Pathogenesis-Related 1 (PR1) Protein Family Genes Involved in Sugarcane Responses to Ustilago scitaminea Stress.

Plant resistance against biotic stressors is significantly influenced by pathogenesis-related 1 (PR1) proteins. This study examines the systematic identification and characterization of PR1 family genes in sugarcane (Saccharum spontaneum Np-X) and the transcript expression of selected genes in two sugarcane cultivars (ROC22 and Zhongtang3) in response to Ustilago scitaminea pathogen infection. A total of 18 SsnpPR1 genes were identified at the whole-genome level and further categorized into four groups. Notably, tandem and segmental duplication occurrences were detected in one and five SsnpPR1 gene pairs, respectively. The SsnpPR1 genes exhibited diverse physio-chemical attributes and variations in introns/exons and conserved motifs. Notably, four SsnpPR1 (SsnpPR1.02/05/09/19) proteins displayed a strong protein-protein interaction network. The transcript expression of three SsnpPR1 (SsnpPR1.04/06/09) genes was upregulated by 1.2-2.6 folds in the resistant cultivar (Zhongtang3) but downregulated in the susceptible cultivar (ROC22) across different time points as compared to the control in response to pathogen infection. Additionally, SsnpPR1.11 was specifically upregulated by 1.2-3.5 folds at 24-72 h post inoculation (hpi) in ROC22, suggesting that this gene may play an important negative regulatory role in defense responses to pathogen infection. The genetic improvement of sugarcane can be facilitated by our results, which also establish the basis for additional functional characterization of SsnpPR1 genes in response to pathogenic stress.

Maize requires arogenate dehydratase 2 for resistance to Ustilago maydis and plant development.

Maize (Zea mays) smut is a common biotrophic fungal disease caused by Ustilago maydis and leads to low maize yield. Maize resistance to U. maydis is a quantitative trait. However, the molecular mechanism underlying the resistance of maize to U. maydis is poorly understood. Here, we reported that a maize mutant caused by a single gene mutation exhibited defects in both fungal resistance and plant development. maize mutant highly susceptible to U. maydis (mmsu) with a dwarf phenotype forms tumors in the ear. A map-based cloning and allelism test demonstrated that 1 gene encoding a putative arogenate dehydratase/prephenate dehydratase (ADT/PDT) is responsible for the phenotypes of the mmsu and was designated as ZmADT2. Combined transcriptomic and metabolomic analyses revealed that mmsu had substantial differences in multiple metabolic pathways in response to U. maydis infection compared with the wild type. Disruption of ZmADT2 caused damage to the chloroplast ultrastructure and function, metabolic flux redirection, and reduced the amounts of salicylic acid (SA) and lignin, leading to susceptibility to U. maydis and dwarf phenotype. These results suggested that ZmADT2 is required for maintaining metabolic flux, as well as resistance to U. maydis and plant development in maize. Meanwhile, our findings provided insights into the maize response mechanism to U. maydis infection.

Itaconic acid production by co-feeding of Ustilago maydis: A combined approach of experimental data, design of experiments, and metabolic modeling.

Itaconic acid is a platform chemical with a range of applications in polymer synthesis and is also discussed for biofuel production. While produced in industry from glucose or sucrose, co-feeding of glucose and acetate was recently discussed to increase itaconic acid production by the smut fungus Ustilago maydis. In this study, we investigate the optimal co-feeding conditions by interlocking experimental and computational methods. Flux balance analysis indicates that acetate improves the itaconic acid yield up to a share of 40% acetate on a carbon molar basis. A design of experiment results in the maximum yield of 0.14 itaconic acid per carbon source from 100 g L - 1 $\,\text{g L}{}^{-1}$ glucose and 12 g L - 1 $\,\text{g L}{}^{-1}$ acetate. The yield is improved by around 22% when compared to feeding of glucose as sole carbon source. To further improve the yield, gene deletion targets are discussed that were identified using the metabolic optimization tool OptKnock. The study contributes ideas to reduce land use for biotechnology by incorporating acetate as co-substrate, a C2-carbon source that is potentially derived from carbon dioxide.

Novel Secreted Effectors Conserved Among Smut Fungi Contribute to the Virulence of Ustilago maydis.

