Transformation of a filamentous fungusCryphonectria parasitica usingAgrobacterium tumefaciens

Park, Seung-Moon; Kim, Dae-Hyuk

doi:10.1007/bf02942296

Cited by 22 publications

(4 citation statements)

References 31 publications

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“…In our study, AGL1, which belonged to the succinamopine type, might be more suitable for transformation with R. toruloides NP11 than LBA4404 and GV3101 belonging to octopine and nopaline, respectively. A similar result was observed in Park and Kim [19], in which transformation efficiency was higher with AGL1 than with GV3101 and LBA4404 for Cryphonectria parasitica . Varied transformation efficiency was also observed even in the same type of Agrobacterium .…”

Section: Resultssupporting

confidence: 89%

“…Similar results were also reported in Harpophora oryzae and Magnaporthe grise , in which the transformation efficiency was increased as the ratio of A. tumefaciens to yeast [22,23]. Excessive growth of A. tumefaciens during cocultivation was reported to inhibit the recipient's growth by Park and Kim [19], and Meyer also observed that fewer transformants were detected as the A. tumefaciens input number increased from 3 × 10 8 cells to 6 × 10 8 cells [24]. This effect was probably due to the nutrient limitation in the cocultivation medium, resulting in a growth disadvantage for the transformants with fewer microbes [24].…”

Section: Resultssupporting

confidence: 78%

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Analysis of carotenoid profile changes and carotenogenic genes transcript levels inRhodosporidium toruloidesmutants from an optimizedAgrobacterium tumefaciens‐mediated transformation method

Sun

et al. 2020

Biotech and App Biochem

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Rhodosporidium toruloides has been reported as a potential biotechnological microorganism to produce carotenoids. The most commonly used molecular and genetic manipulation methods based on Agrobacterium‐mediated transformation (ATMT). However, this method was of relatively lower transformation efficiency. In this study, we optimized the ATMT method for R. toruloides on account of the promoter on T‐DNA, the ratio of A. tumefaciens to R. toruloides NP11, acetosyringone concentration, cocultivation temperature and time, and a transformation efficiency of 2,369 cells per 105 recipient cells was obtained and was 24 times as that of the previous report. With this optimized method, four redder mutants and four yellower mutants were selected out with torularhodin and β‐carotene production preference, respectively. The highest torularhodin production was 1,638.15 µg/g dry cell weight in A1‐13. The yellower mutants were found to divert the metabolic flux from torularhodin and torulene to γ‐carotene and β‐carotene, and the proportion of γ‐carotene and β‐carotene were all over 92%. TAIL‐PCR was carried out to found T‐DNA insertion in these mutants, and insertion hotspot was found. RT‐qPCR results showed that CTA1 genes in these mutants were closely related to the synthesis of total carotenoids, especially torularhodin, and was a potenial metabolic engineering site in the future.

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Section: Resultssupporting

confidence: 89%

Section: Resultssupporting

confidence: 78%

Analysis of carotenoid profile changes and carotenogenic genes transcript levels inRhodosporidium toruloidesmutants from an optimizedAgrobacterium tumefaciens‐mediated transformation method

Sun

et al. 2020

Biotech and App Biochem

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“…The hypervirulent Agrobacterium strain we used for ATMT was not reported to have a preference for multiple insertions in other mutant screens [ 3 , 40 ]. However, it was described as particularly effective for the transformation of filamentous fungi [ 78 ]. This observation is consistent with an increased frequency of T-DNA transfer.…”

