The galactose metabolism genes UGE1 and UGM1 are novel virulence factors of the maize anthracnose fungus Colletotrichum graminicola.

Fungal cell walls represent the frontline contact with the host and play a prime role in pathogenesis. While the roles of the cell wall polymers like chitin and branched ß-glucan are well understood in vegetative and pathogenic development, that of the most prominent galactose-containing polymers galactosaminogalactan and fungal-type galactomannan is unknown in plant pathogenic fungi. Mining the genome of the maize pathogen Colletotrichum graminicola identified the single-copy key galactose metabolism genes UGE1 and UGM1, encoding a UDP-glucose-4-epimerase and UDP-galactopyranose mutase, respectively. UGE1 is thought to be required for biosynthesis of both polymers, whereas UGM1 is specifically required for fungal-type galactomannan formation. Promoter:eGFP fusion strains revealed that both genes are expressed in vegetative and in pathogenic hyphae at all stages of pathogenesis. Targeted deletion of UGE1 and UGM1, and fluorescence-labeling of galactosaminogalactan and fungal-type galactomannan confirmed that Δuge1 mutants were unable to synthesize either of these polymers, and Δugm1 mutants did not exhibit fungal-type galactomannan. Appressoria of Δuge1, but not of Δugm1 mutants, were defective in adhesion, highlighting a function of galactosaminogalactan in the establishment of these infection cells on hydrophobic surfaces. Both Δuge1 and Δugm1 mutants showed cell wall defects in older vegetative hyphae and severely reduced appressorial penetration competence. On intact leaves of Zea mays, both mutants showed strongly reduced disease symptom severity, indicating that UGE1 and UGM1 represent novel virulence factors of C. graminicola.

The serine-threonine protein kinase Snf1 orchestrates the expression of plant cell wall-degrading enzymes and is required for full virulence of the maize pathogen Colletotrichum graminicola.

Colletotrichum graminicola, the causal agent of maize leaf anthracnose and stalk rot, differentiates a pressurized infection cell called an appressorium in order to invade the epidermal cell, and subsequently forms biotrophic and necrotrophic hyphae to colonize the host tissue. While the role of force in appressorial penetration is established (Bechinger et al., 1999), the involvement of cell wall-degrading enzymes (CWDEs) in this process and in tissue colonization is poorly understood, due to the enormous number and functional redundancy of these enzymes. The serine/threonine protein kinase gene SNF1 identified in Sucrose Non-Fermenting yeast mutants mediates de-repression of catabolite-repressed genes, including many genes encoding CWDEs. In this study, we identified and functionally characterized the SNF1 homolog of C. graminicola. Δsnf1 mutants showed reduced vegetative growth and asexual sporulation rates on media containing polymeric carbon sources. Microscopy revealed reduced efficacies in appressorial penetration of cuticle and epidermal cell wall, and formation of unusual medusa-like biotrophic hyphae by Δsnf1 mutants. Severe and moderate virulence reductions were observed on intact and wounded leaves, respectively. Employing RNA-sequencing we show for the first time that more than 2,500 genes are directly or indirectly controlled by Snf1 in necrotrophic hyphae of a plant pathogenic fungus, many of which encode xylan- and cellulose-degrading enzymes. The data presented show that Snf1 is a global regulator of gene expression and is required for full virulence.

CgIPT1 is required for synthesis of cis-zeatin cytokinins and contributes to stress tolerance and virulence in Colletotrichum graminicola.

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.

Molecular Characterization of the Purine Degradation Pathway Genes ALA1 and URE1 of the Maize Anthracnose Fungus Colletotrichum graminicola Identified Urease as a Novel Target for Plant Disease Control.

Fungal pathogenicity is governed by environmental factors, with nitrogen playing a key role in triggering pathogenic development. Spores germinating on the plant cuticle are exposed to a nitrogen-free environment, and reprograming of nitrogen metabolism is required for bridging the time needed to gain access to the nitrogen sources of the host. Although degradation of endogenous purine bases efficiently generates ammonium and may allow the fungus to bridge the preinvasion nitrogen gap, the roles of the purine degradation pathway and of the key genes encoding allantoicase and urease are largely unknown in plant pathogenic fungi. To investigate the roles of the allantoicase and urease genes ALA1 and URE1 of the maize anthracnose fungus Colletotrichum graminicola in pathogenic development, we generated ALA1:eGFP and URE1:eGFP fusion strains as well as allantoicase- and urease-deficient mutants. Virulence assays, live cell, and differential interference contrast imaging, chemical complementation and employment of a urease inhibitor showed that the purine degradation genes ALA1 and URE1 are required for bridging nitrogen deficiency at early phases of the infection process and for full virulence. Application of the urease inhibitor acetohydroxamic acid did not only protect maize from C. graminicola infection, but also interfered with the infection process of the wheat powdery mildew fungus Blumeria graminis f. sp. tritici, the maize and broad bean rusts Puccinia sorghi and Uromyces viciae-fabae, and the potato late blight pathogen Phytophthora infestans. Our data strongly suggest that inhibition of the purine degradation pathway might represent a novel approach to control plant pathogenic fungi and oomycetes.

