Paxilline-negative mutants of Penicillium paxilli generated by heterologous and homologous plasmid integration

Young, Carolyn A.; Itoh, Yasuo; Johnson, Richard D.; Garthwaite, Ian; Miles, Christopher O.; Finch, Sarah C.; Scott, Barry

doi:10.1007/s002940050349

Cited by 58 publications

(79 citation statements)

References 29 publications

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“…Protoplasts of E. festucae were prepared as described previously (Young et al, 1998(Young et al, , 2005. Protoplasts were transformed with 5 mg of either circular (pPN74 and pPN82) or linear PCR-amplified product (primers M13F and M13R for pPN75, pII99-2 and pII99-3 for pPN78) or cotransformed with 5 mg of circular pII99 and 10 mg of pPN83 using the method described previously (Itoh et al, 1994).…”

Section: E Festucae Transformationmentioning

confidence: 99%

Reactive Oxygen Species Play a Role in Regulating a Fungus–Perennial Ryegrass Mutualistic Interaction

et al. 2006

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Although much is known about the signals and mechanisms that lead to pathogenic interactions between plants and fungi, comparatively little is known about fungus-plant mutualistic symbioses. We describe a novel role for reactive oxygen species (ROS) in regulating the mutualistic interaction between a clavicipitaceous fungal endophyte, Epichloe¨festucae, and its grass host, Lolium perenne. In wild-type associations, E. festucae grows systemically in intercellular spaces of leaves as infrequently branched hyphae parallel to the leaf axis. A screen to identify symbiotic genes isolated a fungal mutant that altered the interaction from mutualistic to antagonistic. This mutant has a single-copy plasmid insertion in the coding region of a NADPH oxidase gene, noxA. Plants infected with the noxA mutant lose apical dominance, become severely stunted, show precocious senescence, and eventually die. The fungal biomass in these associations is increased dramatically, with hyphae showing increased vacuolation. Deletion of a second NADPH oxidase gene, noxB, had no effect on the E. festucaeperennial ryegrass symbiosis. ROS accumulation was detected cytochemically in the endophyte extracellular matrix and at the interface between the extracellular matrix and host cell walls of meristematic tissue in wild-type but not in noxA mutant associations. These results demonstrate that fungal ROS production is critical in maintaining a mutualistic fungus-plant interaction.

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Section: E Festucae Transformationmentioning

confidence: 99%

Reactive Oxygen Species Play a Role in Regulating a Fungus–Perennial Ryegrass Mutualistic Interaction

et al. 2006

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“…paxilli ATCC26601 was used as the experimental material (Young et al 1998). Cultivation and cDNA preparation were performed as described previously (Tagami et al 2013).…”

Section: Strain and Plasmidsmentioning

confidence: 99%

Functional analysis of a prenyltransferase gene (paxD) in the paxilline biosynthetic gene cluster

Liu

Noike

Minami

et al. 2013

Appl Microbiol Biotechnol

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Paxilline is an indole diterpene produced by Penicillium paxilli. Six genes (paxB, C, G, M, P, and Q) in paxilline biosynthetic gene cluster were previously shown to be responsible for paxilline biosynthesis. In this study, we have characterized paxD, which is located next to paxQ and has weak similarities to fungal dimethylallyl tryptophan synthase genes. PaxD was overexpressed in E. coli and the purified enzyme was used for in vitro analysis. When paxilline and dimethylallyl diphosphate were used as substrates, one major and one minor product, which were identified as di-prenyl paxilline and mono-prenyl paxilline by liquid chromatography-mass spectrometry analysis, were formed. The structure of the major product was determined to be 21,22-diprenylated paxilline, showing that PaxD catalyzed the successive di-prenylation. Traces of both products were detected in culture broth of P. paxilli by liquid chromatography-mass spectrometry analysis. The enzyme is likely to be a dimer and required no divalent cations. The optimum pH and temperature were 8.0 and 37°C, respectively. The Km values were calculated as 106.4 ± 5.4 µM for paxilline and 0.57 ± 0.02 µM for DMAPP with a kcat of 0.97 ± 0.01/sec.

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“…Protoplasts of E. festucae were prepared as previously described (Young et al, 1998(Young et al, , 2005. Protoplasts were transformed with 5 mg of either circular (pPN109, pPN110, and pPN114) or linear PCR product amplified from pPN108 with primer set pII99-2 and pII99-3 or cotransformed with 5 mg of circular pII99 and 10 mg of pPN83 (Tanaka et al, 2006) using a previously described method (Itoh et al, 1994).…”

Section: E Festucae Transformationmentioning

confidence: 99%

“…Filters were probed with [a-32 P]dCTP-labeled probes (3000 Ci/mmol; Amersham Biosciences). Probe labeling and purification and hybridization conditions were performed as previously described (Young et al, 1998). Standard PCR amplifications of genomic and plasmid DNA templates were performed as previously described (Tanaka et al, 2005).…”

Section: Dna Preparations Hybridizations and Pcrmentioning

confidence: 99%

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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Paxilline-negative mutants of Penicillium paxilli generated by heterologous and homologous plasmid integration

Cited by 58 publications

References 29 publications

Reactive Oxygen Species Play a Role in Regulating a Fungus–Perennial Ryegrass Mutualistic Interaction

Reactive Oxygen Species Play a Role in Regulating a Fungus–Perennial Ryegrass Mutualistic Interaction

Functional analysis of a prenyltransferase gene (paxD) in the paxilline biosynthetic gene cluster

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

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