Fungal pathogens deploy a set of molecules (proteins, specialized metabolites, and sRNAs), so-called effectors, to aid the infection process. In comparison to other plant pathogens, smut fungi have small genomes and secretomes of 20 Mb and around 500 proteins, respectively. Previous comparative genomic studies have shown that many secreted effector proteins without known domains, i.e., novel, are conserved only in the Ustilaginaceae family. By analyzing the secretomes of 11 species within Ustilaginaceae, we identified 53 core homologous groups commonly present in this lineage. By collecting existing mutants and generating additional ones, we gathered 44 Ustilago maydis strains lacking single core effectors as well as 9 strains containing multiple deletions of core effector gene families. Pathogenicity assays revealed that 20 of these 53 mutant strains were affected in virulence. Among the 33 mutants that had no obvious phenotypic changes, 13 carried additional, sequence-divergent, structurally similar paralogs. We report a virulence contribution of seven previously uncharacterized single core effectors and of one effector family. Our results help to prioritize effectors for understanding U. maydis virulence and provide genetic resources for further characterization. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Analysis of the indispensable RAD51 cofactor BRCA2 in Naganishia liquefaciens, a Basidiomycota yeast.

The BRCA2 tumor suppressor plays a critical role in homologous recombination by regulating RAD51, the eukaryotic homologous recombinase. We identified the BRCA2 homolog in a Basidiomycota yeast, Naganishia liquefaciens BRCA2 homologs are found in many Basidiomycota species but not in Ascomycota species. Naganishia BRCA2 (Brh2, for BRCA2 homolog) is about one-third the size of human BRCA2. Brh2 carries three potential BRC repeats with two oligonucleotide/oligosaccharide-binding domains. The homolog of DSS1, a small acidic protein serving as an essential partner of BRCA2 was also identified. The yeast two-hybrid assay shows the interaction of Brh2 with both Rad51 and Dss1. Unlike human BRCA2, Brh2 is not required for normal cell growth, whereas loss of Dss1 results in slow growth. The loss of Brh2 caused pronounced sensitivity to UV and ionizing radiation, and their HR ability, as assayed by gene-targeting efficiency, is compromised. These phenotypes are indistinguishable from those of the rad51 mutant, and the rad51 brh2 double mutant. Naganishia Brh2 is likely the BRCA2 ortholog that functions as an indispensable auxiliary factor for Rad51.

Genome-Wide Characterization of the Maize (Zea mays L.) WRKY Transcription Factor Family and Their Responses to Ustilago maydis.

Members of the WRKY transcription factor (TF) family are unique to plants and serve as important regulators of diverse physiological processes, including the ability of plants to manage biotic and abiotic stressors. However, the functions of specific WRKY family members in the context of maize responses to fungal pathogens remain poorly understood, particularly in response to Ustilago maydis (DC.) Corda (U. maydis), which is responsible for the devastating disease known as corn smut. A systematic bioinformatic approach was herein employed for the characterization of the maize WRKY TF family, leading to the identification of 120 ZmWRKY genes encoded on 10 chromosomes. Further structural and phylogenetic analyses of these TFs enabled their classification into seven different subgroups. Segmental duplication was established as a major driver of ZmWRKY family expansion in gene duplication analyses, while the Ka/Ks ratio suggested that these ZmWRKY genes had experienced strong purifying selection. When the transcriptional responses of these genes to pathogen inoculation were evaluated, seven U. maydis-inducible ZmWRKY genes were identified, as validated using a quantitative real-time PCR approach. All seven of these WKRY proteins were subsequently tested using a yeast one-hybrid assay approach, which revealed their ability to directly bind the ZmSWEET4b W-box element, thereby controlling the U. maydis-inducible upregulation of ZmSWEET4b. These results suggest that these WRKY TFs can control sugar transport in the context of fungal infection. Overall, these data offer novel insight into the evolution, transcriptional regulation, and functional characteristics of the maize WRKY family, providing a basis for future research aimed at exploring the mechanisms through which these TFs control host plant responses to common smut and other fungal pathogens.

A conserved extracellular Ribo1 with broad-spectrum cytotoxic activity enables smut fungi to compete with host-associated bacteria.

