Signal peptide peptidase activity connects the unfolded protein response to plant defense suppression by Ustilago maydis.

The corn smut fungus Ustilago maydis requires the unfolded protein response (UPR) to maintain homeostasis of the endoplasmic reticulum (ER) during the biotrophic interaction with its host plant Zea mays (maize). Crosstalk between the UPR and pathways controlling pathogenic development is mediated by protein-protein interactions between the UPR regulator Cib1 and the developmental regulator Clp1. Cib1/Clp1 complex formation results in mutual modification of the connected regulatory networks thereby aligning fungal proliferation in planta, efficient effector secretion with increased ER stress tolerance and long-term UPR activation in planta. Here we address UPR-dependent gene expression and its modulation by Clp1 using combinatorial RNAseq/ChIPseq analyses. We show that increased ER stress resistance is connected to Clp1-dependent alterations of Cib1 phosphorylation, protein stability and UPR gene expression. Importantly, we identify by deletion screening of UPR core genes the signal peptide peptidase Spp1 as a novel key factor that is required for establishing a compatible biotrophic interaction between U. maydis and its host plant maize. Spp1 is dispensable for ER stress resistance and vegetative growth but requires catalytic activity to interfere with the plant defense, revealing a novel virulence specific function for signal peptide peptidases in a biotrophic fungal/plant interaction.

Aeroallergens in North-Central Nigeria.

INTRODUCTION AND OBJECTIVES: Aeroallergens are airborne organic substances which are responsible for allergenic diseases in hypersensitive individuals. People are exposed to their allergens either directly or after their entrance into the interiors. The spatio-temporal pattern of aeroallergens and their relationship with weather variability in Abuja and Nassarawa, North-Central Nigeria was studied. MATERIALS AND METHODS: Aerosamples were trapped with modified Tauber-like pollen traps. Samples were collected monthly and centrifuged at 2500rpm for 5 min and subjected to acetolysis. Meteorological data were collected from the Nigerian Meteorological Agency. RESULTS AND CONCLUSION: Aeroallergens concentration were unequivocally regulated by weather variables in both locations, indicating the possible use of aeroallergens especially pollen and spores as bio-indicators of weather variations and change. Aeroallergens encountered were fungal spores, pollen, diatom frustules, fern spores, algal cyst/cells in decreasing order of dominance. Among pollen group, Poaceae, Amarathaceae/Chenopodiaceae and Hymenocardia acida dominated. Spores of Smut species, Puccinia, Curvularia and Nigrospora were major contributors among aeromycoflora. Fungal spores morphotype dominated during the rainier months and were major contributors of the aeroallergen spectrum with those belonging to Deuteromycete preponderant. Aeroallergens which were previously identified as triggers of conjunctivitis, asthma, allergic sinusitis and bronchopulmonary allergic diseases were frequently present in both locations. Pollen prevailed more during the harmattan, influenced by northeast trade wind. Pollen component differed and was based on autochthonous source plants, indicating difference in sub-vegetational types.

An immunity-triggering effector from the Barley smut fungus Ustilago hordei resides in an Ustilaginaceae-specific cluster bearing signs of transposable element-assisted evolution.

The basidiomycete smut fungus Ustilago hordei was previously shown to comprise isolates that are avirulent on various barley host cultivars. Through genetic crosses we had revealed that a dominant avirulence locus UhAvr1 which triggers immunity in barley cultivar Hannchen harboring resistance gene Ruh1, resided within an 80-kb region. DNA sequence analysis of this genetically delimited region uncovered the presence of 7 candidate secreted effector proteins. Sequence comparison of their coding sequences among virulent and avirulent parental and field isolates could not distinguish UhAvr1 candidates. Systematic deletion and complementation analyses revealed that UhAvr1 is UHOR_10022 which codes for a small effector protein of 171 amino acids with a predicted 19 amino acid signal peptide. Virulence in the parental isolate is caused by the insertion of a fragment of 5.5 kb with similarity to a common U. hordei transposable element (TE), interrupting the promoter of UhAvr1 and thereby changing expression and hence recognition of UhAVR1p. This rearrangement is likely caused by activities of TEs and variation is seen among isolates. Using GFP-chimeric constructs we show that UhAvr1 is induced only in mated dikaryotic hyphae upon sensing and infecting barley coleoptile cells. When infecting Hannchen, UhAVR1p causes local callose deposition and the production of reactive oxygen species and necrosis indicative of the immune response. UhAvr1 does not contribute significantly to overall virulence. UhAvr1 is located in a cluster of ten effectors with several paralogs and over 50% of TEs. This cluster is syntenous with clusters in closely-related U. maydis and Sporisorium reilianum. In these corn-infecting species, these clusters harbor however more and further diversified homologous effector families but very few TEs. This increased variability may have resulted from past selection pressure by resistance genes since U. maydis is not known to trigger immunity in its corn host.

The Ustilago maydis effector Pep1 suppresses plant immunity by inhibition of host peroxidase activity.

The corn smut Ustilago maydis establishes a biotrophic interaction with its host plant maize. This interaction requires efficient suppression of plant immune responses, which is attributed to secreted effector proteins. Previously we identified Pep1 (Protein essential during penetration-1) as a secreted effector with an essential role for U. maydis virulence. pep1 deletion mutants induce strong defense responses leading to an early block in pathogenic development of the fungus. Using cytological and functional assays we show that Pep1 functions as an inhibitor of plant peroxidases. At sites of Δpep1 mutant penetrations, H2O2 strongly accumulated in the cell walls, coinciding with a transcriptional induction of the secreted maize peroxidase POX12. Pep1 protein effectively inhibited the peroxidase driven oxidative burst and thereby suppresses the early immune responses of maize. Moreover, Pep1 directly inhibits peroxidases in vitro in a concentration-dependent manner. Using fluorescence complementation assays, we observed a direct interaction of Pep1 and the maize peroxidase POX12 in vivo. Functional relevance of this interaction was demonstrated by partial complementation of the Δpep1 mutant defect by virus induced gene silencing of maize POX12. We conclude that Pep1 acts as a potent suppressor of early plant defenses by inhibition of peroxidase activity. Thus, it represents a novel strategy for establishing a biotrophic interaction.

