\textit {P. chrysogenum}$, 748 genes showed differential expression, while 1572 genes from $\textit {A. chrysogenum}$ are differentially expressed in the industrial strain. Common in both fungi is the upregulation of genes belonging to primary and secondary metabolism, notably those involved in precursor supply for $\beta$-lactam production. Other genes not essential for $\beta$-lactam production are downregulated with a preference for those responsible for transport processes or biosynthesis of other secondary metabolites. Transcriptional regulation was shown to be an important parameter during strain improvement in different organisms. We therefore investigated deletion strains of the major transcriptional regulator $\it velvet$ from both production strains. We identified 567 $\textit {P. chrysogenum}$ and 412 $\textit {A. chrysogenum}$ Velvet target genes. In both deletion strains, approximately 50% of all secondary metabolite cluster genes are differentially regulated, including $\beta$-lactam biosynthesis genes. Most importantly, 35-57% of Velvet target genes are among those that showed differential expression in both improved industrial strains. $\textbf {Conclusions:}$ The major finding of our comparative transcriptome analysis is that strain improvement programs in two unrelated fungal $\beta$-lactam antibiotic producers alter the expression of target genes of Velvet, a global regulator of secondary metabolism. From these results, we conclude that regulatory alterations are crucial contributing factors for improved $\beta$-lactam antibiotic titers during strain improvement in both fungi

Construction of a codon-adapted Nourseotricin-resistance marker gene for efficient targeted gene deletion in the mycophenolic acid producer Penicillium brevicompactum\textit {Penicillium brevicompactum}

$\textit {Penicillium brevicompactum}$ is a filamentous ascomycete used in the pharmaceutical industry to produce mycophenolic acid, an immunosuppressant agent. To extend options for genetic engineering of this fungus, we have tested two resistance markers that have not previously been applied to $\textit {P. brevicompactum}$. Although a generally available phleomycin resistance marker $\it (ble)$ was successfully used in DNA-mediated transformation experiments, we were not able to use a commonly applicable nourseothricin resistance cassette $\textit {(nat1)}$. To circumvent this failure, we constructed a new nat gene, considering the codon bias for $\textit {P. brevicompactum}$. We then used this modified nat gene in subsequent transformation experiments for the targeted disruption of two nuclear genes, $\textit {MAT1-2-1}$ and $\it flbA$. For $\textit {MAT1-2-1}$, we obtained deletion strains with a frequency of about 10%. In the case of flbA, the frequency was about 4%, and this disruption strain also showed reduced conidiospore formation. To confirm the deletion, we used $\it ble$ to reintroduce the wild-type genes. This step restored the wild-type phenotype in the $\it flbA$ deletion strain, which had a sporulation defect. The successful transformation system described here substantially extends options for genetically manipulating the biotechnologically relevant fungus $\textit {P. brevicompactum}$

RNA editing during sexual development occurs in distantly related filamentous ascomycetes

RNA editing is a post-transcriptional process that modifies RNA molecules leading to transcript sequences that differ from their template DNA. A-to-I editing was found to be widely distributed in nuclear transcripts of metazoa, but was detected in fungi only recently in a study of the filamentous ascomycete $\textit {Fusarium graminearum}$ that revealed extensive A-to-I editing of mRNAs in sexual structures (fruiting bodies). Here, we searched for putative RNA editing events in RNA-seq data from $\textit {Sordaria macrospora}$ and $\textit {Pyronema confluens}$, two distantly related filamentous ascomycetes, and in data from the Taphrinomycete $\textit {Schizosaccharomyces pombe }$. Like $\textit {F. graminearum, S. macrospora }$ is a member of the Sordariomycetes, whereas $\textit {P. confluens}$ belongs to the early-diverging group of Pezizomycetes. We found extensive A-to-I editing in RNA-seq data from sexual mycelium from both filamentous ascomycetes, but not in vegetative structures. A-to-I editing was not detected in different stages of meiosis of $\textit {S. pombe}$. A comparison of A-to-I editing in $\textit {S. macrospora}$ with $\textit {F. graminearum}$ and $\textit {P. confluens}$, respectively, revealed little conservation of individual editing sites. An analysis of RNA-seq data from two sterile developmental mutants of $\textit {S. macrospora}$ showed that A-to-I editing is strongly reduced in these strains. Sequencing of cDNA fragments containing more than one editing site from $\textit {P. confluens}$ showed that at the beginning of sexual development, transcripts were incompletely edited or unedited, whereas in later stages transcripts were more extensively edited. Taken together, these data suggest that A-to-I RNA editing is an evolutionary conserved feature during fruiting body development in filamentous ascomycetes

Phosphoproteomic analysis of STRIPAK mutants identifies a conserved serine phosphorylation site in PAK kinase CLA4 to be important in fungal sexual development and polarized growth

The highly conserved striatin-interacting phosphatases and kinases (STRIPAK) complex regulates phosphorylation/dephosphorylation of developmental proteins in eukaryotic microorganisms, animals and humans. To first identify potential targets of STRIPAK, we performed extensive isobaric tags for relative and absolute quantification-based proteomic and phosphoproteomic analyses in the filamentous fungus $\textit {Sordaria macrospora}$. In total, we identified 4,193 proteins and 2,489 phosphoproteins, which are represented by 10,635 phosphopeptides. By comparing phosphorylation data from wild type and mutants, we identified 228 phosphoproteins to be regulated in all three STRIPAK mutants, thus representing potential targets of STRIPAK. To provide an exemplarily functional analysis of a STRIPAK-dependent phosphorylated protein, we selected CLA4, a member of the conserved p21-activated kinase family. Functional characterization of the $\Delta$cla4 deletion strain showed that CLA4 controls sexual development and polarized growth. To determine the functional relevance of CLA4 phosphorylation and the impact of specific phosphorylation sites on development, we next generated phosphomimetic and -deficient variants of CLA4. This analysis identified (de)phosphorylation of a highly conserved serine (S685) residue in the catalytic domain of CLA4 as being important for fungal cellular development. Collectively, these analyses significantly contribute to the understanding of the mechanistic function of STRIPAK as a phosphatase and kinase signaling complex

Genome-wide chromatin immunoprecipitation sequencing analysis of the Penicillium chrysogenum\textit {Penicillium chrysogenum} velvet protein PcVelA as a novel downstream regulator of fungal development

$\textit {Penicillium chrysogenum}$ is the sole industrial producer of the $\beta$-lactam antibiotic penicillin, which is the most commonly used drug for treating bacterial infections. In $\textit {P. chrysogenum}$ and other filamentous fungi, secondary metabolism and morphogenesis are controlled by the highly conserved multisubunit velvet complex. Here we present the first chromatin immunoprecipitation next-generation sequencing (ChIP-seq) analysis of a fungal velvet protein, providing experimental evidence that a velvet homologue in $\textit {P. chrysogenum}$ (PcVelA) acts as a direct transcriptional regulator at the DNA level in addition to functioning as a regulator at the protein level in $\textit {P. chrysogenum}$, which was previously described. We identified many target genes that are related to processes known to be dependent on PcVelA, e.g., secondary metabolism as well as asexual and sexual development. We also identified seven PcVelA target genes that encode putative methyltransferases. Yeast two-hybrid and bimolecular fluorescence complementation analyses showed that one of the putative methyltransferases, PcLlmA, directly interacts with PcVelA. Furthermore, functional characterization of PcLlmA demonstrated that this protein is involved in the regulation of conidiosporogenesis, pellet formation, and hyphal morphology, all traits with major biotechnological relevance