Section: Discussionmentioning

confidence: 99%

A Genetic Screen for Pathogenicity Genes in the Hemibiotrophic Fungus Colletotrichum higginsianum Identifies the Plasma Membrane Proton Pump Pma2 Required for Host Penetration

et al. 2015

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We used insertional mutagenesis by Agrobacterium tumefaciens mediated transformation (ATMT) to isolate pathogenicity mutants of Colletotrichum higginsianum. From a collection of 7200 insertion mutants we isolated 75 mutants with reduced symptoms. 19 of these were affected in host penetration, while 17 were affected in later stages of infection, like switching to necrotrophic growth. For 16 mutants the location of T-DNA insertions could be identified by PCR. A potential plasma membrane H+-ATPase Pma2 was targeted in five independent insertion mutants. We genetically inactivated the Ku80 component of the non-homologous end-joining pathway in C. higginsianum to establish an efficient gene knockout protocol. Chpma2 deletion mutants generated by homologous recombination in the ΔChku80 background form fully melanized appressoria but entirely fail to penetrate the host tissue and are non-pathogenic. The ChPMA2 gene is induced upon appressoria formation and infection of A. thaliana. Pma2 activity is not important for vegetative growth of saprophytically growing mycelium, since the mutant shows no growth penalty under these conditions. Colletotrichum higginsianum codes for a closely related gene (ChPMA1), which is highly expressed under most growth conditions. ChPMA1 is more similar to the homologous yeast genes for plasma membrane pumps. We propose that expression of a specific proton pump early during infection may be common to many appressoria forming fungal pathogens as we found ChPMA2 orthologs in several plant pathogenic fungi.

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“…Various Agrobacterium strains have been used to transform Cryphonectria parasitica. In C. parasitica, Agrobacterium AGL-1 was more efficient for transformation than LBA4404 (Park and Kim 2004). These results suggest that the difference in transformation efficiency was due to the genetic background of the A. tumefaciens strains (Krishnamohan et al 2001), the effect of inhibitor from H. mompa, and the strain specificity of A. tumefaciens for H. mompa strains.…”

Section: Optimization Of Transformation Conditionsmentioning

confidence: 91%

<i>Agrobacterium tumefaciens</i>-mediated transformation of the violet root-rot fungus, <i>Helicobasidium mompa</i>, and the effect of activated carbon

Kano¹,

Kurita²,

Kanematsu³

et al. 2011

Mycoscience

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Agrobacterium tumefaciens-mediated transformation (ATMT) has been successfully applied to the violet root-rot fungus Helicobasidium mompa, which is the causal agent of violet root-rot disease. The A. tumefaciens strains carried a binary plasmid vector containing the hygromycin B phosphotransferase gene (hph) controlled by the heterologous fungal Agaricus bisporus P-gpd (glyceraldehyde-3-phosphate dehydrogenase) promoter and the trpC terminator. The transformation system was optimized using defined cocultivation conditions. When H. mompa strain V17 was cocultivated with A. tumefaciens strain AGL-1 using 5% agar, we obtained more hygromycin-resistant colonies than with strains EHA105 or MAFF301222 using 2% agar. In addition, our results suggest that the activated carbon is necessary in ATMT to reduce background growth of H. mompa. The presence of the hph gene in transformants was detected by polymerase chain reaction (PCR), and single-copy integration of the marker gene was demonstrated by Southern blot analysis. Thus, the ATMT system can be considered a promising tool for insertional mutagenesis studies of H. mompa.

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Transformation of a filamentous fungusCryphonectria parasitica usingAgrobacterium tumefaciens

Cited by 22 publications

References 31 publications

Analysis of carotenoid profile changes and carotenogenic genes transcript levels inRhodosporidium toruloidesmutants from an optimizedAgrobacterium tumefaciens‐mediated transformation method

Analysis of carotenoid profile changes and carotenogenic genes transcript levels inRhodosporidium toruloidesmutants from an optimizedAgrobacterium tumefaciens‐mediated transformation method

A Genetic Screen for Pathogenicity Genes in the Hemibiotrophic Fungus Colletotrichum higginsianum Identifies the Plasma Membrane Proton Pump Pma2 Required for Host Penetration

<i>Agrobacterium tumefaciens</i>-mediated transformation of the violet root-rot fungus, <i>Helicobasidium mompa</i>, and the effect of activated carbon

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