Transformation of Rhizoctonia solani

摘要: The aim of this study was to develop a genetic transformation system for the plant pathogenic fungus Rhizoctonia solani (teliomorph, Thanatephprus cucumeris Frank [Donk]). availability would allow us gene exchange, epidemiology, and use techniques such as disruption or silencing investigate role fungal enzymes in pathogenesis. The approach adopted Agrobacterium tumefaciens transform reports literature suggested that most efficient easiest method use. As preliminary test, Fusarium oxysporum transformed using binary vector (pBINAN) containing hygromycin resistance under control an ascomycete promoter terminator. Hygromycin resistant transformants were obtained after co-incubation conidia with bacterium. presence transgene confirmed by analysis DNA. number depended on background A tumefaciens. Strains AGLO AGRO gave higher numbers compared LBA4404. No when basidiomycete (pBINHL1), (CaMV35S) promoter. Since used pBINHL1 originates from Ustilago maydis, tested cynodontis. Stable U cynodontis vector. A series experiments carried out R. mycelium. Both protoplast methods tested. Parameters affecting production regeneration examined. Protoplast varied age mycelium, osmotic stabilizer used, time treatment protoplasting enzymes. Regeneration protoplasts also affected growth medium. Transformation several isolates different anastomosis groups (AG) attempted inducing take up DNA polyethyleneglycol. Two plasmids used; (1) pAN7-1 promoter, (2) pHL-1 which is obtained. Attempts then made mycelium both recipient. small colonies plasmid (pBINHL1) driven On transfer fresh medium these grow about 2cm diameter, stop growing. second they failed show any growth. A. protoplasts. To improve efficiency, constructed fused R laccase sequence. vector. Further specially modified expression basidiomycetes insertion artificial introns 5' 3' untranslated regions, AT CG conversions coding region. Most recipient unstable phenotype. However, one AG 6 isolate stable Of six recovered isolate, five shown PCR southern blotting contain transgene. In four phenotype absence selection.