Quantitative analysis of protein-RNA interactions in fission yeast.

Characterizing the interactions between RNAs and proteins in vivo is key to better understand how organisms regulate gene expression. Here, we describe a robust and quantitative protocol to measure specific RNA-protein interactions in a native context using RNA immunoprecipitation (RIP). We provide a comprehensive experimental framework to detect cotranslational interactions and detail the quantitative analysis of purified RNAs by PCR and high-throughput sequencing. Although we developed the protocol in fission yeast, it can be readily implemented in other yeast species. For complete details on the use and execution of this protocol, please refer to Toullec et al. (2021).

Nuclear ingression of cytoplasmic bodies accompanies a boost in autophagy.

Membrane contact sites are functional nodes at which organelles reorganize metabolic pathways and adapt to changing cues. In Saccharomyces cerevisiae, the nuclear envelope subdomain surrounding the nucleolus, very plastic and prone to expansion, can establish contacts with the vacuole and be remodeled in response to various metabolic stresses. While using genotoxins with unrelated purposes, we serendipitously discovered a fully new remodeling event at this nuclear subdomain: the nuclear envelope partitions into its regular contact with the vacuole and a dramatic internalization within the nucleus. This leads to the nuclear engulfment of a globular, cytoplasmic portion. In spite of how we discovered it, the phenomenon is likely DNA damage-independent. We define lipids supporting negative curvature, such as phosphatidic acid and sterols, as bona fide drivers of this event. Mechanistically, we suggest that the engulfment of the cytoplasm triggers a suction phenomenon that enhances the docking of proton pump-containing vesicles with the vacuolar membrane, which we show matches a boost in autophagy. Thus, our findings unveil an unprecedented remodeling of the nucleolus-surrounding membranes with impact on metabolic adaptation.

The Hsp90 cochaperone TTT promotes cotranslational maturation of PIKKs prior to complex assembly.

Phosphatidylinositol 3-kinase-related kinases (PIKKs) are a family of kinases that control fundamental processes, including cell growth, DNA damage repair, and gene expression. Although their regulation and activities are well characterized, little is known about how PIKKs fold and assemble into active complexes. Previous work has identified a heat shock protein 90 (Hsp90) cochaperone, the TTT complex, that specifically stabilizes PIKKs. Here, we describe a mechanism by which TTT promotes their de novo maturation in fission yeast. We show that TTT recognizes newly synthesized PIKKs during translation. Although PIKKs form multimeric complexes, we find that they do not engage in cotranslational assembly with their partners. Rather, our findings suggest a model by which TTT protects nascent PIKK polypeptides from misfolding and degradation because PIKKs acquire their native state after translation is terminated. Thus, PIKK maturation and assembly are temporally segregated, suggesting that the biogenesis of large complexes requires both dedicated chaperones and cotranslational interactions between subunits.

Chaperone-mediated ordered assembly of the SAGA and NuA4 transcription co-activator complexes in yeast.

Transcription initiation involves the coordinated activities of large multimeric complexes, but little is known about their biogenesis. Here we report several principles underlying the assembly and topological organization of the highly conserved SAGA and NuA4 co-activator complexes, which share the Tra1 subunit. We show that Tra1 contributes to the overall integrity of NuA4, whereas, within SAGA, it specifically controls the incorporation of the de-ubiquitination module (DUB), as part of an ordered assembly pathway. Biochemical and functional analyses reveal the mechanism by which Tra1 specifically interacts with either SAGA or NuA4. Finally, we demonstrate that Hsp90 and its cochaperone TTT promote Tra1 de novo incorporation into both complexes, indicating that Tra1, the sole pseudokinase of the PIKK family, shares a dedicated chaperone machinery with its cognate kinases. Overall, our work brings mechanistic insights into the assembly of transcriptional complexes and reveals the contribution of dedicated chaperones to this process.

New insights into the evolutionary conservation of the sole PIKK pseudokinase Tra1/TRRAP.

