Figure 4 - uploaded by Luis Antelo
Content may be subject to copyright.
Source publication
In filamentous fungi, Sfp-type 4'-phosphopantetheinyl transferases (PPTases) activate enzymes involved in primary (alpha-aminoadipate reductase [AAR]) and secondary (polyketide synthases and nonribosomal peptide synthetases) metabolism. We cloned the PPTase gene PPT1 of the maize anthracnose fungus Colletotrichum graminicola and generated PPTase-de...
Contexts in source publication
Context 1
... HPLC-MS analyses of culture filtrates of the wild-type strain showed that C. graminicola secreted the siderophores coprogen B ( Figure 2B, blue line) and 2-N-methylcoprogen B ( Figure 2B, black line) under iron-limiting conditions; culture filtrates of Dppt1 strains did not contain any siderophore ( Figure 2B, red line). Furthermore, only the wild-type strain but not the mutants contained the intracellular siderophore ferricrocin (see Supplemental Figure 4 online). These results are in agreement with reports of growth defects of siderophore-deficient mutants of G. zeae and C. heterostrophus under iron-limiting conditions (Oide et al., 2006;Greenshields et al., 2007). ...
Context 2
... metabolite analyses in culture filtrates revealed that several secondary metabolites were formed by the wild type but not by the Dppt1 strains (Figure 3). In addition to the extracellular NRPs coprogen B and 2-N-methylcoprogen B (Figure 2B), the intracellular iron storage NRP ferricrocin was detected in hyphal extracts (see Supplemental Figure 4 online). Furthermore, we identified six novel fungal metabolites using HPLC-MS and nuclear magnetic resonance (NMR) spectrometry, including two pyrones (colletopyrone B and C), two anthraquinones (colletoquinone A and B), a 10-hydroxyanthrone (colletoanthrone A), and an 11-membered macrolactone (colletolactone A) (Fig- ure 3; see Supplemental Figure 5 Supplemental Tables 1 to 4 online). ...
Context 3
... on the size and morphology of oval and falcate conidia, wild-type and Dppt1 strains were allowed to sporulate in liquid complete medium. Conidia of the Dppt1 strains were smaller than wild-type conidia and showed drastic morpholog- ical defects (i.e., small conidia had round to spherical shapes and larger spores had central protrusions or round poles; Figure 4A). Interestingly, germination rates of mutant and wild-type conidia were similar on different hydrophobic substrata (i.e., polyester sheets, onion [Allium cepa cv Grano] epidermis, and maize surface; Figure 4B). ...
Context 4
... of the Dppt1 strains were smaller than wild-type conidia and showed drastic morpholog- ical defects (i.e., small conidia had round to spherical shapes and larger spores had central protrusions or round poles; Figure 4A). Interestingly, germination rates of mutant and wild-type conidia were similar on different hydrophobic substrata (i.e., polyester sheets, onion [Allium cepa cv Grano] epidermis, and maize surface; Figure 4B). However, germ tubes of Dppt1 strains frequently branched ( Figure 4C). ...
Context 5
... germination rates of mutant and wild-type conidia were similar on different hydrophobic substrata (i.e., polyester sheets, onion [Allium cepa cv Grano] epidermis, and maize surface; Figure 4B). However, germ tubes of Dppt1 strains frequently branched ( Figure 4C). ...
Context 6
... differentiation and melanization, wild-type and Dppt1 conidia were inoculated onto artificial and natural hydro- phobic substrata (i.e., polyester, onion epidermal layers, and maize leaf surfaces; Figure 5). While the wild-type strain germi- nated and efficiently formed appressoria on all hydrophobic surfaces, Dppt1 strains showed severely reduced rates of ap- pressorium formation ( Figure 5A), although they had no defects in germination rates ( Figure 4B). As expected, wild-type appres- soria were strongly melanized ( Figure 5B, asterisk), but infection cells of Dppt1 strains lacked melanization ( Figure 5C, arrow- head). ...
Citations
... The development of disease symptoms was scored and photographed daily. To analyze appressorial penetration rates by microscopy, leaves were inoculated with 10 µL droplets of a suspension containing 10 5 conidia/mL 0.02% (v/v) Tween 20, and penetration rates were counted at 48 hpi [21]. For B. cinerea infections, strawberry fruits were surface sterilized by incubation in 70% ethanol for 3 min, followed by three washes in sterile water. ...