Two genes in a pathogenicity gene cluster encoding secreted proteins are required for appressorial penetration and infection of the maize anthracnose fungus Colletotrichum graminicola.

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.

Specialized infection strategies of falcate and oval conidia of Colletotrichum graminicola.

For many filamentous fungi with pathogenic lifestyles, the presence of distinct asexual conidia has been described. However, the role of these spore types remains mostly obscure. Colletotrichum graminicola is a hemibiotrophic filamentous fungus, causing anthracnose on maize plants with a high potential of epidemic disease spreading. C. graminicola generates two types of conidia. Falcate shaped conidia formed in necrotic lesions on maize tissues are able to generate appressoria with high efficiency and are considered key disease spreading propagules. The second conidia type, the smaller oval conidia, is formed in the vascular system of the infected plant, probably causing the distribution of the disease in planta. Barely any knowledge exists about how these conidia are able to exhibit their specific functions in the life cycle and pathogenicity of C. graminicola. Here, we show that germlings derived from both falcate and oval conidia differ in the secretion of a germination inhibitor and signals for germling fusion. Germination experiments combined with HPLC and mass spectrometry analyses revealed that germination of falcate conidia is regulated by the self-inhibitor mycosporine-glutamine, whereas this compound is absent from oval conidia cultures. Additionally, germlings derived from oval conidia undergo germling fusions at high frequencies and are able to induce such a fusion when co-incubated with falcate conidia. Falcate conidia germlings alone, however, were never observed to fuse. Plant infection experiments showed a positive correlation between germling fusions and efficient leaf infection by oval conidia. However, this correlation was not observed for infection by falcate conidia. Together, our findings reveal significant differences of two types of conidia derived from the same pathogenic fungus with distinct roles in pathogenesis.

Polyphenols from Rheum Roots Inhibit Growth of Fungal and Oomycete Phytopathogens and Induce Plant Disease Resistance.

A growing world population requires an increase in the quality and quantity of food production. However, field losses due to biotic stresses are currently estimated to be between 10 and 20% worldwide. The risk of resistance and strict pesticide legislation necessitate innovative agronomical practices to adequately protect crops in the future, such as the identification of new substances with novel modes of action. In the present study, liquid chromatography mass spectrometry was used to characterize Rheum rhabarbarum root extracts that were primarily composed of the stilbenes rhaponticin, desoxyrhaponticin, and resveratrol. Minor components were the flavonoids catechin, epicatechin gallate, and procyanidin B1. Specific polyphenolic mixtures inhibited mycelial growth of several phytopathogenic fungi and oomycetes. Foliar spray applications with fractions containing stilbenes and flavonoids inhibited spore germination of powdery mildew in Hordeum vulgare with indications of synergistic interactions. Formulated extracts led to a significant reduction in the incidence of brown rust in Triticum aestivum under field conditions. Arabidopsis thaliana mutant and quantitative reverse-transcription polymerase chain reaction studies suggested that the stilbenes induce salicylic acid-mediated resistance. Thus, the identified substances of Rheum roots represent an excellent source of antifungal agents that can be used in horticulture and agriculture.

Attenuation of PAMP-triggered immunity in maize requires down-regulation of the key ß-1,6-glucan synthesis genes KRE5 and KRE6 in biotrophic hyphae of Colletotrichum graminicola.