Phosphorylation by protein kinases is a fundamental mechanism of signal transduction. Many kinase families contain one or several members that, although evolutionarily conserved, lack the residues required for catalytic activity. Studies combining structural, biochemical, and functional approaches revealed that these pseudokinases have crucial roles in vivo and may even represent attractive targets for pharmacological intervention. Pseudokinases mediate signal transduction by a diversity of mechanisms, including allosteric regulation of their active counterparts, assembly of signaling hubs, or modulation of protein localization. One such pseudokinase, named Tra1 in yeast and transformation/transcription domain-associated protein (TRRAP) in mammals, is the only member lacking all catalytic residues within the phosphatidylinositol 3-kinase related kinase (PIKK) family of kinases. PIKKs are related to the PI3K family of lipid kinases, but function as Serine/Threonine protein kinases and have pivotal roles in diverse processes such as DNA damage sensing and repair, metabolic control of cell growth, nonsense-mediated decay, or transcription initiation. Tra1/TRRAP is the largest subunit of two distinct transcriptional co-activator complexes, SAGA and NuA4/TIP60, which it recruits to promoters upon transcription factor binding. Here, we review our current knowledge on the Tra1/TRRAP pseudokinase, focusing on its role as a scaffold for SAGA and NuA4/TIP60 complex assembly and recruitment to chromatin. We further discuss its evolutionary history within the PIKK family and highlight recent findings that reveal the importance of molecular chaperones in pseudokinase folding, function, and conservation.

Chromatin modification factors in plant pathogenic fungi: Insights from Ustilago maydis.

Adaptation to the environment is a requirement for the survival of every organism. For pathogenic fungi this also implies coping with the different conditions that occur during the infection cycle. After detecting changes to external media, organisms must modify their gene expression patterns in order to accommodate the new circumstances. Control of gene expression is a complex process that involves the coordinated action of multiple regulatory elements. Chromatin modification is a well-known mechanism for controlling gene expression in response to environmental changes in all eukaryotes. In pathogenic fungi, chromatin modifications are known to play crucial roles in controlling host interactions and their virulence capacity, yet little is known about the specific genes they directly target and to which signals they respond. The smut fungus Ustilago maydis is an excellent model system in which multiple molecular and cellular approaches are available to study biotrophic interactions. Many target genes regulated during the infection process have been well studied, however, how they are controlled and specifically how chromatin modifications affect gene regulation in the context of infection is not well known in this organism. Here, we analyse the presence of chromatin modifying enzymes and complexes in U. maydis and discuss their putative roles in this plant pathogen in the context of findings from other organisms, including other plant pathogens such as Magnaporthe oryzae and Fusarium graminearum. We propose U. maydis as a remarkable organism with interesting chromatin features, which would allow finding new functions of chromatin modifications during plant pathogenesis.

The Hos2 Histone Deacetylase Controls Ustilago maydis Virulence through Direct Regulation of Mating-Type Genes.

Morphological changes are critical for host colonisation in plant pathogenic fungi. These changes occur at specific stages of their pathogenic cycle in response to environmental signals and are mediated by transcription factors, which act as master regulators. Histone deacetylases (HDACs) play crucial roles in regulating gene expression, for example by locally modulating the accessibility of chromatin to transcriptional regulators. It has been reported that HDACs play important roles in the virulence of plant fungi. However, the specific environment-sensing pathways that control fungal virulence via HDACs remain poorly characterised. Here we address this question using the maize pathogen Ustilago maydis. We find that the HDAC Hos2 is required for the dimorphic switch and pathogenic development in U. maydis. The deletion of hos2 abolishes the cAMP-dependent expression of mating type genes. Moreover, ChIP experiments detect Hos2 binding to the gene bodies of mating-type genes, which increases in proportion to their expression level following cAMP addition. These observations suggest that Hos2 acts as a downstream component of the cAMP-PKA pathway to control the expression of mating-type genes. Interestingly, we found that Clr3, another HDAC present in U. maydis, also contributes to the cAMP-dependent regulation of mating-type gene expression, demonstrating that Hos2 is not the only HDAC involved in this control system. Overall, our results provide new insights into the role of HDACs in fungal phytopathogenesis.

Histone deacetylases: revealing the molecular base of dimorphism in pathogenic fungi.

