The role of ascospores in further spread of QoI-resistant cytochrome b alleles (G143A) in field populations of Mycosphaerella graminicola

Strobilurin fungicides or quinone outside inhibitors (QoIs) have been used successfully to control Septoria leaf blotch in the United Kingdom since 1997. However, QoI-resistant isolates of Mycosphaerella graminicola were reported for the first time at Rothamsted during the summer of 2002. Sequence analysis of the cytochrome b gene revealed that all resistant isolates carried a mutation resulting in the replacement of glycine by alanine at codon 143 (G143A). Extensive monitoring using real-time polymerase chain reaction (PCR) testing revealed that fungicide treatments based on QoIs rapidly selected for isolates carrying resistant A143 (R) alleles within field populations. This selection is driven mainly by polycyclic dispersal of abundantly produced asexual conidia over short distances. In order to investigate the role of sexually produced airborne ascospores in the further spread of R alleles, a method integrating spore trapping with real-time PCR assays was developed. This method enabled us to both quantify the number of M. graminicola ascospores in air samples as well as estimate the frequency of R alleles in ascospore populations. As expected, most ascospores were produced at the end of the growing season during senescence of the wheat crop. However, a rapid increase in R-allele frequency, from 35 to 80%, was measured immediately in airborne ascospore populations sampled in a wheat plot after the first QoI application at growth stage 32. After the second QoI application, most R-allele frequencies measured for M. graminicola populations present in leaves and aerosols sampled from the treated plot exceeded 90%. Spatial sampling and testing of M. graminicola flag leaf populations derived from ascospores in the surrounding crop showed that ascospores carrying R alleles can spread readily within the crop at distances of up to 85 m. After harvest, fewer ascospores were detected in air samples and the R-allele frequencies measured were influenced by ascospores originating from nearby wheat field

Aberrant protein N- glycosylation impacts upon infection-related growth transitions of the haploid plant-pathogenic fungus Mycosphaerella graminicola

The ascomycete fungus Mycosphaerella graminicola is the causal agent of Septoria Tritici Blotch disease of wheat and can grow as yeast-like cells or as hyphae depending on environmental conditions. Hyphal growth is however essential for successful leaf infection. A T-DNA mutagenesis screen performed on haploid spores identified a mutant, which can undergo yeast-like growth but cannot switch to hyphal growth. For this reason the mutant was non-pathogenic towards wheat leaves. The gene affected, MgAlg2, encoded a homologue of Saccharomyces cerevisiae ScAlg2, an alpha-1,2-mannosyltransferase, which functions in the early stages of asparagine-linked protein (N-) glycosylation. Targeted gene deletion and complementation experiments confirmed that loss of MgAlg2 function prevented the developmental growth switch. MgAlg2 was able to functionally complement the S. cerevisiae ScAlg2-1 temperature sensitive growth phenotype. Spores of Delta MgAlg2 mutants were hypersensitive to the cell wall disrupting agent Calcofluor white and produced abnormally hypo-N-glycosylated proteins. Gene expression, proteome and glycoproteome analysis revealed that Delta MgAlg2 mutant spores show responses typically associated with the accumulation of mis-folded proteins. The data presented highlight key roles for protein N-glycosylation in regulating the switch to hyphal growth, possibly as a consequence of maintaining correct folding and localization of key proteins involved in this process

Inactivation of plant infecting fungal and viral pathogens to achieve biological containment in drainage water using UV treatment

