Iron (Fe) is an essential element for plant pathogens as well as for their host plants. As Fe plays a central role in pathogen virulence, most plants have evolved Fe‐withholding strategies to reduce ...Fe availability to pathogens. On the other hand, plants need Fe for an oxidative burst in their basal defense response against pathogens. To investigate how the plant Fe nutritional status affects plant tolerance to a hemibiotrophic fungal pathogen, we employed the maize–Colletotrichum graminicola pathosystem. Fungal infection progressed rapidly via biotrophic to necrotrophic growth in Fe‐deficient leaves, while an adequate Fe nutritional status suppressed the formation of infection structures of C. graminicola already during the early biotrophic growth phase. As indicated by Prussian blue and 3,3'‐diaminobenzidine (DAB) staining, the retarding effect of an adequate Fe nutritional status on fungal development coincided temporally and spatially with the recruitment of Fe to infection sites and a local production of H₂O₂. A similar coincidence between local Fe and H₂O₂ accumulation was found in a parallel approach employing C. graminicola mutants affected in Fe acquisition and differing in virulence. These results indicate that an adequate Fe nutritional status delays and partially suppresses the fungal infection process and the biotrophic growth phase of C. graminicola, most likely via the recruitment of free Fe to the fungal infection site for a timely oxidative burst.
Summary
The hemibiotrophic maize pathogen Colletotrichum graminicola synthesizes one intracellular and three secreted siderophores. eGFP fusions with the key siderophore biosynthesis gene, SID1, ...encoding l‐ornithine‐N5‐monooxygenase, suggested that siderophore biosynthesis is rigorously downregulated specifically during biotrophic development. In order to investigate the role of siderophores during vegetative development and pathogenesis, SID1, which is required for synthesis of all siderophores, and the non‐ribosomal peptide synthetase gene NPS6, synthesizing secreted siderophores, were deleted. Mutant analyses revealed that siderophores are required for vegetative growth under iron‐limiting conditions, conidiation, ROS tolerance, and cell wall integrity. Δsid1 and Δnps6 mutants were hampered in formation of melanized appressoria and impaired in virulence. In agreement with biotrophy‐specific downregulation of siderophore biosynthesis, Δsid1 and Δnps6 strains were not affected in biotrophic development, but spread of necrotrophic hyphae was reduced. To address the question why siderophore biosynthesis is specifically downregulated in biotrophic hyphae, maize leaves were infiltrated with siderophores. Siderophore infiltration alone did not induce defence responses, but formation of biotrophic hyphae in siderophore‐infiltrated leaves caused dramatically increased ROS formation and transcriptional activation of genes encoding defence‐related peroxidases and PR proteins. These data suggest that fungal siderophores modulate the plant immune system.
Production of marker-free genetically modified (GM) plants is one of the major challenges of molecular fruit breeding. Employing clean vector technologies, allowing the removal of undesired DNA ...sequences from GM plants, this goal can be achieved. The present study describes the establishment of a clean vector system in apple Malus×domestica Borkh., which is based on the use of the neomycin phosphotransferase II gene (nptII) as selectable marker gene and kanamycin/paramomycin as selective agent. The nptII gene can be removed after selection of GM shoots via site-specific excision mediated by heat-shock-inducible expression of the budding yeast FLP recombinase driven by the soybean Gmhsp17.5-E promoter. We created a monitoring vector containing the nptII and the flp gene as a box flanked by two direct repeats of the flp recognition target (FRT) sites. The FRT-flanked box separates the gusA reporter gene from the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter. Consequently, GUS expression does only occur after elimination of the FRT-flanked box. Transformation experiments using the monitoring vector resulted in a total of nine transgenic lines. These lines were investigated for transgenicity by PCR, RT-PCR and Southern hybridization. Among different temperature regimes tested, exposure to 42°C for 3.5 to 4h led to efficient induction of FLP-mediated recombination and removal of the nptII marker gene. A second round of shoot regeneration from leaf explants led to GM apple plants completely free of the nptII gene.
► We produced transgenic apple clones containing the nptII, flp and the gusA genes. ► nptII and flp genes are flanked by two FRT recognition sites. ► Both genes were excised by heat-shock induced expression of the FLP recombinase. ► GusA expression was used to monitor where excision was taking place. ► The system was successful used to produce marker-free apple plants.
In this review article, we show that occurrence of fungicide resistance is one of the most important issues in modern agriculture. Fungicide resistance may be due to mutations of genes encoding ...fungicide targets (qualitative fungicide resistance) or to different mechanisms that are induced by sub-lethal fungicide stress. These mechanisms result in different and varying levels of resistance (quantitative fungicide resistance). We discuss whether or not extensive use of fungicides in agricultural environments is related to the occurrence of fungicide resistance in clinical environments. Furthermore, we provide recommendations of how development of fungicide resistant pathogen populations may be prevented or delayed.
•CgIPT1 encodes a tRNA-isopentenyltransferase of the maize pathogen Colletotrichum graminicola.•Mutants deleted for CgIPT1 have lost the ability to produce cis-zeatin-type cytokinins.•Mutants are ...less tolerant to certain abiotic stress conditions and are less virulent.•Mutants still evoke the phenomenon of green islands on senescing maize leaves, indicating that fungal cytokinin is not essential to cause this phenotype.
