Biology subjects

Barrales, R. R.

Publications and source records attributed to Barrales, R. R..

8 recordsLinked to original sources

bioRxiv2026

Perinuclear anchoring of telomeres enables plant infection by Ustilago maydis

The spatial organization of the genome is critical for cellular function, yet its contribution to the success of host-pathogen interactions remains poorly understood. A conserved hallmark of nuclear architecture is the perinuclear localization of heterochromatin, which includes not only specific chromosomal subdomains but also major structural elements like centromeres and telomeres. To determine the importance of this organization during fungal pathogenesis, we used the maize pathogen Ustilago maydis as an infection model system. We identify the lamina-associated protein homolog, Lem2, as a molecular tether for telomeres and show its deletion abrogates fungal penetration, causing developmental arrest at the appressorium stage. Mechanistically, this infection defect is associated with a failure in nuclear migration. Using hyphal filaments induced under axenic conditions, we observed that mutant nuclei consistently fail to transit from the mother cell into the growing filament. Furthermore, RNA-seq analysis correlates this failure with a marked misregulation of the DNA damage response (DDR) and cell cycle control, including the aberrant expression of multiple checkpoint and signaling proteins. Crucially, artificially tethering telomeres to the nuclear periphery in this mutant partially restores the nuclear migration defect and plant penetration, while simultaneously suppressing the aberrant DDR. This work establishes a functional link between a specific spatial nuclear configuration and fungal infectivity, revealing the conserved telomere-anchoring machinery as a potential target for novel antifungal strategies.

molecular biology↗

bioRxiv2024

A systematic quantitative approach comprehensively defines domain-specific functional pathways linked to Schizosaccharomyces pombe heterochromatin regulation

Heterochromatin plays a critical role in regulating gene expression and maintaining genome integrity. While structural and enzymatic components have been linked to heterochromatin establishment, a comprehensive view of the underlying pathways at diverse heterochromatin domains remains elusive. Here, we developed a systematic approach to identify factors involved in heterochromatin silencing at pericentromeres, subtelomeres, and the silent mating type locus in Schizosaccharomyces pombe. Using quantitative measures, iterative genetic screening, and domain-specific heterochromatin reporters, we identified 369 mutants with different degrees of reduced or enhanced silencing. As expected, mutations in the core heterochromatin machinery globally decreased silencing. However, most other mutants exhibited distinct qualitative and quantitative profiles that indicate domain-specific functions. For example, decreased mating type silencing was linked to mutations in heterochromatin maintenance genes, while compromised subtelomere silencing was associated with metabolic pathways. Furthermore, similar phenotypic profiles revealed shared functions for subunits within complexes. We also discovered that the uncharacterized protein Dhm2 plays a crucial role in maintaining constitutive and facultative heterochromatin, while its absence caused phenotypes akin to DNA replication-deficient mutants. Collectively, our systematic approach unveiled a landscape of domain-specific heterochromatin regulators controlling distinct states and identified Dhm2 as a previously unknown factor linked to heterochromatin inheritance and replication fidelity.

genomics↗

bioRxiv2023

Row1, a member of a new family of conserved fungal proteins involved in infection, is required for appressoria functionality in Ustilago maydis

The appressorium of phytopathogenic fungi is a specific structure with a crucial role in plant cuticle penetration. Pathogens with melanized appressoria break the cuticle through cell wall melanization and intracellular turgor pressure. However, in fungi with non-melanized appressorium, the mechanisms governing cuticle penetration are poorly understood. Here we characterize Row1, a previously uncharacterized appressoria-specific protein of Ustilago maydis that localizes to membrane and secretory vesicles. Deletion of row1 decrease appressoria formation and plant penetration, thereby reducing virulence. Specifically, the {Delta}row1 mutant has a thicker cell wall that is more resistant to glucanase degradation. We also observed that the {Delta}row1 mutant has secretion defects. Our data suggest that Row1 could modify the glucans that form the fungal cell wall and may be involved in unconventional protein secretion, thereby promoting both appressoria maturation and penetration. We show that Row1 is functionally conserved at least among Ustilaginaceae and belongs to the Row family, which consists of five other proteins that are highly conserved among Basidiomycota fungi and are involved in U. maydis virulence. We observed similarities in localization between Row1 and Row2, which is also involved in cell wall remodelling and secretion, suggesting similar molecular functions for members of this protein family.