Transgenic maize plants expressing the Totivirus antifungal protein, KP4, are highly resistant to corn smut.

The corn smut fungus, Ustilago maydis, is a global pathogen responsible for extensive agricultural losses. Control of corn smut using traditional breeding has met with limited success because natural resistance to U. maydis is organ specific and involves numerous maize genes. Here, we present a transgenic approach by constitutively expressing the Totivirus antifungal protein KP4, in maize. Transgenic maize plants expressed high levels of KP4 with no apparent negative impact on plant development and displayed robust resistance to U. maydis challenges to both the stem and ear tissues in the greenhouse. More broadly, these results demonstrate that a high level of organ independent fungal resistance can be afforded by transgenic expression of this family of antifungal proteins.

A method to visualize the actin and microtubule cytoskeleton by indirect immunofluorescence.

The cytoskeleton provides the basic architectural organization and shape of the eukaryotic cell, and plays a key role in segregation of the genetic material. A method to visualize the actin and microtubule cytoskeleton in the fungus Ustilago maydis by indirect immunofluorescence is described here. The method entails growth of cells to early logarithmic phase, fixation with a cross-linking agent or organic solvent, partial digestion of the cell wall and permeabilization of cells with a detergent to allow entry of antibodies, exposure to primary antibody, followed by treatment with secondary antibody conjugated to a fluorophore to allow visualization with fluorescence microscopy.

Yeast killer systems.

The killer phenomenon in yeasts has been revealed to be a multicentric model for molecular biologists, virologists, phytopathologists, epidemiologists, industrial and medical microbiologists, mycologists, and pharmacologists. The surprisingly widespread occurrence of the killer phenomenon among taxonomically unrelated microorganisms, including prokaryotic and eukaryotic pathogens, has engendered a new interest in its biological significance as well as its theoretical and practical applications. The search for therapeutic opportunities by using yeast killer systems has conceptually opened new avenues for the prevention and control of life-threatening fungal diseases through the idiotypic network that is apparently exploited by the immune system in the course of natural infections. In this review, the biology, ecology, epidemiology, therapeutics, serology, and idiotypy of yeast killer systems are discussed.

Hypersensitivity pneumonitis induced by a smut fungus Ustilago esculenta.

A case of hypersensitivity pneumonitis caused by a smut fungus Ustilago esculenta is presented.

Neutralizing monoclonal antibodies against alpha and beta subunits of the Ustilago maydis virus encoded toxin.

The toxins secreted by Ustilago maydis are encoded by dsRNA viruses. The KP6 toxin encoded by subtype P6 consists of two polypeptides alpha and beta, which are not covalently bound. Neutralizing monoclonal antibodies (MoAbs) were raised against each subunit. Some of the anti-beta MoAbs identify different epitopes in the antigen. The MoAbs were used to affinity purify alpha and beta polypeptides from culture media and to detect the precursor of the mature toxin.

A study of the aeromycoflora of Cádiz: relationship to anthropogenic activity.

We describe a quantitative and qualitative study of the fungal spores found in the air of Cádiz during 1989 using a Cour-type trap. The results of this study can be extrapolated to other coastal cities of southern Europe with a Mediterranean climate. The spores identified have been classified into 25 taxonomic categories. The most abundant were Cladosporium, Chaetomium and Ustilago, and the most frequent, in addition to those mentioned, were Alternaria, Ascophyta and Venturia. The great abundance of Cladosporium is in accordance with the coastal situation of the city. Cladosporium, Alternaria, Curvularia and Stemphylium reached maximum concentrations jointly in October, 1989. They showed mutual cross-reactions. Ustilago and Nigrospora appeared during the period of cereal harvesting and storage.

Individual patterns of immediate skin reactivity to mold extracts.

One hundred atopic patients were skin tested intradermally over a 2-year period with 30 different mold extracts. Subjects were monitored for immediate reactions. Data suggest that to evaluate mold sensitivity in atopic patients one must use multiple mold extracts.

Virus-like particles in Ustilago maydis: mutants with partial genomes.

Mutants with partial genomes for the virus-like particles of U. maydis were recovered following treatment with nitrosoguanidine. Examination of the properties retained by progeny of genetic crosses indicates that the 2.9 X 10(6) dalton component of double-stranded RNA contains the information for capsid formation and dsRNA replication. Other components appear to contain the information for killer function and immunity to killer. The use of such mutants for studies on the evolution of viruses with segmented genomes is discussed.

Inheritance of killer phenotypes and double-stranded RNA in Ustilago maydis.

Three different killer specificities in U. maydis are inherited cytoplasmically and transmitted by cell fusion. Each killer generates low frequencies of specifically immune forms in crosses with sensitive strains. The properties of immunity to each killer are also inherited cytoplasmically and transmitted by cell fusion. Killer strains carry virus-like particles about 41 nm in diameter. Each killer possesses distinct double-stranded RNA components that range in molecular weight from 0.46 X 10(6) to 2.9 X 10(6). Two components are shared by all three killers. Immune strains possess new forms. Crosses and heterokaryons between different killers revealed unilateral or mutual restrictions that prevent inclusion of two killer specificities in the same cell.