Ribotoxins are secreted ribonucleases that specifically target and cleave the universally conserved sarcin-ricin loop sequence of rRNA, which leads to inhibition of protein biosynthesis and subsequently to cell death. We have identified and characterized a secreted Ribo1 protein of plant pathogenic smut fungi. Heterologous expression in different model systems showed that smut Ribo1 has cytotoxic activity against bacteria, yeast, host and nonhost plants. Recombinant expression of Ribo1 in Nicotiana benthamiana induced plant cell death; however, an active site mutant induced cell death only when expressed as a secreted protein. In the maize smut Ustilago maydis, transcription of Ribo1 is specifically induced in early infection stages. While a knockout mutant revealed that Ribo1 is dispensable for U. maydis virulence, the overexpression of Ribo1 in planta had a strong dominant negative effect on virulence and induced host defense responses including cell death. Our findings suggest a function of Ribo1 during the epiphytic development rather than for invasive colonization of the host. Accordingly, in the presence of the biocontrol bacteria Pantoea sp., which were isolated from maize leaves, the ribo1 knockout mutant was significantly impaired in virulence. Together, we conclude that Ribo1 enables smut fungi to compete with host-associated bacteria during epiphytic development.

Insight into the biochemical and cell biological function of an intrinsically unstructured heat shock protein, Hsp12 of Ustilago maydis.

Small heat shock proteins (sHsps) play diverse roles in the stress response and maintenance of cellular functions. The Ustilago maydis genome codes for few sHsps. Among these, Hsp12 has previously been demonstrated to be involved in the pathogenesis of the fungus by our group. In the present study we further investigated the biological function of the protein in the pathogenic development of U. maydis. Analysis of the primary amino acid sequence of Hsp12 in combination with spectroscopic methods to analyse secondary protein structures revealed an intrinsically disordered nature of the protein. We also carried out detailed analysis on the protein aggregation prevention activity associated with Hsp12. Our data suggest Hsp12 has trehalose-dependent protein aggregation prevention activity. Through assaying the interaction of Hsp12 with lipid membranes in vitro we also showed the ability of U. maydis Hsp12 to induce stability in lipid vesicles. U. maydis hsp12 deletion mutants exhibited defects in the endocytosis process and delayed completion of the pathogenic life cycle. Therefore, U. maydis Hsp12 contributes to the pathogenic development of the fungus through its ability to relieve proteotoxic stress during infection as well as its membrane-stabilizing function.

Combination of in vivo proximity labeling and co-immunoprecipitation identifies the host target network of a tumor-inducing effector in the fungal maize pathogen Ustilago maydis.

Plant pathogens secrete effectors, which target host proteins to facilitate infection. The Ustilago maydis effector UmSee1 is required for tumor formation in the leaf during infection of maize. UmSee1 interacts with maize SGT1 (suppressor of G2 allele of skp1) and blocks its phosphorylation in vivo. In the absence of UmSee1, U. maydis cannot trigger tumor formation in the bundle sheath. However, it remains unclear which host processes are manipulated by UmSee1 and the UmSee1-SGT1 interaction to cause the observed phenotype. Proximity-dependent protein labeling involving the turbo biotin ligase tag (TurboID) for proximal labeling of proteins is a powerful tool for identifying the protein interactome. We have generated transgenic U. maydis that secretes biotin ligase-fused See1 effector (UmSee1-TurboID-3HA) directly into maize cells. This approach, in combination with conventional co-immunoprecipitation, allowed the identification of additional UmSee1 interactors in maize cells. Collectively, our data identified three ubiquitin-proteasome pathway-related proteins (ZmSIP1, ZmSIP2, and ZmSIP3) that either interact with or are close to UmSee1 during host infection of maize with U. maydis. ZmSIP3 represents a cell cycle regulator whose degradation appears to be promoted in the presence of UmSee1. Our data provide a possible explanation of the requirement for UmSee1 in tumor formation during U. maydis-Zea mays interaction.

Determinants governing BRC function evaluated by mutational analysis of Brh2 in Ustilago maydis.

BRC is a short evolutionarily conserved sequence motif generally arranged in multiple tandem repeats that is present as a defining feature in members of the BRCA2 tumor suppressor protein family. From crystallographic studies of a co-complex, the human BRC4 was found to form a structural element that interacts with RAD51, a key component in the DNA repair machinery directed by homologous recombination. The BRC is distinguished by two tetrameric sequence modules with characteristic hydrophobic residues separated by an intervening spacer region marked by certain highly conserved residues forming a hydrophobic surface for interaction with RAD51. It is present as a single copy in Brh2 of Ustilago maydis, the only reported example of a fungal BRCA2 ortholog. By comparative sequence analysis, examples of BRCA2 orthologs were identified in other fungal phyla, some of which featured multiple tandem repeats like those found in mammals. An expeditious biological assay system was developed for evaluating the two-tetramer module model and assessing the importance of particular conserved amino acid residues of BRC contributing to Brh2 functionality in DNA repair. This work was aided by the finding that the human BRC4 repeat could substitute completely for the endogenous BRC element in Brh2, while the human BRC5 repeat could not. In a survey of point mutations of certain residues, certain BRC mutant variants termed antimorphs were identified that caused a DNA repair phenotype more severe than the null.

