Abstract
Fusarium graminearum is a destructive phytopathogen that infects major cereal crops such as wheat, maize and barley. Double or triple mutants are often very useful in the phenotypic and genetic analysis of genes that function redundantly or within similar pathways. When single gene mutants are available, double or triple mutants can be generated by crossing heterothallic strains or multiple rounds of protoplast transformation. When individual mutants carry different antibiotic resistance, it is convenient to use the sexual crossing to generate desired recombinant strains. Here, we present a protocol for generating double or triple mutants by sexual crossing in one homothallic strain with further antibiotic resistance and genomic DNA PCR screening of recombinant progenies.
Keywords: Fusarium graminearum, Sexual crossing, Double mutants, Triple mutants
Background
The ascomycete fungus Fusarium graminearum is a devastating phytopathogen that causes head blight, ear rot, stalk rot and crown in cereals. It can produce perithecia on carrot agar in vitro. This assay can be used for studying perithecia development, ascospore discharge and sexual recombination (Nicholson, 2007). F. graminearum is homothallic and has both MAT1-1 and MAT1-2-1 locus; each of these locus deletion mutants is sterile in self-crosses (Zheng et al., 2013). To generate double gene mutants, one single gene mutant has traditionally been outcrossed with MAT deletion mutants and further outcrossed with another single mutant (Bowden and Leslie, 1999; Lee et al., 2011; Son et al., 2012). Another strategy of double or triple mutants construct is deleting a gene in the other mutant strain with protoplast transformation (Oide et al., 2007 and 2014).Here, we adapt and simplify this method, and present the details of the protocol to generate double and triple mutants using sexual crosses in one homothallic strain with further antibiotic resistance screening.
Materials and Reagents
Equipment
Procedure
To generate triple mutants, we screened for three antibiotic resistance genes as selective markers: hygromycin B phosphotransferase (hph) gene conferring resistance to the antibiotic hygromycin, neomycin phosphotransferase (npt) gene for G418, and nourseothricin acetyltransferase (nat) gene for nourseothricin.
Note: There is a video presenting the method of generating recombinant perithecia of F. graminearum (https://www.jove.com/video/3895/sexual-development-and-ascospore-discharge-in-fusarium-graminearum). Our protocol was adapted from the video with modifications in fungal culture conditions, aerial mycelium press, perithecia induction conditions and ascospores discharge. In our protocol, we also improve the method of screening recombinants with antibiotics screening. The video can be a visual reference to this protocol.
Notes
Data analysis
The false positive rates in recombinant progeny screening for double mutants and triple mutants were found to be zero when we conducted the experiments (Figure 3). Therefore, this is a highly efficient method for generating double and triple mutants using the sexual crossing and antibiotic screening. Figure 3. PCR validation of all recombinant progenies in the triple mutants screening. All individual colonies were found to be positive recombinant progeny and the false positive rate was zero. The used primers were to amplify a sequence within the targeted genes.
Recipes
Acknowledgments
The plasmid of nourseothricin acetyltransferase (nat) gene was kindly provided by Dr. Ling Xu. This protocol was adapted from the previous publication (Cavinder et al., 2012). This work was supported by the Ministry of Science and Technology of China (Grant 2016YFD0100600), the Natural Science Foundation of China (Grant 31730077) and the Ministry of Agriculture of China (Grant 2016ZX08009-003). The authors declare that there is no conflict of interests.
References
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