Expression of Green Fluorescent Protein in
            <i>Aureobasidium pullulans</i>
            and Quantification of the Fungus on Leaf Surfaces

Wymelenberg, Amber J. Vanden; Cullen, Daniel; Spear, R. N.; Schoenike, Barry; Andrews, John H.

doi:10.2144/97234st01

Cited by 109 publications

(80 citation statements)

References 3 publications

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“…To further elucidate the role of NoxR in fungal growth and branching, we generated an E. festucae noxR overexpressing strain, containing noxR under the control of a translation elongation factor (TEF) promoter (Vanden Wymelenberg et al, 1997). Ptef-noxR transformants of E. festucae formed smaller, more compact colonies than the wild type and had hyphae that were more frequently branched, phenotypes similar to that observed for the wild type treated with DPI ( Figures 7A, 7B, and 7I).…”

Section: Overexpression Of Noxr Causes Hyphal Hyperbranchingmentioning

confidence: 74%

A p67Phox-Like Regulator Is Recruited to Control Hyphal Branching in a Fungal–Grass Mutualistic Symbiosis

2006

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Key requirements for microbes to initiate and establish mutualistic symbiotic interactions with plants are evasion of potential host defense responses and strict control of microbial growth. Reactive oxygen species (ROS) produced by a specific NADPH oxidase isoform, NoxA, regulate hyphal growth in the mutualistic interaction between the fungal endophyte Epichloe¨festucae and its grass host Lolium perenne. Unlike mammalian systems, little is known about the fungal NADPH oxidase complex and its response to differentiation signals. We identify an E. festucae p67 phox -like regulator, NoxR, dispensable in culture but essential in planta for the symbiotic interaction. Plants infected with a noxR deletion mutant show severe stunting and premature senescence, whereas hyphae in the meristematic tissues show increased branching leading to increased fungal colonization of pseudostem and leaf blade tissue. Inhibition of ROS production or overexpression of noxR recapitulates the hyperbranching phenotype in culture. NoxR interacts in vitro with the small GTP binding protein RacA and requires a functional RacA binding site to complement the noxR mutant and restore the wild-type plant interaction phenotype. These results show that NoxR is a key regulator of NoxA in symbiosis, where it acts together with RacA to spatially regulate ROS production and control hyphal branching and patterning.

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Section: Overexpression Of Noxr Causes Hyphal Hyperbranchingmentioning

confidence: 74%

A p67Phox-Like Regulator Is Recruited to Control Hyphal Branching in a Fungal–Grass Mutualistic Symbiosis

2006

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“…Bialaphos-resistant transformants were selected on regeneration medium containing bialaphos (250 mg/mL). To visualize pathogens by expression of enhanced (E) GFP, pTEFEGFP was introduced into the tested pathogens with pCB1636 carrying a hygromycin-resistant gene (Sweigard et al, 1997;Vanden Wymelenberg et al, 1997). Hygromycin-resistant transformants were selected on regeneration medium containing hygromycin (100 mg/mL).…”

Section: Fungal Transformationmentioning

confidence: 99%

Entry Mode–Dependent Function of an Indole Glucosinolate Pathway in Arabidopsis for Nonhost Resistance against Anthracnose Pathogens

