A Novel Potent Carrier for Unconventional Protein Export in Ustilago maydis

Recombinant proteins are ubiquitously applied in fields like research, pharma, diagnostics or the chemical industry. To provide the full range of useful proteins, novel expression hosts need to be established for proteins that are not sufficiently produced by the standard platform organisms. Unconventional secretion in the fungal model Ustilago maydis is an attractive novel option for export of heterologous proteins without N-glycosylation using chitinase Cts1 as a carrier. Recently, a novel factor essential for unconventional Cts1 secretion termed Jps1 was identified. Here, we show that Jps1 is unconventionally secreted using a fusion to bacterial β-glucuronidase as an established reporter. Interestingly, the experiment also demonstrates that the protein functions as an alternative carrier for heterologous proteins, showing about 2-fold higher reporter activity than the Cts1 fusion in the supernatant. In addition, Jps1-mediated secretion even allowed for efficient export of functional firefly luciferase as a novel secretion target which could not be achieved with Cts1. As an application for a relevant pharmaceutical target, export of functional bi-specific synthetic nanobodies directed against the SARS-CoV2 spike protein was demonstrated. The establishment of an alternative efficient carrier thus constitutes an excellent expansion of the existing secretion platform.

Ustilago maydis Secreted Endo-Xylanases Are Involved in Fungal Filamentation and Proliferation on and Inside Plants

Plant pathogenic fungi must be able to degrade host cell walls in order to penetrate and invade plant tissues. Among the plant cell wall degrading enzymes (PCWDEs) produced, xylanases are of special interest since its degradation target, xylan, is one of the main structural polysaccharides in plant cell walls. In the biotrophic fungus Ustilago maydis, attempts to characterize PCWDEs required for virulence have been unsuccessful, most likely due to functional redundancy. In previous high-throughput screening, we found one xylanase to be important for U. maydis infection. Here, we characterize the entire U. maydis endo-xylanase family, comprising two enzymes from the glycoside hydrolase (GH) 10 family, Xyn1 and Xyn2, one from GH11, Xyn11A, and one from GH43, Xyn3. We show that all endo-xylanases except Xyn3 are secreted and involved in infection in a non-redundant manner, suggesting different roles for each xylanase in this process. Taking a closer look inside the plant during the pathogenic process, we observed that all secreted xylanases were necessary for fungal proliferation. Finally, we found that at least Xyn11A accumulated in the apoplast of the infected plant after three days, highlighting the role of these enzymes as important secreted proteins during fungal proliferation inside plant tissues.

Dissection of the Complex Transcription and Metabolism Regulation Networks Associated with Maize Resistance to Ustilago maydis

The biotrophic fungal pathogen Ustilago maydis causes common smut in maize, forming tumors on all aerial organs, especially on reproductive organs, leading to significant reduction in yield and quality defects. Resistance to U. maydis is thought to be a quantitative trait, likely controlled by many minor gene effects. However, the genes and the underlying complex mechanisms for maize resistance to U. maydis remain largely uncharacterized. Here, we conducted comparative transcriptome and metabolome study using a pair of maize lines with contrast resistance to U. maydis post-infection. WGCNA of transcriptome profiling reveals that defense response, photosynthesis, and cell cycle are critical processes in maize response to U. maydis, and metabolism regulation of glycolysis, amino acids, phenylpropanoid, and reactive oxygen species are closely correlated with defense response. Metabolomic analysis supported that phenylpropanoid and flavonoid biosynthesis was induced upon U. maydis infection, and an obviously higher content of shikimic acid, a key compound in glycolysis and aromatic amino acids biosynthesis pathways, was detected in resistant samples. Thus, we propose that complex gene co-expression and metabolism networks related to amino acids and ROS metabolism might contribute to the resistance to corn smut.

