Search

Leaf senescence, the final stage of leaf development, is influenced by numerous internal and environmental signals. However, how biotic stresses such as pathogen infection regulate leaf senescence remains largely unclear. In this study, we found that the premature leaf senescence in Arabidopsis caused by the soil-borne vascular fungus Verticillium dahliae was impaired by disruption of a protein elicitor from V. dahliae 1 named PevD1. Constitutive or inducible overexpression of PevD1 accelerated Arabidopsis leaf senescence. Interestingly, a senescence-associated NAC transcription factor, ORE1, was targeted by PevD1. PevD1 could interact with and stabilize ORE1 protein by disrupting its interaction with the RING-type ubiquitin E3 ligase NLA. Mutation of ORE1 suppressed the premature senescence caused by overexpressing PevD1, whereas overexpression of ORE1 or PevD1 led to enhanced ethylene production and thereby leaf senescence. We showed that ORE1 directly binds the promoter of ACS6 and promotes its expression for mediating PevD1-induced ethylene biosynthesis. Loss-of-function of ACSs could suppress V. dahliae-induced leaf senescence in ORE1-overexpressing plants. Furthermore, we found thatPevD1 also interacts with Gossypium hirsutum ORE1 (GhORE1) and that virus-induced gene silencing of GhORE1 delays V. dahliae-triggered leaf senescence in cotton, indicating a possibly conserved mechanism in plants. Taken together, these results suggest that V. dahliae induces leaf senescence by secreting the effector PevD1 to manipulate the ORE1-ACS6 cascade, providing new insights into biotic stress-induced senescence in plants., (Copyright © 2021 The Author. Published by Elsevier Inc. All rights reserved.)

Pseudogymnoascus destructans (Pd), the causative agent of white-nose syndrome in North America, has decimated bat populations within a decade. The fungus impacts bats during hibernation when physiological functions, including immune responses, are down-regulated. Studies have shown that Pd is native to Europe, where it is not associated with mass mortalities. Moreover, genomic and proteomic studies indicated that European bats may have evolved an effective immune defence, which is lacking in North American bats. However, it is still unclear which defence strategy enables European bats to cope with the pathogen. Here, we analyzed selected physiological and immunological parameters in torpid, Pd infected European greater mouse-eared bats (Myotis myotis) showing three different levels of infection (asymptomatic, mild and severe symptoms). From a subset of the studied bats we tracked skin temperatures during one month of hibernation. Contrasting North American bats, arousal patterns remained unaffected by Pd infections in M. myotis. In general, heavier M. myotis aroused more often from hibernation and showed less severe disease symptoms than lean individuals; most likely because heavy bats were capable of reducing the Pd load more effectively than lean individuals. In the blood of severely infected bats, we found higher gene expression levels of an inflammatory cytokine (IL-1β), but lower levels of an acute phase protein (haptoglobin), reactive oxygen metabolites (ROMs) and plasma non-enzymatic antioxidant capacity (OXY) compared to conspecifics with lower levels of infection. We conclude that M. myotis, and possibly also other European bat species, tolerate Pd infections during torpor by using selected acute phase response parameters at baseline levels, yet without arousing from torpor and without synthesizing additional immune molecules., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The present study demonstrated that chitin-based nanofibers (CNFs) trigger the chitinase genes (PGIP1 and CaChi2), while elevating salicylic acid that can protect plants against pathogens. Cross-talk between this genetic induction and salicylic-acid-mediated immune response was also observed, which may arm a plant against multiple pathovars. Crab and mushroom based CNFs were synthesized by electrospinning and ball milling techniques. Plants (mung bean, Vigna radiata) (pepper, Capsicum annuum) were pre-inoculated with CNFs and treated with the pathogens Scrolotium rolfsii for pepper and Macrophomina phaseolina for mung bean and shrimp-based CNFs were used as a control. Treated plants had elevated levels of chitinase genes in response to CNFs at inoculation concentrations <10 mg/mL both in soil and media, to protect them against the pathogenic fungal disease. After 24 h of exposure to the pathogens, qRT-PCR showed genes class II chitinase gene (CaChi2) and polygalacturonase inhibitor protein 1 (PGIP1) to be up-regulated in both root and shoot at 0.1 and 1 mg/mL of inoculation, respectively. The ball milled mushroom CNFs were sufficient to trigger the membrane based enzymes with less diameter (≥15 nm) to be most efficient versus others. In vitro analysis showed IC 50 of ball milled mushroom CNFs to be most efficient in limiting the growth of fungal biomass. Further trigger-like effects were prominent in reducing pathogenic fungal spread in both species., (Copyright © 2021. Published by Elsevier B.V.)

Northern corn leaf blight, caused by the fungal pathogen Setosphaeria turcica (anamorph Exserohilum turcicum), is one of the most devastating foliar diseases of maize (Zea mays). Four genes Ht1, Ht2, Ht3 and Htn1 represent the major sources of genetic resistance against the hemibiotrophic fungus S. turcica. Differential maize lines containing these genes also form the basis to classify S. turcica races. Here, we show that Ht2 and Ht3 are identical and allelic to the previously cloned Htn1 gene. Using a map-based cloning approach and Targeting Induced Local Lesions in Genomes (TILLING), we demonstrate that Ht2/Ht3 is an allele of the wall-associated receptor-like kinase gene ZmWAK-RLK1. The ZmWAK-RLK1 variants encoded by Htn1 and Ht2/Ht3 differ by multiple amino acid polymorphisms that particularly affect the putative extracellular domain. A diversity analysis in maize revealed the presence of dozens of ZmWAK-RLK1 alleles. Ht2, Ht3 and Htn1 have been described over decades as independent resistance loci with different race spectra and resistance responses. Our work demonstrates that these three genes are allelic, which has major implications for northern corn leaf blight resistance breeding and nomenclature of S. turcica pathotypes. We hypothesize that genetic background effects have confounded the classical description of these disease resistance genes in the past., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Background: Sesame (Sesamum indicum) charcoal rot, a destructive fungal disease caused by Macrophomina phaseolina (Tassi) Goid (MP), is a great threat to the yield and quality of sesame. However, there is a lack of information about the gene-for-gene relationship between sesame and MP, and the molecular mechanism behind the interaction is not yet clear. The aim of this study was to interpret the molecular mechanism of sesame resistance against MP in disease-resistant (DR) and disease-susceptible (DS) genotypes based on transcriptomics. This is the first report of the interaction between sesame and MP using this method., Results: A set of core genes that response to MP were revealed by comparative transcriptomics and they were preferentially associated with GO terms such as ribosome-related processes, fruit ripening and regulation of jasmonic acid mediated signalling pathway. It is also exhibited that translational mechanism and transcriptional mechanism could co-activate in DR so that it can initiate the immunity to MP more rapidly. According to weighted gene co-expression network analysis (WGCNA) of differentially expressed gene sets between two genotypes, we found that leucine-rich repeat receptor-like kinase (LRR-RLK) proteins may assume an important job in sesame resistance against MP. Notably, compared with DS, most key genes were induced in DR such as pattern recognition receptors (PRRs) and resistance genes, indicating that DR initiated stronger pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). Finally, the study showed that JA/ET and SA signalling pathways all play an important role in sesame resistance to MP., Conclusions: The defence response to MP of sesame, a complex bioprocess involving many phytohormones and disease resistance-related genes, was illustrated at the transcriptional level in our investigation. The findings shed more light on further understanding of different responses to MP in resistant and susceptible sesame.

