Factors affecting the production and regeneration of protoplasts from Colletotrichum lindemuthianum

Ishikawa, Francine Hiromi; Barcelos, Quélen de Lima; Souza, Elaine Aparecida de; Dias, Eustáquio Souza

doi:10.1590/s1413-70542010000100009

Cited by 6 publications

(3 citation statements)

References 13 publications

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“…Conidia were harvested from 10–15 days-old cultures of the C. lindemuthianum strains ( Table 1 ), which were washed twice in water, and used to inoculate 100 ml M 3 S medium [43] . Protoplasts were obtained [47] and transformation was carried out as previously described [44] . Briefly, protoplasts in STC (1.2 M sorbitol, 10 mM Tris-HCl and 50 mM CaCl 2 , pH 7.5) at a concentration of 10 7 protoplasts in 100 µl were combined with 3–5 µg of plasmid DNA (either pMF357 or pGR02) in a 50-ml polypropylene centrifuge tube.…”

Section: Methodsmentioning

confidence: 99%

Heterokaryon Incompatibility Is Suppressed Following Conidial Anastomosis Tube Fusion in a Fungal Plant Pathogen

et al. 2012

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It has been hypothesized that horizontal gene/chromosome transfer and parasexual recombination following hyphal fusion between different strains may contribute to the emergence of wide genetic variability in plant pathogenic and other fungi. However, the significance of vegetative (heterokaryon) incompatibility responses, which commonly result in cell death, in preventing these processes is not known. In this study, we have assessed this issue following different types of hyphal fusion during colony initiation and in the mature colony. We used vegetatively compatible and incompatible strains of the common bean pathogen Colletotrichum lindemuthianum in which nuclei were labelled with either a green or red fluorescent protein in order to microscopically monitor the fates of nuclei and heterokaryotic cells following hyphal fusion. As opposed to fusion of hyphae in mature colonies that resulted in cell death within 3 h, fusions by conidial anastomosis tubes (CAT) between two incompatible strains during colony initiation did not induce the vegetative incompatibility response. Instead, fused conidia and germlings survived and formed heterokaryotic colonies that in turn produced uninucleate conidia that germinated to form colonies with phenotypic features different to those of either parental strain. Our results demonstrate that the vegetative incompatibility response is suppressed during colony initiation in C. lindemuthianum. Thus, CAT fusion may allow asexual fungi to increase their genetic diversity, and to acquire new pathogenic traits.

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Section: Methodsmentioning

confidence: 99%

Heterokaryon Incompatibility Is Suppressed Following Conidial Anastomosis Tube Fusion in a Fungal Plant Pathogen

et al. 2012

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“…Stable protoplasts in sufficient quantity for transformation were obtained, observing that there are several influencing factors in obtaining it, for example, the enzymatic preparation, the osmotic stabilizer, the age of mycelium, and the microorganism to be used (Almeida et al, 2008;Ishikawa et al, 2010). Even for S. macrospora, which was the first attempt to produce protoplasts, the quantities were favorable, ranging between 30-45 x 10 5 protoplasts/mL, and to S. maydis, of 40-45 x 10 5 protoplast/mL.…”

Section: Resultsmentioning

confidence: 99%

Colonization of maize seeds by two species of Stenocarpella transformed with fluorescent proteins and assessed through scanning electron microscopy