Aim: To explore whether ultraviolet (UV) light treatment within a closed circulating and filtered water drainage system can kill plant pathogenic species. Methods and Results: Ultraviolet experiments at 254 nm were conducted to determine the inactivation coefficients for seven plant pathogenic species. At 200 mJ cm-2, the individual species log reductions obtained for six Ascomycete fungi and a cereal virus were as follows: Leptosphaeria maculans (9 center dot 9-log), Leptosphaeria biglobosa (7 center dot 1-log), Barley stripe mosaic virus (BSMV) (4 center dot 1-log), Mycosphaerella graminicola (2 center dot 9-log), Fusarium culmorum (1 center dot 2-log), Fusarium graminearum (0 center dot 6-log) and Magnaporthe oryzae (0 center dot 3-log). Dilution experiments showed that BSMV was rendered noninfectious when diluted to > 1/512. Follow-up large-scale experiments using up to 400 l of microbiologically contaminated waste water revealed that the filtration of drainage water followed by UV treatment could successfully be used to inactivate several plant pathogens. Conclusions: By combining sedimentation, filtration and UV irradiation within a closed system, plant pathogens can be successfully removed from collected drainage water. Significance and Impact of the Study: Ultraviolet irradiation is a relatively low cost, energy efficient and labour nonintensive method to decontaminate water arising from a suite of higher biological containment level laboratories and plant growth rooms where genetically modified and/or quarantine fungal and viral plant pathogenic organisms are being used for research purposes

A conserved fungal glycosyltransferase facilitates pathogenesis of plants by enabling hyphal growth on solid surfaces

Article Authors Metrics Comments Related Content Abstract Author summary Introduction Results and discussion Conclusions Methods Supporting information Acknowledgments References Reader Comments (0) Media Coverage (0) Figures Abstract Pathogenic fungi must extend filamentous hyphae across solid surfaces to cause diseases of plants. However, the full inventory of genes which support this is incomplete and many may be currently concealed due to their essentiality for the hyphal growth form. During a random T-DNA mutagenesis screen performed on the pleomorphic wheat (Triticum aestivum) pathogen Zymoseptoria tritici, we acquired a mutant unable to extend hyphae specifically when on solid surfaces. In contrast “yeast-like” growth, and all other growth forms, were unaffected. The inability to extend surface hyphae resulted in a complete loss of virulence on plants. The affected gene encoded a predicted type 2 glycosyltransferase (ZtGT2). Analysis of >800 genomes from taxonomically diverse fungi highlighted a generally widespread, but discontinuous, distribution of ZtGT2 orthologues, and a complete absence of any similar proteins in non-filamentous ascomycete yeasts. Deletion mutants of the ZtGT2 orthologue in the taxonomically un-related fungus Fusarium graminearum were also severely impaired in hyphal growth and non-pathogenic on wheat ears. ZtGT2 expression increased during filamentous growth and electron microscopy on deletion mutants (ΔZtGT2) suggested the protein functions to maintain the outermost surface of the fungal cell wall. Despite this, adhesion to leaf surfaces was unaffected in ΔZtGT2 mutants and global RNAseq-based gene expression profiling highlighted that surface-sensing and protein secretion was also largely unaffected. However, ΔZtGT2 mutants constitutively overexpressed several transmembrane and secreted proteins, including an important LysM-domain chitin-binding virulence effector, Zt3LysM. ZtGT2 likely functions in the synthesis of a currently unknown, potentially minor but widespread, extracellular or outer cell wall polysaccharide which plays a key role in facilitating many interactions between plants and fungi by enabling hyphal growth on solid matrices

Unraveling incompatibility between wheat and the fungal pathogen Zymoseptoria tritici through apoplastic proteomics

Background: Hemibiotrophic fungal pathogen Zymoseptoria tritici causes severe foliar disease in wheat. However, current knowledge of molecular mechanisms involved in plant resistance to Z. tritici and Z. tritici virulence factors is far from being complete. The present work investigated the proteome of leaf apoplastic fluid with emphasis on both host wheat and Z. tritici during the compatible and incompatible interactions. Results: The proteomics analysis revealed rapid host responses to the biotrophic growth, including enhanced carbohydrate metabolism, apoplastic defenses and stress, and cell wall reinforcement, might contribute to resistance. Compatibility between the host and the pathogen was associated with inactivated plant apoplastic responses as well as fungal defenses to oxidative stress and perturbation of plant cell wall during the initial biotrophic stage, followed by the strong induction of plant defenses during the necrotrophic stage. To study the role of anti-oxidative stress in Z. tritici pathogenicity in depth, a YAP1 transcription factor regulating antioxidant expression was deleted and showed the contribution to anti-oxidative stress in Z. tritici ,but was not required for pathogenicity. This result suggests the functional redundancy of antioxidants in the fungus. Conclusions: The data demonstrate that incompatibility is probably resulted from the proteome-level activation of host apoplastic defenses as well as fungal incapability to adapt to stress and interfere with host cell at the biotrophic stage of the interaction