In plants, pathogen defense is initiated by recognition of pathogen-associated molecular patterns (PAMPs) via plasma membrane-localized pattern-recognition receptors (PRRs). Fungal structural cell wall polymers such as branched ß-glucans are essential for infection structure rigidity and pathogenicity, but at the same time represent PAMPs. Kre5 and Kre6 are key enzymes in ß-1,6-glucan synthesis and formation of branch points of the ß-glucan network. In spite of the importance of branched ß-glucan for hyphal rigidity and plant-fungus interactions, neither the role of KRE5 and KRE6 in pathogenesis nor mechanisms allowing circumventing branched ß-glucan-triggered immune responses are known. We functionally characterized KRE5 and KRE6 of the ascomycete Colletotrichum graminicola, a hemibiotroph that infects maize (Zea mays). After appressorial plant invasion, this fungus sequentially differentiates biotrophic and highly destructive necrotrophic hyphae. RNAi-mediated reduction of KRE5 and KRE6 transcript abundance caused appressoria to burst and swelling of necrotrophic hyphae, indicating that ß-1,6-glucosidic bonds are essential in these cells. Live cell imaging employing KRE5:mCherry and KRE6:mCherry knock-in strains and probing of infection structures with a YFP-conjugated ß-1,6-glucan-binding protein showed expression of these genes and exposure of ß-1,6-glucan in conidia, appressoria and necrotrophic, but not in biotrophic hyphae. Overexpression of KRE5 and KRE6 in biotrophic hyphae led to activation of broad-spectrum plant defense responses, including papilla and H2 O2 formation, as well as transcriptional activation of several defense-related genes. Collectively, our results strongly suggest that down-regulation of synthesis and avoidance of exposure of branched ß-1,3-ß-1,6-glucan in biotrophic hyphae is required for attenuation of plant immune responses.

The Glycosylphosphatidylinositol Anchor Biosynthesis Genes GPI12, GAA1, and GPI8 Are Essential for Cell-Wall Integrity and Pathogenicity of the Maize Anthracnose Fungus Colletotrichum graminicola.

Glycosylphosphatidylinositol (GPI) anchoring of proteins is one of the most common posttranslational modifications of proteins in eukaryotic cells and is important for associating proteins with the cell surface. In fungi, GPI-anchored proteins play essential roles in cross-linking of ß-glucan cell-wall polymers and cell-wall rigidity. GPI-anchor synthesis is successively performed at the cytoplasmic and the luminal face of the ER membrane and involves approximately 25 proteins. While mutagenesis of auxiliary genes of this pathway suggested roles of GPI-anchored proteins in hyphal growth and virulence, essential genes of this pathway have not been characterized. Taking advantage of RNA interference (RNAi) we analyzed the function of the three essential genes GPI12, GAA1 and GPI8, encoding a cytoplasmic N-acetylglucosaminylphosphatidylinositol deacetylase, a metallo-peptide-synthetase and a cystein protease, the latter two representing catalytic components of the GPI transamidase complex. RNAi strains showed drastic cell-wall defects, resulting in exploding infection cells on the plant surface and severe distortion of in planta-differentiated infection hyphae, including formation of intrahyphal hyphae. Reduction of transcript abundance of the genes analyzed resulted in nonpathogenicity. We show here for the first time that the GPI synthesis genes GPI12, GAA1, and GPI8 are indispensable for vegetative development and pathogenicity of the causal agent of maize anthracnose, Colletotrichum graminicola.

Correspondence between symptom development of Colletotrichum graminicola and fungal biomass, quantified by a newly developed qPCR assay, depends on the maize variety.

BACKGROUND: Penetration attempts of the hemibiotroph Colletotrichum graminicola may activate PAMP-triggered immunity (PTI) on different cultivars of Zea mays to different extent. However, in most events, this does not prevent the establishment of a compatible pathogenic interaction. In this study, we investigate the extent to which the host variety influences PTI. Furthermore, we assess whether visual disease symptoms occurring on different maize varieties reliably reflect fungal biomass development in planta as determined by qPCR and GFP tracing. RESULTS: Employing a set of four maize varieties, which were selected from a panel of 27 varieties, for in-depth assessment of pathogenesis of the wild type strain of C. graminicola, revealed considerable differences in susceptibility as evidenced by symptom severity that decreased from variety Golden Jubilee to Mikado to Farmtop to B73. However, a newly developed qPCR assay and microscopical observation of a GFP-labelled strain showed that disease symptoms are in some instances inconsistent when compared with other indicators of susceptibility. Of the four varieties assessed, either Golden Jubilee, Mikado and B73, or Golden Jubilee, Farmtop and B73 showed a direct correlation between symptom and fungal biomass development. In a pairwise comparison, however, Mikado and Farmtop showed an inverse correlation for these features. CONCLUSIONS: The genotype of maize contributes to the severity of symptoms resulting from an infection with C. graminicola. Partially, this may be attributed to the extent of PTI activated in different varieties, as reflected by papilla formation. Furthermore, when evaluating the susceptibility of a variety, it should be considered that symptom severity must not have to reflect the extent of fungal growth in the infected tissue.