Crop breeding for resistance to pathogens largely relies on genes encoding receptors that confer race-specific immunity. Here, we report the identification of the wheat Pm4 race-specific resistance gene to powdery mildew. Pm4 encodes a putative chimeric protein of a serine/threonine kinase and multiple C2 domains and transmembrane regions, a unique domain architecture among known resistance proteins. Pm4 undergoes constitutive alternative splicing, generating two isoforms with different protein domain topologies that are both essential for resistance function. Both isoforms interact and localize to the endoplasmatic reticulum when co-expressed. Pm4 reveals additional diversity of immune receptor architecture to be explored for breeding and suggests an endoplasmatic reticulum-based molecular mechanism of Pm4-mediated race-specific resistance.

A novel fungal immunomodulatory protein (FIP) was found in the precious medical and edible mushroom Morchella conica SH, defined as FIP-mco, which belongs to the FIP family. Phylogenetic analyses of FIPs from different origins were performed using Neighbor-Joining method. It was found that FIP-mco belonged to a new branch of the FIP family and may evolved from a different ancestor compared with most other FIPs. The cDNA sequence of FIP-mco was cloned and expressed in the yeast Pichia Pastoris X33. The recombinant protein of FIP-mco (rFIP-mco) was purified by agarose Ni chromatography and determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The protein rFIP-mco could significantly suppress the proliferation of A549 and HepG2 cells at the concentration of 15 and 5 μg/ml, respectively, and inhibited the migration and invasion of human A549 and HepG2 cells at the concentration of 15 and 30 μg/ml respectively in vitro . Further, rFIP-mco can significantly reduce the expression levels of TNF-α, IL-1β, and IL-6 in the THP1 cells (human myeloid leukemia mononuclear cells). In order to explore the potential mechanism of the cytotoxicity effect of rFIP-mco on A549 and HepG2 cells, cell cycle and apoptosis assay in the two cancer cells were conducted. The results demonstrated that G0/G1 to S-phase arrest and increased apoptosis may contribute to the proliferation inhibition by rFIP-mco in the two cancer cells. Molecular mechanism of rFIP-mco's reduction effect on the inflammatory cytokines was also studied by suppression of the NF-κB signaling pathway. It showed that suppression of NF-κB signaling is responsible for the reduction of inflammatory cytokines by rFIP-mco. The results indicated the prospect of FIP-mco from M. conica SH as an effective and feasible source for cancer therapeutic studies and medical applications., (Copyright © 2020 Wu, Sun, Deng, Song, Li, Zeng and Tang.)

Plant immunity is often defined by the immunity hormones: salicylic acid (SA), jasmonic acid (JA), and ethylene (ET). These hormones are well known for differentially regulating defence responses against pathogens. In recent years, the involvement of other plant growth hormones such as auxin, gibberellic acid, abscisic acid, and cytokinins (CKs) in biotic stresses has been recognized. Previous reports have indicated that endogenous and exogenous CK treatment can result in pathogen resistance. We show here that CK induces systemic immunity in tomato (Solanum lycopersicum), modulating cellular trafficking of the pattern recognition receptor (PRR) LeEIX2, which mediates immune responses to Xyn11 family xylanases, and promoting resistance to Botrytis cinerea and Oidium neolycopersici in an SA- and ET-dependent mechanism. CK perception within the host underlies its protective effect. Our results support the notion that CK promotes pathogen resistance by inducing immunity in the host., (© 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Chromoblastomycosis (CBM) is a chronic worldwide subcutaneous mycosis, caused by several dimorphic, pigmented dematiaceous fungi. It is difficult to treat patients with the disease, mainly because of its recalcitrant nature. The correct activation of host immune response is critical to avoid fungal persistence in the tissue and disease chronification. CD4+ T cells are crucial for the development of protective immunity to F. pedrosoi infection. Here, we investigated T helper cell response dynamics during experimental CBM. Following footpad injection with F. pedrosoi hyphae and conidia, T cells were skewed towards a Th17 and Th1 phenotype. The Th17 population was the main Th cell subset found in the infected area during the early stages of experimental murine CBM, followed by Th1 predominance in the later stages, coinciding with the remission phase of the disease in this experimental model. Depletion of CD25+ cells, which leads to a reduction of Treg cells in the draining lymph node, resulted in decline in fungal burden after 14 days of infection. However, fungal cells were not cleared in the later stages of the disease, prolonging CBM clinical features in those animals. IL-17A and IFN-γ neutralization hindered fungal cell elimination in the course of the disease. Similarly, in dectin-2 KO animals, Th17 contraction in the course of experimental CBM was accompanied by fungal burden decrease in the first 14 days of infection, although it did not affect disease resolution. In this study, we gained insight into T helper subsets' dynamics following footpad injections of F. pedrosoi propagules and uncovered their contribution to disease resolution. The Th17 population proved to be important in eliminating fungal cells in the early stages of infection. The Th1 population, in turn, closely assisted by Treg cells, proved to be relevant not only in the elimination of fungal cells at the beginning of infection but also essential for their complete elimination in later stages of the disease in a mouse experimental model of CBM., Competing Interests: The authors have declared that no competing interests exist.

The chromoblastomycosis is a subcutaneous mycosis with a high morbidity rate, Fonsecaea pedrosoi being the largest etiologic agent of this mycosis, usually confined to the skin and subcutaneous tissues. Rarely people get the cure, because the therapies shown to be deficient and few studies report the host-parasite relationship. Dendritic cells (DCs) are specialized in presenting antigens to naïve T lymphocytes inducing primary immune responses. Therefore, we propose to study the migratory capacity of DCs after infection with conidia of F. pedrosoi. The phenotype of DCs was evaluated using cells obtained from footpad and lymph nodes of BALB/c mice after 12, 24 and 72 h of infection. After 24 and 72 h of infection, we found a significant decrease in DCs in footpad and a significant increase in the lymph nodes after 72 h. The expression of surface markers and co-stimulatory molecules were reduced in cells obtained from footpad. To better assess the migratory capacity of DCs migration from footpad, CFSE-stained conidia were injected subcutaneously. We found that after 12 and 72 h, CD11c + cells were increased in regional lymph nodes, leading us to believe that DCs (CD11c + ) were able to phagocytic conidia present in footpad and migrated to regional lymph nodes.

Background: Tree moss (Pseudevernia furfuracea [L.] Zopf.), a lichen growing on conifers, is a frequent fragrance sensitizer. Previous studies have shown two subgroups of tree moss-allergic patients: a group sensitized to common allergens of tree and oak moss (Evernia prunastri), and another group sensitized to colophonium-derived allergens, which may contaminate tree moss extract., Objectives: To report the results of including tree moss extract in the baseline series and discuss the clinical implications., Methods: Tree moss extract was included in the baseline series and sensitized patients were assessed for concomitant allergy to colophonium and oak moss, and the relevance of these reactions was analyzed., Results: Altogether, 22 of 632 patients (3.5%) had positive reactions to tree moss. Eight patients were sensitized to tree moss only (among fragrance allergens) and 75% had relevant reactions to colophonium. Fourteen patients were sensitized to other fragrance allergens as well and 28.5% had relevant colophonium reactions., Conclusions: The prevalence of positive tree moss reactions is high enough to justify its inclusion in the baseline series. If tree moss is not included, patients with positive colophonium reactions should be informed of possible (false) cross-reactivity to tree moss to avoid this labeled fragrance allergen., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Key Message: Modification of the poplar defense pathway through pathogen-induced expression of an amphibian host defense peptide modulates plant innate immunity and confers robust and reliable resistance against a major poplar pathogen, Septoria musiva. Host defense peptides (HDPs), also known as cationic antimicrobial peptides, represent a diverse group of small membrane-active molecules that are part of the innate defense system of their hosts against pathogen invasion. Here we describe a strategy for development of poplar plants with enhanced HDP production and resistance to the commercially significant fungal pathogen Septoria musiva. The naturally occurring linear amphipathic α-helical HDP dermaseptin B1, which has 31 residues and originated from the skin secretion of arboreal frogs, was N-terminally modified (MsrA2) and evaluated in vitro for antifungal activity and phytotoxicity. The MsrA2 peptide inhibited germination of S. musiva conidia at physiologically relevant low micromolar concentrations that were non-toxic to poplar protoplasts. The nucleotide sequence of MsrA2, optimized for expression in plants, was introduced into the commercial hybrid poplar Populus nigra L. × P. maximowiczii A. Henry (NM6) via Agrobacterium-mediated transformation. Transgene expression was regulated by the pathogen-inducible poplar promoter win3.12T, a part of the poplar innate defense system. Most importantly, the induced accumulation of MsrA2 peptide in poplar leaves was sufficient to confer resistance against S. musiva. The antifungal resistance of plants with high MsrA2 expression and MsrA2 accumulation was strong and reproducible, and without deleterious effects on plant growth and development. These results provide an insight into development of new technologies for engineering durable disease resistance against major pathogens of poplar and other plants.

