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 nonmelanized 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 decreases appressoria formation and plant penetration, thereby reducing virulence. Specifically, the Δrow1 mutant has a thicker cell wall that is more resistant to glucanase degradation. We also observed that the Δrow1 mutant has secretion defects. 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. Our data suggest that Row1 could modify the chitin-glucan matrix of the fungal cell wall and may be involved in unconventional protein secretion, thereby promoting both appressoria maturation and penetration.

N-glycosylation of the protein disulfide isomerase Pdi1 ensures full 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 important for normal pathogenic development in U. maydis. Our results provide new insights into the role of glycosylation in fungal pathogenesis.

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.

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.

Doppler ultrasound: a powerful tool for vascular access surveillance.

National Kidney Foundation Kidney Disease Outcomes Quality Initiative (NKF KDOQI) guidelines recommend Doppler ultrasound (DU) for surveillance of vascular access (VA), but trials have not been unanimous about its benefit on VA patency. The aim of this study was to evaluate the accuracy of DU for patency, as well as to highlight additional data provided by this method. A transversal study was conducted to evaluate DU method in correlation with BTM using paired t-test and Pearson test. Ultrasonography evaluation was performed with a Siemens Acuson X150 Ultrasound device and BTM-Qa with the Blood Temperature Monitor BTM(®). Access blood flow (Qa) values were correlated with several factors by nonparametric tests. Fifty hemodialysis patients were included, with mean age of 64.5 ± 13.7 years; durations of hemodialysis and VA were 51.4 ± 47.3 and 47.6 ± 42.1 months, respectively. The mean difference between DU and BTM flows was 20.5 ml/minute (p 0.624). Pearson correlation was 0.851 (p < 0.001). DU-Qa values varied significantly with several factors: type of VA, reason for DU referral, the presence of artery stenosis, and the location and number of stenosis. BTM-Qa values only varied significantly with the presence and number of stenosis. Various silent abnormalities were detected with DU. DU provides accurate anatomic and hemodynamic data to further knowledge regarding the etiology of stenosis and other abnormalities that compromise VA well functioning.

Doppler ultrasound and calcification score: improving vascular access surveillance.

AIM: Vascular access (VA) dysfunction limits hemodialysis delivery, which increases morbidity and mortality. The most com mon cause of VA failure is thrombosis, due to flow limiting stenosis resulting from neointimal hyperplasia. This occurs not only due to hemodynamic factors but also by systemic ones related to vascular atherosclerosis, inflammation and calcification, which has developed a simple vascular calcification score (SVCS) predictor of vascular calcification and arterial stiffness. The NKF-K/DOQ recommends several diagnostic procedures for VA surveillance. Blood access flow (Qa) has predictive power for the detection of stenosis. Our aim was to evaluate the role of systemic factors, especially SCVS, on Qa. MATERIAL AND METHODS: Transversal study in 50 patients. Qa value was obtained with Blood Temperature Monitor and Doppler method. Pearson coefficient evaluated correlation between them. Clinical, lab and radiological variables were recorded and non-parametric tests evaluated how both Qa varied with them. RESULTS: Pearson's corelation between DU-Qa and TD-Qa was 0.851 (p-value <0.001). DU-Qa varied significantly with age (p = 0.012), VA type (p = 0.021), SCVS (p = 0.030), intra-access arterial pressure (p = 0.015) and time on dialysis (p = 0.002). BTM-Qa varied significantly with diabetes status (p = 0.027), age (p = 0.017), first VA status (p = 0.036), intra-access arterial pressure (p = 0.028) and dialysis time (p = 0.001). Nevertheless, gender, hypertensive status and analitical parameters did not change the flow values. CONCLUSION: Higher SVCS was associated only with lower DU-Qas, giving this method an advantage towards the indirect one. Additionally, a simple method like SVCS may be used to guide new surveillance recommendations accordingly to risk stratification.

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.

Nephrology Partnership for Advancing Technology in Healthcare (N-PATH) program: the teachers' perspective.