Host-induced silencing of Fusarium culmorum genes protects wheat from infection.

Plants producing antisense or double-stranded RNA molecules that target specific genes of eukaryotic pests or pathogens can become protected from their attack. This beneficial effect was also reported for plant-fungus interactions and is believed to reflect uptake of the RNAs by the fungus via an as yet unknown mechanism, followed by target gene silencing. Here we report that wheat plants pre-infected with Barley stripe mosaic virus (BSMV) strains containing antisense sequences against target genes of the Fusarium head blight (FHB) fungus F. culmorum caused a reduction of corresponding transcript levels in the pathogen and reduced disease symptoms. Stable transgenic wheat plants carrying an RNAi hairpin construct against the ß-1, 3-glucan synthase gene FcGls1 of F. culmorum or a triple combination of FcGls1 with two additional, pre-tested target genes also showed enhanced FHB resistance in leaf and spike inoculation assays under greenhouse and near-field conditions, respectively. Microscopic evaluation of F. culmorum development in plants transiently or stably expressing FcGls1 silencing constructs revealed aberrant, swollen fungal hyphae, indicating severe hyphal cell wall defects. The results lead us to propose host-induced gene silencing (HIGS) as a plant protection approach that may also be applicable to highly FHB-susceptible wheat genotypes.

Infection structure-specific expression of ß-1,3-glucan synthase is essential for pathogenicity of Colletotrichum graminicola and evasion of ß-glucan-triggered immunity in maize.

ß-1,3-Glucan and chitin are the most prominent polysaccharides of the fungal cell wall. Covalently linked, these polymers form a scaffold that determines the form and properties of vegetative and pathogenic hyphae. While the role of chitin in plant infection is well understood, the role of ß-1,3-glucan is unknown. We functionally characterized the ß-1,3-glucan synthase gene GLS1 of the maize (Zea mays) pathogen Colletotrichum graminicola, employing RNA interference (RNAi), GLS1 overexpression, live-cell imaging, and aniline blue fluorochrome staining. This hemibiotroph sequentially differentiates a melanized appressorium on the cuticle and biotrophic and necrotrophic hyphae in its host. Massive ß-1,3-glucan contents were detected in cell walls of appressoria and necrotrophic hyphae. Unexpectedly, GLS1 expression and ß-1,3-glucan contents were drastically reduced during biotrophic development. In appressoria of RNAi strains, downregulation of ß-1,3-glucan synthesis increased cell wall elasticity, and the appressoria exploded. While the shape of biotrophic hyphae was unaffected in RNAi strains, necrotrophic hyphae showed severe distortions. Constitutive expression of GLS1 led to exposure of ß-1,3-glucan on biotrophic hyphae, massive induction of broad-spectrum defense responses, and significantly reduced disease symptom severity. Thus, while ß-1,3-glucan synthesis is required for cell wall rigidity in appressoria and fast-growing necrotrophic hyphae, its rigorous downregulation during biotrophic development represents a strategy for evading ß-glucan-triggered immunity.

Biotrophy-specific downregulation of siderophore biosynthesis in Colletotrichum graminicola is required for modulation of immune responses of maize.

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-N(5) -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.

Melanin is not required for turgor generation but enhances cell-wall rigidity in appressoria of the corn pathogen Colletotrichum graminicola.

The ascomycete and causative agent of maize anthracnose and stem rot, Colletotrichum graminicola, differentiates melanized infection cells called appressoria that are indispensable for breaching the plant cell wall. High concentrations of osmolytes accumulate within the appressorium, and the internal turgor pressure of up to 5.4 MPa provides sufficient force to penetrate the leaf epidermis directly. In order to assess the function of melanin in C. graminicola appressoria, we identified and characterized the polyketide synthase 1 (CgPKS1) gene which displayed high similarity to fungal polyketide synthases (PKS) involved in synthesis of 1,3,6,8-tetrahydronaphthalene, the first intermediate in melanin biosynthesis. Cgpks1 albino mutants created by targeted gene disruption were unable to penetrate intact leaves and ruptured frequently but, surprisingly, were able to penetrate ultrathin polytetrafluoroethylene membranes mimicking the plant surface. Nonmelanized Cgpks1 appressoria were sensitive to externally applied cell-wall-degrading enzymes whereas melanized appressoria were not affected. Expression studies using a truncated CgPKS1 fused to green fluorescent protein revealed fluorescence in immature appressoria and in setae, which is in agreement with transcript data obtained by RNA-Seq and quantitative polymerase chain reaction. Unexpectedly, surface scans of mutant and wild-type appressoria revealed considerable differences in cell-wall morphology. Melanization of appressoria is indispensable for successful infection of intact leaves. However, cell collapse experiments and analysis of the appressorial osmolyte content by Mach-Zehnder interferometry convincingly showed that melanin is not required for solute accumulation and turgor generation, thus questioning the role of melanin as a barrier for osmolytes in appressoria of C. graminicola.