Field-collected US strain C18 of Cryphonectria parasitica, the chestnut blight fungus, was earlier reported to be infected by a double-stranded RNA virus, mycoreovirus 2 (MyRV2). Next-generation sequencing has revealed co-infection of C18 by a positive-strand RNA virus, hypovirus 4 (CHV4). The current molecular and genetic analyses showed interesting commensal interactions between the two viruses. CHV4 facilitated the stable infection and enhanced vertical transmission of MyRV2, which was readily lost during subculturing and showed reduced vertical transmission in single infections. Deletion of a key antiviral RNA silencing gene, dcl2, in isolate C18 increased stability of MyRV2 in single infections. The ability of CHV4 to facilitate stable infection with MyRV2 appears to be associated with the inhibitory effect of CHV4 on RNA silencing via compromising the induction of transcriptional up-regulation of dcl2. These results suggest that natural infection of isolate C18 by MyRV2 in the field was facilitated by CHV4 co-infection., (Copyright © 2019. Published by Elsevier Inc.)

Recently, we have reported serological cross-reactivity between paracoccidioidomycosis ceti and paracoccidioidomycosis, histoplasmosis, and coccidioidomycosis. However, data on the interaction of Arthrographis kalrae with the above pathogenic fungal infections are lacking. A. kalrae is a widely occurring ascomycetous fungus; causes superficial and deep mycoses; shows thermally dependent dimorphism; and has a genomic profile related to the above-mentioned fungal species. Our study aims to investigate cross-reactivity using eight murine sera, obtained from experimental infection with two A. kalrae isolates. The murine sera were incubated with fungal cells of A. kalrae, Coccidioides posadasii, Histoplasma capsulatum, Paracoccidioides sp., and P. brasiliensis. Thirty murine sera, obtained from experimental infection with six isolates of H. capsulatum, sera from three cases of dolphin paracoccidioidomycosis ceti, two human sera from patients with paracoccidioidomycosis, and a serum sample from a healthy person with a history of coccidioidomycosis, were also incubated with A. kalrae fungal cells and the respective fungal cells that caused the infection as positive controls. Sera derived from the mice infected with A. kalrae reacted strongly when incubated with the Paracoccidioides sp., P. brasiliensis, and C. posadasii, but no positive reaction was observed against the fungal cells of H. capsulatum. The murine sera infected with three out of six isolates of H. capsulatum, and all cetacean and human serum samples reacted positively with the fungal cells of A. kalrae. The present study demonstrated serological cross-reactions among A. kalrae infection, coccidioidomycosis, paracoccidioidomycosis, paracoccidioidomycosis ceti, and histoplasmosis.

White-nose syndrome (WNS) caused by the fungus, Pseudogymnoascus destructans (Pd) has killed millions of North American hibernating bats. Currently, methods to prevent the disease are limited. We conducted two trials to assess potential WNS vaccine candidates in wild-caught Myotis lucifugus. In a pilot study, we immunized bats with one of four vaccine treatments or phosphate-buffered saline (PBS) as a control and challenged them with Pd upon transfer into hibernation chambers. Bats in one vaccine-treated group, that received raccoon poxviruses (RCN) expressing Pd calnexin (CAL) and serine protease (SP), developed WNS at a lower rate (1/10) than other treatments combined (14/23), although samples sizes were small. The results of a second similar trial provided additional support for this observation. Bats vaccinated orally or by injection with RCN-CAL and RCN-SP survived Pd challenge at a significantly higher rate (P = 0.01) than controls. Using RT-PCR and flow cytometry, combined with fluorescent in situ hybridization, we determined that expression of IFN-γ transcripts and the number of CD4 + T-helper cells transcribing this gene were elevated (P < 0.10) in stimulated lymphocytes from surviving vaccinees (n = 15) compared to controls (n = 3). We conclude that vaccination with virally-vectored Pd antigens induced antifungal immunity that could potentially protect bats against WNS.

Background: Members of the Pleosporaceae family are known as important sources of airborne allergens which are responsible for asthma and allergic diseases., Aims: The purpose of this study was to investigate the gene profiling and expression pattern of Alt a 1 in Alternaria alternata and other members of the Pleosporaceae family including Stemphylium botryosum, Ulocladium chartarum, Curvularia lunata, Cladosporium cladosporioides, and Epicoccum nigrum., Methods: Alternaria alternata and related genera were cultured on Czapek-Dox broth medium at 25°C for 21 days. The presence of Alt a 1 was assessed in fungal culture filtrates by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then confirmed by immunoblot analysis. Real-time PCR was carried out for quantitation of the Alt a 1 gene encoding corresponding protein at the transcriptional level using cDNA prepared from fungal RNA., Results: SDS-PAGE showed protein bands ranging from 14 to 100kDa. A 14kDa band corresponding to Alt a 1 was present in A. alternata, S. botryosum and U. chartarum. The gene expression of Alt a 1 was reported in A. alternata and some other related genera. The C t mean value recorded for A. alternata strains ranged from 24.70 to 27.84 while it was in the range 23.62-32.09 for other related taxa. No apparent transcription or expression was revealed in C. cladosporioides., Conclusions: The presence and efficient expression of Alt a 1 gene in A. alternata and other related taxa indicate that Alt a 1 protein is a major component of the secretory machinery of Pleosporaceae family members, and it may play a crucial role in its allergenicity., (Copyright © 2018 Asociación Española de Micología. Publicado por Elsevier España, S.L.U. All rights reserved.)

Purpose: CARD9 deficiency is an inborn error of immunity that predisposes otherwise healthy humans to mucocutaneous and invasive fungal infections, mostly caused by Candida, but also by dermatophytes, Aspergillus, and other fungi. Phaeohyphomycosis are an emerging group of fungal infections caused by dematiaceous fungi (phaeohyphomycetes) and are being increasingly identified in patients with CARD9 deficiency. The Corynespora genus belongs to phaeohyphomycetes and only one adult patient with CARD9 deficiency has been reported to suffer from invasive disease caused by C. cassiicola. We identified a Colombian child with an early-onset, deep, and destructive mucocutaneous infection due to C. cassiicola and we searched for mutations in CARD9., Methods: We reviewed the medical records and immunological findings in the patient. Microbiologic tests and biopsies were performed. Whole-exome sequencing (WES) was made and Sanger sequencing was used to confirm the CARD9 mutations in the patient and her family. Finally, CARD9 protein expression was evaluated in peripheral blood mononuclear cells (PBMC) by western blotting., Results: The patient was affected by a large, indurated, foul-smelling, and verrucous ulcerated lesion on the left side of the face with extensive necrosis and crusting, due to a C. cassiicola infectious disease. WES led to the identification of compound heterozygous mutations in the patient consisting of the previously reported p.Q289* nonsense (c.865C > T, exon 6) mutation, and a novel deletion (c.23&#95;29del; p.Asp8Alafs10*) leading to a frameshift and a premature stop codon in exon 2. CARD9 protein expression was absent in peripheral blood mononuclear cells from the patient., Conclusion: We describe here compound heterozygous loss-of-expression mutations in CARD9 leading to severe deep and destructive mucocutaneous phaeohyphomycosis due to C. cassiicola in a Colombian child.

