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.

4.5SH RNA counteracts deleterious exonization of SINE B1 in mice.

4.5SH RNA is a highly abundant, small rodent-specific noncoding RNA that localizes to nuclear speckles enriched in pre-mRNA-splicing regulators. To investigate the physiological functions of 4.5SH RNA, we have created mutant mice that lack the expression of 4.5SH RNA. The mutant mice exhibited embryonic lethality, suggesting that 4.5SH RNA is an essential species-specific noncoding RNA in mice. RNA-sequencing analyses revealed that 4.5SH RNA protects the transcriptome from abnormal exonizations of the antisense insertions of the retrotransposon SINE B1 (asB1), which would otherwise introduce deleterious premature stop codons or frameshift mutations. Mechanistically, 4.5SH RNA base pairs with complementary asB1-containing exons via the target recognition region and recruits effector proteins including Hnrnpm via its 5' stem loop region. The modular organization of 4.5SH RNA allows us to engineer a programmable splicing regulator to induce the skipping of target exons of interest. Our results also suggest the general existence of splicing regulatory noncoding RNAs.

Mating Types of Ustilago esculenta Infecting Zizania latifolia Cultivars in Japan Are Biased towards MAT-2 and MAT-3.

Zizania latifolia cultivars infected by the endophytic fungus Ustilago esculenta develop an edible stem gall. Stem gall development varies among cultivars and individuals and may be affected by the strain of U. esculenta. To isolate haploids from two Z. latifolia cultivars in our paddy fields, Shirakawa and Ittenkou, we herein performed the sporadic isolation of U. esculenta strains from stem gall tissue, a PCR-based assessment of the mating type, and in vitro mating experiments. As a result, we obtained heterogametic strains of MAT-2 and MAT-3 as well as MAT-2, but not MAT-3, haploid strains. Another isolation method, in which we examined poorly growing small clusters of sporidia derived from teliospores, succeeded in isolating a MAT-3 haploid strain. We also identified the mating types of 10 U. esculenta strains collected as genetic resources from different areas in Japan. All strains, except for one MAT-1 haploid strain, were classified as MAT-2 haploid strains or heterogametic strains of MAT-2 and MAT-3. The isolated strains of MAT-1, MAT-2, and MAT-3 mated with each other to produce hyphae. Collectively, these results indicate that the mating types of U. esculenta infecting Z. latifolia cultivars in Japan are biased towards MAT-2 and MAT-3 and that U. esculenta populations in these Japanese cultivars may be characterized by the low isolation efficiency of the MAT-3 haploid.

Wear of a Highly Cross-Linked Polyethylene Liner of the Acetabular Component Placed With Excessive Acetabular Inclination.

Few studies have investigated the influence of excessive acetabular inclination during total hip arthroplasty (THA) on the wear of highly cross-linked polyethylene (HXLPE). The goal of this study was to determine whether excessive acetabular inclination during THA causes prominent wear of the HXLPE liner. We retrospectively evaluated 62 hips of 54 women and 8 men who underwent primary THA with an HXLPE liner between January 2006 and September 2011. Postoperative anteroposterior pelvic radiographs were used to measure acetabular inclination and analyze polyethylene wear. Patients were divided into the following groups: the excessive acetabular inclination group (acetabular inclination angle ≥50°; n=20) and the control group (acetabular inclination angle <50°; n=42). Clinical information and imaging findings were compared and examined between the 2 groups. Further, we evaluated the correlation between the acetabular inclination angle and the polyethylene wear rate. In all cases, mean follow-up duration was 6.3 years. The annual liner wear was 0.00446 and 0.0254 mm/y in the control and excessive acetabular inclination groups, respectively. The excessive acetabular inclination group had significantly higher polyethylene liner wear compared with the control group (P=.00991). A weak correlation was seen between acetabular inclination angle and polyethylene wear rate in all cases (Spearman's rank correlation, r=0.283, P=.0258). Excessive acetabular inclination may increase HXLPE liner wear in the long term. [Orthopedics. 2022;45(2):e96-e100.].

The production and secretion of tRNA-derived RNA fragments in the corn smut fungus Ustilago maydis.