Siqueira¹,

Machado²,

Corrêa³

et al. 2014

J. Seed Sci.

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-Stenocarpella maydis and Stenocarpella macrospora species causing leaf spots and stem and ear rots, can be transported and disseminated between cultivating areas through seeds. The objective was to transform isolates of species of Stenocarpella with GFP and DsRed and to correlate different inoculum potentials with the effect caused by the presence of these pathogens in the tissues of maize seeds. The isolates were transformed with introduction of the genes in their nuclei, employing the technique of protoplast transformation. Seeds were inoculated by osmotic conditioning method with transformed and not transformed isolates, with different periods of exposition of seeds to those isolates, characterizing the inoculum potentials, P1 (24 h), P2 (48 h), P3 (72 h) and P4 (96 h). The seeds inoculated with isolates expressing GFP and DsRed in both species elucidated by means of the intensities of the emitted fluorescence, the ability of those organisms to cause infection and colonization in different inoculum potentials. The potentials P3 and P4 caused the highest levels of emitted fluorescence for the colonization by both pathogens. A comprehensive and abundant mycelial growth in the colonized seed structures were well visualized at potential P3 and P4 by means of SEM.Index terms: seed pathology, genetic transformation, GFP, DsRed protein, fungus.Colonização de sementes de milho por duas espécies de Stenocarpella transformados com proteínas fluorescentes e avaliadas por microscopia eletrônica de varredura RESUMO -Stenocarpella maydis e Stenocarpella macrospora, espécies causadoras de manchas foliares, podridões em plantas e grãos ardidos de milho, podem ser transportadas e dispersas para áreas produtoras através das sementes. O objetivo foi transformar isolados de espécies de Stenocarpella com GFP e DsRed e correlacionar diferentes potenciais de inóculo com o efeito causado pela presença desses patógenos nos tecidos de sementes de milho. Os isolados foram transformados por meio da introdução dos genes nos seus núcleos, empregando a técnica de transformação de protoplastos. Sementes foram inoculadas pelo método de condicionamento osmótico com isolados transformados e não transformados, com diferentes tempos de exposição das sementes a estes isolados, caracterizando os potenciais de inóculo, P1 (24 h), P2 (48 h), P3 (72 h) e P4 (96 h). As sementes inoculadas, com isolados transformados expressando GFP e DsRed, de ambas as espécies, elucidaram por meio das intensidades das fluorescências emitidas, a capacidade desses organismos de causarem infecção e colonização em diferentes potenciais de inóculo, tendo P3 e P4, provocado as maiores intensidades de fluorescência pelas colonizações dos patógenos. Um abrangente e abundante crescimento micelial nas estruturas colonizadas das sementes foi observado nos potenciais P3 e P4, por meio da MEV.Termos para indexação: patologia de sementes, transformação genética, proteína GFP, proteína DsRed, fungo.1

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“…Lophiotrema sp. F6932 is a slow-growing and heavly-melanized fungus; two characteristics that have been shown to contribute significantly to recalcitrant to protoplast generation and amenability to genetic manipulation in filamentous fungi ( Ishikawa et al, 2010 ; Voigt et al, 2020 ). No protoplast isolation protocol exists in literature for fungi belonging to Lophiotrema genus or even the closely related phylogenetic group Lophiostoma.…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9 RNP-assisted validation of palmarumycin biosynthetic gene cluster in Lophiotrema sp. F6932

Gakuubi

Ching²,

Munusamy³

et al. 2022

Front. Microbiol.

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Lophiotrema is a genus of ascomycetous fungi within the family Lophiotremataceae. Members of this genus have been isolated as endophytes from a wide range of host plants and also from plant debris within terrestrial and marine habitats, where they are thought to function as saprobes. Lophiotrema sp. F6932 was isolated from white mangrove (Avicennia officinalis) in Pulau Ubin Island, Singapore. Crude extracts from the fungus exhibited strong antibacterial activity, and bioassay-guided isolation and structure elucidation of bioactive constituents led to the isolation of palmarumycin C8 and a new analog palmarumycin CP30. Whole-genome sequencing analysis resulted in the identification of a putative type 1 iterative PKS (iPKS) predicated to be involved in the biosynthesis of palmarumycins. To verify the involvement of palmarumycin (PAL) gene cluster in the biosynthesis of these compounds, we employed ribonucleoprotein (RNP)-mediated CRISPR-Cas9 to induce targeted deletion of the ketosynthase (KS) domain in PAL. Double-strand breaks (DSBs) upstream and downstream of the KS domain was followed by homology-directed repair (HDR) with a hygromycin resistance cassette flanked by a 50 bp of homology on both sides of the DSBs. The resultant deletion mutants displayed completely different phenotypes compared to the wild-type strain, as they had different colony morphology and were no longer able to produce palmarumycins or melanin. This study, therefore, confirms the involvement of PAL in the biosynthesis of palmarumycins, and paves the way for implementing a similar approach in the characterization of other gene clusters of interest in this largely understudied fungal strain.

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Factors affecting the production and regeneration of protoplasts from Colletotrichum lindemuthianum

Cited by 6 publications

References 13 publications

Heterokaryon Incompatibility Is Suppressed Following Conidial Anastomosis Tube Fusion in a Fungal Plant Pathogen

Heterokaryon Incompatibility Is Suppressed Following Conidial Anastomosis Tube Fusion in a Fungal Plant Pathogen

Colonization of maize seeds by two species of Stenocarpella transformed with fluorescent proteins and assessed through scanning electron microscopy

CRISPR/Cas9 RNP-assisted validation of palmarumycin biosynthetic gene cluster in Lophiotrema sp. F6932

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