Agrobacterium tumefaciens-mediated transformation of Lasiodiplodia theobromae, the causal agent of gummosis in cashew nut plants

Muniz, Celli Rodrigues; Silva, G.F. da; Júnior, Manoel Teixeira Souza; Freire, F. C. O.; Kema, G.H.J.; Guedes, Maria Izabel Florindo

doi:10.4238/2014.february.21.8

Cited by 5 publications

(7 citation statements)

References 18 publications

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“…We employed an established method, Agrobacterium tumefaciens -mediated transformation, to extend barcoded insertion library techniques ( Wetmore et al, 2015 ) into a non-model basidiomycetous fungus. The efficiency of A. tumefaciens transformation in diverse fungal species ( Michielse et al, 2005 ; Martínez-Cruz et al, 2017 ; Wu et al, 2016 ; Zhang et al, 2015 ; Liu et al, 2013 ; Zhang et al, 2014 ; Li et al, 2013 ; Han et al, 2012 ; Muniz et al, 2014 ; Rodrigues et al, 2013 ; Celis et al, 2017 ) will enable use of RB-TDNAseq in many fungal species with limited genetic tools. We used RB-TDNAseq to simultaneously track mutants in over 6,500 genes for altered lipid catabolism and neutral lipid accumulation using a simple, scalable BarSeq protocol.…”

Section: Discussionmentioning

confidence: 99%

Functional genomics of lipid metabolism in the oleaginous yeast Rhodosporidium toruloides

Coradetti

Pinel

Geiselman

et al. 2018

eLife

115

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The basidiomycete yeast Rhodosporidium toruloides (also known as Rhodotorula toruloides) accumulates high concentrations of lipids and carotenoids from diverse carbon sources. It has great potential as a model for the cellular biology of lipid droplets and for sustainable chemical production. We developed a method for high-throughput genetics (RB-TDNAseq), using sequence-barcoded Agrobacterium tumefaciens T-DNA insertions. We identified 1,337 putative essential genes with low T-DNA insertion rates. We functionally profiled genes required for fatty acid catabolism and lipid accumulation, validating results with 35 targeted deletion strains. We identified a high-confidence set of 150 genes affecting lipid accumulation, including genes with predicted function in signaling cascades, gene expression, protein modification and vesicular trafficking, autophagy, amino acid synthesis and tRNA modification, and genes of unknown function. These results greatly advance our understanding of lipid metabolism in this oleaginous species and demonstrate a general approach for barcoded mutagenesis that should enable functional genomics in diverse fungi.

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Section: Discussionmentioning

confidence: 99%

Functional genomics of lipid metabolism in the oleaginous yeast Rhodosporidium toruloides

Coradetti

Pinel

Geiselman

et al. 2018

eLife

115

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“…Griffon & Maubl. is a pathogen with a worldwide distribution in tropical and subtropical regions, with low host specificity, causing many diseases in several species of plants (Lima et al, 2013;Muniz et al, 2014). According to the Systematic Mycology and Microbiology Laboratory Fungus-Host Distributions Database, there are around 638 literature records distributed in 71 locations around the world and 360 hosts (Farr and Rossman, 2015) associated with this pathogen.…”

Section: Introductionmentioning

confidence: 99%

Phylogeography and population structure analysis reveals diversity by mutations in Lasiodiplodia theobromae with distinct sources of selection

Santos¹,

Carvalho²,

Aguiar³

et al. 2017

Genet. Mol. Res.

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ABSTRACT. Lasiodiplodia theobromae is a plant pathogen with a worldwide distribution, with low host specificity, causing stem cankers, dieback diseases, and fruit rot in several species of plants. In coconut, this pathogen is reported as the etiological agent of "coconut leaf blight" (CLB) disease, causing several losses in fruit production. The CLB is an important disease for this crop in Brazil. In our study, we used a phylogeographic approach through the molecular characterization of the translation elongation factor 1-α (TEF1-α) to elucidate the pathogen distribution in Brazil and other countries, besides, search information about diversity sources of this pathogen in coconut palm tree at Brazilian northern, northeast, and southeast. We found that L. theobromae diversity is within populations (locations), and populations that are located closest to the center of the tropical zone have more variability as Central Africa, Brazilian Southeast, and Northeast. The widespread distribution could be in part related with long-distance dispersal via global trade of plants and plant products. The entrance route of L. theobromae in Brazil probably occurred from Africa route and not occurred once. In Brazil, the diversity of this pathogen in coconut tree could be linked to two agents of selection: high host diversity (in Northeast) and distinct management measures adopted in Southeast. These different sources of selection, mainly the mutations, could be one of the reasons that we found distinct reactions to "coconut leaf blight" chemical control in these regions.