The development of new anti-ureolytic compounds is of great interest due to the newly discovered role of urease inhibitors in crop protection. Purine degradation and the generation of ammonium by urease are required for the full virulence of biotrophic and hemibiotrophic fungal plant pathogens. Accordingly, chemicals displaying urease inhibitor activity may be used as a novel class of fungicides. Several urease inhibitors belonging to different chemical classes are known, and some compounds have been developed as urea fertilizer additives. We tested whether the natural urease inhibitors p-benzoquinone (p-HQ) and hydroquinone (HQ), as well as the synthetic inhibitors isopropoxy carbonyl phosphoric acid amide (iCPAA), benzyloxy carbonyl phosphoric acid amide (bCPAA), and dipropyl-hexamino-1,3 diphosphazenium chloride (DDC), prevent or delay plant infection caused by pathogens differing in lifestyles and host plants. p-BQ, HQ, and DCC not only protected maize from infection by the hemibiotroph C. graminicola, but also inhibited the infection process of biotrophs such as the wheat powdery mildew fungus Blumeria graminis f. sp. tritici and the broad bean rust fungus Uromyces viciae-fabae. Interestingly, the natural quinone-based compounds even reduced the symptom severity of the necrotrophic fungi, i.e., the grey mold pathogen B. cinerea and the Southern Leaf Spot fungus C. heterostrophus, to some extent. The urease inhibitors p-BQ, HQ, and DCC interfered with appressorial penetration and confirmed the appropriateness of urease inhibitors as novel fungicidal agents.
... One example is the 4-phosphopantetheinyl transferase protein (CgPPT1). CgPPT1 is functionally involved in and required for the biotrophy-necrotrophy transition of Colletotrichum graminicola [117]. In accordance with the hypothesis that N. parvum is an hemibiotroph, its genome contains a CgPPT1 gene. ...
Neofusicoccum parvum is a fungal plant pathogen of a wide range of hosts but knowledge about the virulence factors of N. parvum and host–pathogen interactions is rather limited. The molecules involved in the interaction between N. parvum and Eucalyptus are mostly unknown, so we used a multi-omics approach to understand pathogen–host interactions. We present the first comprehensive characterization of the in vitro secretome of N. parvum and a prediction of protein–protein interactions using a dry-lab non-targeted interactomics strategy. We used LC-MS to identify N. parvum protein profiles, resulting in the identification of over 400 proteins, from which 117 had a different abundance in the presence of the Eucalyptus stem. Most of the more abundant proteins under host mimicry are involved in plant cell wall degradation (targeting pectin and hemicellulose) consistent with pathogen growth on a plant host. Other proteins identified are involved in adhesion to host tissues, penetration, pathogenesis, or reactive oxygen species generation, involving ribonuclease/ribotoxin domains, putative ricin B lectins, and necrosis elicitors. The overexpression of chitosan synthesis proteins during interaction with the Eucalyptus stem reinforces the hypothesis of an infection strategy involving pathogen masking to avoid host defenses. Neofusicoccum parvum has the molecular apparatus to colonize the host but also actively feed on its living cells and induce necrosis suggesting that this species has a hemibiotrophic lifestyle.
... Indeed, appressoria of ∆pks1 and ∆ppt1 mutants of C. graminicola, the latter lacking the enzyme 4 -phosphopanthetenyl transferase required for post-translational activation of Pks1, form non-melanized appressoria. These appressoria generate turgor pressure comparable to the WT strain, but disrupt due to cell wall weakening [43,54]. ...
The genus Colletotrichum harbors many plant pathogenic species, several of which cause significant yield losses in the field and post harvest. Typically, in order to infect their host plants, spores germinate, differentiate a pressurized infection cell, and display a hemibiotrophic lifestyle after plant invasion. Several factors required for virulence or pathogenicity have been identified in different Colletotrichum species, and adaptation of cell wall biogenesis to distinct stages of pathogenesis has been identified as a major pre-requisite for the establishment of a compatible parasitic fungus-plant interaction. Here, we highlight aspects of fungal cell wall biogenesis during plant infection, with emphasis on the maize leaf anthracnose and stalk rot fungus, Colletotrichum graminicola.
... There are very few reports on PPTases in fungi and, so far, include those involved in lysine biosynthesis in yeasts, e.g., Lys5p/Lys7p, or those involved in both lysine biosynthesis and polyketide synthesis in filamentous fungi, i.e., CfwA/NpgA [58], and PPT1 from various species [59][60][61][62][63]. These PPTases have been investigated in terms of their roles in primary and secondary metabolism and the effects on fungal virulence. ...
