Tools for Botrytis cinerea: New expression vectors make the gray mold fungus more accessible to cell biology approaches

Schumacher, Julia

doi:10.1016/j.fgb.2012.03.005

Cited by 175 publications

(268 citation statements)

References 76 publications

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“…Complementation of the ⌬bcgpx3 strain was achieved via directed integration of bcgpx3 with the native promoter and terminator at the bcniaD gene locus (primers 39/40) (43). The complementation vector pNDN-OGG was cut with NcoI and NotI, and bcgpx3 was integrated via yeast recombination.…”

Section: Methodsmentioning

confidence: 99%

“…Via yeast recombination the fusion construct was introduced in the NotI-linearized vector pNDN-OGG (42). The vector comprises gene flanks for the B. cinerea nitrate reductase, ensuring directed integration at the bcniaD locus, a nourseothricin resistance cassette, and gfp with the oliC promoter for constitutive expression of the fusion protein (43). The vectors pNDN-OG bcskn7 G and pNDN-OG bcgpx3 G were cut with ApaI and SacI, and the replacement fragments were used for transformation.…”

Section: Methodsmentioning

confidence: 99%

“…For this purpose the vector pNDH-OGG was cut with NcoI, and the amplified gene bap1 (primers 44/45) with overhangs to gfp and the oliC promoter were introduced via homologous recombination. The vector contains gene flanks for the B. cinerea nitrate reductase, ensuring directed integration at the bcniaD locus, a hygromycin resistance cassette, and gfp with the oliC promoter for constitutive expression of the fusion protein (43). The GFP vector pNDH-O Bap1 GG was digested with the enzymes ApaI and SacII and used for transformation.…”

Section: Methodsmentioning

confidence: 99%

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Unraveling the Function of the Response Regulator BcSkn7 in the Stress Signaling Network of Botrytis cinerea

et al. 2015

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Important for the lifestyle and survival of every organism is the ability to respond to changing environmental conditions. The necrotrophic plant pathogen Botrytis cinerea triggers an oxidative burst in the course of plant infection and therefore needs efficient signal transduction to cope with this stress. The factors involved in this process and their precise roles are still not well known. Here, we show that the transcription factor Bap1 and the response regulator (RR) B. cinerea Skn7 (BcSkn7) are two key players in the oxidative stress response (OSR) of B. cinerea; both have a major influence on the regulation of classical OSR genes. A yeast-one-hybrid (Y1H) approach proved direct binding to the promoters of gsh1 and grx1 by Bap1 and of glr1 by BcSkn7. While the function of Bap1 is restricted to the regulation of oxidative stress, analyses of ⌬bcskn7 mutants revealed functions beyond the OSR. Involvement of BcSkn7 in development and virulence could be demonstrated, indicated by reduced vegetative growth, impaired formation of reproductive structures, and reduced infection cushion-mediated penetration of the host by the mutants. Furthermore, ⌬bcskn7 mutants were highly sensitive to oxidative, osmotic, and cell wall stress. Analyses of ⌬bap1 bcskn7 double mutants indicated that loss of BcSkn7 uncovers an underlying phenotype of Bap1. In contrast to Saccharomyces cerevisiae, the ortholog of the glutathione peroxidase Gpx3p is not required for nuclear translocation of Bap1. The presented results contribute to the understanding of the OSR in B. cinerea and prove that it differs substantially from that of yeast, demonstrating the complexity and versatility of components involved in signaling pathways.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Unraveling the Function of the Response Regulator BcSkn7 in the Stress Signaling Network of Botrytis cinerea

et al. 2015

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“…They were then ligated into the BamHI-NotI-or Xho-NotI-digested pGEX4T1 expression vector, giving rise to pGEX-Cprac, pGEX-Cpcdc42, pGEX-CpRas1, pGEX-Cdc24DH, pGEX-CpCdc24DHPH, pGEXCpDock180DOCK, and pGEX-CpDock180DHR2. 3HA vectors for coimmunoprecipitation were created by yeast recombinational cloning (59). For this purpose, the codon-modified 3HA tag, the gpd promoter, and a HindIII site were integrated into pRS426-Hph, giving rise to p3HA-GPD-Hyg.…”

Section: Methodsmentioning

confidence: 99%

Small-GTPase-Associated Signaling by the Guanine Nucleotide Exchange Factors CpDock180 and CpCdc24, the GTPase Effector CpSte20, and the Scaffold Protein CpBem1 in Claviceps purpurea

et al. 2014

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bMonomeric GTPases of the Rho subfamily are important mediators of polar growth and NADPH (Nox) signaling in a variety of organisms. These pathways influence the ability of Claviceps purpurea to infect host plants. GTPase regulators contribute to the nucleotide loading cycle that is essential for proper functionality of the GTPases. Scaffold proteins gather GTPase complexes to facilitate proper function. The guanine nucleotide exchange factors (GEFs) CpCdc24 and CpDock180 activate GTPase signaling by triggering nucleotide exchange of the GTPases. Here we show that CpCdc24 harbors nucleotide exchange activity for both Rac and Cdc42 homologues. The GEFs partly share the cellular distribution of the GTPases and interact with the putative upstream GTPase CpRas1. Interaction studies show the formation of higher-order protein complexes, mediated by the scaffold protein CpBem1. Besides the GTPases and GEFs, these complexes also contain the GTPase effectors CpSte20 and CpCla4, as well as the regulatory protein CpNoxR. Functional characterizations suggest a role of CpCdc24 mainly in polarity, whereas CpDock180 is involved in stress tolerance mechanisms. These findings indicate the dynamic formation of small GTPase complexes and improve the model for GTPase-associated signaling in C. purpurea.

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“…For construction of the gfp-mid1 fragment, the coding region of mid1 was amplified using primers 23 and 24, which contain overlapping sequences homologous to the glucanase terminator and gfp (encoding codon-optimized GFP) (46), respectively, of the pNAN-OGG vector (47). This vector contains flanks mediating the replacement of the gene encoding the nitrite reductase and therefore ensuring integration at a known locus.…”

Section: Methodsmentioning

confidence: 99%

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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Tools for Botrytis cinerea: New expression vectors make the gray mold fungus more accessible to cell biology approaches

Cited by 175 publications

References 76 publications

Unraveling the Function of the Response Regulator BcSkn7 in the Stress Signaling Network of Botrytis cinerea

Unraveling the Function of the Response Regulator BcSkn7 in the Stress Signaling Network of Botrytis cinerea

Small-GTPase-Associated Signaling by the Guanine Nucleotide Exchange Factors CpDock180 and CpCdc24, the GTPase Effector CpSte20, and the Scaffold Protein CpBem1 in Claviceps purpurea

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

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