microbiology↗

bioRxiv2023

Systematic characterization of Ustilago maydis sirtuins shows Sir2 as a modulator of pathogenic gene expression

Phytopathogenic fungi must adapt to the different environmental conditions found during infection and avoid the immune response of the plant. For these adaptations, fungi must tightly control gene expression, allowing sequential changes in transcriptional programs. In addition to transcription factors, chromatin modification is used by eukaryotic cells as a different layer of transcriptional control. Specifically, the acetylation of histones is one of the chromatin modifications with a strong impact on gene expression. Hyperacetylated regions usually correlate with high transcription and hypoacetylated areas with low transcription. Thus, histone deacetylases (HDACs) commonly act as repressors of transcription. One member of the family of HDACs is represented by sirtuins, which are deacetylases dependent on NAD+, and, thus, their activity is considered to be related to the physiological stage of the cells. This property makes sirtuins good regulators during environmental changes. However, only a few examples exist, and with differences in the extent of the implication of the role of sirtuins during fungal phytopathogenesis. In this work, we have performed a systematic study of sirtuins in the maize pathogen Ustilago maydis, finding Sir2 to be involved in the dimorphic switch from yeast cell to filament and pathogenic development. Specifically, the deletion of sir2 promotes filamentation, whereas its overexpression highly reduces tumor formation in the plant. Moreover, transcriptomic analysis revealed that Sir2 represses genes that are expressed during biotrophism development. Interestingly, our results suggest that this repressive effect is not through histone deacetylation, indicating a different target of Sir2 in this fungus.

molecular biology↗

bioRxiv2021

The inner nuclear membrane protein Lem2 coordinates RNA degradation at the nuclear periphery

Transcriptionally silent chromatin often localizes to the nuclear periphery. However, whether the nuclear envelope (NE) is a site for post-transcriptional gene repression is unknown. Here we demonstrate that S. pombe Lem2, an NE protein, regulates nuclear exosome-mediated RNA degradation. Lem2 deletion causes accumulation of non-coding RNAs and meiotic transcripts. Indeed, an engineered exosome substrate RNA shows Lem2-dependent localization to the nuclear periphery. Lem2 does not directly bind RNA, but instead physically interacts with the exosome-targeting MTREC complex and promotes RNA recruitment. The Lem2-assisted pathway acts independently of nuclear bodies where exosome factors assemble, revealing that multiple spatially distinct degradation pathways exist. The Lem2 pathway is environmentally responsive: nutrient availability modulates Lem2 regulation of meiotic transcripts. Our data indicate that Lem2 recruits exosome co-factors to the nuclear periphery to coordinate RNA surveillance and regulates transcripts during the mitosis-to-meiosis switch.

molecular biology↗

bioRxiv2020

Local chromatin context dictates the genetic determinants of the heterochromatin spreading reaction

Heterochromatin spreading, the expansion of gene-silencing structures from DNA-encoded nucleation sites, occurs in distinct settings. Spreading re-establishes gene-poor constitutive heterochromatin every cell cycle, but also invades gene-rich euchromatin de novo to steer cell fate decisions. How chromatin context, i.e. euchromatic, heterochromatic, or different nucleator types, influences the determinants of this process remains poorly understood. By screening a nuclear function gene deletion library in fission yeast using a previously established heterochromatin spreading sensor system, we identified regulators that positively or negatively alter the propensity of a nucleation site to spread heterochromatin. We find that different chromatin contexts are dependent on unique sets of genes for the regulation of heterochromatin spreading. Further, we find that spreading in constitutive heterochromatin requires Clr6 histone deacetylase complexes containing the Fkh2 transcription factor, while the Clr3 deacetylase is globally required for silencing. Fkh2 acts by recruiting Clr6 to nucleation-distal chromatin sites. Our results segregate the pathways that control lateral heterochromatin spreading from those that instruct DNA-directed assembly in nucleation.