Microbial natural products have had phenomenal success in drug discovery and development yet form distinct classes based on the origin of their native producer. Methods that enable metabolic engineers to combine the most useful features of the different classes of natural products may lead to molecules with enhanced biological activities. In this study, we modified the metabolism of the fungus Aspergillus oryzae to enable the synthesis of triketide lactone (TKL), the product of the modular polyketide synthase DEBS1-TE engineered from bacteria. We established (2S)-methylmalonyl-CoA biosynthesis via introducing a propionyl-CoA carboxylase complex (PCC); reassembled the 11.2 kb DEBS1-TE coding region from synthetic codon-optimized gene fragments using yeast recombination; introduced bacterial phosphopantetheinyltransferase SePptII; investigated propionyl-CoA synthesis and degradation pathways; and developed improved delivery of exogenous propionate. Depending on the conditions used titers of TKL ranged from <0.01–7.4 mg/L. In conclusion, we have demonstrated that A. oryzae can be used as an alternative host for the synthesis of polyketides from bacteria, even those that require toxic or non-native substrates. Our metabolically engineered A. oryzae may offer advantages over current heterologous platforms for producing valuable and complex natural products.
... However, adding lysine to spores did not rescue the pathogenicity defect of PPT1 mutants and the lysine auxotroph mutant AAR1. This is in contrast with similar experiments performed on other plant pathogenic fungi, in which the pathogenicity defects of PPT1 null mutants and lysine auxotrophs were partially rescued by exogenous lysine or wounding (Horbach et al., 2009;Zainudin et al., 2015). These first results await additional experiments, but it is tempting to speculate that, as in other fungi, the pathogenicity defect of the Z. tritici PPT1 mutant is due to a lack of production of yet unknown secondary metabolites, independently of its lysine auxotrophy, and DHN melanin/siderophore deficiencies. ...
... However, adding lysine to spores did not rescue the pathogenicity defect of PPT1 mutants and the lysine auxotroph mutant AAR1. This is in contrast with similar experiments performed on other plant pathogenic fungi, in which the pathogenicity defects of PPT1 null mutants and lysine auxotrophs were partially rescued by exogenous lysine or wounding (Horbach et al., 2009;Zainudin et al., 2015). These first results await additional experiments, but it is tempting to speculate that, as in other fungi, the pathogenicity defect of the Z. tritici PPT1 mutant is due to a lack of production of yet unknown secondary metabolites, independently of its lysine auxotrophy, and DHN melanin/siderophore deficiencies. ...
Zymoseptoria is a major fungal pathogen of wheat, responsible for the Septoria Tritici Blotch (STB) disease. Recently, STB has been the subject of intensive molecular studies. Notably, massive transcriptomic analyses have helped to explore this particular bi-phasic (asymptomatic/necrotrophic) infection process. Cytological analyses have also improved our understanding of the asymptomatic phase. These advances suggest that Zymoseptoria behaves as a hemi-biotrophic fungus, acting like an endophyte during its asymptomatic phase. STB is still difficult to control. The emergence of fungicide-resistant isolates has reduced the efficacy of many fungicides requiring the development of novel fungicides and methods to counteract/reduce fungicide resistance. Likewise, because Stb-resistant wheat cultivars have all been successively defeated by virulent isolates, there is a need to identify new resistance genes in wheat, and to develop better disease resistance management methods (pyramiding, mixture/alternation) to sustainably control this pathogen.
... For time lapse studies of young germlings, a recording interval of 5 min was used. To track Cgarp1-TagRFP-T localization during plant penetration, a heat-inactivated onion epidermis covering water agar (1% agarose and 1% Serva agar) was inoculated with 10 μL drops containing 10 3 oval conidia in 0.01% Tween for 29 h at 23 °C [68]. Different layers of an infected onion epidermis were recorded at a fixed distance of 0.5 μm. ...
Polar growth is a key characteristic of all filamentous fungi. It allows these eukaryotes to not only effectively explore organic matter but also interact within its own colony, mating partners, and hosts. Therefore, a detailed understanding of the dynamics in polar growth establishment and maintenance is crucial for several fields of fungal research. We developed a new marker protein, the actin-related protein 1 (Arp1) fused to red and green fluorescent proteins, which allows for the tracking of polar axis establishment and active hyphal growth in microscopy approaches. To exclude a probable redundancy with known polarity markers, we compared the localizations of the Spitzenkörper (SPK) and Arp1 using an FM4-64 staining approach. As we show in applications with the coprophilous fungus Sordaria macrospora and the hemibiotrophic plant pathogen Colletotrichum graminicola, the monitoring of Arp1 can be used for detailed studies of hyphal growth dynamics and ascospore germination, the interpretation of chemotropic growth processes, and the tracking of elongating penetration pegs into plant material. Since the Arp1 marker showed the same dynamics in both fungi tested, we believe this marker can be broadly applied in fungal research to study the manifold polar growth processes determining fungal life.