Fungi, as every living organism, interact with the external world and have to adapt to its fluctuations. For pathogenic fungi, such interaction involves adapting to the hostile environment of their host. Survival depends on the capacity of fungi to detect and respond to external stimuli, which is achieved through a tight and efficient genetic control. Chromatin modifications represent a well-known layer of regulation that controls gene expression in response to environmental signals. However, less is known about the chromatin modifications that are involved in fungal virulence and the specific cues and signalling pathways that target chromatin modifications to specific genes. In a recently published study, our research group identified one such regulatory pathway. We demonstrated that the histone deacetylase (HDAC) Hos2 is involved in yeast-to-hyphal transition (dimorphism) and it is associated with the virulence of the maize pathogen Ustilago maydis, the causative agent of smut disease in corn. Hos2 activates mating-type genes by directly binding to their gene bodies. Furthermore, Hos2 acts downstream of the nutrient-sensing cyclic AMP-Protein Kinase A pathway. We also found that another HDAC, Clr3, contributes to this regulation, possibly in cooperation with Hos2. As a whole, our data suggest that there is a direct link between changes in the environment and acetylation of nucleosomes within certain genes. We propose that histone acetylation is critical to the proper timing and induction of transcription of the genes encoding factors that coordinate changes in morphology with pathogenesis.

Endoplasmic reticulum glucosidases and protein quality control factors cooperate to establish biotrophy in Ustilago maydis.

Secreted fungal effectors mediate plant-fungus pathogenic interactions. These proteins are typically N-glycosylated, a common posttranslational modification affecting their location and function. N-glycosylation consists of the addition, and subsequent maturation, of an oligosaccharide core in the endoplasmic reticulum (ER) and Golgi apparatus. In this article, we show that two enzymes catalyzing specific stages of this pathway in maize smut (Ustilago maydis), glucosidase I (Gls1) and glucosidase II ß-subunit (Gas2), are essential for its pathogenic interaction with maize (Zea mays). Gls1 is required for the initial stages of infection following appressorium penetration, and Gas2 is required for efficient fungal spreading inside infected tissues. While U. maydis Δgls1 cells induce strong plant defense responses, Δgas2 hyphae are able to repress them, showing that slight differences in the N-glycoprotein processing can determine the extent of plant-fungus interactions. Interestingly, the calnexin protein, a central element of the ER quality control system for N-glycoproteins in eukaryotic cells, is essential for avoiding plant defense responses in cells with defective N-glycoproteins processing. Thus, N-glycoprotein maturation and this conserved checkpoint appear to play an important role in the establishment of an initial biotrophic state with the plant, which allows subsequent colonization.

Identification of O-mannosylated virulence factors in Ustilago maydis.

The O-mannosyltransferase Pmt4 has emerged as crucial for fungal virulence in the animal pathogens Candida albicans or Cryptococcus neoformans as well as in the phytopathogenic fungus Ustilago maydis. Pmt4 O-mannosylates specific target proteins at the Endoplasmic Reticulum. Therefore a deficient O-mannosylation of these target proteins must be responsible for the loss of pathogenicity in pmt4 mutants. Taking advantage of the characteristics described for Pmt4 substrates in Saccharomyces cerevisiae, we performed a proteome-wide bioinformatic approach to identify putative Pmt4 targets in the corn smut fungus U. maydis and validated Pmt4-mediated glycosylation of candidate proteins by electrophoretic mobility shift assays. We found that the signalling mucin Msb2, which regulates appressorium differentiation upstream of the pathogenicity-related MAP kinase cascade, is O-mannosylated by Pmt4. The epistatic relationship of pmt4 and msb2 showed that both are likely to act in the same pathway. Furthermore, constitutive activation of the MAP kinase cascade restored appressorium development in pmt4 mutants, suggesting that during the initial phase of infection the failure to O-mannosylate Msb2 is responsible for the virulence defect of pmt4 mutants. On the other hand we demonstrate that during later stages of pathogenic development Pmt4 affects virulence independently of Msb2, probably by modifying secreted effector proteins. Pit1, a protein required for fungal spreading inside the infected leaf, was also identified as a Pmt4 target. Thus, O-mannosylation of different target proteins affects various stages of pathogenic development in U. maydis.

The general transcriptional repressor Tup1 is required for dimorphism and virulence in a fungal plant pathogen.