Quantitative proteomics analysis reveals important roles of N-glycosylation on ER quality control system for development and pathogenesis in Magnaporthe oryzae

The fungal pathogen Magnaporthe oryzae can cause rice blast and wheat blast diseases, which threatens worldwide food production. During infection, M. oryzae follows a sequence of distinct developmental stages adapted to survival and invasion of the host environment. M. oryzae attaches onto the host by the conidium, and then develops an appressorium to breach the host cuticle. After penetrating, it forms invasive hyphae to quickly spread in the host cells. Numerous genetic studies have focused on the mechanisms underlying each step in the infection process, but systemic approaches are needed for a broader, integrated understanding of regulatory events during M. oryzae pathogenesis. Many infection-related signaling events are regulated through post-translational protein modifications within the pathogen. N-linked glycosylation, in which a glycan moiety is added to the amide group of an asparagine residue, is an abundant modification known to be essential for M. oryzae infection. In this study, we employed a quantitative proteomics analysis to unravel the overall regulatory mechanisms of N-glycosylation at different developmental stages of M. oryzae. We detected changes in N-glycosylation levels at 559 glycosylated residues (N-glycosites) in 355 proteins during different stages, and determined that the ER quality control system is elaborately regulated by N-glycosylation. The insights gained will help us to better understand the regulatory mechanisms of infection in pathogenic fungi. These findings may be also important for developing novel strategies for fungal disease control. Genetic studies have shown essential functions of N-glycosylation during infection of the plant pathogenic fungi, however, systematic roles of N-glycosylation in fungi is still largely unknown. Biological analysis demonstrated N-glycosylated proteins were widely present at different development stages of Magnaporthe oryzae and especially increased in the appressorium and invasive hyphae. A large-scale quantitative proteomics analysis was then performed to explore the roles of N-glycosylation in M. oryzae. A total of 559 N-glycosites from 355 proteins were identified and quantified at different developmental stages. Functional classification to the N-glycosylated proteins revealed N-glycosylation can coordinate different cellular processes for mycelial growth, conidium formation, and appressorium formation. N-glycosylation can also modify key components in N-glycosylation, O-glycosylation and GPI anchor pathways, indicating intimate crosstalk between these pathways. Interestingly, we found nearly all key components of the endoplasmic reticulum quality control (ERQC) system were highly N-glycosylated in conidium and appressorium. Phenotypic analyses to the gene deletion mutants revealed four ERQC components, Gls1, Gls2, GTB1 and Cnx1, are important for mycelial growth, conidiation, and invasive hyphal growth in host cells. Subsequently, we identified the Gls1 N-glycosite N497 was important for invasive hyphal growth and partially required for conidiation, but didn't affect colony growth. Mutation of N497 resulted in reduction of Gls1 in protein level, and localization from ER into the vacuole, suggesting N497 is important for protein stability of Gls1. Our study showed a snapshot of the N-glycosylation landscape in plant pathogenic fungi, indicating functions of this modification in cellular processes, developments and pathogenesis

The Predicted Secretome of the Plant Pathogenic Fungus Fusarium graminearum: A Refined Comparative Analysis