et al. 2010

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When faced with nonadapted fungal pathogens, Arabidopsis thaliana mounts nonhost resistance responses, which typically result in the termination of early pathogenesis steps. We report that nonadapted anthracnose fungi engage two alternative entry modes during pathogenesis on leaves: turgor-mediated invasion beneath melanized appressoria, and a previously undiscovered hyphal tip-based entry (HTE) that is independent of appressorium formation. The frequency of HTE is positively regulated by carbohydrate nutrients and appears to be subject to constitutive inhibition by the fungal mitogenactivated protein kinase (MAPK) cascade of MAPK ESSENTIAL FOR APPRESSORIUM FORMATION1. The same MAPK cascade is essential for appressorium formation. Unexpectedly, the Arabidopsis indole glucosinolate pathway restricts entry of the nonadapted anthracnose fungi only when these pathogens employ HTE. Arabidopsis mutants defective in indole glucosinolate biosynthesis or metabolism support the initiation of postinvasion growth of nonadapted Colletotrichum gloeosporioides and Colletotrichum orbiculare. However, genetic disruption of Colletotrichum appressorium formation does not permit HTE on host plants. Thus, Colletotrichum appressoria play a critical role in the suppression of preinvasion plant defenses, in addition to their previously described role in turgor-mediated plant cell invasion. We also show that HTE is the predominant morphogenetic response of Colletotrichum at wound sites. This implies the existence of a fungal sensing system to trigger appropriate morphogenetic responses during pathogenesis at wound sites and on intact leaf tissue.

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“…The amplified product was digested with XbaI and BamHI and introduced into the XbaIBamHI sites of pCB3ATG26 to produce pGDATG26. To express RFP under the Aureobasidium pullulans TEF promoter (Vanden Wymelenberg et al, 1997), the TEF promoter was amplified from pTEFEGFP using the primers TEFNS1 and TEFXAS1 and introduced into pBAT, resulting in pBATTEFP (see Supplemental Table 3 online). The entire ORF of mRFP1 was amplified with the primers MRFPXKZ and MRFPSTOPB (Campbell et al, 2002;Asakura et al, 2006) and introduced into pBATTEFP, resulting in pBATTEFPMR.…”

Section: Plasmid Constructsmentioning

confidence: 99%

Atg26-Mediated Pexophagy Is Required for Host Invasion by the Plant Pathogenic Fungus Colletotrichum orbiculare

et al. 2009

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The number of peroxisomes in a cell can change rapidly in response to changing environmental and physiological conditions. Pexophagy, a type of selective autophagy, is involved in peroxisome degradation, but its physiological role remains to be clarified. Here, we report that cells of the cucumber anthracnose fungus Colletotrichum orbiculare undergo peroxisome degradation as they infect host plants. We performed a random insertional mutagenesis screen to identify genes involved in cucumber pathogenesis by C. orbiculare. In this screen, we isolated a homolog of Pichia pastoris ATG26, which encodes a sterol glucosyltransferase that enhances pexophagy in this methylotrophic yeast. The C. orbiculare atg26 mutant developed appressoria but exhibited a specific defect in the subsequent host invasion step, implying a relationship between pexophagy and fungal phytopathogenicity. Consistent with this, its peroxisomes are degraded inside vacuoles, accompanied by the formation of autophagosomes during infection-related morphogenesis. The autophagic degradation of peroxisomes was significantly delayed in the appressoria of the atg26 mutant. Functional domain analysis of Atg26 suggested that both the phosphoinositide binding domain and the catalytic domain are required for pexophagy and pathogenicity. In contrast with the atg26 mutant, which is able to form appressoria, the atg8 mutant, which is defective in the entire autophagic pathway, cannot form normal appressoria in the earlier steps of morphogenesis. These results indicate a specific function for Atg26-enhanced pexophagy during host invasion by C. orbiculare.

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Expression of Green Fluorescent Protein in Aureobasidium pullulans and Quantification of the Fungus on Leaf Surfaces

Cited by 109 publications

References 3 publications

A p67Phox-Like Regulator Is Recruited to Control Hyphal Branching in a Fungal–Grass Mutualistic Symbiosis

A p67Phox-Like Regulator Is Recruited to Control Hyphal Branching in a Fungal–Grass Mutualistic Symbiosis

Entry Mode–Dependent Function of an Indole Glucosinolate Pathway in Arabidopsis for Nonhost Resistance against Anthracnose Pathogens

Atg26-Mediated Pexophagy Is Required for Host Invasion by the Plant Pathogenic Fungus Colletotrichum orbiculare

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