Enzymes from Fungal and Plant Origin Required for Chemical Diversification of Insecticidal Loline Alkaloids in Grass-Epichloë Symbiota
Figure 3
Replacement of lolN and lolM with hph maker gene.
(A) Schematic representation of lolN-lolM replacement by the hph marker gene via homologous recombination. Shown are maps of the wild-type lolN and lolM in Epichloë festucae E2368 (WT), targeting vector (pKAES323), and the locus after homologous recombination (KO). Black bars represent DNA sequence, and filled arrows represent genes. Bent blue lines on the bars represent HindIII digestion sites. Colored arrowheads represent primers used to generate pKAES323 and to screen the transformants. (B) Southern-blot analysis of E. festucae strains. Wild-type E2368 and transformants were probed with a lolN fragment or lolM gene amplified from E2368 (old probe was stripped off the membrane before new hybridization). Lanes contained HindIII-digested genomic DNA from E2368 (WT), lolN-lolM knockout transformant (KO), ectopic transformant of E2368 with pKAES323 (Ect), and E2368 transformed with the empty vector pKAES173 (WT+vec).