molecular biology↗

bioRxiv2019

Set1/COMPASS repels heterochromatin invasion at euchromatic sites by disrupting Suv39/Clr4 activity and nucleosome stability

Protection of euchromatin from invasion by gene-repressive heterochromatin is critical for cellular health and viability. In addition to constitutive loci such as pericentromeres and subtelomeres, heterochromatin can be found interspersed in gene-rich euchromatin, where it regulates gene expression pertinent to cell fate. While hetero- and euchromatin are globally poised for mutual antagonism, the mechanisms underlying precise spatial encoding of heterochromatin containment within euchromatic sites remain opaque. We investigated ectopic heterochromatin invasion by manipulating the fission yeast mating type locus boundary, using a single-cell spreading reporter system. We found that heterochromatin repulsion is locally encoded by Set1/COMPASS on certain actively transcribed genes and that this protective role is most prominent at heterochromatin islands, small domains interspersed in euchromatin that regulate cell fate specifiers. Interestingly, this effect can be gene orientation dependent. Sensitivity to invasion by heterochromatin, surprisingly, is not dependent on Set1 altering overall gene expression levels. At least two independent pathways direct this Set1 activity-inhibition of catalysis by Suv39/Clr4 and disruption of nucleosome stability. Taken together, these results describe a mechanism for spatial encoding of euchromatic signals that repel heterochromatin invasion.

genetics↗

bioRxiv2019

N-glycosylation of the protein disulfide isomerase Pdi1 ensures Ustilago maydis virulence

Fungal pathogenesis depends on accurate secretion and location of virulence factors which drive host colonization. Protein glycosylation is a common posttranslational modification of cell wall components and other secreted factors, typically required for correct protein localization, secretion and function. Thus, the absence of glycosylation is associated with animal and plant pathogen avirulence. While the relevance of protein glycosylation for pathogenesis has been well established, the main glycoproteins responsible for the loss of virulence observed in glycosylation-defective fungi have not been identified. Here, we devise a proteomics approach to identify such proteins and use it to demonstrate a role for the highly conserved protein disulfide isomerase Pdi1 in virulence. We show that efficient Pdi1 N-glycosylation, which promotes folding into the correct protein conformation, is required for full pathogenic development of the corn smut fungus Ustilago maydis. Remarkably, the observed virulence defects are reminiscent of those seen in glycosylation-defective cells suggesting that the N-glycosylation of Pdi1 is necessary for the full secretion of virulence factors. All these observations, together with the fact that Pdi1 protein and RNA expression levels rise upon virulence program induction, suggest that Pdi1 glycosylation is a crucial event for pathogenic development in U. maydis. Our results provide new insights into the role of glycosylation in fungal pathogenesis.\n\nAuthor summaryFungal pathogens require virulence factors to be properly secreted and localized to guarantee complete infection. In common with many proteins, virulence factors must be post-translationally modified by glycosylation for normal localization, secretion and function. This is especially important for virulence factors, which are mainly comprised of cell wall and secreted proteins. Aberrant glycosylation leads to a loss of virulence in both animal and plant pathogenic fungi. We have previously demonstrated that glycosylation is important for virulence of the corn smut fungus, Ustilago maydis. However, the glycoproteins involved and their specific roles in the infection process have not yet been reported. Here, we describe a proteomic assay designed to identify glycoproteins involved in plant infection. Using this method, we define the role of Pdi1 protein disulfide isomerase in virulence. Interestingly, abolishing Pdi1 N-glycosylation mimics {Delta}pdi1 defects observed during infection, suggesting that Pdi1 N-glycosylation is required for the secretion of virulence factors. We hypothesize that Pdi1 N-glycosylation is crucial for maintaining proper effector protein folding during the infection process, especially in the harsh conditions found inside the maize plant.

microbiology↗