The biogenesis of small non-coding RNAs is a molecular event that contributes to cellular functions. The basidiomycete fungus Ustilago maydis is a biotrophic pathogen parasitizing maize. A hallmark of its genome structure is an absence of RNAi machinery including Dicer and Argonaute proteins, which are responsible for the production of small RNAs in other organisms. However, it remains unclear whether U. maydis produces small RNAs during fungal growth. Here we found that U. maydis cells accumulate approximately 20-30 nucleotides of small RNA fragments during growth in the axenic culture condition. The RNA-seq analysis of these fragments identified that these small RNAs are originated from tRNAs and 5.8S ribosomal RNA. Interestingly, majority of their sequences are generated from tRNAs responsible for asparagine, glutamine and glycine, suggesting a bias of origin. The cleavage of tRNAs mainly occurs at the position near anticodon-stem-loop. We generated the deletion mutants of two genes nuc1 and nuc2 encoding RNase T2, which is a candidate enzyme that cleaves tRNAs. The deletion mutants of two genes largely fail to accumulate tRNA-derived RNA fragments. Nuc1 and tRNA are co-localized at the tip of budding cells and tRNA fragment could be detected in culture supernatant. Our results suggest that specific tRNAs would be cleaved during secretory processes and tRNA fragments might have extracellular functions.

Cell wall-associated effectors of plant-colonizing fungi.

Plant-colonizing fungi secrete a cocktail of effector proteins during colonization. After secretion, some of these effectors are delivered into plant cells to directly dampen the plant immune system or redirect host processes benefitting fungal growth. Other effectors function in the apoplastic space either as released proteins modulating the activity of plant enzymes associated with plant defense or as proteins bound to the fungal cell wall. For such fungal cell wall-bound effectors, we know particularly little about their molecular function. In this review, we describe effectors that are associated with the fungal cell wall and discuss how they contribute to colonization.

The functionally conserved effector Sta1 is a fungal cell wall protein required for virulence in Ustilago maydis.

The biotrophic fungus Ustilago maydis causes the smut disease of maize. The interaction with its host and induction of characteristic tumors are governed largely by secreted effectors whose function is mostly unknown. To identify effectors with a prominent role in virulence, we used RNA sequencing and found that the gene sta1 is upregulated during early stages of infection. We characterized Sta1 by comparative genomics, reverse genetics, protein localization, stress assays, and microscopy. sta1 mutants show a dramatic reduction of virulence and show altered colonization of tissue neighboring the vascular bundles. Functional orthologues of Sta1 are found in related smut pathogens infecting monocot and dicot plants. Sta1 is secreted by budding cells but is attached to the cell wall of filamentous hyphae. Upon constitutive expression of Sta1, fungal filaments become susceptible to Congo red, ß-glucanase, and chitinase, suggesting that Sta1 alters the structure of the fungal cell wall. Constitutive or delayed expression of sta1 during plant colonization negatively impacts on virulence. Our results suggest that Sta1 is a novel kind of effector, which needs to modify the hyphal cell wall to allow hyphae to be accommodated in tissue next to the vascular bundles.

Neofunctionalization of the secreted Tin2 effector in the fungal pathogen Ustilago maydis.

Plant-pathogenic fungi hijack their hosts by secreting effector proteins. Effectors serve to suppress plant immune responses and modulate the host metabolism to benefit the pathogen. Smut fungi are biotrophic pathogens that also parasitize important cereals, including maize1. Symptom development is usually restricted to the plant inflorescences. Ustilago maydis is an exception in its ability to cause tumours in both inflorescences and leaves of maize, and in inducing anthocyanin biosynthesis through the secreted Tin2 effector2,3. How the unique lifestyle of U. maydis has evolved remains to be elucidated. Here we show that Tin2 in U. maydis has been neofunctionalized. We functionally compared Tin2 effectors of U. maydis and the related smut Sporisorium reilianum, which results in symptoms only in the inflorescences of maize and fails to induce anthocyanin. We show that Tin2 effectors from both fungi target distinct paralogues of a maize protein kinase, leading to stabilization and inhibition, respectively. An ancestral Tin2 effector functionally replaced the virulence function of S. reilianum Tin2 but failed to induce anthocyanin, and was unable to substitute for Tin2 in U. maydis. This shows that Tin2 in U. maydis has acquired a specialized function, probably connected to the distinct pathogenic lifestyle of this fungus.