A critical step in the life cycle of many fungal pathogens is the transition between yeast-like growth and the formation of filamentous structures, a process known as dimorphism. This morphological shift, typically triggered by multiple environmental signals, is tightly controlled by complex genetic pathways to ensure successful pathogenic development. In animal pathogenic fungi, one of the best known regulators of dimorphism is the general transcriptional repressor, Tup1. However, the role of Tup1 in fungal dimorphism is completely unknown in plant pathogens. Here we show that Tup1 plays a key role in orchestrating the yeast to hypha transition in the maize pathogen Ustilago maydis. Deletion of the tup1 gene causes a drastic reduction in the mating and filamentation capacity of the fungus, in turn leading to a reduced virulence phenotype. In U. maydis, these processes are controlled by the a and b mating-type loci, whose expression depends on the Prf1 transcription factor. Interestingly, Δtup1 strains show a critical reduction in the expression of prf1 and that of Prf1 target genes at both loci. Moreover, we observed that Tup1 appears to regulate Prf1 activity by controlling the expression of the prf1 transcriptional activators, rop1 and hap2. Additionally, we describe a putative novel prf1 repressor, named Pac2, which seems to be an important target of Tup1 in the control of dimorphism and virulence. Furthermore, we show that Tup1 is required for full pathogenic development since tup1 deletion mutants are unable to complete the sexual cycle. Our findings establish Tup1 as a key factor coordinating dimorphism in the phytopathogen U. maydis and support a conserved role for Tup1 in the control of hypha-specific genes among animal and plant fungal pathogens.

Protein glycosylation in the phytopathogen Ustilago maydis: From core oligosaccharide synthesis to the ER glycoprotein quality control system, a genomic analysis.

The corn smut fungus Ustilago maydis has, over recent decades, become established as a robust pathogenic model for studying fungi-plant relationships. This use of U. maydis can be attributed to its biotrophic host interaction, easy culture and genetic manipulation in the laboratory, and the severe disease symptoms it induces in infected maize. Recent studies have shown that normal protein glycosylation is essential for pathogenic development, but dispensable for the saprophytic growth or mating. Given the relevance of protein glycosylation for U. maydis virulence, and consequently its role in the plant pathogenesis, here we review the main actors and events implicated in protein glycosylation. Furthermore, we describe the results of an in silico search, where we identify all the conserved members of the N- and O-glycosylation pathways in U. maydis at each stage: core oligosaccharide synthesis, addition of the core oligosaccharide to nascent target proteins, maturation and extension of the core oligosaccharide, and the quality control system used by the cell to avoid the presence of unfolded glycoproteins. Finally, we discuss how these genes could affect U. maydis virulence and their biotechnological implications.

The requirement for protein O-mannosylation for Ustilago maydis virulence seems to be linked to intrinsic aspects of the infection process rather than an altered plant response.

Fungal plant pathogenesis involves complex crosstalk between fungi and their plant hosts. In the case of biotrophic fungi, the host interaction is finely controlled to maintain plant viability during infection since the fungus depends on the survival of colonized plant cells. Many proteins which participate in this process are thought to be glycosylated. Thus, defects in the glycosylation of fungal proteins might alter the normally attenuated plant response and consequently affect fungal progression. O-mannosyltransferases are responsible for adding mannose residues onto target proteins, with each O-mannosyltransferase having individual target specificities. In an earlier study, we showed that O-mannosylation is essential for Ustilago maydis virulence. We found that the loss of O-mannosyltransferase PMT4 was associated with a reduced formation frequency of the invasive morphogenic structure known as the appressorium, combined with a loss in their ability to penetrate plant cuticle. Here, we discuss the possible molecular causes of these phenotypes and present additional evidence, which argue against an alteration of plant response to fungal infection as the primary cause of the Δpmt4 phenotype.

The O-mannosyltransferase PMT4 is essential for normal appressorium formation and penetration in Ustilago maydis.

In Saccharomyces cerevisiae, the PMT, KRE2/MNT1, and MNN1 mannosyltransferase protein families catalyze the steps of the O-mannosylation pathway, sequentially adding mannoses to target proteins. We have identified members of all three families and analyzed their roles in pathogenesis of the maize smut fungus Ustilago maydis. Furthermore, we have shown that PMT4, one of the three PMT family members in U. maydis, is essential for tumor formation in Zea mays. Significantly, PMT4 seems to be required only for pathogenesis and is dispensable for other aspects of the U. maydis life cycle. We subsequently show that the deletion of pmt4 results in a strong reduction in the frequency of appressorium formation, with the few appressoria that do form lacking the capacity to penetrate the plant cuticle. Our findings suggest that the O-mannosylation pathway plays a key role in the posttranslational modification of proteins involved in the pathogenic development of U. maydis. The fact that PMT homologs are not found in plants may open new avenues for the development of fungal control strategies. Moreover, the discovery of a highly specific requirement for a single O-mannosyltransferase should aid in the identification of the proteins directly involved in fungal plant penetration, thus leading to a better understanding of plant-fungi interactions.