Restriction enzyme improves the efficiency of genetic transformations in Moniliophthora perniciosa, the causal agent of witches’ broom disease in Theobroma cacao

Lopes, Francis Julio Fagundes; Queiroz, Marisa Vieira de; Lima, Juliana Oliveira; Silva, Viviane Aline Oliveira; Araújo, Elza Fernandes de

doi:10.1590/s1516-89132008000100004

Cited by 7 publications

(7 citation statements)

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“…This promoter, besides A. bisporus and L. bicolor, has been shown to be functional at least in Suillus bovinus, Hebeloma cylindsrosporum, Coprinus cinereus, Hypholoma sublateritium and Moniliophthora perniciosa. [6][7][8][9][10] Since the tryptophan synthetase terminator of Aspergillus nidulans (TtrpC) is present in the cloning cassette of pSILBAγ, it is suited not only for ihpRNA triggering but also for transgene expression studies in basidiomycetes with the possibility to incorporate an intronic sequence before or after the gene coding region of interest ( Fig. 3C).…”

Section: E Ctomycorrhiza (Ecm) Is a Mutualistic Association Between Fmentioning

confidence: 99%

Gene knockdown by ihpRNA-triggering in the ectomycorrhizal basidiomycete fungusLaccaria bicolor

Kemppainen

Pardo

2010

Bioengineered Bugs

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Ectomycorrhiza (ECM) is a mutualistic association between fungi and the roots of the vast majority of trees. These include numerous ecologically and economically relevant species and the participating fungal symbionts are predominantly filamentous basidiomycetes. In natural ecosystems the plant nutrient uptake from soil takes place via the extraradical mycelia of these ECM mycosimbionts as a trade for plant photosyntates. The symbiotic phase in the life cycle of ECM basidiomycetes is the dikaryotic hyphae. Therefore, studies on symbiotic relevant gene functions require the inactivation of both gene copies in these dikaryotic fungi. RNA silencing is a eukaryotic sequence homology-dependent degradation of target RNAs which is believed to have evolved as a protection mechanism against invading nucleic acids. In different eukaryotic organisms, including fungi, the RNA silencing pathway can be artificially triggered to target and degrade gene transcripts of interest, resulting in gene knock-down. Most importantly, RNA silencing can act at the cytosolic level affecting mRNAs originating from several gene copies and different nuclei thus offering an efficient means of altering gene expression in dikaryotic organisms. Therefore, the pHg/pSILBAγ silencing vector was constructed for efficient RNA silencing triggering in the model mycorrhizal fungus Laccaria bicolor. This cloning vector carries the Agaricus bisporus gpdII-promoter, two multiple cloning sites separated by a L. bicolor nitrate reductase intron and the Aspergillus nidulans trpC terminator. pSILBAγ allows an easy two-step PCR-cloning of hairpin sequences to be expressed in basidiomycetes. With one further cloning step into pHg, a pCAMBIA1300-based binary vector carrying a hygromycin resistance cassette, makes the pHg/pSILBAγ plasmid compatible with Agrobacterium-mediated transformation. The pHg/pSILBAγ-system results in predominantly single integrations of RNA silencing triggering T-DNAs in the fungal genome and the integration sites of the transgenes can be resolved by plasmid rescue. Besides the optimized use in L. bicolor, general consideration was taken to build a vector system with maximum compatibility with other homobasidiomycetes and different transformation techniques.

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Section: E Ctomycorrhiza (Ecm) Is a Mutualistic Association Between Fmentioning

confidence: 99%

Gene knockdown by ihpRNA-triggering in the ectomycorrhizal basidiomycete fungusLaccaria bicolor

