Abstract
Aspergillus flavus often invade many important corps and
produce harmful aflatoxins both in preharvest and during
storage stages. The regulation mechanism of aflatoxin biosynthesis
in this fungus has not been well explored mainly
due to the lack of an efficient transformation method for
constructing a genome-wide gene mutant library. This challenge
was resolved in this study, where a reliable and efficient
Agrobacterium tumefaciens-mediated transformation (ATMT)
protocol for A. flavus NRRL 3357 was established. The results
showed that removal of multinucleate conidia, to collect
a homogenous sample of uninucleate conidia for use as the
transformation material, is the key step in this procedure.
A. tumefaciens strain AGL-1 harboring the ble gene for zeocin
resistance under the control of the gpdA promoter from
A. nidulans is suitable for genetic transformation of this fungus.
We successfully generated A. flavus transformants with
an efficiency of ~ 60 positive transformants per 106 conidia
using our protocol. A small-scale insertional mutant library
(~ 1,000 mutants) was constructed using this method and
the resulting several mutants lacked both production of conidia
and aflatoxin biosynthesis capacity. Southern blotting
analysis demonstrated that the majority of the transformants
contained a single T-DNA insert on the genome. To the best
of our knowledge, this is the first report of genetic transformation
of A. flavus via ATMT and our protocol provides an
effective tool for construction of genome-wide gene mutant
libraries for functional analysis of important genes in A.
flavus.
Citations
Citations to this article as recorded by

- Agrobacterium tumefaciens-mediated transformation for the genetic modification of the biotechnologically relevant fungus Aspergillus vadensis through synthetic biology
Carolina Ropero-Pérez, Paloma Manzanares, Jose F. Marcos, Sandra Garrigues
Current Research in Biotechnology.2024; 7: 100178. CrossRef - Development of Green Fluorescent Protein-Tagged Strains of Fusarium acuminatum via PEG-Mediated Genetic Transformation
Fangyi Ju, Zhongqiang Qi, Jiajin Tan, Tingting Dai
Microorganisms.2024; 12(12): 2427. CrossRef - An efficient targeted gene deletion approach for Cochliobolus heterostrophus using Agrobacterium tumefaciens-mediated transformation
Jiaying Sun, Rui Yang, Yujia Liu, Zengran Zhou, Jiaqi Jia, Hongming Huang, Shuqin Xiao, Chunsheng Xue
Journal of Microbiological Methods.2024; 216: 106863. CrossRef - Establishment of an Agrobacterium tumefaciens-Mediated Transformation System for Hirsutella sinensis
Lijuan Wu, Xinkun Hu, Shen Yan, Zenglin Wu, Xuzhong Tang, Lei Xie, Yujie Qiu, Rui Li, Ji Chen, Mengliang Tian
Current Issues in Molecular Biology.2024; 46(9): 10618. CrossRef - Role of Flavohemoglobins in the Development and Aflatoxin Biosynthesis of Aspergillus flavus
Xiaoling Zhou, Dongyue Chen, Min Yu, Yuan Jiao, Fang Tao
Journal of Fungi.2024; 10(6): 437. CrossRef - HacA, a key transcription factor for the unfolded protein response, is required for fungal development, aflatoxin biosynthesis and pathogenicity of Aspergillus flavus
Min Yu, Xiaoling Zhou, Dongyue Chen, Yuan Jiao, Guomin Han, Fang Tao
International Journal of Food Microbiology.2024; 417: 110693. CrossRef - Synthetic Biology Tools for Engineering Aspergillus oryzae