Maize AFP1 confers antifungal activity by inhibiting chitin deacetylases from a broad range of fungi

Adapted plant pathogenic fungi deacetylate chitin to chitosan to avoid host perception and disarm the chitin-triggered plant immunity. Whether plants have evolved factors to counteract this fungal evasion mechanism in the plant-pathogen interface remains obscure. Here, we decipher the underlying mechanism of maize cysteine-rich receptor-like secreted proteins (CRRSPs)- AFP1, which exhibits mannose-binding dependent antifungal activity. AFP1 initials the action by binding to specific sites on the surface of yeast-like cells, filaments, and germinated spores of the biotrophic fungi Ustilago maydis. This could result in fungal cell growth and cell budding inhibition, delaying spore germination and subsequently reducing fungal viability in a mannose-binding dependence manner. The antifungal activity of AFP1 is conferred by its interaction with the PMT-dependent mannosylated chitin deacetylases (CDAs) and interfering with the conversion of chitin. Our finding that AFP1 targets CDAs from pathogenic fungi and nonpathogenic budding yeast suggests a potential application of the CRRSP in combating fungal diseases and reducing threats posed by the fungal kingdom.

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

Spatiotemporal expression is mostly achieved by transport and translation of mRNAs at defined subcellular sites. An emerging mechanism mediating mRNA trafficking is microtubule-dependent co-transport of mRNAs 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 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 recognizes 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 that exhibit a variant binding mode for interaction with currently unknown partners. Thus, endosomal attachment of the mRNA transporter is orchestrated by a sophisticated MLLE domain binding platform.

Characterization of Fungal FAD-Dependent AA3_2 Glucose Oxidoreductases from Hitherto Unexplored Phylogenetic Clades

The CAZy auxiliary activity family 3 (AA3) comprises FAD-dependent enzymes belonging to the superfamily of glucose-methanol-choline (GMC) oxidoreductases. Glucose oxidase (GOx; EC 1.1.3.4) and glucose dehydrogenase (GDH; EC 1.1.5.9) are part of subfamily AA3_2 and catalyze the oxidation of β-D-glucose at its anomeric carbon to D-glucono-1,5-lactone. Recent phylogenetic analysis showed that AA3_2 glucose oxidoreductases can be grouped into four major clades, GOx I and GDH I–III, and in minor clades such as GOx II or distinct subclades. This wide sequence space of AA3_2 glucose oxidoreductases has, however, not been studied in detail, with mainly members of GOx I and GDH I studied biochemically or structurally. Here, we report the biochemical characterization of four fungal glucose oxidoreductases from distinct, hitherto unexplored clades or subclades. The enzyme from Aureobasidium subglaciale, belonging to the minor GOx II clade, showed a typical preference for oxygen and glucose, confirming the correct annotation of this clade. The other three enzymes exhibited strict dehydrogenase activity with different substrate specificities. GDH II from Trichoderma virens showed an almost six-fold higher catalytic efficiency for maltose compared to glucose. The preferred substrate for the two GDH III enzymes from Rhizoctonia solani and Ustilago maydis was gentiobiose, a β(1→6) disaccharide, as judged from the catalytic efficiency. Overall, the newly studied AA3_2 glucose oxidoreductases showed a much broader substrate spectrum than the archetypal GOx from Aspergillus niger, which belongs to clade GOx I.

Identificación y expresión del efector central fúngico Pep1 en Thecaphora frezii

Thecaphora frezii es un hongo fitopatógeno perteneciente a la clase Ustilaginomicetes, que produce laenfermedad del carbón de maní. En su ciclo biológico presenta tres estructuras, las teliosporas (es laestructura de resistencia) y las basidiosporas e hifas. El micelio (hifas) es la estructura infectiva, que penetraen el ginóforo de la planta e inicia la infección. Para dicha acción, sería necesaria la expresión de la proteínaPep1 ya que la misma fue identificada en otros Ustilaginomicetes como el Ustilago maydis y el Ustilagohordei, que infectan al maíz y a la cebada, respectivamente, y su expresión es fundamental para dichaacción.Pudimos amplificar el ADN copia de Pep1 de Thecaphora frezii cuya secuencia traducida codificaría parauna proteína de 180 aminoácidos. Se observaron grandes homologías con ortólogas de otras especies y lapresencia de cuatro cisteínas conservadas. Paralelamente, medimos los niveles de expresión de estetranscripto, encontrándose muy elevado en las hifas, coincidiendo con el estadio infectivo del hongo.Futuros estudios funcionales de inactivación génica del gen pep1 deberán realizarse para comprobarfenotípicamente el efecto que este gen provoca en cultivos de maní.