We have previously shown that the maize pathogen Colletotrichum graminicola is able to synthesise cytokinins (CKs). However, it remained unsettled whether fungal CK production is essential for virulence in this hemibiotrophic fungus. Here, we identified a candidate gene, CgIPT1, that is homologous to MOD5 of Saccharomyces cerevisiae and genes from other fungi and plants, which encode tRNA-isopentenyltransferases (IPTs). We show that the wild type strain mainly synthesises cis-zeatin-type (cisZ) CKs whereas ΔCgipt1 mutants are severely impeded to do so. The spectrum of CKs produced confirms bioinformatical analyses predicting that CgIpt1 is a tRNA-IPT. The virulence of the ΔCgipt1 mutants is moderately reduced. Furthermore, the mutants exhibit increased sensitivities to osmotic stress imposed by sugar alcohols and salts, as well as cell wall stress imposed by Congo red. Amendment of media with CKs did not reverse this phenotype suggesting that fungal-derived CKs do not explain the role of CgIpt1 in mediating abiotic stress tolerance. Moreover, the mutants still cause green islands on senescing maize leaves indicating that the cisZ-type CKs produced by the fungus do not cause this phenotype.
When inoculated onto maize leaves at the onset of senescence, the hemibiotroph Colletotrichum graminicola causes green islands that are surrounded by senescing tissue. Taking advantage of green ...islands as indicators of sites of the establishment of successful infection and of advanced high-performance liquid chromatography tandem mass spectrometry methodology, we analyzed changes in the patterns and levels of cytokinins (CK) at high spatial and analytical resolution. Twenty individual CK were detected in green islands. Levels of cis-zeatin-9-riboside and cis-zeatin-9-riboside-5'-monophosphate increased drastically, whereas that of the most prominent CK, cis-zeatin-O-glucoside, decreased. The fungus likely performed these conversions because corresponding activities were also detected in in vitro cultures amended with CK. We found no evidence that C. graminicola is able to synthesize CK entirely de novo in minimal medium but, after adding dimethylallyl diphosphate, a precursor of CK biosynthesis occurring in plants, a series of trans-zeatin isoforms (i.e., trans-zeatin-9-riboside-5'-monophosphate, trans-zeatin-9-riboside, and trans-zeatin) was formed. After applying CK onto uninfected leaves, transcripts of marker genes for senescence, photosynthesis, and assimilate distribution were measured by quantitative reverse-transcribed polymerase chain reaction; furthermore, pulse-amplitude modulation chlorophyll fluorometry and single-photon avalanche diode analyses were conducted. These experiments suggested that modulation of CK metabolism at the infection site affects host physiology.
Summary
To avoid pathogen‐associated molecular pattern recognition, the hemibiotrophic maize pathogen Colletotrichum graminicola secretes proteins mediating the establishment of biotrophy. Targeted ...deletion of 26 individual candidate genes and seven gene clusters comprising 32 genes of C. graminicola identified a pathogenicity cluster (CLU5) of five co‐linear genes, all of which, with the exception of CLU5b, encode secreted proteins. Targeted deletion of all genes of CLU5 revealed that CLU5a and CLU5d are required for full appressorial penetration competence, with virulence deficiencies independent of the host genotype and organ inoculated. Cytorrhysis experiments and microscopy showed that Δclu5a mutants form pressurized appressoria, but they are hampered in forming penetration pores and fail to differentiate a penetration peg. Whereas Δclu5d mutants elicited WT‐like papillae, albeit at increased frequencies, papillae induced by Δclu5a mutants were much smaller than those elicited by the WT. Synteny of CLU5 is not only conserved in Colletotrichum spp. but also in additional species of Sordariomycetes including insect pathogens and saprophytes suggesting importance of CLU5 for fungal biology. Since CLU5a and CLU5d also occur in non‐pathogenic fungi and since they are expressed prior to plant invasion and even in vegetative hyphae, the encoded proteins probably do not act primarily as effectors.
Many plant pathogenic fungi, such as the cereal pathogen Colletotrichum graminicola, differentiate highly specialized infection structures called appressoria, which send a penetration peg into the ...underlying plant cell. Appressoria have been shown to generate enormous turgor pressure, but direct evidence for mechanical infection of plants by fungi is lacking. A microscopic method was developed that uses elastic optical waveguides to visualize and measure forces locally exerted by single appressoria. By this method, the force exerted by appressoria of C. graminicola was found to be about 17 micronewtons.
Pathogens have the potential to shape plant community structure, and thus, it is important to understand the factors that determine pathogen diversity and infection in communities. The abundance, ...origin, and evolutionary relationships of plant hosts are all known to influence pathogen patterns and are typically studied separately. We present an observational study that examined the influence of all three factors and their interactions on the diversity of and infection of several broad taxonomic groups of foliar, floral, and stem pathogens across three sites in a temperate grassland in the central United States. Despite that pathogens are known to respond positively to increases in their host abundances in other systems, we found no relationship between host abundance and either pathogen diversity or infection. Native and exotic plants did not differ in their infection levels, but exotic plants hosted a more generalist pathogen community compared to native plants. There was no phylogenetic signal across plants in pathogen diversity or infection. The lack of evidence for a role of abundance, origin, and evolutionary relationships in shaping patterns of pathogens in our study might be explained by the high generalization and global distributions of our focal pathogen community, as well as the high diversity of our plant host community. In general, the community‐level patterns of aboveground pathogen infections have received less attention than belowground pathogens, and our results suggest that their patterns might not be explained by the same drivers.
We investigated the effects of plant abundance, origin, and phylogenetic community context on the diversity and infection levels of aboveground pathogens in a temperate grassland in the central United States. We found no relationship between host abundance, plant origin or phylogeny, and our observed infection levels. However, exotic host species harbored a more generalist pathogen community.
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