The fungus Fusarium graminearum forms an intimate association with the host species wheat whilst infecting the floral tissues at anthesis. During the prolonged latent period of infection, extracellular communication between live pathogen and host cells must occur, implying a role for secreted fungal proteins. The wheat cells in contact with fungal hyphae subsequently die and intracellular hyphal colonisation results in the development of visible disease symptoms. Since the original genome annotation analysis was done in 2007, which predicted the secretome using TargetP, the F. graminearum gene call has changed considerably through the combined efforts of the BROAD and MIPS institutes. As a result of the modifications to the genome and the recent findings that suggested a role for secreted proteins in virulence, the F. graminearum secretome was revisited. In the current study, a refined F. graminearum secretome was predicted by combining several bioinformatic approaches. This strategy increased the probability of identifying truly secreted proteins. A secretome of 574 proteins was predicted of which 99% was supported by transcriptional evidence. The function of the annotated and unannotated secreted proteins was explored. The potential role(s) of the annotated proteins including, putative enzymes, phytotoxins and antifungals are discussed. Characterisation of the unannotated proteins included the analysis of Pfam domains and features associated with known fungal effectors, for example, small size, cysteine-rich and containing internal amino acid repeats. A comprehensive comparative genomic analysis involving 57 fungal and oomycete genomes revealed that only a small number of the predicted F. graminearum secreted proteins can be considered to be either species or sequenced strain specific

Apoplastic recognition of multiple candidate effectors from the wheat pathogen Zymoseptoria tritici in the nonhost plant Nicotiana benthamiana

The fungus Zymoseptoria tritici is a strictly apoplastic, host-specific pathogen of wheat leaves and causal agent of septoria tritici blotch (STB) disease. All other plants are considered nonhosts, but the mechanism of nonhost resistance (NHR) to Z. tritici has not been addressed previously. We sought to develop Nicotiana benthamiana as a system to study NHR against Z. tritici. Fluorescence microscopy and quantitative reverse transcription polymerase chain reactions were used to establish the interaction between Z. tritici and N. benthamiana. Agrobacteriummediated transient expression was used to screen putative Z. tritici effector genes for recognition in N. benthamiana, and virus-induced gene silencing (VIGS) was employed to determine the role of two receptor-like kinases (RLKs), NbBAK1 and NbSOBIR1, in Z. tritici effector recognition. Numerous Z. tritici putative effectors (14 of 63 tested) induced cell death or chlorosis in N. benthamiana. For most, phenotypes were light-dependent and required effector secretion to the leaf apoplastic space. Moreover, effector-induced host cell death was dependent on NbBAK1 and NbSOBIR1. Our results indicate widespread recognition of apoplastic effectors from a wheat-infecting fungal pathogen in a taxonomically distant nonhost plant species presumably by cell surface immune receptors. This suggests that apoplastic recognition of multiple nonadapted pathogen effectors may contribute to NHR

Considerations for the development of Computer-assisted Language Learning (CALL) teacher training course: a practical experience from a CALL course development in Indonesia

The need for technology training for teachers will keep on growing in line with the development of technology itself. Although technology nowadays is more and more user friendly and may need no specific training on how to use it, teachers need to possess the knowledge that underpins the idea of using it for teaching and learning process. Teachers need to have solid pedagogical knowledge on how to use the technology to deliver contents to their students. Therefore, a technology-training course for teachers is always necessary. This paper presents the partial results of a design based study/research (DBR) on the development of online technology training for teachers with focus on CALL in Indonesia. Questions regarding factors affecting online CALL course and ways to improve the course in terms of training materials, activities, as well as the administration of the training are addressed in the study. Based on the study, some considerations on how to design such technology-training course are proposed. The considerations are ranging from aspects associated with technology competence for teacher standards, constructivism in online learning, adult learning theory, online instructional models, the technology, pedagogy and content knowledge (TPACK) framework and open educational resources (OER). Information regarding those aspects will be useful to assist other CALL teacher training course developers later to inform their decision in the development of the course which is based on a good theoretical understanding as well as highly practical in learning activitie