An efficient method for DNA extraction from Cladosporioid fungi

We developed an efficient method for DNA extraction from Cladosporioid fungi, which are important fungal plant pathogens. The cell wall of Cladosporioid fungi is often melanized, which makes it difficult to extract DNA from their cells. In order to overcome this we grew these fungi for three days on agar plates and extracted DNA from mycelium mats after manual or electric homogenization. High-quality DNA was isolated, with an A260/A280 ratio ranging between 1.6 and 2.0. Isolated genomic DNA was efficiently digested with restriction enzymes and produced distinct banding patterns on agarose gels for the different Cladosporium species. Clear DNA fragments from the isolated DNA were amplified by PCR using small and large subunit rDNA primers, demonstrating that this method provides DNA of sufficiently high quality for molecular analyse

Fungal plant pathogens and the plant immune system

Fungi are notorious plant pathogens and continuously threat global food production. In the last decades we have obtained a better understanding of infection strategies of fungi and the plant immune system. This has facilitated more efficient introduction of disease resistance genes in crop plants by plant breeders. A brief overview of progress in research and applications will be provided as well as a glimpse into the future

Physiological studies on cultivar-specific resistance of tomato plants to Cladosporium fulvum

Ultrastructural and physiological aspects of cultivar-specific resistance of tomato against Cladosporium fulvum (syn. Fulvia fulva) are subject of this thesis.The ultrastructural study described in the first paper was meant as an introduction to a physiological study of cultivar-specific resistance. Light, fluorescence and scanning electron microscopy revealed no differences between virulent and avirulent races of Cladosporium fulvum with respect to conidial germination and stomatal penetration. In incompatible interactions fungal growth was arrested very soon after stomatal penetration and the fungus became confined to stomata and surrounding cells, which showed callose deposition, cell browning and cell collapse. In addition, extracellular material was deposited on the outer surface of mesophyll cells. In compatible interactions, however, fungal growth was not inhibited and the above- mentioned host reactions did hardly occur.In compatible and incompatible interactions differential changes in soluble leaf proteins were found which might be correlated with cultivar-specific resistance (second paper). A de novo synthezised protein of host origin appeared more rapidly in incompatible than in compatible interactions. After some time this protein also appeared in non-inoculated leaflets opposite the inoculated ones. Although the appearance of the protein was strongly associated with the hypersensitive response in incompatible interactions, its presence, at the concentrations tested, was not correlated with inhibition of Cladosporium fulvum in tomato leaf.In the third paper, a possible mechanism of fungal growth inhibition of Cladosporium fulvum in incompatible interactions is described. Two phytoalexins were found in leaves which accumulated more rapidly in incompatible than in compatible interactions. Fungal growth inhibition in incompatible interactions coincided with accumulation of phytoalexins. However, in fruits neither differential accumulation of these nor of the sesquiterpene rishitin took place.The fourth paper describes the identification of the two above-mentioned phytoalexins of inoculated tomato leaves and fruits and one, which only occurred in tomato fruits, as being falcarindiol, falcarinol and cis -tetradeca-6-ene-1,3diyne-5,8-diol, respectively. Falcarindiol and falcarinol had not been found in Solanaceae before, while cis -tetradeca-6-ene-1,3-diyne-5,8-diol was a completely novel compound. Although the level to which the polyacetylenes accumulated in vivo nearly reached the ED 50 -values of hyphal and mycelial growth in vitro it is difficult to conclude that these phytoalexins can explain fungal growth inhibition in incompatible interactions completely.In the fifth paper, the induction of phytoalexins was studied, mainly of rishitin which can be readily quantified. In culture filtrates of young cultures of Cladosporium fulvum high-molecular weight glycoproteins were found which were inducers of rishitin accumulation. These elicitors contained many glucose and only a few mannose and galactose residues. They appeared not to be host-specific, as they induced also glyceollin and pisatin in soybean cotyledons and pea pods, respectively.In the sixth paper, elicitors occurring in the cell wall of Cladosporium fulvum were compared with those in culture filtrates of varying age. Elicitors present in culture filtrates of young cultures are probably other macromolecules than those in culture filtrates of old cultures. The former contain nearly only glucose, while the latter, in addition to glucose, contain many mannose and galactose residues, which are likely derived from the cell wall.Rishitin-inducing activity always appeared to be positively correlated with the mannose and galactose content of the elicitor. The elicitor from the cell wall is a peptido galactoglucomannan which occurs on the cell surface and is very similar to the peptido phosphogalactorunnan, proteo galactoglucomannan, and galactomannan of Cladosporium werneckii, Piricularia oryzae and Penicillium charlesii, respectively. Presence of terminal mannose and/or galactose residues of the peptido galactoglucomannan is likely a prerequisite for its rishitin and necrosis-inducing activity. Like the culture filtrate elicitor, the cell wall elicitor is neither race, nor cultivar or host-specific. Although the accumulation of phytoalexins in leaves was correlated with the incompatible interaction (third paper), no evidence was obtained for the induction of phytoalexins being race or cultivar-specific.Probably elicitors of phytoalexin accumulation are merely inducers of a general defence reaction (including callose deposition, lignification and cell necrosis). This mans that in the future we should rather look for substances which specifically suppress the resistance response than for those which specifically induce such a reaction

Ga and Gß Proteins Regulate the Cyclic AMP Pathway That Is Required for Development and Pathogenicity of the Phytopathogen Mycosphaerella graminicola

