Calcium measurement in living filamentous fungi expressing codon‐optimized aequorin

Nelson, Glyn; Kozlova-Zwinderman, O.; Collis, A. J.; Knight, Marc R.; Fincham, J. R. S.; Stanger, C.; Renwick, A. G.; Hessing, J. G. M.; Punt, Peter J.; Hondel, Cees A. M. J. J. van den; Read, Nick D.

doi:10.1111/j.1365-2958.2004.04066.x

Cited by 97 publications

(126 citation statements)

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“…In fungi, Nelson et al (20) showed that mechanical perturbation provokes a transient increase in intracellular calcium. In full agreement with these reports, our results show a series of induced genes involved in calcium signaling and transport.…”

Section: Discussionmentioning

confidence: 99%

An injury-response mechanism conserved across kingdoms determines entry of the fungus Trichoderma atroviride into development

Hernández‐Oñate¹,

Esquivel-Naranjo²,

Mendoza‐Mendoza

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

100

139

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A conserved injury-defense mechanism is present in plants and animals, in which the production of reactive oxygen species (ROS) and lipid metabolism are essential to the response. Here, we describe that in the filamentous fungus Trichoderma atroviride, injury results in the formation of asexual reproduction structures restricted to regenerating cells. High-throughput RNA-seq analyses of the response to injury in T. atroviride suggested an oxidative response and activation of calcium-signaling pathways, as well as the participation of lipid metabolism, in this phenomenon. Gene-replacement experiments demonstrated that injury triggers NADPH oxidase (Nox)-dependent ROS production and that Nox1 and NoxR are essential for asexual development in response to damage. We further provide evidence of H 2 O 2 and oxylipin production that, as in plants and animals, may act as signal molecules in response to injury in fungi, suggesting that the three kingdoms share a conserved defense-response mechanism.conidiation | stress | transcriptome

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Section: Discussionmentioning

confidence: 99%

An injury-response mechanism conserved across kingdoms determines entry of the fungus Trichoderma atroviride into development

Hernández‐Oñate¹,

Esquivel-Naranjo²,

Mendoza‐Mendoza

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

100

139

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“…GB5 medium containing 12% EtOH was used as a control for AMD treatment. For calculation of the cytosolic Ca 2ϩ -concentration at the end of each measurement, the total luminescence was measured for 120 s after complete discharge of the present aequorin by injection of 100 l 4 M CaCl 2 in 10% EtOH (48,51). The RLU values were multiplied by 1.24 to correct for quenching by ethanol.…”

Section: Methodsmentioning

confidence: 99%

“…The RLU values were multiplied by 1.24 to correct for quenching by ethanol. RLU values were then converted to [Ca 2ϩ ] cyt by using the following empirically derived equations: pCa ϭ 0.332588 ϫ (Ϫlog 10 K) ϩ 5.5593, and K ϭ luminescence (in RLU) s Ϫ1 /total luminescence (in RLU) (48,51).…”

Section: Methodsmentioning

confidence: 99%

“…In Aspergillus awamori, the calcium ionophore A23187 was shown to increase intracellular Ca 2ϩ levels by acting as a mobile ion carrier, as demonstrated by increased aequorin luminescence (48). To study the effect of this ionophore in B. cinerea, the growth of the wild type and all mutants was compared on a medium amended with A23187.…”

Section: Identification Of B Cinerea Mid1 and Cch1 Homologsmentioning

confidence: 99%

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Cch1 and Mid1 Are Functionally Required for Vegetative Growth under Low-Calcium Conditions in the Phytopathogenic Ascomycete Botrytis cinerea

Harren

Tudzynski

2013

Eukaryot Cell

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In the filamentous phytopathogen Botrytis cinerea, the Ca 2؉ /calcineurin signaling cascade has been shown to play an important role in fungal growth, differentiation, and virulence. This study deals with the functional characterization of two components of this pathway, the putative calcium channel proteins Cch1 and Mid1. The cch1 and mid1 genes were deleted, and single and double knockout mutants were analyzed during different stages of the fungal life cycle. Our data indicate that Cch1 and Mid1 are functionally required for vegetative growth under conditions of low extracellular calcium, since the growth of both deletion mutants is strongly impaired when they are exposed to the Ca 2؉ -chelating agents EGTA and 1,2-bis(o-aminophenoxy)ethane-N,N,N=,N=-tetraacetic acid (BAPTA). The impact of external Ca 2؉ was investigated by supplementing with CaCl 2 and the ionophore A23187, both of which resulted in elevated growth for all mutants. However, deletion of either gene had no impact on germination, sporulation, hyphal morphology, or virulence. By use of the aequorin reporter system to measure intracellular calcium levels, no differences between the mutant strains and the wild type were obtained. Localization studies revealed a subcellular distribution of the Mid1-green fluorescent protein (GFP) fusion protein in network-like filaments, probably the endoplasmic reticulum (ER) membranes, indicating that Mid1 is not a plasma membrane-located calcium channel in B. cinerea.