Sclerotinia stem rot, caused by the fungal pathogen Sclerotinia sclerotiorum, is a destructive disease of canola and many other broadleaf crops. The primary inoculum responsible for initiating Sclerotinia epidemics is airborne ascospores released from the apothecia of sclerotia. Timely detection of the presence of airborne ascospores can serve as an early-warning system for forecasting and management of the disease. A major challenge is to develop a portable and automated device which can be deployed onsite to detect and quantify the presence of minute quantities of ascospores in the air and serves as a unit in a network of systems for forecasting of the epidemic. In this communication, we present the development of an impedimetric non-Faradaic biosensor based on anti-S. sclerotiorum polyclonal antibodies as probes to selectively capture the ascospores and sense their binding by an impedance based interdigitated electrode which was found to directly and unambiguously correlate the number of ascospores on sensor surface with the impedance response.

Aim: To study the pathogenesis of chromoblastomycosis using the alternative model host Galleria mellonella., Methodology: We analyzed the virulence of different dematiaceous fungal strains and the host immune responses (hemocytes density and morphological changes) to Fonsecaea monophora by the alternative infection model. Then detected the development of the pathogenic muriform cells within larvae under microscope., Results: Increasing inocula resulted in greater larval mortality and Cladophialophora carrionii was the most virulent. Low inocula activated the humoral immune response significantly. Moreover, the conidia underwent morphological transition to muriform cells within larvae., Conclusion: We developed an invertebrate host model that can be used to evaluate the virulence of dematiaceous fungi, which may provide further insights into overcoming current limitations in studying chromoblastomycosis in vivo.

Ascomycetes and zygomycetes account for the majority of (i) fungi responsible for cutaneous, subcutaneous, and invasive human fungal infections, (ii) plant fungal pathogens, (iii) fungi that threaten global biodiversity, (iv) fungal agents of agricultural spoilage, and (v) fungi in water-damaged buildings. Rapid recognition of fungal infection (or contamination) enables early treatment (or remediation). A bioinformatics search found homologues of Saccharomyces cerevisiae Mnn9p present in members of the Zygomycota and Ascomycota phyla and absent in members of the Chytridiomycota and Basidiomycota. Mnn9p is a component of the yeast mannan polymerization complex and is necessary for α-1,6 mannan production. A monoclonal antibody (2DA6) was produced that was reactive with purified mannans of Mucor , Rhizopus , Aspergillus , Fusarium , and Candida species. Experimentation using a 2DA6 antigen capture enzyme-linked immunosorbent assay (ELISA) and extracts of fungi from the four phyla found agreement between the presence or absence of Mnn9p homologues and production or lack of production of mannan reactive with 2DA6. Studies of cell extracts from yeast mannan mutants identified α-1,6 mannan as the epitope recognized by 2DA6. To translate this finding into a point-of-use diagnostic, a 2DA6 lateral flow immunoassay was constructed that detected mannan in (i) extracts of dermatophytes and fungi that produce trauma-related infection and (ii) tissue from plants infected with Grosmannia clavigera or Sclerotium cepivorum These studies (i) revealed that the conservation of α-1,6-linked mannan in fungi of the Zygomycota and Ascomycota can be exploited as a broad diagnostic target and (ii) have provided a means to detect that target in an immunoassay platform that is well suited for clinic or field use. IMPORTANCE A key question asked when faced with an infection, an infestation, or environmental damage is whether it is a fungus. Identification of fungi as the cause of the problem can lead to remediation or treatment. Zygomycetes and ascomycetes account for the vast majority of fungal causes of human, animal, and plant disease, large-scale biodiversity loss, agricultural spoilage, and contamination of water-damaged buildings. These studies revealed the conservation of a common cell wall structural component of zygomycetes and ascomycetes to be a diagnostic target applicable to multiple pathogenic fungi and have leveraged that insight for practical use. Monoclonal antibodies reactive with this pan-fungal structure were produced and used to construct immunoassays (including ELISA and lateral flow assay) for detection of a broad range of pathogenic fungi., (Copyright © 2018 Burnham-Marusich et al.)

Fonsecaea pedrosoi (F. pedrosoi) is the most common agent of chromoblastomycosis. Transformation of this fungus from its saprophytic phase into pathogenic sclerotic cells in tissue is an essential link to the refractoriness of this infection. Experimental studies in murine models have shown that the absence of CD4+ T cells impairs host defense against F. pedrosoi infection. Clinical research has also suggested that a relatively low level of the Th1 cytokine INF-γ and inefficient T cell proliferation are simultaneously present in patients with severe chromoblastomycosis upon in vitro stimulation with ChromoAg, an antigen prepared from F. pedrosoi. In the present study, we show that in mice intraperitoneally infected with F. pedrosoi-spores, -hyphae or in vitro-induced sclerotic cells respectively, the transformation of this causative agent into sclerotic cells contributes to a compromised Th1 cytokine production in the earlier stage of infection with impaired generation of neutrophil reactive oxygen species (ROS) and pan-inhibition of Th1/Th2/Th17 cytokine production with disseminated infection in the later stage by using a CBA murine Th1/Th2/Th17 cytokine kit. In addition, we have further demonstrated that intraperitoneal administration of recombinant mouse IFN-γ (rmIFN-γ) effectively reduces the fungal load in the infected mouse spleen, and dampens the peritoneal dissemination of F. pedrosoi-sclerotic cells. Meanwhile, exogeneous rmIFN-γ contributes to the formation and maintenance of micro-abscess and restores the decrease in neutrophil ROS generation in the mouse spleen infected with F. pedrosoi-sclerotic cells. Of note, we have once again demonstrated that it is a chitin-like component, but not β-glucans or mannose moiety, that exclusively accumulates on the outer cell wall of F. pedrosoi-sclerotic cells which were induced in vitro or isolated from the spleens of intraperitoneally infected BALB/c mice. In addition, our results indicate that decreased accumulation of chitin on the surface of live F. pedrosoi-sclerotic cells after chitinase treatment can be self-compensated in a time-dependent manner. Importantly, we have for the first time demonstrated that exclusive accumulation of chitin on the transformed sclerotic cells of F. pedrosoi is involved in an impaired murine Th1 cytokine profile, therefore promoting the refractoriness of experimental murine chromoblastomycosis.

Background: Ascochyta blight, caused by the fungus Ascochyta lentis, is one of the most destructive lentil diseases worldwide, resulting in over $16 million AUD annual loss in Australia alone. The use of resistant cultivars is currently considered the most effective and environmentally sustainable strategy to control this disease. However, little is known about the genes and molecular mechanisms underlying lentil resistance against A. lentis., Results: To uncover the genetic basis of lentil resistance to A. lentis, differentially expressed genes were profiled in lentil plants during the early stages of A. lentis infection. The resistant 'ILL7537' and susceptible 'ILL6002' lentil genotypes were examined at 2, 6, and 24 h post inoculation utilising high throughput RNA-Sequencing. Genotype and time-dependent differential expression analysis identified genes which play key roles in several functions of the defence response: fungal elicitors recognition and early signalling; structural response; biochemical response; transcription regulators; hypersensitive reaction and cell death; and systemic acquired resistance. Overall, the resistant genotype displayed an earlier and faster detection and signalling response to the A. lentis infection and demonstrated higher expression levels of structural defence-related genes., Conclusions: This study presents a first-time defence-related transcriptome of lentil to A. lentis, including a comprehensive characterisation of the molecular mechanism through which defence against A. lentis is induced in the resistant lentil genotype.