Kemppainen

Pardo

2010

Bioengineered Bugs

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“…However, it should be applicable to any homobasidiomycete with functional RNA silencing machinery, sensitivity to hygromycin and capacity to recognize the Agaricus bisporus gpdII promoter. The latter requirement has been documented to be fulfilled at least by Suillus bovinus, Hebeloma cylindrosporum, Coprinus cinereus, Hypholoma sublateritium and Moniliophthora perniciosa Combier et al, 2003;Godio et al, 2004;Burns et al, 2005;Fagundes Lopes et al, 2008) pHg/pSILBAg silencing vector system for homobasidiomycetes 179 indicating that this promoter is widely active in homobasidiomycetes. pSILBAg also allows an easy construction of expression cassettes extending its use to transgene expression.…”

Section: Introductionmentioning

confidence: 99%

pHg/pSILBAγ vector system for efficient gene silencing in homobasidiomycetes: optimization of ihpRNA – triggering in the mycorrhizal fungus Laccaria bicolor

Kemppainen

Pardo²

2010

Microbial Biotechnology

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SummarypSILBAγ silencing vector was constructed for efficient RNA silencing triggering in the model mycorrhizal fungus Laccaria bicolor. This cloning vector carries the Agaricus bisporus gpdII promoter, two multiple cloning sites separated by a L. bicolor nitrate reductase intron and the Aspergillus nidulans trpC terminator. pSILBAγ allows an easy oriented two‐step PCR cloning of hairpin sequences to be expressed in basidiomycetes. With one further cloning step into pHg, a pCAMBIA1300‐based binary vector carrying a hygromycin resistance cassette, the pHg/pSILBAγ plasmid is used for Agrobacterium‐mediated transformation. The pHg/pSILBAγ system results in predominantly single integrations of RNA silencing triggering T‐DNAs in the fungal genome and the integration sites of the transgenes can be resolved by plasmid rescue. pSILBAγ construct and two other pSILBA plasmid variants (pSILBA and pSILBAα) were evaluated for their capacity to silence Laccaria nitrate reductase gene. While all pSILBA variants tested resulted in up to 65–76% of transformants with reduced growth on nitrate, pSILBAγ produced the highest number (65%) of strongly affected fungal strains. The strongly silenced phenotype was shown to correlate with T‐DNA integration in transcriptionally active genomic sites. pHg/pSILBAγ was shown to produce T‐DNAs with minimum CpG methylation in transgene promoter regions which assures the maximum silencing trigger production in Laccaria. Methylation of the target endogene was only slight in RNA silencing triggered with constructs carrying an intronic spacer hairpin sequence. The silencing capacity of the pHg/pSILBAγ was further tested with Laccaria inositol‐1,4,5‐triphosphate 5‐phosphatase gene. Besides its use in silencing triggering, the herein described plasmid system can also be used for transgene expression in Laccaria. pHg/pSILBAγ silencing system is optimized for L. bicolor but it should be highly useful also for other homobasidiomycetes, group of fungi currently lacking molecular tools for RNA silencing.

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“…brasiliensis (Barbosa et al, 2004), A. nidulans (Lima et al, 2003) and A. bisporus (Lopes et al, 2008). Silencing with the hpRNA and as plasmid constructs resulted in significant decrease in the relative transcription level in most of the transformants, from which the most prominent decrease was observed in the case of using the hpRNA plasmids (Fig.…”

Section: Characterization Of the Mevalonate-isoprenoid Biosynthesismentioning

confidence: 89%

“…GPD is usually considered to be efficiently and constitutively expressed; for example, in S. cerevisiaes 2 -5% of the poly (A) + RNA may constitute gpd mRNA (Ringel et al, 2013). On the basis of this feature, promoter of GPDencoding genes were found to be efficient for expression of heterologous genes in several yeasts and filamentous fungi, and have been used to construct efficient transformation systems in numerous fungal species at various taxonomic positions, e.g., Rhizomucor miehei, Paracoccidioides brasiliensis, A. nidulans and A. bisporus (Lima et al, 2003;Barbosa et al, 2004;Vastag et al, 2004;Lopes et al, 2008). The characterization of three individual genes (gpd1, gpd2, and gpd3) encoding GPD together with the use of the promoter of the gpd1 gene for recombinant protein production in M. circinelloides is well describe by Wolff and Arnau (Wolff and Arnau 2002).…”