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“…S5). Growth of the Dmid1 mutant was also inhibited by elevated intracellular Ca 2+ concentrations, as shown by growth assays using the ionophore A23187, which increases intracellular Ca 2+ levels (Nelson et al, 2004). Filter papers soaked with 9.5 mM ionophore in ethanol were applied to MM agar plates supplemented with 4.2, 10, 30, 40, 50 and 100 mM CaCl 2 and inoculated with 20.1Dku70 and Dmid1.…”

Section: Mid1 Affects Ca 2+ Homeostasismentioning

confidence: 99%

Deletion of Mid1, a putative stretch-activated calcium channel in Claviceps purpurea, affects vegetative growth, cell wall synthesis and virulence

Bormann

Tudzynski

2009

Microbiology

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Deletion of Mid1, a putative stretch-activated calcium channel in Claviceps purpurea, affects vegetative growth, cell wall synthesis and virulence The putative Claviceps purpurea homologue of the Saccharomyces cerevisiae stretch-activated calcium ion channel Mid1 was investigated for its role in vegetative growth, differentiation and pathogenicity on rye (Secale cereale). Gene replacement mutants of Cl. purpurea mid1 were not affected in polar growth and branching in axenic culture but showed a significantly reduced growth rate. The growth defect could not be complemented by Ca 2+ supplementation, in contrast to mid1 mutants in yeast, but the altered sensitivity of the mutants to changes in external and internal Ca 2+ concentrations indicates some role of Mid1 in Ca 2+ homeostasis. The major effect of mid1 deletion, however, was the complete loss of virulence: infected rye plants showed no disease symptoms at all. Detailed analyses of in vitro-infected rye ovaries demonstrated that the Dmid1 mutants had multiple apical branches and were unable to infect the host tissue, suggesting that Mid1 is essential for generating the necessary mechanical force for penetration. This is believed to be the first report of an essential role for a Mid1 homologue in the virulence of a plantpathogenic fungus. INTRODUCTIONThe phytopathogenic biotrophic ascomycete Claviceps purpurea (Fries ex Fries) Tulasne parasitizes a large number of Poaceae species (Tudzynski & Scheffer, 2004). It colonizes ovaries of its main host, rye (Secale cereale), by penetrating the cuticle and growing through the ovarian tissue in a strictly directed and unbranched manner until it reaches the vascular bundles. By tapping into the vascular bundles, the fungus assures a stable nutrition supply. This is a prerequisite for the subsequent colonization of the entire ovary. During this process the fungus produces masses of conidia which are secreted in a viscous fluid called honeydew. Spores are dispersed to other blooming florets mainly zoochorically and hydrochorically. The period of conidia formation ends when sclerotium formation is initiated. The sclerotia represent resting structures consisting of a solid plectenchymatous structure of pigmented storage cells (Tenberge, 1999). They contain the so-called ergot alkaloids, which represent a class of pharmacologically important mycotoxins (Schardl et al., 2006).The fungus grows mainly intercellularly and therefore decomposes the middle lamella between the plant cells. This lamella predominantly consists of pectin; degradation of pectin by fungal polygalacturonases is essential for full pathogenicity (Oeser et al., 2002). It is possible that Ca 2+ complexed in this pectin could act as a trail for the hyphae on their way through the ovary. A tip-high gradient of cytoplasmic calcium ([Ca 2+ ] cyt. ) has been observed in a wide range of eukaryotes that grow in a polarized and oriented manner, including fungal hyphae Silverman-Gavrila & Lew, 2003), neurons (Mattson, 1999), and pollen tubes and root hairs of vascula...

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Calcium measurement in living filamentous fungi expressing codon‐optimized aequorin

Cited by 97 publications

References 80 publications

An injury-response mechanism conserved across kingdoms determines entry of the fungus Trichoderma atroviride into development

An injury-response mechanism conserved across kingdoms determines entry of the fungus Trichoderma atroviride into development

Cch1 and Mid1 Are Functionally Required for Vegetative Growth under Low-Calcium Conditions in the Phytopathogenic Ascomycete Botrytis cinerea

Deletion of Mid1, a putative stretch-activated calcium channel in Claviceps purpurea, affects vegetative growth, cell wall synthesis and virulence

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