Lysine acetylation is a key posttranslational modification that regulates diverse proteins involved in a range of biological processes. The role of histone acetylation in plant defense is well established, and it is known that pathogen effector proteins encoding acetyltransferases can directly acetylate host proteins to alter immunity. However, it is unclear whether endogenous plant enzymes can modulate protein acetylation during an immune response. Here, we investigate how the effector molecule HC-toxin (HCT), a histone deacetylase inhibitor produced by the fungal pathogen Cochliobolus carbonum race 1, promotes virulence in maize through altering protein acetylation. Using mass spectrometry, we globally quantified the abundance of 3,636 proteins and the levels of acetylation at 2,791 sites in maize plants treated with HCT as well as HCT-deficient or HCT-producing strains of C. carbonum Analyses of these data demonstrate that acetylation is a widespread posttranslational modification impacting proteins encoded by many intensively studied maize genes. Furthermore, the application of exogenous HCT enabled us to show that the activity of plant-encoded enzymes (histone deacetylases) can be modulated to alter acetylation of nonhistone proteins during an immune response. Collectively, these results provide a resource for further mechanistic studies examining the regulation of protein function by reversible acetylation and offer insight into the complex immune response triggered by virulent C. carbonum ., Competing Interests: The authors declare no conflict of interest., (Copyright © 2017 the Author(s). Published by PNAS.)

Ascochyta blight causes severe losses in field pea production and the search for resistance traits towards the causal agent Didymella pinodes is of particular importance for farmers. Various microsymbionts have been reported to shape the plants' immune response. However, regardless their contribution to resistance, they are hardly included in experimental designs. We delineate the effect of symbionts (rhizobia, mycorrhiza) on the leaf proteome and metabolome of two field pea cultivars with varying resistance levels against D. pinodes and, furthermore, show cultivar specific symbiont colonisation efficiency. The pathogen infection showed a stronger influence on the interaction with the microsymbionts in the susceptible cultivar, which was reflected in decreased nodule weight and root mycorrhiza colonisation. Vice versa, symbionts induced variation of the host's infection response which, however, was overruled by genotypic resistance associated traits of the tolerant cultivar such as maintenance of photosynthesis and provision of sugars and carbon back bones to fuel secondary metabolism. Moreover, resistance appears to be linked to sulphur metabolism, a functional glutathione-ascorbate hub and fine adjustment of jasmonate and ethylene synthesis to suppress induced cell death. We conclude that these metabolic traits are essential for sustainment of cell vitality and thus, a more efficient infection response., Significance: The infection response of two Pisum sativum cultivars with varying resistance levels towards Didymella pinodes was analysed most comprehensively at proteomic and metabolomic levels. Enhanced tolerance was linked to newly discovered cultivar specific metabolic traits such as hormone synthesis and presumably suppression of cell death., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Dematiaceous molds are found ubiquitously in the environment and cause a wide spectrum of human disease, including infections associated with high rates of mortality. Despite this, the mechanism of the innate immune response has been less well studied, although it is key in the clearance of fungal pathogens. Here, we focus on Exserohilum rostratum , a dematiaceous mold that caused 753 infections during a multistate outbreak due to injection of contaminated methylprednisolone. We show that macrophages are incapable of phagocytosing Exserohilum Despite a lack of phagocytosis, macrophage production of tumor necrosis factor alpha is triggered by hyphae but not spores and depends upon Dectin-1, a C-type lectin receptor. Dectin-1 is specifically recruited to the macrophage-hyphal interface but not the macrophage-spore interface due to differences in carbohydrate antigen expression between these two fungal forms. Corticosteroid and antifungal therapy perturb this response, resulting in decreased cytokine production. In vivo soft tissue infection in wild-type mice demonstrated that Exserohilum provokes robust neutrophilic and granulomatous inflammation capable of thwarting fungal growth. However, coadministration of methylprednisolone acetate results in robust hyphal tissue invasion and a significant reduction in immune cell recruitment. Our results suggest that Dectin-1 is crucial for macrophage recognition and the macrophage response to Exserohilum and that corticosteroids potently attenuate the immune response to this pathogen., (Copyright © 2017 American Society for Microbiology.)

Mycosphaerella graminicola (Zymoseptoria tritici commonly known as Septoria), the causal agent of Septoria Leaf Blotch (STB), is considered one of the major threats to European wheat production. Previous studies have shown the importance of ubiquitination in plant defence against a multitude of pathogens. However the ubiquitination machinery in wheat is under studied, particularly E2 enzymes that have the ability to control the ubiquitination and thereby the fate of many different target proteins. In this study we identify an E2 enzyme, Triticum aestivum Ubiquitin conjugating enzyme 4 (TaU4) that functions in wheat defence against Septoria. We demonstrate TaU4 to be a bona fide E2 enzyme through an E2 charging assay. TaU4 localises in both the cytoplasm and nucleus, therefore potentially interacting with E3 ligases and substrate proteins in multiple compartments. Virus Induced Gene Silencing of TaU4 in wheat leaves resulted in delayed development of disease symptoms, reduced Septoria growth and reproduction. We conclude that TaU4 is a novel negative regulator of defence against Septoria.

Disease-resistance genes encoding intracellular nucleotide-binding domain and leucine-rich repeat proteins (NLRs) are key components of the plant innate immune system and typically detect the presence of isolate-specific avirulence (AVR) effectors from pathogens. NLR genes define the fastest-evolving gene family of flowering plants and are often arranged in gene clusters containing multiple paralogs, contributing to copy number and allele-specific NLR variation within a host species. Barley mildew resistance locus a (Mla) has been subject to extensive functional diversification, resulting in allelic resistance specificities each recognizing a cognate, but largely unidentified, AVR a gene of the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). We applied a transcriptome-wide association study among 17 Bgh isolates containing different AVR a genes and identified AVR a1 and AVR a13 , encoding candidate-secreted effectors recognized by Mla1 and Mla13 alleles, respectively. Transient expression of the effector genes in barley leaves or protoplasts was sufficient to trigger Mla1 or Mla13 allele-specific cell death, a hallmark of NLR receptor-mediated immunity. AVR a1 and AVR a13 are phylogenetically unrelated, demonstrating that certain allelic MLA receptors evolved to recognize sequence-unrelated effectors. They are ancient effectors because corresponding loci are present in wheat powdery mildew. AVR A1 recognition by barley MLA1 is retained in transgenic Arabidopsis, indicating that AVR A1 directly binds MLA1 or that its recognition involves an evolutionarily conserved host target of AVR A1 Furthermore, analysis of transcriptome-wide sequence variation among the Bgh isolates provides evidence for Bgh population structure that is partially linked to geographic isolation., Competing Interests: The authors declare no conflict of interest.

Innate immune responses in host plants begin with the recognition of pathogen-specific nonself molecules and terminate with the secretion of immune molecules. In the dicotyledonous model plant, Arabidopsis thaliana, two distinct secretory pathways required for disease resistance to powdery mildew fungi have been identified so far. One is an exocytic pathway consisting of PEN1, SNAP33 and VAMP721/722 SNARE proteins, but the other is an efflux-mediated one composed of PEN2 atypical myrosinase and PEN3 ABC transporter. Based on the conservation of the mechanically same exocytic pathway in the monocotyledonous plant barely, the former is regarded as an ancient secretory pathway, whereas the latter is considered as a newly evolved one in the Brassicaceae family including Arabidopsis. We recently identified synaptotagmin 1 as an additional regulator of these two secretory pathways. With current results, we discuss how these two secretory pathways contribute to Arabidopsis immunity depending on fungal adaptedness to Arabidopsis.