Engineered Sucrose Metabolism Improves the Smut Disease Suppression Potency of Pseudomonas sp. ST4.

Sporisorium scitamineum and Ustilago maydis are two fungal pathogens causing severe sugarcane and maize diseases, respectively. Sexual mating of compatible sporidia is essential for these pathogens to form infections dikaryotic mycelia and cause smut diseases. We showed recently that in the presence of exogenous glucose, the Pseudomonas sp. strain ST4 could block the fungal mating and display a strong disease suppression potency on S. scitamineum. With the aim of conferring strain ST4 the ability to metabolize sucrose in plants for glucose production, we identified a strong native promoter pSsrA in strain ST4 and additional promoter elements to facilitate translation and peptide translocation for the construction of a fusion gene encoding sucrose metabolism. The cscA gene encoding sucrose hydrolase from Pseudomonas protegens Pf-5 was fused to the promoter pSsrA, a translational coupler bicistronic design and a Tat signal peptide, which was then cloned into mini-Tn7 transposon. This synthetic gene cassette was integrated into the chromosome of strain ST4, and the resultant engineered strain ST4E was able to hydrolyze sucrose with high efficiency and displayed elevated inhibitory activity on the mating and virulence of S. scitamineum and U. maydis. The findings from this study provide a valuable device and useful clues for the engineering of sucrose metabolism in non- or weak-sucrose-utilizing bacterial strains and present an improved biocontrol agent against plant smut pathogens. IMPORTANCE Sporisorium scitamineum and Ustilago maydis are typical dimorphic fungi causing severe sugarcane and maize smut diseases, respectively. Sexual mating of compatible sporidia is essential for these pathogens to form infections dikaryotic mycelia and cause smut diseases. We previously demonstrated that the biocontrol strain Pseudomonas sp. ST4 could block the fungal mating and displays a strong suppression potency on smut diseases, while it was unable to utilize the host-sourced sucrose for glucose production critical for antifungus efficiency. In this study, we constructed a high-expression gene cassette for minitransposon-mediated genome integration and sucrose hydrolysis in the bacterial periplasmic space. The resultant engineered strain ST4E was able to hydrolyze sucrose and inhibit the mating and hyphal growth of S. scitamineum and U. maydis. These findings provide a valuable tool and useful clues for the engineering of sucrose metabolism in non- or weak-sucrose-utilizing bacterial strains and present an improved biocontrol agent against plant smut pathogens.

Efficient virus detection utilizing chitin-immobilized nanobodies synthesized in Ustilago maydis.

The COVID-19 pandemic has greatly impacted the global economy and health care systems, illustrating the urgent need for timely and inexpensive responses to pandemic threats in the form of vaccines and antigen tests. Currently, antigen testing is mostly conducted by qualitative flow chromatography or via quantitative ELISA-type assays. The latter mostly utilize materials like protein-adhesive polymers and gold or latex particles. Here we present an alternative ELISA approach using inexpensive, biogenic materials and permitting quick detection based on components produced in the microbial model Ustilago maydis. In this fungus, heterologous proteins like biopharmaceuticals can be exported by fusion to unconventionally secreted chitinase Cts1. As a unique feature, the carrier chitinase binds to chitin allowing its additional use as a purification or immobilization tag. Recent work has demonstrated that nanobodies are suitable target proteins. These proteins represent a very versatile alternative antibody format and can quickly be adapted to detect novel antigens by camelidae immunization or synthetic libraries. In this study, we exemplarily produced different mono- and bivalent SARS-CoV-2 nanobodies directed against the spike protein receptor binding domain (RBD) as Cts1 fusions and screened their antigen binding affinity in vitro and in vivo. Functional nanobody-Cts1 fusions were immobilized on chitin forming an RBD tethering surface. This provides a solid base for future development of inexpensive antigen tests utilizing unconventionally secreted nanobodies as antigen trap and a matching ubiquitous and biogenic surface for immobilization.