Ustilago maydis effectors and their impact on virulence.

Biotrophic fungal plant pathogens establish an intimate relationship with their host to support the infection process. Central to this strategy is the secretion of a range of protein effectors that enable the pathogen to evade plant immune defences and modulate host metabolism to meet its needs. In this Review, using the smut fungus Ustilago maydis as an example, we discuss new insights into the effector repertoire of smut fungi that have been gained from comparative genomics and discuss the molecular mechanisms by which U. maydis effectors change processes in the plant host. Finally, we examine how the expression of effector genes and effector secretion are coordinated with fungal development in the host.

An assay for entry of secreted fungal effectors into plant cells.

Successful colonization of plants by prokaryotic and eukaryotic pathogens requires active effector-mediated suppression of defense responses and host tissue reprogramming. Secreted effector proteins can either display their activity in the apoplast or translocate into host cells and function therein. Although characterized in bacteria, the molecular mechanisms of effector delivery by fungal phytopathogens remain elusive. Here we report the establishment of an assay that is based on biotinylation of effectors in the host cytoplasm as hallmark of uptake. The assay exploits the ability of the bacterial biotin ligase BirA to biotinylate any protein that carries a short peptide (Avitag). It is based on the stable expression of BirA in the cytoplasm of maize plants and on engineering of Ustilago maydis strains to secrete Avitagged effectors. We demonstrate translocation of a number of effectors in the U. maydis-maize system and show data that suggest that the uptake mechanism could be rather nonspecific The assay promises to be a powerful tool for the classification of effectors as well as for the functional study of effector uptake mechanism not only in the chosen system but more generally for systems where biotrophic interactions are established.

A Patient-Specific Instrument for Femoral Stem Placement During Total Hip Arthroplasty.

To ensure that the femoral stem is placed in the proper position during total hip arthroplasty, the authors developed a patient-specific instrument. A total of 10 total hip arthroplasties were performed with the assistance of the patient-specific instrument during this study. The mean accuracy of stem tilt, varus/valgus, and anteversion was 2.1°±4.1°, 1.0°±0.7°, and 4.7°±1.2°, respectively. No complications were observed and no reoperations were required for any of the patients who underwent surgery included in this study. The results support the feasibility of this patient-specific instrument for use during stem placement in total hip arthroplasty. [Orthopedics. 2017; 40(2):e374-e377.].

Accuracy of using Diagnosis Procedure Combination administrative claims data for estimating the amount of opioid consumption among cancer patients in Japan.

OBJECTIVE: The state of opioid consumption among cancer patients has never been comprehensively investigated in Japan. The Diagnosis Procedure Combination claims data may be used to measure and monitor opioid consumption among cancer patients, but the accuracy of using the Diagnosis Procedure Combination data for this purpose has never been tested. METHODS: We aimed to ascertain the accuracy of using the Diagnosis Procedure Combination claims data for estimating total opioid analgesic consumption by cancer patients compared with electronic medical records at Aomori Prefectural Central Hospital. We calculated percent differences between estimates obtained from electronic medical records and Diagnosis Procedure Combination claims data by month and drug type (morphine, oxycodone, fentanyl, buprenorphine, codeine and tramadol) between 1 October 2012 and 30 September 2013, and further examined the causes of discrepancy by reviewing medical and administrative charts between April and July 2013. RESULTS: Percent differences varied by month for drug types with small prescription volumes, but less so for drugs with larger prescription volumes. Differences also tended to diminish when consumption was compared for a year instead of a month. Total percent difference between electronic medical records and Diagnosis Procedure Combination data during the study period was -0.1% (4721 mg per year per hospital), as electronic medical records as baseline. Half of the discrepancy was caused by errors in data entry. CONCLUSION: Our study showed that Diagnosis Procedure Combination claims data can be used to accurately estimate opioid consumption among a population of cancer patients, although the same conclusion cannot be made for individual estimates or when making estimates for a group of patients over a short period of time.