Section: Genetic Modification Of Mucormycotina Fungimentioning

confidence: 99%

“…promoter is well characterized: it is strongly expressing and can be induced with increased glucose concentration (Wolf and Arnau, 2002), moreover it has been used successfully in several studies (Hirano et al, 2000;Lima et al, 2003;Lopes et al, 2008;Csernetics et al, 2011, 2015, Papp et al, 2013.…”

Section: Effect Of Cultivation Time On the Transcription Of M Circinmentioning

confidence: 99%

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Functional characterization of the mevalonate-isoprenoid biosynthesis pathway genes in Mucor circinelloides

Kumar¹

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Alvarez et al., 2009). In addition, species of Rhizomucor, Apophysomyces, Saksenaea, Cunninghamella, Cokeromyces, and Syncephalastrum have also been described as caustaive agents of mucormycosis, but less cases have been reported (Gomes et al., 2011). Five major forms of mucormycosis occur, including rhino-orbito-cerebral, pulmonary, disseminated, cutaneous, and gastrointestinal, while species of Entomophthoromycotina generally cause locally manifested-cutaneous and subcutaneousand slowly progressive infections (Prabhu and Patel, 2004; Pfaller and Diekema, 2005). An increase in the number of mucormycosis has been observed in the last few decades, mainly because of the modern surgical interventions, immunosuppressive therapies and irresponsible use of antimicrobial agents (Ribes et al., 2000; Chayakulkeeree, et al., 2006; Ibrahim et al., 2012). According to Centers for Disease Control and Prevention USA (CDC, 2015) the overall mortality rate of mucormycosis found to be 54%. It was 46% among people with sinus infections, 76% for pulmonary infections, and 96% for disseminated mucormycosis (CDC, 2015). The most vulnerable group of these infections are immunocompromised patients, the major risk factors include neutropenia, cancer, corticosteroid treatment, diabetes mellitus in ketoacidosis, deferoxamine treatment, organ transplantation, and burn injury (Sugar, 2000; Sugar and Liu, 2000; Prabhu and Patel, 2004; Ibrahim et al., 2008). Phagocytes-primarily macrophages and neutrophils-have the most important role in host immune defence against fungi causing mucormycosis, thus longer neutropenia is one of the major risk for developing infection (Ibrahim et al, 2012; Morace and Borghi, 2012). Pulmonary alveolar macrophages play important role in defence against these fungi, because inhalation of the sporangiospores is one of the most common way of infection (van de Veerdonk et al., 2010). 3.3. Antifungal agents applied in clinics to treat fungal infections Only few antimycotic drugs were reported which are able to kill the Mucoromycotina fungi, whereas others merely only inhibit the growth of pathogens. The proper antifungal therapy and selection of drug should be based on several criteria, such as immune capability of the host, site of infection, characteristics of the infection (the fungal species and its susceptibility to different antifungal drugs), and pharmacokinetic characteristics of the antifungal drug (e.g., absorption, elimination, and toxicity) (Lepak et al., 2015). Mucorales are mostly resistant against widely used antifungal drugs used in clinics (such as against different azoles and echinocandins), thus generally, antifungal therapy need to be combined with surgical debridement of the necrotic regions.

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Restriction enzyme improves the efficiency of genetic transformations in Moniliophthora perniciosa, the causal agent of witches’ broom disease in Theobroma cacao

Abstract: The presence of restriction enzymes in the transformation mixture improved the efficiency of transformation in

Cited by 7 publications

References 31 publications

Gene knockdown by ihpRNA-triggering in the ectomycorrhizal basidiomycete fungusLaccaria bicolor

Gene knockdown by ihpRNA-triggering in the ectomycorrhizal basidiomycete fungusLaccaria bicolor

pHg/pSILBAγ vector system for efficient gene silencing in homobasidiomycetes: optimization of ihpRNA – triggering in the mycorrhizal fungus Laccaria bicolor

Functional characterization of the mevalonate-isoprenoid biosynthesis pathway genes in Mucor circinelloides

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