Efficient insertional mutagenesis system for the dimorphic pathogenic fungus Sporothrix schenckii using Agrobacterium tumefaciens

ABSTRACT The function of calcium as a signaling molecule is conserved in eukaryotes from fungi to humans. Previous studies have identified the calcium-activated phosphatase calcineurin as a critical factor in governing growth of the human pathogenic fungus Cryptococcus neoformans at mammalian body temperature. Here, we employed insertional mutagenesis to identify new genes required for growth at 37°C. One insertion mutant, cam1-ts, that displayed a growth defect at 37°C and hypersensitivity to the calcineurin inhibitor FK506 at 25°C was isolated. Both phenotypes were linked to the dominant marker in genetic crosses, and molecular analysis revealed that the insertion occurred in the 3′ untranslated region of the gene encoding the calcineurin activator calmodulin (CAM1) and impairs growth at 37°C by significantly reducing calmodulin mRNA abundance. The CAM1 gene was demonstrated to be essential using genetic analysis of a CAM1/cam1Δ diploid strain. In the absence of calcineurin function, the cam1-ts mutant displayed a severe morphological defect with impaired bud formation. Expression of a calmodulin-independent calcineurin mutant did not suppress the growth defect of the cam1-ts mutant at 37°C, indicating that calmodulin promotes growth at high temperature via calcineurin-dependent and -independent pathways. In addition, a Ca2+-binding-defective allele of CAM1 complemented the 37°C growth defect, FK506 hypersensitivity, and morphogenesis defect of the cam1-ts mutant. Our findings reveal that calmodulin performs Ca2+- and calcineurin-independent and -dependent roles in controlling C. neoformans morphogenesis and high-temperature growth.

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Agrobacterium tumefaciens-mediated transformation: An efficient tool for insertional mutagenesis and targeted gene disruption in Harpophora oryzae

Microbiological Research ◽

10.1016/j.micres.2015.09.008 ◽

2016 ◽

Vol 182 ◽

pp. 40-48 ◽

Cited By ~ 6

Author(s):

Ning Liu ◽

Guo-Qing Chen ◽

Guo-Ao Ning ◽

Huan-Bin Shi ◽

Chu-Long Zhang ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Gene Disruption ◽

Insertional Mutagenesis ◽

Efficient Tool ◽

Targeted Gene Disruption

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Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in the symbiotic ectomycorrhizal fungusHebeloma cylindrosporum

FEMS Microbiology Letters ◽

10.1016/s0378-1097(03)00089-2 ◽

2003 ◽

Vol 220 (1) ◽

pp. 141-148 ◽

Cited By ~ 119

Author(s):

Jean-Philippe Combier ◽

Delphine Melayah ◽

Colette Raffier ◽

Gilles Gay ◽

Roland Marmeisse

Keyword(s):

Agrobacterium Tumefaciens ◽

Insertional Mutagenesis

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Identification of ENA1 as a Virulence Gene of the Human Pathogenic Fungus Cryptococcus neoformans through Signature-Tagged Insertional Mutagenesis

Eukaryotic Cell ◽

10.1128/ec.00375-08 ◽

2009 ◽

Vol 8 (3) ◽

pp. 315-326 ◽

Cited By ~ 63

Author(s):

Alexander Idnurm ◽

Felicia J. Walton ◽

Anna Floyd ◽

Jennifer L. Reedy ◽

Joseph Heitman

Keyword(s):

Cryptococcus Neoformans ◽

Fungal Pathogens ◽

Insertional Mutagenesis ◽

Pathogenic Fungus ◽

Mammalian Host ◽

New Genes ◽

Strain Collection ◽

Mutant Strains ◽

Human Pathogenic Fungus

ABSTRACT A library of more than 4,500 signature-tagged insertion mutants of the human pathogenic fungus Cryptococcus neoformans was generated, and a subset was screened in a murine inhalation model to identify genes required for virulence. New genes that regulate aspects of C. neoformans virulence were also identified by screening the entire library for in vitro phenotypes related to the ability to cause disease, including melanin production, growth at high temperature, and growth under conditions of nutrient limitation. A screen of 10% of the strain collection in mice identified an avirulent mutant strain with an insertion in the ENA1 gene, which is predicted to encode a fungus-specific sodium or potassium P-type ATPase. The results of the deletion of the gene and complementation experiments confirmed its key role in mammalian virulence. ena1 mutant strains exhibited no change in sensitivity to high salt concentrations but were sensitive to alkaline pH conditions, providing evidence that the fungus may have to survive at elevated pH during infection of the mammalian host. The mutation of the well-characterized virulence factor calcineurin (CNA1) also rendered C. neoformans strains sensitive to elevated pH. ENA1 transcripts in wild-type and cna1 mutant strains were upregulated in response to high pH, and cna1 ena1 double mutant strains exhibited increased sensitivity to elevated pH, indicating that at least two pathways in the fungus mediate survival under alkaline conditions. Signature-tagged mutagenesis is an effective strategy for the discovery of new virulence genes in fungal pathogens of animals.

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Colonization of Fusarium oxysporum transformed with the red fluorescence protein gene (tdTomato) mediated by Agrobacterium tumefaciens in roots of two avocado cultivars

Research Society and Development ◽

10.33448/rsd-v10i2.12554 ◽

2021 ◽

Vol 10 (2) ◽

pp. e22010212554

Author(s):

Grace Estefanía Uribe Hurtado ◽

William Fernando Viera Arroyo ◽

Álvaro Xavier Sampedro Lema ◽

Kang Jin Cho ◽

Alicia Beatriz Villavicencio Pazos ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Fusarium Oxysporum ◽

Protein Gene ◽

Phytophthora Cinnamomi ◽

Pathogenic Fungus ◽

Persea Americana ◽

Soil Pathogens ◽

Fluorescence Protein ◽

Red Fluorescence Protein ◽

Low Incidence

Avocado (Persea americana) is a fruit crop of economic importance in Ecuador. Currently, a low incidence of Phytophthora cinnamomi has been reported, however, there are other soil pathogens that can affect this crop, even at the initial stages of plant multiplication (nursery), for this reason the use of rootstocks that tolerate these biotic adversities is recommended. In this research, the fungus Fusarium oxysporum was isolated from roots of nursery seedlings with symptoms of necrosis. In addition, an isolate of this pathogenic fungus modified with a strain of Agrobacterium tumefaciens was used to determine the infection of F. oxysporum in the roots of the Fuerte (commercial) and Criollo (local) cultivars. The results allowed to infer that the cultivar Criollo presented a greater tolerance to F. oxysporum than the cultivar Fuerte, which corroborates its use as a rootstock for commercial avocado varieties. Furthermore, to our knowledge, this is the first report of F. oxysporum affecting avocado nursery seedlings in Ecuador.

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