Telomerase RNA plays a major role in the completion of the life cycle in Ustilago maydis and shares conserved domains with other Ustilaginales.

The RNA subunit of telomerase is an essential component whose primary sequence and length are poorly conserved among eukaryotic organisms. The phytopathogen Ustilago maydis is a dimorphic fungus of the order Ustilaginales. We analyzed several species of Ustilaginales to computationally identify the TElomere RNA (TER) gene ter1. To confirm the identity of the TER gene, we disrupted the gene and characterized telomerase-negative mutants. Similar to catalytic TERT mutants, ter1Δ mutants exhibit phenotypes of growth delay, telomere shortening and low replicative potential. ter1-disrupted mutants were unable to infect maize seedlings in heterozygous crosses and showed defects such as cell cycle arrest and segregation failure. We concluded that ter1, which encodes the TER subunit of the telomerase of U. maydis, have similar and perhaps more extensive functions than trt1.

Chemical Constituents and Bioactive Principles from the Mexican Truffle and Fermented Products of the Derived Fungus Ustilago maydis MZ496986.

To look in-depth into the traditional Mexican truffle, this study investigated the phytochemical and pharmacological properties of field-collected corn galls and the fermentate of its pathogen Ustilago maydis MZ496986. Here, we established the chemical profiles of both materials via the gradient HPLC-UV method and successfully identified six previously unreported chemical entities, ustilagols A-F (1-6), and 17 known components. Compounds 3, 5, and 9 exhibited potent nitric oxide production inhibitory activities in murine brain microglial BV-2 cells (IC50 = 6.7 ± 0.5, 5.8 ± 0.9, and 3.9 ± 0.1 µM) without cytotoxic effects. DIMBOA (9) also attenuates lipopolysaccharide (LPS)-stimulated NF-κB activation in RAW 264.7 macrophages (IC50 = 58.1 ± 7.2 µM). Ustilagol G (7) showed potent antiplatelet aggregation in U46619-stimulated human platelets (IC50 = 16.5 ± 5.3 µM). These findings highlighted the potential of corn galls and U. maydis MZ496986 fermentate as functional foods for improving inflammation-related discomforts and vascular obstruction.

Ger1 is a secreted aspartic acid protease essential for spore germination in Ustilago maydis.

Ustilago maydis expresses a number of proteases during its pathogenic lifecycle. Some of the proteases including both intracellular and extracellular ones have previously been shown to influence the virulence of the pathogen. However, any role of secreted proteases in the sporulation process of U. maydis have not been explored earlier. In this study we have investigated the biological function of one such secreted protease, Ger1 belonging to aspartic protease A1 family. An assessment of the real time expression of ger1 revealed an infection specific expression of the protein especially during late phases of infection. We also evaluated any contribution of the protein in the pathogenicity of the fungus. Our data revealed an involvement of Ger1 in the sporulation and spore germination processes of U. maydis. Ger1 also showed positive influence on the pathogenicity of the fungus and accordingly the ger1 deletion mutant exhibited reduced pathogenicity. The study also demonstrated the protease activity associated with Ger1 to be essential for its biological function. Fluorescence microscopy of maize plants infected with U. maydis cells expressing Ger1-mcherry-HA also revealed that Ger1 is efficiently secreted within maize apoplast.

The Rheb GTPase promotes pheromone blindness via a TORC1-independent pathway in the phytopathogenic fungus Ustilago maydis.

The target of the rapamycin (TOR) signaling pathway plays a negative role in controlling virulence in phytopathogenic fungi. However, the actual targets involved in virulence are currently unknown. Using the corn smut fungus Ustilago maydis, we tried to address the effects of the ectopic activation of TOR on virulence. We obtained gain-of-function mutations in the Rheb GTPase, one of the conserved TOR kinase regulators. We have found that unscheduled activation of Rheb resulted in the alteration of the proper localization of the pheromone receptor, Pra1, and thereby pheromone insensitivity. Since pheromone signaling triggers virulence in Ustilaginales, we believe that the Rheb-induced pheromone blindness was responsible for the associated lack of virulence. Strikingly, although these effects required the concourse of the Rsp5 ubiquitin ligase and the Art3 α-arrestin, the TOR kinase was not involved. Several eukaryotic organisms have shown that Rheb transmits environmental information through TOR-dependent and -independent pathways. Therefore, our results expand the range of signaling manners at which environmental conditions could impinge on the virulence of phytopathogenic fungi.