New gene models and alternative splicing in the maize pathogen Colletotrichum graminicola revealed by RNA-Seq analysis.

BACKGROUND: An annotated genomic sequence of the corn anthracnose fungus Colletotrichum graminicola has been published previously, but correct identification of gene models by means of automated gene annotation remains a challenge. RNA-Seq offers the potential for substantially improved gene annotations and for the identification of posttranscriptional RNA modifications, such as alternative splicing and RNA editing. RESULTS: Based on the nucleotide sequence information of transcripts, we identified 819 novel transcriptionally active regions (nTARs) and revised 906 incorrectly predicted gene models, including revisions of exon-intron structure, gene orientation and sequencing errors. Among the nTARs, 146 share significant similarity with proteins that have been identified in other species suggesting that they are hitherto unidentified genes in C. graminicola. Moreover, 5'- and 3'-UTR sequences of 4378 genes have been retrieved and alternatively spliced variants of 69 genes have been identified. Comparative analysis of RNA-Seq data and the genome sequence did not provide evidence for RNA editing in C. graminicola. CONCLUSIONS: We successfully employed deep sequencing RNA-Seq data in combination with an elaborate bioinformatics strategy in order to identify novel genes, incorrect gene models and mechanisms of transcript processing in the corn anthracnose fungus C. graminicola. Sequence data of the revised genome annotation including several hundreds of novel transcripts, improved gene models and candidate genes for alternative splicing have been made accessible in a comprehensive database. Our results significantly contribute to both routine laboratory experiments and large-scale genomics or transcriptomic studies in C. graminicola.

Compositions of fungal secretomes indicate a greater impact of phylogenetic history than lifestyle adaptation.

BACKGROUND: Since the first fungal genome sequences became available, investigators have been employing comparative genomics to understand how fungi have evolved to occupy diverse ecological niches. The secretome, i.e. the entirety of all proteins secreted by an organism, is of particular importance, as by these proteins fungi acquire nutrients and communicate with their surroundings. RESULTS: It is generally assumed that fungi with similar nutritional lifestyles have similar secretome compositions. In this study, we test this hypothesis by annotating and comparing the soluble secretomes, defined as the sets of proteins containing classical signal peptides but lacking transmembrane domains of fungi representing a broad diversity of nutritional lifestyles. Secretome size correlates with phylogeny and to a lesser extent with lifestyle. Plant pathogens and saprophytes have larger secretomes than animal pathogens. Small secreted cysteine-rich proteins (SSCPs), which may comprise many effectors important for the interaction of plant pathogens with their hosts, are defined here to have a mature length of ≤ 300 aa residues, at least four cysteines, and a total cysteine content of ≥5%. SSCPs are found enriched in the secretomes of the Pezizomycotina and Basidiomycota in comparison to Saccharomycotina. Relative SSCP content is noticeably higher in plant pathogens than in animal pathogens, while saprophytes were in between and closer to plant pathogens. Expansions and contractions of gene families and in the number of occurrences of functional domains are largely lineage specific, e.g. contraction of glycoside hydrolases in Saccharomycotina, and are only weakly correlated with lifestyle. However, within a given lifestyle a few general trends exist, such as the expansion of secreted family M14 metallopeptidases and chitin-binding proteins in plant pathogenic Pezizomycotina. CONCLUSIONS: While the secretomes of fungi with similar lifestyles share certain characteristics, the expansion and contraction of gene families is largely lineage specific, and not shared among all fungi of a given lifestyle.

A modular plasmid system for protein co-localization and bimolecular fluorescence complementation in filamentous fungi.