The N-PATH (Nephrology Partnership for Advancing Technology in Healthcare) program concluded with the 60th European Renal Association 2023 Congress in Milan, Italy. This collaborative initiative aimed to provide advanced training in interventional nephrology to young European nephrologists. Funded by Erasmus+ Knowledge Alliance, N-PATH addressed the global burden of chronic kidney disease (CKD) and the shortage of nephrologists. CKD affects >850 million people worldwide, yet nephrology struggles to attract medical talent, leading to unfilled positions in residency programs. To address this, N-PATH focused on enhancing nephrology education through four specialized modules: renal expert in renal pathology (ReMAP), renal expert in vascular access (ReVAC), renal expert in medical ultrasound (ReMUS) and renal expert in peritoneal dialysis (RePED). ReMAP emphasized the importance of kidney biopsy in nephrology diagnosis and treatment, providing theoretical knowledge and hands-on training. ReVAC centred on vascular access in haemodialysis, teaching trainees about different access types, placement techniques and managing complications. ReMUS recognized the significance of ultrasound in nephrology, promoting interdisciplinary collaboration and preparing nephrologists for comprehensive patient care. RePED addressed chronic peritoneal dialysis, offering comprehensive training in patient selection, prescription, monitoring, complications and surgical techniques for catheter insertion. Overall, N-PATH's strategy involved collaborative networks, hands-on training, mentorship, an interdisciplinary approach and the integration of emerging technologies. By bridging the gap between theoretical knowledge and practical skills, N-PATH aimed to revitalize interest in nephrology and prepare proficient nephrologists to tackle the challenges of kidney diseases. In conclusion, the N-PATH program aimed to address the shortage of nephrologists and improve the quality of nephrology care in Europe. By providing specialized training, fostering collaboration and promoting patient-centred care, N-PATH aimed to inspire future nephrology professionals to meet the growing healthcare demands related to kidney diseases and elevate the specialty's status within the medical community.

Genes involved in protein glycosylation determine the activity and cell internalization of the antifungal peptide PAF26 in Saccharomyces cerevisiae.

We have previously characterized the synthetic hexapeptide PAF26 as a cell-penetrating and non-lytic antifungal peptide that is active against Saccharomyces cerevisiae and filamentous fungi. Numerous cell wall (CW) proteins are glycosylated in fungi and many of these play important roles in fungal pathogenesis. In this study, we screened a collection of S. cerevisiae deletion mutants for protein glycosylation genes whose deletion altered the sensitivity to PAF26. Increased tolerance to PAF26 was observed in mutants with the following disrupted genes: PMT1-6, EOS1, ALG5, MNN1, MNN4 and MNN5. Significantly, genes coding for protein O-mannosyltransferase 2 (Pmt2p), which is responsible for the addition of the first mannosyl residue of O-linked carbohydrates, and for Eos1p, an enzyme involved in N-linked glycosylation of proteins, showed resistance to PAF26 and defects in CW integrity. Microscopic studies on the S. cerevisiae Δeos1 deletion mutant demonstrated a blockage of peptide internalization by cells. Protoplasts lacking CWs interacted with the peptide, but were more resistant to peptide killing than cells possessing CWs due to a blockage in PAF26 internalization. Interestingly, protoplasts obtained from Δeos1 behaved similarly to those of the parental strain. Collectively, these observations demonstrate that the CW is a positive factor that determines the internalization of the PAF26, and that Eos1p exerts its activity through the glycosylation of specific protein(s) involved in peptide internalization.

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.

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.

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.

Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis.