Germicide Fenaminosulf Promots Gall Formation of Zizania latifolia without directly affecting the growth of endophytic fungus Ustilago esculenta.

Zizania latifolia is a popular aquatic vegetable in China because of its enlarged edible stems resulting from persistent infection by a fungal endophyte, Ustilago esculenta. Fenaminosulf (FM) is a germicide that can be used to improve agricultural crop yields. In Z. latifolia fields, appropriate spraying of FM not just controls diseases, but also promotes an earlier harvest of Z. latifolia. In this study, we show that the timing of gall formation was advanced and the plant's yield was increased significantly under a high concentration treatment of FM. Yet FM had a strong inhibitory effect on the growth of U. esculenta in vitro, while the transcript levels of mating-type alleles, cell metabolism-related genes and chitin synthase genes were all substantially downregulated. Through a transcriptome analysis, we investigated changes in gene expression of the host Z. latifolia and fungal endophyte U. esculenta in response to FM. FM directly affected the growth of Z. latifolia by altering the expression level of genes involved in plant-pathogen interactions, plant hormone signal transduction and some metabolism pathways. By contrast, FM had little effect on U. esculenta growing inside of Z. latifolia. Collectively, our results provide a more in-depth understanding of the molecular processes that promote gall formation in Z. latifolia, while also identifying potential targets for genetic manipulation to improve the yield and quality of Z. latifolia, in a safer and more effective way.

Many ways to TOPLESS - manipulation of plant auxin signalling by a cluster of fungal effectors.

Plant biotrophic pathogens employ secreted molecules, called effectors, to suppress the host immune system and redirect the host's metabolism and development in their favour. Putative effectors of the gall-inducing maize pathogenic fungus Ustilago maydis were analysed for their ability to induce auxin signalling in plants. Using genetic, biochemical, cell-biological, and bioinformatic approaches we functionally elucidate a set of five, genetically linked effectors, called Topless (TPL) interacting protein (Tips) effectors that induce auxin signalling. We show that Tips induce auxin signalling by interfering with central corepressors of the TPL family. CRISPR-Cas9 mutants and deletion strain analysis indicate that the auxin signalling inducing subcluster effectors plays a redundant role in virulence. Although none of the Tips seem to have a conserved interaction motif, four of them bind solely to the N-terminal TPL domain and, for Tip1 and Tip4, we demonstrate direct competition with auxin/indole-3-acetic acid transcriptional repressors for their binding to TPL class of corepressors. Our findings reveal that TPL proteins, key regulators of growth-defence antagonism, are a major target of the U. maydis effectome.

Organic acids and glucose prime late-stage fungal biotrophy in maize.

Many plant-associated fungi are obligate biotrophs that depend on living hosts to proliferate. However, little is known about the molecular basis of the biotrophic lifestyle, despite the impact of fungi on the environment and food security. In this work, we show that combinations of organic acids and glucose trigger phenotypes that are associated with the late stage of biotrophy for the maize pathogen Ustilago maydis. These phenotypes include the expression of a set of effectors normally observed only during biotrophic development, as well as the formation of melanin associated with sporulation in plant tumors. U. maydis and other hemibiotrophic fungi also respond to a combination of carbon sources with enhanced proliferation. Thus, the response to combinations of nutrients from the host may be a conserved feature of fungal biotrophy.

A MademoiseLLE domain binding platform links the key RNA transporter to endosomes.

Spatiotemporal expression can be achieved by transport and translation of mRNAs at defined subcellular sites. An emerging mechanism mediating mRNA trafficking is microtubule-dependent co-transport on shuttling endosomes. Although progress has been made in identifying various components of the endosomal mRNA transport machinery, a mechanistic understanding of how these RNA-binding proteins are connected to endosomes is still lacking. Here, we demonstrate that a flexible MademoiseLLE (MLLE) domain platform within RNA-binding protein Rrm4 of Ustilago maydis is crucial for endosomal attachment. Our structure/function analysis uncovered three MLLE domains at the C-terminus of Rrm4 with a functionally defined hierarchy. MLLE3 recognises two PAM2-like sequences of the adaptor protein Upa1 and is essential for endosomal shuttling of Rrm4. MLLE1 and MLLE2 are most likely accessory domains exhibiting a variable binding mode for interaction with currently unknown partners. Thus, endosomal attachment of the mRNA transporter is orchestrated by a sophisticated MLLE domain binding platform.