Experimental approaches to investigate effector translocation into host cells in the Ustilago maydis/maize pathosystem.

The fungus Ustilago maydis is a pathogen that establishes a biotrophic interaction with Zea mays. The interaction with the plant host is largely governed by more than 300 novel, secreted protein effectors, of which only four have been functionally characterized. Prerequisite to examine effector function is to know where effectors reside after secretion. Effectors can remain in the extracellular space, i.e. the plant apoplast (apoplastic effectors), or can cross the plant plasma membrane and exert their function inside the host cell (cytoplasmic effectors). The U. maydis effectors lack conserved motifs in their primary sequences that could allow a classification of the effectome into apoplastic/cytoplasmic effectors. This represents a significant obstacle in functional effector characterization. Here we describe our attempts to establish a system for effector classification into apoplastic and cytoplasmic members, using U. maydis for effector delivery.

Microbial effectors target multiple steps in the salicylic acid production and signaling pathway.

Microbes attempting to colonize plants are recognized through the plant immune surveillance system. This leads to a complex array of global as well as specific defense responses, which are often associated with plant cell death and subsequent arrest of the invader. The responses also entail complex changes in phytohormone signaling pathways. Among these, salicylic acid (SA) signaling is an important pathway because of its ability to trigger plant cell death. As biotrophic and hemibiotrophic pathogens need to invade living plant tissue to cause disease, they have evolved efficient strategies to downregulate SA signaling by virulence effectors, which can be proteins or secondary metabolites. Here we review the strategies prokaryotic pathogens have developed to target SA biosynthesis and signaling, and contrast this with recent insights into how plant pathogenic eukaryotic fungi and oomycetes accomplish the same goal.

Fungal effectors and plant susceptibility.

Plants can be colonized by fungi that have adopted highly diverse lifestyles, ranging from symbiotic to necrotrophic. Colonization is governed in all systems by hundreds of secreted fungal effector molecules. These effectors suppress plant defense responses and modulate plant physiology to accommodate fungal invaders and provide them with nutrients. Fungal effectors either function in the interaction zone between the fungal hyphae and host or are transferred to plant cells. This review describes the effector repertoires of 84 plant-colonizing fungi. We focus on the mechanisms that allow these fungal effectors to promote virulence or compatibility, discuss common plant nodes that are targeted by effectors, and provide recent insights into effector evolution. In addition, we address the issue of effector uptake in plant cells and highlight open questions and future challenges.

Characterization of the largest effector gene cluster of Ustilago maydis.

In the genome of the biotrophic plant pathogen Ustilago maydis, many of the genes coding for secreted protein effectors modulating virulence are arranged in gene clusters. The vast majority of these genes encode novel proteins whose expression is coupled to plant colonization. The largest of these gene clusters, cluster 19A, encodes 24 secreted effectors. Deletion of the entire cluster results in severe attenuation of virulence. Here we present the functional analysis of this genomic region. We show that a 19A deletion mutant behaves like an endophyte, i.e. is still able to colonize plants and complete the infection cycle. However, tumors, the most conspicuous symptoms of maize smut disease, are only rarely formed and fungal biomass in infected tissue is significantly reduced. The generation and analysis of strains carrying sub-deletions identified several genes significantly contributing to tumor formation after seedling infection. Another of the effectors could be linked specifically to anthocyanin induction in the infected tissue. As the individual contributions of these genes to tumor formation were small, we studied the response of maize plants to the whole cluster mutant as well as to several individual mutants by array analysis. This revealed distinct plant responses, demonstrating that the respective effectors have discrete plant targets. We propose that the analysis of plant responses to effector mutant strains that lack a strong virulence phenotype may be a general way to visualize differences in effector function.

A secreted Ustilago maydis effector promotes virulence by targeting anthocyanin biosynthesis in maize.