We identified and functionally characterized genes encoding three G alpha proteins and one G beta protein in the dimorphic fungal wheat pathogen Mycosphaerella graminicola, which we designated MgGpa1, MgGpa2, MgGpa3, and MgGpb1, respectively. Sequence comparisons and phylogenetic analyses showed that MgGPA1 and MgGPA3 are most related to the mammalian G alpha(i) and G alpha(s) families, respectively, whereas MgGPA2 is not related to either of these families. On potato dextrose agar (PDA) and in yeast glucose broth (YGB), MgGpa1 mutants produced significantly longer spores than those of the wild type (WT), and these developed into unique fluffy mycelia in the latter medium, indicating that this gene negatively controls filamentation. MgGpa3 mutants showed more pronounced yeast-like growth accompanied with hampered filamentation and secreted a dark-brown pigment into YGB. Germ tubes emerging from spores of MgGpb1 mutants were wavy on water agar and showed a nested type of growth on PDA that was due to hampered filamentation, numerous cell fusions, and increased anastomosis. Intracellular cyclic AMP (cAMP) levels of MgGpb1 and MgGpa3 mutants were decreased, indicating that both genes positively regulate the cAMP pathway, which was confirmed because the WT phenotype was restored by adding cAMP to these mutant cultures. The cAMP levels in MgGpa1 mutants and the WT were not significantly different, suggesting that this gene might be dispensable for cAMP regulation. In planta assays showed that mutants of MgGpa1, MgGpa3, and MgGpb1 are strongly reduced in pathogenicity. We concluded that the heterotrimeric G proteins encoded by MgGpa3 and MgGpb1 regulate the cAMP pathway that is required for development and pathogenicity in M. graminicola

Novel Introner-Like Elements in fungi are involved in parallel gains of spliceosomal introns

Spliceosomal introns are key components of the eukaryotic gene structure. Although they contributed to the emergence of eukaryotes, their origin remains elusive. In fungi, they might originate from the multiplication of invasive introns named Introner-Like Elements (ILEs). However, so far ILEs have been observed in six fungal species only, including Fulvia fulva and Dothistroma septosporum (Dothideomycetes), arguing against ILE insertion as a general mechanism for intron gain. Here, we identified novel ILEs in eight additional fungal species that are phylogenetically related to F. fulva and D. septosporum using PCR amplification with primers derived from previously identified ILEs. The ILE content appeared unique to each species, suggesting independent multiplication events. Interestingly, we identified four genes each containing two gained ILEs. By analysing intron positions in orthologues of these four genes in Ascomycota, we found that three ILEs had inserted within a 15 bp window that contains regular spliceosomal introns in other fungal species. These three positions are not the result of intron sliding because ILEs are newly gained introns. Furthermore, the alternative hypothesis of an inferred ancestral gain followed by independent losses contradicts the observed degeneration of ILEs. These observations clearly indicate three parallel intron gains in four genes that were randomly identified. Our findings suggest that parallel intron gain is a phenomenon that has been highly underestimated in ILE-containing fungi, and likely in the whole fungal kingdom

Functional Analysis of Cladosponum fulvum Effector Catalog

Onlangs is de DNA-sequentie van het genoom van Cladosporium fulvum bepaald. Het voornaamste doel daarvan is de identificatie en karakterisering van nieuwe effectors

The tomato Orion locus comprises a unique class of Hcr9 genes

Resistance against the tomato fungal pathogen Cladosporium fulvum is often conferred by Hcr9 genes (Homologues of the C. fulvum resistance gene Cf-9) that are located in the Milky Way cluster on the short arm of chromosome 1. These Hcr9 genes mediate recognition of fungal avirulence gene products. In contrast, the resistance gene Cf-Ecp2 mediates recognition of the virulence factor Ecp2 and is located in the Orion (OR) cluster on the short arm of chromosome 1. Here, we report the map- and homology-based cloning of the OR Hcr9 cluster. A method was optimised to generate clone-specific fingerprint data that were subsequently used in the efficient calculation of genomic DNA contigs. Three Hcr9s were identified as candidate Cf-Ecp2 genes. By PCR-based cloning using specific OR sequences, orthologous Hcr9 genes were identified from different Lycopersicon species and haplotypes. The OR Hcr9s are very homologous. However, based on the relative low sequence homology to other Hcr9s, the OR Hcr9s are classified as a new subgrou

Synergistic action of serine- and metallo-proteases from Fusarium oxysporum f. sp. lycopersici cleaves chitin-binding tomato chitinases, reduces their antifungal activity and enhances fungal virulence

As part of their defence strategy against fungal pathogens, plants secrete chitinases that degrade chitin, the major structural component of fungal cell walls. Some fungi are not sensitive to plant chitinases because they secrete chitin-binding effector proteins that protect their cell wall against these enzymes. However, it is not known how fungal pathogens that lack chitin-binding effectors overcome this plant defence barrier. Here, we investigated the ability of fungal tomato pathogens to cleave chitin-binding domain (CBD)-containing chitinases and its effect on fungal virulence. Four tomato CBD-chitinases were produced in Pichia pastoris and incubated with secreted proteins isolated from seven fungal tomato pathogens. Of these, Fusarium oxysporum f. sp. lycopersici, Verticillium dahliae and Botrytis cinerea were able to cleave the extracellular tomato chitinases SlChi1 and SlChi13. Cleavage by F. oxysporum removed the CBD from the N-terminus, as shown by mass spectrometry, and significantly reduced the chitinase and antifungal activity of both chitinases. Both secreted metallo-protease FoMep1 and serine protease FoSep1 were responsible for this cleavage. Double deletion mutants of FoMep1 and FoSep1 of F. oxysporum lacked chitinase cleavage activity on SlChi1 and SlChi13 and showed reduced virulence on tomato. These results demonstrate the importance of plant chitinase cleavage in fungal virulence