Hui Yang, Chaonan Song, Chengwei Liu, Pengchao Wang
Journal of Fungi.2024; 10(1): 34. CrossRef - Construction of Cordycepin High-Production Strain and Optimization of Culture Conditions
Hui Zhang, Ping Chen, Lin Xu, De Xu, Wendi Hu, Yong Cheng, Shengli Yang
Current Microbiology.2023;[Epub] CrossRef - Agrobacterium tumefaciens-mediated transformation of Nigrospora sp. isolated from switchgrass leaves and antagonistic toward plant pathogens
Summi Dutta, Gabriella Houdinet, Gitanjali NandaKafle, Arjun Kafle, Christine V. Hawkes, Kevin Garcia
Journal of Microbiological Methods.2023; 215: 106849. CrossRef - Systematic Characterization of bZIP Transcription Factors Required for Development and Aflatoxin Generation by High-Throughput Gene Knockout in Aspergillus flavus
Qianqian Zhao, Hao Pei, Xiaoling Zhou, Kai Zhao, Min Yu, Guomin Han, Jun Fan, Fang Tao
Journal of Fungi.2022; 8(4): 356. CrossRef - Homologous Expression and Characterization of α-L-rhamnosidase from Aspergillus niger for the Transformation of Flavonoids
Hangyu Ye, Xiaojun Li, Luyuan Li, Yinjun Zhang, Jianyong Zheng
Applied Biochemistry and Biotechnology.2022; 194(8): 3453. CrossRef - Genetic Manipulation and Transformation Methods for Aspergillus spp.
Ye-Eun Son, Hee-Soo Park
Mycobiology.2021; 49(2): 95. CrossRef - Homologous overexpression of genes in Cordyceps militaris improves the production of polysaccharides
Yifeng Wang, Xi Yang, Ping Chen, Shengli Yang, Hui Zhang
Food Research International.2021; 147: 110452. CrossRef - A Novel Site-Specific Integration System for Genetic Modification of Aspergillus flavus
Fang Tao, Kai Zhao, Qianqian Zhao, Fangzhi Xiang, Guomin Han
G3 Genes|Genomes|Genetics.2020; 10(2): 605. CrossRef - Identification of antibiotics for use in selection of the chytrid fungi Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans
Kristyn A. Robinson, Mallory Dunn, Shane P. Hussey, Lillian K. Fritz-Laylin, Louise A. Rollins-Smith
PLOS ONE.2020; 15(10): e0240480. CrossRef - Aromatic Polyketides from a Symbiotic Strain Aspergillus fumigatus D and Characterization of Their Biosynthetic Gene D8.t287
Yi Hua, Rui Pan, Xuelian Bai, Bin Wei, Jianwei Chen, Hong Wang, Huawei Zhang
Marine Drugs.2020; 18(6): 324. CrossRef - An optimized Agrobacterium tumefaciens-mediated transformation system for random insertional mutagenesis in Fonsecaea monophora
Xing Xiao, Yu Li, JingLin Qin, Ya He, Wenying Cai, Zhiwen Chen, Liyan Xi, Junmin Zhang
Journal of Microbiological Methods.2020; 170: 105838. CrossRef - Genome-wide association study leads to novel genetic insights into resistance to Aspergillus flavus in maize kernels
Guomin Han, Cuiping Li, Fangzhi Xiang, Qianqian Zhao, Yang Zhao, Ronghao Cai, Beijiu Cheng, Xuewen Wang, Fang Tao
BMC Plant Biology.2020;[Epub] CrossRef - The Efficacy of Composite Essential Oils against Aflatoxigenic Fungus Aspergillus flavus in Maize
Fangzhi Xiang, Qianqian Zhao, Kai Zhao, Hao Pei, Fang Tao
Toxins.2020; 12(9): 562. CrossRef - Ethylene and Benzaldehyde Emitted from Postharvest Tomatoes Inhibit Botrytis cinerea via Binding to G-Protein Coupled Receptors and Transmitting with cAMP-Signal Pathway of the Fungus
Yongwen Lin, Hongchun Ruan, Komivi Senyo Akutse, Baochun Lai, Yizhang Lin, Youming Hou, Fenglin Zhong
Journal of Agricultural and Food Chemistry.2019; 67(49): 13706. CrossRef