Arthrographis kalrae is occasionally described as an opportunistic human pathogen. This study investigated the immune response to A. kalrae during murine experimental infection (7, 14, 28 and 56 days post infection). The fungal load was higher in the early phase and mice presented with neurological syndrome over the course of the infection. There was a gradual increase in the level of anti-A. kalrae IgG and increased levels of DTH at 14 days. There was decreased IFN-γ (14-56 days) and an increase in IL-4 (7 and 56 days). Decreased levels of cytokines (IFN-γ, IL-4, IL-10 and IL-17) were observed in the brain at 56 days p.i. The results suggest that the immune response during murine A. kalrae infection modulates to the pattern of Th2 response. This study shows for the first time the cytokines and cellular immunomodulation that occur in response to an experimental infection with A. kalrae in mice., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The effect of chitosan- and vanillin-based immune modulators on the development of the phytopathogen Cochliobolus sativus (S. Ito & Kurib.) Drechsler ex Dastur, which induces dark-brown blotch (helminthosporiosis) in wheat, has been studied. It was shown that treatment with these substances led to a decreased injured area in leaves and an increase in the biotrophic period of pathogen development. It was found that vanillin-modified chitosan effectively provided wheat resistance to hemibiotrophic pathogen C. sativus. Changes in leaf peroxidase activity correlated with the manifestation of disease symptoms.

Bananas and plantains are considered an important crop around the world. Banana production is affected by several constraints, of which Black Sigatoka Disease, caused by the fungus Mycosphaerella fijiensis, is considered one of the most important diseases in banana plantations. The banana accession 'Calcutta-4' has a natural resistance to Black Sigatoka; however, the fruit is not valuable for commercialization. Gene identification and expression studies in 'Calcutta-4' might reveal possible gene candidates for resistant to the disease and elucidate mechanisms for resistance. A subtracted cDNA library was generated from leaves after 6, 9 and 12 days inoculated with M. fijiensis conidia on greenhouse banana plants of the accession 'Calcutta-4'. Bioinformatic analysis revealed 99 good quality sequences. Blast2go analysis revealed that 31% of the sequences could not be categorized and, according to the Biological Process Category, 32 and 28 ESTs are related to general metabolic and cellular processes, respectively; while 10 ESTs response to stimulus. Seven sequences were redundant and one was similar to genes that may be involved in pathogen resistance including the putative disease resistance protein RGA1. Genes encoding zinc finger domains were identified and may play an important role in pathogen resistance by inducing the expression of downstream genes. Expression analysis of four selected genes was performed using RT-qPCR during the early stage of the disease development at 6, 9, 12 and 15 days post inoculation showing a peak of up regulation at 9 or 12 days post inoculation. Three of the four genes showed an up-regulation of expression in 'Calcutta-4' when compared to 'Williams' after inoculation with M. fijiensis, suggesting a fine regulation of specific gene candidates that may lead to a resistance response. The genes identified in early responses in a plant-pathogen interaction may be relevant for the resistance response of 'Calcutta-4' to Black Sigatoka. Genes with different functions may play a role in plant response to the disease. The present study suggests a fine up regulation of these genes that might be needed to perform an incompatible interaction. Further gene functional studies need to be performed to validate their use as candidate resistance genes in susceptible banana cultivars.

The worldwide prevalence of fungal allergy in recent years has augmented mining allergens from yet unexplored ones. Curvularia pallescens (CP) being a dominant aerospore in India and a major sensitiser on a wide range of allergic population, pose a serious threat to human health. Therefore, we aimed to identify novel allergens from CP in our present study. A cohort of 22 CP-sensitised patients was selected by positive Skin prick grade. Individual sera exhibited elevated specific IgE level and significant histamine release on a challenge with antigenic extract of CP. First gel-based profiling of CP proteome was done by 1- and 2-dimensional gel. Parallel 1- and 2-dimensional immunoblot were performed applying individual as well as pooled patient sera. Identification of the sero-reactive spots from the 2-dimensional gel was found to be challenging as CP was not previously sequenced. Hence, mass spectrometry-based proteomic workflow consisting of conventional database search was not alone sufficient. Therefore, de novo sequencing preceded homology search was implemented for further identification. Altogether 11 allergenic proteins including Brn-1, vacuolar protease, and fructose-bis-phosphate aldolase were identified with high statistical confidence (p<0.05). This is the first study to report on any allergens from CP. This kind of proteome-based analysis provided a catalogue of CP allergens that would lead an improved way of diagnosis and therapy of CP-related allergy., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Unlabelled: The white mould disease, caused by Sclerotinia sclerotiorum, is one of the most important diseases in the vital oil crop Brassica napus. Nevertheless, the defense mechanisms of B. napus against S. sclerotiorum are poorly understood. In this study, we performed comparative quantitative proteomics analyses to reveal B. napus defense mechanisms against S. sclerotiorum. The proteomes of B. napus leaves inoculated with S. sclerotiorum wild-type strain 1980 and nonpathogenic mutant strain Ep-1PB as well as empty agar plug as the control were analyzed using TMT label-based quantitative analysis technique. A total of 79, 299 and 173 proteins consistently differentially expressed between Ep-1PB- and mock-inoculated leaves, 1980- and mock-inoculated leaves, as well as 1980- and Ep-1PB-inoculated leaves, respectively, were identified. The differential expression of 12 selected proteins was confirmed by qRT-PCR analyses. The Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein-protein interaction prediction analyses revealed that redox homeostasis, lipid signaling, calcium signaling, histone and DNA methylation-mediated transcription regulation and defense-related proteins such as defensin and defensin-like proteins and cyanate lyase, contribute to defense against S. sclerotiorum. Our results provide new insights into molecular mechanisms that may be involved in defense responses of B. napus to S. sclerotiorum., Significance: The Sclerotinia white mould disease is one of the most important diseases in the significant oil crop Brassica napus. Nevertheless, the defense mechanisms of B. napus against S. sclerotiorum are still largely unknown to date. In this study, we addressed this issue by performing TMT label-based comparative quantitative analyses of the proteomes of B. napus leaves inoculated with S. sclerotiorum wild-type strain 1980 and nonpathogenic mutant strain Ep-1PB as well as empty agar plug as the control. Through comparative analyses on 79, 299, and 173 proteins that are consistently differentially expressed in between Ep-1PB-inoculated and the control leaves, 1980-inoculated and the control leaves, as well as 1980-inoculated and Ep-1PB-inoculated leaves, respectively, we revealed that redox homeostasis, lipid signaling, calcium signaling, histone and DNA methylation-mediated transcription regulation and defense-related proteins such as defensin and defensin-like proteins as well as cyanate lyase, contribute to B. napus defenses against S. sclerotiorum. Notably, the potential role of lipid signaling, calcium signaling, histone and DNA methylation-mediated transcription regulation and cyanate lyase in B. napus defense against S. sclerotiorum are not reported previously but rather unveiled for the first time in this study. The current study represents the most extensive analysis of the protein profile of B. napus in response to S. sclerotiorum inoculation and includes for the first time the results from comparison between plants inoculated with the wild-type strain and a nonpathogenic mutant strain of S. sclerotiorum. Collectively, our results provide new insights into the molecular mechanisms of interactions between B. napus and S. sclerotiorum., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Unlabelled: Oxalic acid (OA) plays dual role in fungal pathogenicity in a concentration dependent manner. While at higher concentration it induces programmed cell death leading to fungal invasion, low oxalate build resistance in plant. Although OA has been identified as a virulence determinant for rot disease caused by Sclerotinia sp., our understanding of how oxalate downregulation impart host immunity is limited. We have earlier shown that ectopic expression of oxalate decarboxylase (FvOXDC) specifically degrades OA in tomato (Solanum lycopersicum). To elucidate low oxalate regulated molecular mechanism imparting immunity, a comparative proteomics approach has been applied to E8.2-OXDC tomato fruit displaying fungal resistance. Mass spectrometric analyses identified 92 OXDC-responsive immunity related protein spots (ORIRPs) presumably associated with acid metabolism, defense signaling and endoplasmic reticulum stress. Metabolome study indicated increased abundance of some of the organic acids paralleling the proteomic analysis. Further, we interrogated the proteome data using network analysis that identified modules enriched in known and novel immunity-related prognostic proteins centered around 14-3-3, translationally controlled tumor protein, annexin and chaperonin. Taken together, our data demonstrate that low oxalate may act as metabolic and immunity determinant through translational reprogramming., Biological Significance: Although OA plays critical role as fungal elicitor, our understanding of how oxalate downregulation by decarboxylative degradation impart immunity is limited. Our study confirms the impact of oxalate down-regulation on overall cellular physiology and provides new perspectives to study plant immunity. The network representation may facilitate the prioritization of candidate proteins for patho-stress tolerance in crop plant. These findings are of great importance for future work towards functional determination and exploitation of target proteins in crop improvement program., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A comparative investigation of humoral and cellular immune response in larvae of Colorado potato beetle Leptinotarsa decemlineata was conducted under development of mycoses caused by entomopatho- genic fungi Metarhizium robertsii, M. brunneum and M. pemphigi. The larvae were found highly suscep- tible to M. robertsii, M. brunneum and less susceptible to M. pemphigi. The susceptibility to the fungi was not correlated with the rate of conidia germination in epicuticular extracts of larvae. A non-specific for Colorado beetle pathogen M. pemphigi did not cause significant changes in the immune response and did not result in colonization of larvae hemocoel. Infection with M. robertsi and M. brunneum led to an increase in total hemocyte count at the initial stages of mycoses (day 2) followed by a sharp decrease on day 3. The strongest decrease was observed for the immunocompetent cells - plasmatocytes and granu- locytes. Enhanced phenoloxidase activity in hemolymph and cuticle was found on days 2 and 3 after in- fection. These changes in immune reactions correlated with the level of virulence of the strains. Thus, the immune response in Colorado potato beetle larvae is an important factor determining differences in the development of mycoses caused by different Metarhizium species.