To elucidate the function of a protein, it is crucial to know its subcellular location and its interaction partners. Common approaches to resolve those questions rely on the genetic tagging of the gene-of-interest (GOI) with fluorescent reporters. To determine the location of a tagged protein, it may be co-localized with tagged marker proteins. The interaction of two proteins under investigation is often analysed by tagging both with the C- and N-terminal halves of a fluorescent protein. In fungi, the tagged GOI are commonly introduced by serial transformation with plasmids harbouring a single tagged GOI and subsequent selection of suitable strains. In this study, a plasmid system is presented that allows the tagging of several GOI on a single plasmid. This novel double tagging plasmid system (DTPS) allows a much faster and less laborious generation of double-labelled fungal strains when compared with conventional approaches. The DTPS also enables the combination of as many tagged GOI as desired and a simple exchange of existing tags. Furthermore, new tags can be introduced smoothly into the system. In conclusion, the DTPS allows an efficient tagging of GOI with a high degree of flexibility and therefore accelerates functional analysis of proteins in vivo.

An adequate Fe nutritional status of maize suppresses infection and biotrophic growth of Colletotrichum graminicola.

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 H2 O2 . A similar coincidence between local Fe and H2 O2 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.

Further Limitations of Synthetic Fungicide Use and Expansion of Organic Agriculture in Europe Will Increase the Environmental and Health Risks of Chemical Crop Protection Caused by Copper-Containing Fungicides.

Copper-containing fungicides have been used in agriculture since 1885. The divalent copper ion is a nonbiodegradable multisite inhibitor that has a strictly protective, nonsystemic effect on plants. Copper-containing plant protection products currently approved in Germany contain copper oxychloride, copper hydroxide, and tribasic copper sulfate. Copper is primarily used to control oomycete pathogens in grapevine, hop, potato, and fungal diseases in fruit production. In the environment, copper is highly persistent and toxic to nontarget organisms. The latter applies for terrestric and aquatic organisms such as earthworms, insects, birds, fish, Daphnia, and algae. Hence, copper fungicides are currently classified in the European Union as candidates for substitution. Pertinently, copper also exhibits significant mammalian toxicity (median lethal dose oral = 300-2500 mg/kg body wt in rats). To date, organic production still profoundly relies on the use of copper fungicides. Attempts to reduce doses of copper applications and the search for copper substitutes have not been successful. Copper compounds compared with modern synthetic fungicides with similar areas of use display significantly higher risks for honey bees (3- to 20-fold), beneficial insects (6- to 2000-fold), birds (2- to 13-fold), and mammals (up to 17-fold). These data contradict current views that crop protection in organic farming is associated with lower environmental or health risks. Further limitations in the range and use of modern single-site fungicides may force conventional production to fill the gaps with copper fungicides to counteract fungicide resistance. In contrast to the European Union Green Deal goals, the intended expansion of organic farming in Europe would further enhance the use of copper fungicides and hence increase the overall risks of chemical crop protection in Europe. Environ Toxicol Chem 2024;43:19-30. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Infection structure-specific reductive iron assimilation is required for cell wall integrity and full virulence of the maize pathogen Colletotrichum graminicola.

Ferroxidases are essential components of the high-affinity reductive iron assimilation pathway in fungi. Two ferroxidase genes, FET3-1 and FET3-2, have been identified in the genome of the maize anthracnose fungus Colletotrichum graminicola. Complementation of growth defects of the ferroxidase-deficient Saccharomyces cerevisiae strain Δfet3fet4 showed that both Fet3-1 and Fet3-2 of C. graminicola represent functional ferroxidases. Expression of enhanced green fluorescent protein fusions in yeast and C. graminicola indicated that both ferroxidase proteins localize to the plasma membrane. Transcript abundance of FET3-1 increased dramatically under iron-limiting conditions but those of FET3-2 were hardly detectable. Δfet3-1 and Δfet3-2 single as well as Δfet3-1/2 double-deletion strains were generated. Under iron-sufficient or deficient conditions, vegetative growth rates of these strains did not significantly differ from that of the wild type but Δfet3-1 and Δfet3-1/2 strains showed increased sensitivity to reactive oxygen species. Furthermore, under iron-limiting conditions, appressoria of Δfet3-1 and Δfet3-1/2 strains showed significantly reduced transcript abundance of a class V chitin synthase and exhibited severe cell wall defects. Infection assays on intact and wounded maize leaves, quantitative data of infection structure differentiation, and infection stage-specific expression of FET3-1 showed that reductive iron assimilation is required for appressorial penetration, biotrophic development, and full virulence.