Establishment of Agrobacterium tumefaciens – mediated genetic transformation of apple pathogen Marssonina coronaria using marker genes under the control of CaMV 35S promoter

Chauhan, Arjun; Modgil, Manju; Rajam, Manchikatla Venkat

doi:10.1016/j.micres.2021.126878

Cited by 8 publications

(4 citation statements)

References 40 publications

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“…In our previous study, 44 P. brocae transformants per 10 6 spores were obtained by optimization of spore concentration, acetosyringone concentration and co-cultivation time [38]. In addition, the type of the co-cultivation membrane may affect the AtMT efficiency of fungi [24,39,50]. For example, three different membranes (nitrocellulose, cellulose and Hybond N) were used for co-cultivation of A. fumigatus and A. tumefaciens and the best material was Hybond N, which increased the number of transformants by 2-3 times compared to nitrocellulose and cellulose [51].…”

Section: Discussionmentioning

confidence: 99%

T-DNA insertion mutagenesis in Penicillium brocae results in identification of an enolase gene mutant impaired in secretion of organic acids and phosphate solubilization

Zhang

Han²,

Shao‐Horn

et al. 2023

Microbiology

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Penicillium brocae strain P6 is a phosphate-solubilizing fungus isolated from farmland in Guangdong Province, China. To gain better insights into the phosphate solubilization mechanisms of strain P6, a T-DNA insertion population containing approximately 4500 transformants was generated by Agrobacterium tumefaciens -mediated transformation. The transformation procedure was optimized by using a Hybond N membrane for co-cultivation of A. tumefaciens and P. brocae. A mutant impaired in phosphate solubilization (named MT27) was obtained from the T-DNA insertion population. Thermal asymmetric interlaced PCR was then used to identify the nucleotide sequences flanking the T-DNA insertion site. The T-DNA in MT27 was inserted into the fourth exon of an enolase gene, which shows 90.8 % nucleotide identity with enolase mRNA from Aspergillus neoniger. Amino acid sequence homology analysis indicated that the enolase is well conserved among filamentous fungi and Saccharomyces cerevisiae. Complementation tests with the MT27 mutant confirmed that the enolase gene is involved in phosphate solubilization. Analysis of organic acids in culture supernatants indicated reduced levels of oxalic acid and lactic acid for the MT27 mutant compared to the parent strain P6 or the complementation strain. In conclusion, we suggest that the identified enolase gene of P. brocae is involved in production of specific organic acids, which, when secreted, act as phosphate solubilizing agents.

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Section: Discussionmentioning

confidence: 99%

T-DNA insertion mutagenesis in Penicillium brocae results in identification of an enolase gene mutant impaired in secretion of organic acids and phosphate solubilization

Zhang

Han²,

Shao‐Horn

et al. 2023

Microbiology

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“…Understanding the mechanisms of its pathogenesis will facilitate disease management. ATMT is an effective method for exploring pathogenicity associated genes in pathogenic fungi ( Fitzgerald et al, 2003 ; Leclerque et al, 2004 ; White and Chen, 2006 ; Zhang et al, 2013 ; Li et al, 2019 ; Villena et al, 2020 ; Binh et al, 2021 ; Casado-Del Castillo et al, 2021 ; Chauhan et al, 2021 ; Liu et al, 2021 ). Howerever, this method has not been applied to C. magnum .…”

Section: Discussionmentioning

confidence: 99%

“…However, little is known about the pathogenicity of C. magnum . Agrobacterium tumefaciens -mediated transformation (ATMT) allows the study of numerous fungi to discover candidate pathogenic genes ( Tsuji et al, 2003 ; Lawrence et al, 2010 ; Zhang et al, 2013 ; Martinez-Cruz et al, 2017 ; Li et al, 2019 ; Binh et al, 2021 ; Chauhan et al, 2021 ). Compared to conventional transformation methods, this approach provides a high percentage of T-DNA single-copy integration ( De Groot et al, 1998 ; Münch et al, 2011 ) and is suitable for random insertional mutagenesis.…”

Section: Introductionmentioning

confidence: 99%

Identifying pathogenicity-related genes in the pathogen Colletotrichum magnum causing watermelon anthracnose disease via T-DNA insertion mutagenesis

Guo

Peng

et al. 2023

Front. Microbiol.