The filamentous fungus Lomentospora prolificans is an emerging pathogen causing severe infections mainly among the immunocompromised population. These diseases course with high mortality rates due to great virulence of the fungus, its inherent resistance to available antifungals, and absence of specific diagnostic tools. Despite being widespread in humanized environments, L. prolificans rarely causes infections in immunocompetent individuals likely due to their developed protective immune response. In this study, conidial and hyphal immunomes against healthy human serum IgG were analyzed, identifying immunodominant antigens and establishing their prevalence among the immunocompetent population. Thirteen protein spots from each morph were detected as reactive against at least 70% of serum samples, and identified by liquid chromatography tandem mass spectrometry (LC-MS/MS). Hence, the most seroprevalent antigens were WD40 repeat 2 protein, malate dehydrogenase, and DHN1, in conidia, and heat shock protein (Hsp) 70, Hsp90, ATP synthase β subunit, and glyceraldehyde-3-phosphate dehydrogenase, in hyphae. More interestingly, the presence of some of these seroprevalent antigens was determined on the cell surface, as Hsp70, enolase, or Hsp90. Thus, we have identified a diverse set of antigenic proteins, both in the entire proteome and cell surface subproteome, which may be used as targets to develop innovative therapeutic or diagnostic tools.

A comprehensive exploration of common and specific plant responses to biotrophs and necrotrophs is necessary for a better understanding of plant immunity. Here, we compared the Arabidopsis defense responses evoked by the biotrophic fungus Golovinomyces orontii and the necrotrophic fungus Botrytis cinerea through integrative network analysis. Two time-course transcriptional datasets were integrated with an Arabidopsis protein-protein interaction (PPI) network to construct a G. orontii conditional PPI sub-network (gCPIN) and a B. cinerea conditional PPI sub-network (bCPIN). We found that hubs in gCPIN and bCPIN played important roles in disease resistance. Hubs in bCPIN evolved faster than hubs in gCPIN, indicating the different selection pressures imposed on plants by different pathogens. By analyzing the common network from gCPIN and bCPIN, we identified two network components in which the genes were heavily involved in defense and development, respectively. The co-expression relationships between interacting proteins connecting the two components were different under G. orontii and B. cinerea infection conditions. Closer inspection revealed that auxin-related genes were overrepresented in the interactions connecting these two components, suggesting a critical role of auxin signaling in regulating the different co-expression relationships. Our work may provide new insights into plant defense responses against pathogens with different lifestyles.

Nonhost resistance (NHR) pertains to the most common form of plant resistance against pathogenic microorganisms of other species. Bipolaris maydis is a non-adapted pathogen affecting soybeans, particularly of maize/soybean intercropping systems. However, no experimental evidence has described the immune response of soybeans against B. maydis. To elucidate the molecular mechanism underlying NHR in soybeans, proteomics analysis based on two-dimensional polyacrylamide gel electrophoresis (2-DE) was performed to identify proteins involved in the soybean response to B. maydis. The spread of B. maydis spores across soybean leaves induced NHR throughout the plant, which mobilized almost all organelles and various metabolic processes in response to B. maydis. Some enzymes, including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), mitochondrial processing peptidase (MPP), oxygen evolving enhancer (OEE), and nucleoside diphosphate kinase (NDKs), were found to be related to NHR in soybeans. These enzymes have been identified in previous studies, and STRING analysis showed that most of the protein functions related to major metabolic processes were induced as a response to B. maydis, which suggested an array of complex interactions between soybeans and B. maydis. These findings suggest a systematic NHR against non-adapted pathogens in soybeans. This response was characterized by an overlap between metabolic processes and response to stimulus. Several metabolic processes provide the soybean with innate immunity to the non-adapted pathogen, B. maydis. This research investigation on NHR in soybeans may foster a better understanding of plant innate immunity, as well as the interactions between plant and non-adapted pathogens in intercropping systems.

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins characterized by the presence of two domains of unknown function 26 (DUF26) in their ectodomain. The CRKs form one of the largest groups of receptor-like protein kinases in plants, but their biological functions have so far remained largely uncharacterized. We conducted a large-scale phenotyping approach of a nearly complete crk T-DNA insertion line collection showing that CRKs control important aspects of plant development and stress adaptation in response to biotic and abiotic stimuli in a non-redundant fashion. In particular, the analysis of reactive oxygen species (ROS)-related stress responses, such as regulation of the stomatal aperture, suggests that CRKs participate in ROS/redox signalling and sensing. CRKs play general and fine-tuning roles in the regulation of stomatal closure induced by microbial and abiotic cues. Despite their great number and high similarity, large-scale phenotyping identified specific functions in diverse processes for many CRKs and indicated that CRK2 and CRK5 play predominant roles in growth regulation and stress adaptation, respectively. As a whole, the CRKs contribute to specificity in ROS signalling. Individual CRKs control distinct responses in an antagonistic fashion suggesting future potential for using CRKs in genetic approaches to improve plant performance and stress tolerance.