... To further support this assumption, we deleted the putative 4 ′ -phosphopantetheinyl transferase (Ppt) encoded by PPT1 in F. mangiferae. Functionality of most PKSs (and non-ribosomal peptide synthetases, NRPSs) depends on post-translationally modified acyl carrier protein (ACP) domains by 4 ′ -phosphopantetheinyl transferases (PPTases) (Neville et al., 2005;Horbach et al., 2009;Zainudin et al., 2015;Derbyshire et al., 2019). The putative Ppt homolog in F. mangiferae, FmPpt1, was identified by determining the ortholog using QuartetS (Yu et al., 2011). ...
The phytopathogenic fungus Fusarium mangiferae belongs to the Fusarium fujikuroi species complex (FFSC). Members of this group cause a wide spectrum of devastating diseases on diverse agricultural crops. F. mangiferae is the causal agent of the mango malformation disease (MMD) and as such detrimental for agriculture in the southern hemisphere. During plant infection, the fungus produces a plethora of bioactive secondary metabolites (SMs), which most often lead to severe adverse defects on plants health. Changes in chromatin structure achieved by posttranslational modifications (PTM) of histones play a key role in regulation of fungal SM biosynthesis. Posttranslational tri-methylation of histone 3 lysine 9 (H3K9me3) is considered a hallmark of heterochromatin and established by the SET-domain protein Kmt1. Here, we show that FmKmt1 is involved in H3K9me3 in F. mangiferae. Loss of FmKmt1 only slightly though significantly affected fungal hyphal growth and stress response and is required for wild type-like conidiation. While FmKmt1 is largely dispensable for the biosynthesis of most known SMs, removal of FmKMT1 resulted in an almost complete loss of fusapyrone and deoxyfusapyrone, γ-pyrones previously only known from Fusarium semitectum. Here, we identified the polyketide synthase (PKS) FmPKS40 to be involved in fusapyrone biosynthesis, delineate putative cluster borders by co-expression studies and provide insights into its regulation.
... Compte tenu de l'importance de l'adaptation aux conditions de carence martiale, il n'est pas étonnant que les régulateurs du métabolisme du fer HapX et SrbA, ainsi que PptA (4'-phosphopantéthéinyl transférase activatrice des NRPSs) soient essentiels à la virulence de divers champignons producteurs de sidérophores [228,325]. ...
La physiopathologie des infections déterminées par les Scedosporium est encore mal connue. Cependant, l’aptitude de ces champignons à accéder aux ressources en fer constitue vraisemblablement un facteur clé de leur virulence, le fer étant impliqué dans de nombreux processus biologiques essentiels. Le premier objectif de cette thèse était donc d’identifier, par une analyse in silico, les gènes potentiellement impliqués dans le métabolisme du fer chez S. apiospermum, en particulier dans l’acquisition du fer médiée par les sidérophores. La biosynthèse de ces petites molécules chélatrices du fer fait intervenir des mégaenzymes appelées NRPSs (non-ribosomal peptide synthetases). Etant donné l’importance des NRPSs dans le métabolisme secondaire fongique, nous avons ensuite étendu l’analyse bio-informatique à l’ensemble des clusters de gènes comportant une NRPS, révélant ainsi le potentiel de synthèse des peptides nonribosomiques chez S. apiospermum. Par ailleurs, des mutants défectifs pour le gène codant la NRPS chargée d’orchestrer la production d’un sidérophore extracellulaire ont été générés. L’étude de ces mutants a permis d’identifier la structure exacte du sidérophore produit, et de démontrer son importance pour la croissance et la virulence du champignon. Nos expériences ont également dévoilé la capacité de ce sidérophore à pirater le fer lié à d’autres sidérophores comme la pyoverdine, ce qui pourrait expliquer l’antagonisme rapporté entre les Scedosporium et le bacille pyocyanique dans le contexte de mucoviscidose. Au total, ces résultats indiquent un rôle majeur du système sidérophore chez Scedosporium, qui pourrait donc constituer une nouvelle cible thérapeutique pour ces champignons très peu sensibles aux antifongiques actuels.
... Conidia of C. graminicola that had formed on Petri dishes at 14 days postinoculation (dpi) were suspended in distilled H 2 O containing 0.02% (vol/vol) Tween 20 and adjusted to a concentration of 10 6 conidia/ml to study symptom severity or 10 5 conidia/ml for microscopy. Leaves were inoculated using 10 µl droplets as described (Horbach et al. 2009). For rust inoculations, 25 ml of suspensions containing 0.1 mg of urediniospores per ml of 0.02% (vol/vol) Tween 20 per plant was sprayed onto the lower epidermis of leaves. ...
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.