Ustilago maydis is a ubiquitous pathogen of maize and a well-established model organism for the study of plant-microbe interactions. This basidiomycete fungus does not use aggressive virulence strategies to kill its host. U. maydis belongs to the group of biotrophic parasites (the smuts) that depend on living tissue for proliferation and development. Here we report the genome sequence for a member of this economically important group of biotrophic fungi. The 20.5-million-base U. maydis genome assembly contains 6,902 predicted protein-encoding genes and lacks pathogenicity signatures found in the genomes of aggressive pathogenic fungi, for example a battery of cell-wall-degrading enzymes. However, we detected unexpected genomic features responsible for the pathogenicity of this organism. Specifically, we found 12 clusters of genes encoding small secreted proteins with unknown function. A significant fraction of these genes exists in small gene families. Expression analysis showed that most of the genes contained in these clusters are regulated together and induced in infected tissue. Deletion of individual clusters altered the virulence of U. maydis in five cases, ranging from a complete lack of symptoms to hypervirulence. Despite years of research into the mechanism of pathogenicity in U. maydis, no 'true' virulence factors had been previously identified. Thus, the discovery of the secreted protein gene clusters and the functional demonstration of their decisive role in the infection process illuminate previously unknown mechanisms of pathogenicity operating in biotrophic fungi. Genomic analysis is, similarly, likely to open up new avenues for the discovery of virulence determinants in other pathogens.

Atypical presentation of fistula dysfunction due to brachial arterial embolization mimicking stroke.

Vascular access thrombosis is an important complication with great impact on access patency and, consequently, on a patient's quality of life and survival. We report the case of a 73-year-old woman with chronic kidney disease on hemodialysis with a radiocephalic arteriovenous fistula on the right arm that was brought to the emergency department with decreased strength in her right arm, ipsilateral hypoesthesia and facial hemi-hypoesthesia. The patient was given a brain computed tomographic scan that did not confirm suspicion of stroke. On re-examination, the patient had new-onset pain at arteriovenous fistula level, and her right arm was cold and pale. The nephrology department was called for arteriovenous fistula evaluation. On physical examination, her forearm fistula had a decreased thrill and arm elevation exacerbated its paleness. A bedside ultrasound was performed for arteriovenous fistula assessment. Doppler ultrasound revealed: partial thrombosis at brachial bifurcation, a flow of 80-105 mL/min at brachial artery level and a radial artery with a damped waveform. Anastomosis and draining vein were permeable. In this case, the diagnosis of acute embolic brachial artery occlusion was made by a fast bedside ultrasound evaluation. The patient underwent thromboembolectomy with Fogarty technique, recovering fistula thrill, radial and cubital pulses. Thromboembolism of the fistula feeding artery is a rare cause of vascular access thrombosis and it is rarely mentioned in the literature. In this report, failure to recognize the upper limb ischemia would have led to delayed treatment, potentially resulting in the fistula's complete thrombosis and further limb ischemia. We highlight the importance of a diagnosis method like Doppler ultrasound, which allows for rapid evaluation at the patient's bedside.

Upper limb anatomy and preoperative mapping.

Vascular access is absolutely essential for haemodialysis due to its relationship with quality of dialysis and associated morbidity. Therefore, it must be monitored and continuously surveilled from the moment it is created to prevent failure in maturation and thrombosis. Multidisciplinary collaboration is necessary when the main aim is to achieve the adequate vascular access flow with the fewest possible complications. The starting point, and probably the main one, is vascular access planning. This planning requires both a deep understanding of the anatomy of the upper limb and enough skill to examine it by Doppler ultrasound. The aim of this article is to review the anatomical and haemodynamical concepts of the arterial and venous vascular tree and explain how to perform ultrasound mapping, optimising the technical resources provided by this tool. Likewise, adequate access creation criteria that minimise the risk of failure and associated complications will be discussed.

Current role of ultrasound in hemodialysis access evaluation.

Physical examination (PE) is considered the backbone before vascular access (VA) placement, during maturation period and for follow-up. However, it may be inadequate in identifying suitable vasculature, mainly in comorbid patients, or in detecting complications. This review highlights the advantages of ultrasound imaging to manage VA before placement, during maturation and follow-up. Furthermore, it analyses the future perspectives in evaluating early and late VA complications thank to the availability of multiparametric platforms, point of care of ultrasound, and portable/wireless systems. Technical improvements and low-cost systems should favor the widespread ultrasound-based VA surveillance programs. This significant turning point needs an adequate training of nephrologists and dialysis nurses and the standardization of exams, parameters, and procedures.