Virus-induced gene silencing (VIGS) has emerged as a powerful reverse genetic technology in plants supplementary to stable transgenic RNAi and, in certain species, as a viable alternative approach for gene functional analysis. The RNA virus Barley stripe mosaic virus (BSMV) was developed as a VIGS vector in the early 2000s and since then it has been used to study the function of wheat genes. Several variants of BSMV vectors are available, with some requiring in vitro transcription of infectious viral RNA, while others rely on in planta production of viral RNA from DNA-based vectors delivered to plant cells either by particle bombardment or Agrobacterium tumefaciens. We adapted the latest generation of binary BSMV VIGS vectors for the identification and study of wheat genes of interest involved in interactions with Zymoseptoria tritici and here present detailed and the most up-to-date protocols., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

This paper reviews current knowledge about genes for resistance to Septoria tritici blotch (STB) of wheat, caused by Zymoseptoria tritici (formerly Mycosphaerella graminicola). These genes can be placed into two classes, although a few may have characteristics of both classes. Qualitative resistance is controlled by genes which control large fractions of genetic variation, 21 of which have been discovered and mapped so far. Most of them have been shown to be genotype-specific, being effective against the minority of Z. tritici isolates which are avirulent, and Stb6 has been shown to control a gene-for-gene relationship. Most qualitative resistances are unlikely to be durable and some formerly effective genes have been overcome by the evolution of pathogen virulence. Quantitative resistance is generally controlled by genes with small-to-moderate effects on STB. They have generally weaker specificity than qualitative genes and have provided more durable resistance. 89 genome regions carrying quantitative trait loci (QTL) or meta-QTL have been identified to date. Some QTL have been mapped at or near loci of qualitative genes, especially Stb6, which is present in several sources of resistance. Another gene of particular interest is Stb16q, which has been effective against all Z. tritici isolates tested so far. In addition to resistance, the susceptibility of wheat cultivars to STB can also be reduced by disease escape traits, some of which may be undesirable in breeding. The fundamental requirements for breeding for STB-resistance are genetic diversity for resistance in wheat germplasm and a field trial site at which STB epidemics occur regularly and effective selection can be conducted for resistance combined with other desirable traits. If these are in place, knowledge of resistance genes can be applied to improving control of STB., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

The xanthophyll cycle is involved in dissipating excess light energy to protect the photosynthetic apparatus in a process commonly assessed from non-photochemical quenching (NPQ) of chlorophyll fluorescence. Here, it is shown that the xanthophyll cycle is modulated by the necrotrophic pathogen Sclerotinia sclerotiorum at the early stage of infection. Incubation of Sclerotinia led to a localized increase in NPQ even at low light intensity. Further studies showed that this abnormal change in NPQ was closely correlated with a decreased pH caused by Sclerotinia-secreted oxalate, which might decrease the ATP synthase activity and lead to a deepening of thylakoid lumen acidification under continuous illumination. Furthermore, suppression (with dithiothreitol) or a defect (in the npq1-2 mutant) of violaxanthin de-epoxidase (VDE) abolished the Sclerotinia-induced NPQ increase. HPLC analysis showed that the Sclerotinia-inoculated tissue accumulated substantial quantities of zeaxanthin at the expense of violaxanthin, with a corresponding decrease in neoxanthin content. Immunoassays revealed that the decrease in these xanthophyll precursors reduced de novo abscisic acid (ABA) biosynthesis and apparently weakened tissue defense responses, including ROS induction and callose deposition, resulting in enhanced plant susceptibility to Sclerotinia. We thus propose that Sclerotinia antagonizes ABA biosynthesis to suppress host defense by manipulating the xanthophyll cycle in early pathogenesis. These findings provide a model of how photoprotective metabolites integrate into the defense responses, and expand the current knowledge of early plant-Sclerotinia interactions at infection sites.

Vaccines are needed to combat the global rise in fungal diseases. In this issue of Cell Host & Microbe, Wüthrich et al. (2015) identify calnexin as a target of antigen-specific CD4 T cell responses against multiple fungal pathogens. This study illustrates that a conserved epitope triggers convergent cell-mediated immune responses to confer heterologous antifungal immunity., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Fungal infections remain a threat due to the lack of broad-spectrum fungal vaccines and protective antigens. Recent studies showed that attenuated Blastomyces dermatitidis confers protection via T cell recognition of an unknown but conserved antigen. Using transgenic CD4(+) T cells recognizing this antigen, we identify an amino acid determinant within the chaperone calnexin that is conserved across diverse fungal ascomycetes. Calnexin, typically an ER protein, also localizes to the surface of yeast, hyphae, and spores. T cell epitope mapping unveiled a 13-residue sequence conserved across Ascomycota. Infection with divergent ascomycetes, including dimorphic fungi, opportunistic molds, and the agent causing white nose syndrome in bats, induces expansion of calnexin-specific CD4(+) T cells. Vaccine delivery of calnexin in glucan particles induces fungal antigen-specific CD4(+) T cell expansion and resistance to lethal challenge with multiple fungal pathogens. Thus, the immunogenicity and conservation of calnexin make this fungal protein a promising vaccine target., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Exserohilum rostratum caused a multistate fungal meningitis outbreak following iatrogenic inoculation of contaminated methylprednisolone in the United States. To gain insight into the immunopathogenesis of this infection, we studied the innate host responses of human neutrophils against E. rostratum conidia and hyphae with or without methylprednisolone. The neutrophil-induced percentage fungal damage against conidia and hyphae was effector-to-target ratio dependent (≤55%). While methylprednisolone did not affect neutrophil-induced fungal damage by treatment of Exserohilum or neutrophils, it compromised phagocytosis of conidia (P < 0.05). These findings suggest that methylprednisolone-treated neutrophils may have altered phagocytic clearance of Exserohilum conidia, reducing host capacity to contain the invasive process., (© The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Platelet-activating factor acetylhydrolase (PAF-AH) derived from Trichoderma harzianum was upregulated by the interaction of T. harzianum with maize roots or the foliar pathogen Curvularia lunata. PAF-AH was associated with chitinase and cellulase expressions, but especially with chitinase, because its activity in the KO40 transformant (PAF-AH disruption transformant) was lower, compared with the wild-type strain T28. The result demonstrated that the colonization of maize roots by T. harzianum induced systemic protection of leaves inoculated with C. lunata. Such protection was associated with the expression of inducible jasmonic acid pathway-related genes. Moreover, the data from liquid chromatography-mass spectrometry confirmed that the concentration of jasmonic acid in maize leaves was associated with the expression level of defense-related genes, suggesting that PAF-AH induced resistance to the foliar pathogen. Our findings showed that PAF-AH had an important function in inducing systemic resistance to maize leaf spot pathogen.

Dematiaceous fungi constitute a large and heterogeneous group, characterized by having a dark pigment, the dihydroxynaftalen melanin-DHN, inside their cell walls. In nature they are found mainly as soil microbiota or decomposing organic matter, and are spread in tropical and subtropical regions. The fungus Fonsecaea monophora causes chromoblastomycosis in humans, and possesses essential mechanisms that may enhance pathogenicity, proliferation and dissemination inside the host. Glycoconjugates confer important properties to these pathogenic microorganisms. In this work, structural characterization of glycan structures present in two different strains of F. monophora MMHC82 and FE5p4, from clinical and environmental origins, respectively, was performed. Each one were grown on Minimal Medium (MM) and Czapeck-Dox (CD) medium, and the water soluble cell wall glycoconjugates and exopolysaccharides (EPS) were evaluated by NMR, methylation and principal component analysis (PCA). By combining the methylation and 2D NMR analyses, it was possible to visualize the glycosidic profiles of the complex carbohydrate mixtures. Significant differences were observed in β-D-Galf-(1→5) and (1→6) linkages, α- and β-D-Glcp-(1→3), (1→4), and (1→6) units, as well as in α-D-Manp. PCA from (1)H-NMR data showed that MMHC82 from CD medium showed a higher variation in the cell wall carbohydrates, mainly related to O-2 substituted β-D-Galf (δ 106.0/5.23 and δ 105.3/5.23) units. In order to investigate the antigenic response of the glycoconjugates, these were screened against serum from chromoblastomycosis patients. The antigen which contained the cell wall of MMHC82 grown in MM had β-D-Manp units that promoted higher antigenic response. The distribution of these fungal species in nature and the knowledge of how cell wall polysaccharides and glycoconjugates structure vary, may contribute to the better understanding and the elucidation of the pathology caused by this fungus.