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“…Here, we first established a PEG-mediated genetic transformation system with an efficient protoplast preparation protocol for L. theobromae . Compared to the ATMT system for L. theobromae transformation, which employs pycnidiospores as transformation targets ( 21 ), the PEG-mediated genetic transformation system here directly targets fungal protoplasts. Yet, sporulation is a difficult task, and sporulation efficiency can be negatively compromised by temperature, light, and substrate regimes ( 21 ).…”

Section: Discussionmentioning

confidence: 99%

“…Compared to the ATMT system for L. theobromae transformation, which employs pycnidiospores as transformation targets ( 21 ), the PEG-mediated genetic transformation system here directly targets fungal protoplasts. Yet, sporulation is a difficult task, and sporulation efficiency can be negatively compromised by temperature, light, and substrate regimes ( 21 ). Thus, the employment of protoplasts, rather than pycnidiospores, as the biological target in transformation is more appropriate, which is an improvement in advancing the transformation efficiency.…”

Section: Discussionmentioning

confidence: 99%

“…However, among the above-reported Chinese hickory pathogens, the high-quality genome sequence data of four Botryosphaeria strains, including B. dothidea strain BDLA16-7, B. cortices strain BCTK16-35, B. fabicerciana strain BFLG18-2, and B. qingyuanensis strain BQTK16-30, have been announced ( 19 , 20 ), but only the genome sequence resource of L. theobromae has not been reported. Additionally, transformation protocols for Lasiodiplodia species have not been described in detail, and only one research showed an Agrobacterium tumefaciens -mediated transformation (ATMT) system for transferring the genes of green fluorescent protein (GFP) and hygromycin B phosphotransferase to L. theobromae ( 21 ). In consideration of the fact that ATMT systems are tedious to prepare ( 18 , 22 ), developing a more efficient transformation system for L. theobromae is important for further elucidation of its life strategies and pathogenicity mechanisms.…”

Section: Introductionmentioning

confidence: 99%

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High-quality genome assembly and genetic transformation system of Lasiodiplodia theobromae strain LTTK16-3, a fungal pathogen of Chinese hickory

Ma,

Yan,

Zhang

et al. 2024

Microbiol Spectr

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Lasiodiplodia theobromae, as one of the causative agents associated with Chinese hickory trunk cankers, has caused huge economic losses to the Chinese hickory industry. Although the biological characteristics of this pathogen and the occurrence pattern of this disease have been well studied, few studies have addressed the related mechanisms due to the poor molecular and genetic study basis of this fungus. In this study, we sequenced and assembled L. theobromae strain LTTK16-3, isolated from a Chinese hickory tree (cultivar of Linan) in Linan, Zhejiang province, China. Phylogenetic analysis and comparative genomics analysis presented crucial cues in the prediction of LTTK16-3, which shared similar regulatory mechanisms of transcription, DNA replication, and DNA damage response with the other four Chinese hickory trunk canker-associated Botryosphaeria strains including, Botryosphaeria dothidea , Botryosphaeria fabicerciana , Botryosphaeria qingyuanensis, and Botryosphaeria corticis . Moreover, it contained 18 strain-specific protein clusters (not conserved in the other L. theobromae strains, AM2As and CITRA15), with potential roles in specific host-pathogen interactions during the Chinese hickory infection. Additionally, an efficient system for L. theobromae protoplast preparation and polyethylene glycol (PEG) -mediated genetic transformation was firstly established as the foundation for its future mechanisms study. Collectively, the high-quality genome data and the efficient transformation system of L. theobromae here set up the possibility of targeted molecular improvements for Chinese hickory canker control. IMPORTANCE Fungi with disparate genomic features are physiologically diverse, possessing species-specific survival strategies and environmental adaptation mechanisms. The high-quality genome data and related molecular genetic studies are the basis for revealing the mechanisms behind the physiological traits that are responsible for their environmental fitness. In this study, we sequenced and assembled the LTTK16-3 strain, the genome of Lasiodiplodia theobromae first obtained from a diseased Chinese hickory tree (cultivar of Linan) in Linan, Zhejiang province, China. Further phylogenetic analysis and comparative genomics analysis provide crucial cues in the prediction of the proteins with potential roles in specific host-pathogen interactions during the Chinese hickory infection. An efficient PEG-mediated genetic transformation system of L. theobromae was established as the foundation for the future mechanisms exploration. The above genetic information and tools set up valuable clues to study L. theobromae pathogenesis and assist in Chinese hickory canker control.

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Agrobacterium tumefaciens-mediated transformation of Lasiodiplodia theobromae, the causal agent of gummosis in cashew nut plants

Cited by 5 publications

References 18 publications

Functional genomics of lipid metabolism in the oleaginous yeast Rhodosporidium toruloides

Functional genomics of lipid metabolism in the oleaginous yeast Rhodosporidium toruloides

Phylogeography and population structure analysis reveals diversity by mutations in Lasiodiplodia theobromae with distinct sources of selection

High-quality genome assembly and genetic transformation system of Lasiodiplodia theobromae strain LTTK16-3, a fungal pathogen of Chinese hickory

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