Construction of pDESTR, a GATEWAY Vector for Gene Disruption in Filamentous Fungi

Abstract

We have constructed pDESTR, a destination vector of gateway system especially for gene targeting and disruption in filamentous fungi. The vector was constructed by removing the multicloning site of pGEM-T easy vector, and inserting hygromycin phosphotransferase gene construct from pCB1004, and a gateway vector conversion cassette. In order to construct a DNA for gene disruption, only an inverse-polymerase chain reaction (PCR) amplification of the restricted, target sequence is needed. After the amplification with a 5′CACC-tagged primer and an ordinary primer, the DNA fragment will be inserted into pENTR/D-TOPO vector and then transferred into pDESTR through LR-recombination reaction. The resulting vector has the disruption construct, after being digested with the restriction enzyme used for the inverse-PCR. The effectiveness of this vector was assessed in Neurospora crassa. The use of pDESTR will therefore simplify the construction of a targeting vector, where multiple ligation steps are usually neede

Flexible gateway constructs for functional analyses of genes in plant pathogenic fungi

“…To date a number of molecular tools for genetic manipulation in fungi have been described (Abe et al, 2006;Catlett et al, 2003;Frandsen et al, 2008;García-Pedrajas et al, 2008;Geu-Flores et al, 2007;Paz et al, 2011;Shafran et al, 2008). However, development of high-throughput approaches is still one of the challenges for the functional genomics in filamentous fungi.…”

“…Among these, the Gateway Ò cloning technology has attracted molecular biologists from different disciplines due to its amenability and robustness (Schoberle et al, 2013). To date, a few methods or constructs have been developed using this technology for the functional analyses of genes in plant pathogenic fungi (Abe et al, 2006;Nakagawa et al, 2007;Paz et al, 2011;Shafran et al, 2008;Zhu et al, 2009). For instance, the One Step Construction of Agrobacterium-Recombination-ready-plasmids (OSCAR) has been developed to create deletion constructs for Agrobacterium tumefaciens mediated transformation (Paz et al, 2011).…”

Reverse Genetics for Functional Genomics of Phytopathogenic Fungi and Oomycetes

“…The Gateway cloning system exploits the precise, site- specific recombination system utilized by bacteriophage lambda in order to shuttle DNA fragments between entry and destination vectors bearing compatible recombination sites while maintaining the ORF [28]. This technology allows researchers to construct multiple destination vectors for different purposes, such as expression in Escherichia coli or S. cerevisiae , from a single entry vector with inserted gene of interest [29]. Since its inception in functional genomics analyses, GATEWAY technology has been widely used to characterize many genes.…”

A directional recombination cloning system for restriction- and ligation-free construction of GFP, DsRed, and lacZ transgenic Drosophila reporters

“…However, some researchers may wish to examine larger elements. Previous studies have demonstrated that pENTR/D-TOPO can successfully accept inserts from 3 to N5 kb in length (Gou et al, 2004;Herr et al, 2005;Abe et al, 2006; see also the vector manual at http://invitrogen.com). Similarly, Gateway-ready recombination vectors for use in other model systems have been used with very high efficiency to clone DNAs up to 5 kb in length (Hope et al, 2004;Johnson et al, 2005); more anecdotal reports suggest that inserts of up to 10 kb can be routinely cloned (www.invitrogen.com; chien.neuro.utah.edu).…”