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Fruit rot caused by Colletotrichum magnum is a crucial watermelon disease threatening the production and quality. To understand the pathogenic mechanism of C. magnum, we optimized the Agrobacterium tumefaciens-mediated transformation system (ATMT) for genetic transformation of C. magnum. The transformation efficiency of ATMT was an average of around 245 transformants per 100 million conidia. Southern blot analysis indicated that approximately 75% of the mutants contained a single copy of T-DNA. Pathogenicity test revealed that three mutants completely lost pathogenicity. The T-DNA integration sites (TISs) of three mutants were Identified. In mutant Cm699, the TISs were found in the intron region of the gene, which encoded a protein containing AP-2 complex subunit σ, and simultaneous gene deletions were observed. Two deleted genes encoded the transcription initiation protein SPT3 and a hypothetical protein, respectively. In mutant Cm854, the TISs were found in the 5′-flanking regions of a gene that was similar to the MYO5 encoding Myosin I of Pyricularia oryzae (78%). In mutant Cm1078, the T-DNA was integrated into the exon regions of two adjacent genes. One was 5′-3′ exoribonuclease 1 encoding gene while the other encoded a WD-repeat protein retinoblastoma binding protein 4, the homolog of the MSl1 of Saccharomyces cerevisiae.

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“…Agrobacterium tumefaciens is a soil-borne, Gram-negative bacterium that thrives in the rhizosphere and readily forms biofilms and thus colonizes plant surfaces [ 3 ]. A. tumefaciens exerts its pathogenicity by transferring T-DNA from its tumor-inducing plasmid to host plant cells [ 4 , 5 , 6 ]. Studies have established that various strains of the genus A. tumefaciens are responsible for the neoplastic condition of plants referred to as crown gall [ 7 ].…”

Section: Introductionmentioning

confidence: 99%

Inhibition of Agrobacterium tumefaciens Growth and Biofilm Formation by Tannic Acid

et al. 2022

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Agrobacterium tumefaciens underlies the pathogenesis of crown gall disease and is characterized by tumor-like gall formation on the stems and roots of a wide variety of economically important plant species. The bacterium initiates infection by colonizing and forming biofilms on plant surfaces, and thus, novel compounds are required to prevent its growth and biofilm formation. In this study, we investigated the ability of tannic acid, which is ubiquitously present in woody plants, to specifically inhibit the growth and biofilm formation of A. tumefaciens. Tannic acid showed antibacterial activity and significantly reduced the biofilm formation on polystyrene and on the roots of Raphanus sativus as determined by 3D bright-field and scanning electron microscopy (SEM) images. Furthermore, tannic acid dose-dependently reduced the virulence features of A. tumefaciens, which are swimming motility, exopolysaccharide production, protease production, and cell surface hydrophobicity. Transcriptional analysis of cells (Abs600 nm = 1.0) incubated with tannic acid for 24 h at 30 °C showed tannic acid most significantly downregulated the exoR gene, which is required for adhesion to surfaces. Tannic acid at 100 or 200 µg/mL limited the iron supply to A. tumefaciens and similarly reduced the biofilm formation to that performed by 0.1 mM EDTA. Notably, tannic acid did not significantly affect R. sativus germination even at 400 µg/mL. The findings of this study suggest that tannic acid has the potential to prevent growth and biofilm formation by A. tumefaciens and thus infections resulting from A. tumefaciens colonization.

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Establishment of Agrobacterium tumefaciens – mediated genetic transformation of apple pathogen Marssonina coronaria using marker genes under the control of CaMV 35S promoter

Cited by 8 publications

References 40 publications

T-DNA insertion mutagenesis in Penicillium brocae results in identification of an enolase gene mutant impaired in secretion of organic acids and phosphate solubilization

T-DNA insertion mutagenesis in Penicillium brocae results in identification of an enolase gene mutant impaired in secretion of organic acids and phosphate solubilization

Identifying pathogenicity-related genes in the pathogen Colletotrichum magnum causing watermelon anthracnose disease via T-DNA insertion mutagenesis

Inhibition of Agrobacterium tumefaciens Growth and Biofilm Formation by Tannic Acid

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