Thin PTFE-like membranes allow characterizing germination and mechanical penetration competence of pathogenic fungi

Kuster, Stefan P.; Ludwig, Nancy; Willers, Guido; Hoffmann, Jens; Deising, H. B.; Kiesow, Andreas

doi:10.1016/j.actbio.2008.05.011

Cited by 12 publications

(11 citation statements)

References 30 publications

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“…PTFE membranes of defined thickness were prepared as described (Küster et al 2008). In order to separate films from glass slides, the potassium bromide layer was dissolved in distilled water, and PTFE layers floated as a membrane film carried by the surface tension of the water.…”

Section: Ptfe Penetration Cytorrhysis Assays Andmentioning

confidence: 99%

Melanin Is Not Required for Turgor Generation but Enhances Cell-Wall Rigidity in Appressoria of the Corn Pathogen Colletotrichum graminicola

Ludwig¹,

Loehrer²,

Hempel³

et al. 2014

MPMI

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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.

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Section: Ptfe Penetration Cytorrhysis Assays Andmentioning

confidence: 99%

Melanin Is Not Required for Turgor Generation but Enhances Cell-Wall Rigidity in Appressoria of the Corn Pathogen Colletotrichum graminicola

Ludwig¹,

Loehrer²,

Hempel³

et al. 2014

MPMI

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“…PTFE membrane fabrication, including film deposition and extensive quality control, was performed as described by Kuster et al (2008). Spores of P. pachyrhizi and M. oryzae were harvested as described above.…”

Section: Penetration Of Polytetrafluoroethylene (Ptfe) Membranesmentioning

confidence: 99%

“…In order to demonstrate the enormous force generated by a non-melanized appressorium, we examined whether P. pachyrhizi and M. oryzae differ in their capability to penetrate non-biodegradable artificial membranes. We used PTFE-like membranes of highly defined thickness because they are known to simulate the cuticle and epidermal cell wall with respect to hydrophobicity and penetration resistance (Kuster et al, 2008). Surprisingly, P. pachyrhizi was able to penetrate all membranes, even those with the highest thickness of 130 lm (Table 1).…”

Section: Estimation Of Force Exerted During Penetrationmentioning

confidence: 99%

In vivo assessment by Mach–Zehnder double‐beam interferometry of the invasive force exerted by the Asian soybean rust fungus (Phakopsora pachyrhizi)

et al. 2014

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SummaryAsian soybean rust (Phakopsora pachyrhizi) causes a devastating disease in soybean (Glycine max). We tested the hypothesis that the fungus generates high turgor pressure in its hyaline appressoria to mechanically pierce epidermal cells.Turgor pressure was determined by a microscopic technique, called transmitted light double-beam interference Mach-Zehnder microscopy (MZM), which was developed in the 1960s as a forefront of live cell imaging. We revitalized some original microscopes and equipped them for modern image capturing. MZM data were corroborated by cytorrhysis experiments.Incipient cytorrhysis determined the turgor pressure in appressoria of P. pachyrhizi to be equivalent to 5.13 MPa. MZM data revealed that osmotically active sugar alcohols only accounted for 75% of this value. Despite having a lower turgor pressure, hyaline rust appressoria were able to penetrate non-biodegradable polytetrafluoroethylene (PTFE) membranes more efficiently than do melanized appressoria of the anthracnose fungus Colletotrichum graminicola or the rice blast fungus Magnaporthe oryzae.Our findings challenge the hypotheses that force-based penetration is a specific hallmark of fungi differentiating melanized appressoria and that this turgor-driven process is solely caused by metabolic degradation products. The appressorial turgor pressure may explain the capability of P. pachyrhizi to forcefully invade a wide range of different plants and may pave the way to novel plant protection approaches.

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“…During maturation, appressoria form rigid cell walls which melanize, and the synthesis of high concentrations of compatible solutes (De Jong et al ., 1997) results in the generation of enormous appressorial turgor pressure (Howard et al ., 1991). Turgor is translated into force, which is exerted at the appressorial base, supporting penetration into the host (Bastmeyer et al ., 2002; Bechinger et al ., 1999; Küster et al ., 2008). In the host, C. graminicola forms biotrophic infection structures, i.e.…”

Section: Introductionmentioning

confidence: 99%

Identification of virulence genes in the corn pathogenColletotrichum graminicolabyAgrobacterium tumefaciens‐mediated transformation

Münch

Ludwig

Floß

et al. 2010

Molecular Plant Pathology

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A previously developed Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for the plant pathogenic fungus Colletotrichum graminicola led to high rates of tandem integration of the whole Ti-plasmid, and was therefore considered to be unsuitable for the identification of pathogenicity and virulence genes by insertional mutagenesis in this pathogen. We used a modified ATMT protocol with acetosyringone present only during the co-cultivation of C. graminicola and A. tumefaciens. Analysis of 105 single-spore isolates randomly chosen from a collection of approximately 2000 transformants, indicated that almost 70% of the transformants had single T-DNA integrations. Of 500 independent transformants tested, 10 exhibited attenuated virulence in infection assays on whole plants. Microscopic analyses primarily revealed defects at different pre-penetration stages of infection-related morphogenesis. Three transformants were characterized in detail. The identification of the T-DNA integration sites was performed by amplification of genomic DNA ends after endonuclease digestion and polynucleotide tailing. In one transformant, the T-DNA had integrated into the 5'-flank of a gene with similarity to allantoicase genes of other Ascomycota. In the second and third transformants, the T-DNA had integrated into an open reading frame (ORF) and into the 5'-flank of an ORF. In both cases, the ORFs have unknown function.

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Thin PTFE-like membranes allow characterizing germination and mechanical penetration competence of pathogenic fungi

Cited by 12 publications

References 30 publications

Melanin Is Not Required for Turgor Generation but Enhances Cell-Wall Rigidity in Appressoria of the Corn Pathogen Colletotrichum graminicola

Melanin Is Not Required for Turgor Generation but Enhances Cell-Wall Rigidity in Appressoria of the Corn Pathogen Colletotrichum graminicola

In vivo assessment by Mach–Zehnder double‐beam interferometry of the invasive force exerted by the Asian soybean rust fungus (Phakopsora pachyrhizi)

Identification of virulence genes in the corn pathogenColletotrichum graminicolabyAgrobacterium tumefaciens‐mediated transformation

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