Transformation of Rhizoctonia solani
Wu, Jiang (2003) Transformation of Rhizoctonia solani. PhD thesis, Murdoch University.
The aim of this study was to develop a genetic transformation system for the plant pathogenic fungus Rhizoctonia solani (teliomorph, Thanatephprus cucumeris Frank [Donk]). The availability of a transformation system would allow us to study gene exchange, epidemiology, and to use techniques such as gene disruption or gene silencing to investigate the role of fungal enzymes in pathogenesis.
The approach adopted was to use Agrobacterium tumefaciens to transform the fungus as reports in the literature suggested that this was the most efficient and easiest method to use. As a preliminary test, Fusarium oxysporum was transformed using a binary vector (pBINAN) containing a hygromycin resistance gene under control of an ascomycete promoter and terminator. Hygromycin resistant transformants were obtained after co-incubation of fungal conidia with the bacterium. The presence of the transgene was confirmed by analysis of DNA. The number of transformants depended on the genetic background of the A tumefaciens. Strains AGLO or AGRO gave higher numbers of transformants compared to LBA4404. No transformants were obtained when the hygromycin gene was under control of a basidiomycete (pBINHL1), or a plant (CaMV35S) promoter. Since the basidiomycete promoter used in pBINHL1 originates from Ustilago maydis, the vector was tested by transformation of Ustilago cynodontis. Stable transformants of U cynodontis were obtained with this vector.
A series of experiments were carried out on transformation of R. solani mycelium. Both the protoplast and the Agrobacterium transformation methods were tested. Parameters affecting protoplast production and regeneration were examined. Protoplast production varied with the age of the mycelium, with the osmotic stabilizer used, and with time of treatment with protoplasting enzymes. Regeneration of protoplasts was also affected by the osmotic stabilizer and the growth medium. Transformation of several isolates from different anastomosis groups (AG) was attempted by inducing protoplasts to take up DNA using polyethyleneglycol. Two plasmids were used; (1) pAN7-1 containing the resistance gene under control of an ascomycete promoter, and (2) pHL-1 in which the resistance gene is under control of a basidiomycete promoter. No transformants were obtained.
Attempts were then made to transform mycelium and protoplasts using A tumefaciens. The experiments used both mycelium and protoplasts as the recipient. A number of small resistant colonies were obtained using binary plasmid (pBINHL1) in which mycelium was transformed with the resistance gene was driven by the basidiomycete promoter. On transfer to fresh medium these colonies would grow to about 2cm diameter, and then stop growing. On a second transfer to fresh medium they failed to show any growth. No resistant colonies were obtained from A. tumefaciens transformation of protoplasts.
To improve transformation efficiency, a vector was constructed in which the hygromycin resistance gene was fused to an R solani laccase promoter sequence. No resistant colonies were obtained using this vector. Further experiments were carried out using a hygromycin resistance gene specially modified for expression in basidiomycetes by the insertion of artificial introns in the 5' and 3' untranslated regions, and a number of AT to CG conversions in the coding region. Most of the recipient isolates gave transformants with the unstable resistance phenotype. However, one AG 6 isolate gave transformants with a stable resistance phenotype. Of six transformants recovered from this isolate, five were shown by PCR and southern blotting to contain the transgene. In four of these transformants the resistance phenotype was stable in the absence of selection.
|Publication Type:||Thesis (PhD)|
|Murdoch Affiliation:||School of Biological Sciences and Biotechnology|
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