EFS12- Book of abstracts - Contact

SESSION 1: FUSARIUM – GENETICS, GENOMICS AND SYSTEMS BIOLOGY
P9 - Agrobacterium-mediated insertional mutagenesis
of Fusarium oxysporum f. sp. cubense for
identification of key genes in the infection cycle of the
pathogen
A. A. Rabie 1 , T. Meyer 2 , L. J. Rose 1 , G. Mostert 1 , I. Beukes 1 , A. C. L.
Churchill 3 , A. Viljoen 1
1 University of Stellenbosch, Department of Plant Pathology, Private Bag X1, Matieland 7602, South
Africa, 2 University of Pretoria, Forestry and Agricultural Biotechnology Institute, Department of
Microbiology and Plant Pathology, Pretoria 0002, South Africa, 3 Cornell University, Department of
Plant Pathology, Ithaca, NY, USA.
E-mail: ankia@sun.ac.za
The causal agent for Fusarium wilt of bananas, Fusarium oxysporum f. sp.
cubense (Foc), is regarded as one of the most destructive pathogens in the world.
The most effective way in controlling the disease is by planting resistant banana
cultivars. Frequent studies on the infection biology of Foc have been conducted,
but the genetic basis for pathogenicity is still poorly understood. Forward and
reverse genetics have proven to be a valuable tool for identifying in vivo essential
genes involved in the molecular mechanisms of pathogenicity and virulence of a
pathogen. In this study, random vector integration was conducted through the
implementation of Agrobacterium-mediated insertional mutagenesis (ATMT).
Transformation was achieved with four A. tumefaciens strains using a vector
conferring Hygromycin B resistance and expression of the green fluorescent
protein gene. Vector insertion was confirmed with molecular methods and
fluorescent microscopy. Transformants were screened for sporulation potential,
alterations in growth rate and pigmentation, the number of T-DNA inserts and in
planta alterations in virulence and pathogenicity. Results indicated the best
transformation efficiency was obtained with A. tumefaciens strain EHA105/S. The
majority of transformants contained one or two insertions. On-going studies
suggest a significant reduced growth rate by some transformants, as well as
irregularities in pigmentation. TAIL-PCR will be carried out on mutants showing a
reduced virulence in order to isolate and identify DNA sequences flanking the T-
DNA. The identification of pathogenicity genes could lead to an improved
understanding of disease development and the development of novel
management strategies.
Key words: Fusarium oxysporum f. sp. cubense, ATMT, GFP, pathogenicity
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