The biotrophic fungus Ustilago maydis causes smut disease in maize with characteristic tumor formation and anthocyanin induction. Here, we show that anthocyanin biosynthesis is induced by the virulence promoting secreted effector protein Tin2. Tin2 protein functions inside plant cells where it interacts with maize protein kinase ZmTTK1. Tin2 masks a ubiquitin-proteasome degradation motif in ZmTTK1, thus stabilizing the active kinase. Active ZmTTK1 controls activation of genes in the anthocyanin biosynthesis pathway. Without Tin2, enhanced lignin biosynthesis is observed in infected tissue and vascular bundles show strong lignification. This is presumably limiting access of fungal hyphae to nutrients needed for massive proliferation. Consistent with this assertion, we observe that maize brown midrib mutants affected in lignin biosynthesis are hypersensitive to U. maydis infection. We speculate that Tin2 rewires metabolites into the anthocyanin pathway to lower their availability for other defense responses. DOI: http://dx.doi.org/10.7554/eLife.01355.001.

Long-term hip simulator testing of the artificial hip joint bearing surface grafted with biocompatible phospholipid polymer.

To prevent periprosthetic osteolysis and subsequent aseptic loosening of artificial hip joints, we recently developed a novel acetabular highly cross-linked polyethylene (CLPE) liner with graft polymerization of 2-methacryloyloxyethyl phosphorylcholine (MPC) on its surface. We investigated the wear resistance of the poly(MPC) (PMPC)-grafted CLPE liner during 20 million cycles in a hip joint simulator. We extended the simulator test of one liner to 70 million cycles to investigate the long-term durability of the grafting. Gravimetric, surface, and wear particle analyses revealed that PMPC grafting onto the CLPE liner surface markedly decreased the production of wear particles and showed that the effect of PMPC grafting was maintained through 70 million cycles. We believe that PMPC grafting can significantly improve the wear resistance of artificial hip joints.

HvCEBiP, a gene homologous to rice chitin receptor CEBiP, contributes to basal resistance of barley to Magnaporthe oryzae.

BACKGROUND: Rice CEBiP recognizes chitin oligosaccharides on the fungal cell surface or released into the plant apoplast, leading to the expression of plant disease resistance against fungal infection. However, it has not yet been reported whether CEBiP is actually required for restricting the growth of fungal pathogens. Here we evaluated the involvement of a putative chitin receptor gene in the basal resistance of barley to the ssd1 mutant of Magnaporthe oryzae, which induces multiple host defense responses. RESULTS: The mossd1 mutant showed attenuated pathogenicity on barley and appressorial penetration was restricted by the formation of callose papillae at attempted entry sites. When conidial suspensions of mossd1 mutant were spotted onto the leaves of HvCEBiP-silenced plants, small brown necrotic flecks or blast lesions were produced but these lesions did not expand beyond the inoculation site. Wild-type M. oryzae also produced slightly more severe symptoms on the leaves of HvCEBiP-silenced plants. Cytological observation revealed that these lesions resulted from appressorium-mediated penetration into plant epidermal cells. CONCLUSIONS: These results suggest that HvCEBiP is involved in basal resistance against appressorium-mediated infection and that basal resistance might be triggered by the recognition of chitin oligosaccharides derived from M. oryzae.

Peroxisome biogenesis factor PEX13 is required for appressorium-mediated plant infection by the anthracnose fungus Colletotrichum orbiculare.

Peroxisomes are ubiquitous organelles of eukaryotic cells that fulfill a variety of biochemical functions, including beta-oxidation of fatty acids. Here, we report that an ortholog of the Saccharomyces cerevisiae peroxisome biogenesis gene PEX13 is required for pathogenicity of Colletotrichum orbiculare. CoPEX13 was identified by screening random insertional mutants for deficiency in fatty acid utilization. Targeted knockout mutants of CoPEX13 were unable to utilize fatty acids as a carbon source. Expression analysis using green fluorescent protein fused to the peroxisomal targeting signals PTS1 and PTS2 revealed that the import machinery for peroxisomal matrix proteins was impaired in copex13 mutants. Appressoria formed by the copex13 mutants were defective in both melanization and penetration ability on host plants, had thin cell walls, and lacked peroxisomes. Moreover, the concentration of intracellular glycerol was lower in copex13 appressoria than those of the wild type. These findings indicate that fatty acid oxidation in peroxisomes is required not only for appressorium melanization but also for cell wall biogenesis and metabolic processes involved in turgor generation, all of which are essential for appressorium penetration ability.