| Northern com leaf blight is one of the important leaf diseases in maize, caused by Exserohilum turcicum which is an important filamentous fungus and can lead to destructive damage to corn in the epidemic years. It is necessary to understand the pathogenesis of the fungus for fungicide development and disease management. It's well-known that identifying the pathogenicity-related genes is a key to understand the mechanism of the pathogenesis in pathogenic fungi. REMI(restriction enzyme-mediated integration), has been increasingly applied to gene tagged and coloned technique. It is an efficient method to apply to tag and clone the pathogenic related genes in some filamentous fungi.In this paper, REMI transformation system of Exserohilum turcicum was established by optimized some parameters. The results showed that the release and regeneration of protoplasts were influenced by the fungal cultural time, the category of cell wall degradation enzymes, digesting time and regeneration media. According to the result, we find a more optimized protocol of the protoplast preparation of Exserohilum turcicum. The protocol is follows, (1) Washing the spores of E. turcicum, which is cultured for 15d, in the PDA medium. (2) Incubating the spores to the Fries medium by 10~5/mL at 25℃with shaked for 20h, then wash filtrate and collect the hypha. (3) Digest the hypha with 12.5mg/mL Lywallzyme, 12.5mg/mL Drislase and 12.5mg/mL Snailase at 30℃and 100rpm for 4～5h. (4) The digesting solution is filtrated by 3 filters. (5) Then, wash by 0.7M NaCl osmotic buffer for 3 times, and then the solution is centrifuged for 10min at 3000rpm and 4℃, after that throw away the upper solution then wash the rest by STC solution 2 times and gain protoplasts which has been needed to concentration. Normally we can isolate protoplasts of Exserohilum turcicum with 1.44×10~6 cell/mL in the 100mL hypha solution.Based on REMI transformation techniques, we obtained 215 REMI transformants of Exserohilum turcicum wild isolate 01-23. To examine the stablility of transformants, these transformants were randomly selected to check the hygromycin-resistance stability for 5 asexual generations on PDA containing 50μg/mL hygromycin B. The result showed that all tested transformants could grow on PDA containing hygromycin B, but the wild isolate 01-23 can not survive on it. So the result showed that these transformants were stable in genetic. We amplified the genome DNA from these mutants by PCR used the hph gene sequence as primers. These amplified fragments were the same sequence as the hph gene which suggested that the hph gene had been inserted into these genomes. We had analysed some phenotypes of these transformants, such as growth velocity and sporulation. Three transformants were reduced in sporulation compared with the wild isolate 01-23, in these transformants M25 lossed sporulation. Meanwhile, the results of pathagenicity assay showed that M25 had reduced virulence. Besides the three transformants were more in sporulation compared with the wild isolate. Five transformants grew less slowly on PDA than wild isolate 01-23. |
