Agrobacterium tumefaciens-mediated transformation of Fusarium oxysporum f. sp. cubense for pathogenicity gene analysis

Abstract

Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most destructive plant diseases in recorded history. The disease was first discovered in Australia in 1874 but became renowned for the severe losses it caused to export banana plantations during the 1960s in Central America. The banana export industry was saved only by replacing Gros Michel bananas, the dessert banana grown for the export market, with highly resistant Cavendish banana cultivars. Despite this apparent solution, the fungus was found to attack Cavendish bananas in the sub-tropics, where plants were believed to be predisposed to the disease by the cool winter climate. Good management practices and conventional disease management strategies have not been sufficient to reduce losses and stop the disease from spreading, and today Fusarium wilt can be found in almost all banana-producing countries of the world. Since 1988, Foc has been responsible for significant losses of Cavendish bananas in tropical Asia. The only sustainable control measure, the use of resistant varieties, is not always popular as people prefer to eat locally adopted varieties that, unfortunately, are susceptible to Foc. Sustainable Fusarium wilt management in banana depends on the improvement of existing banana cultivars or the development of novel disease management strategies. Molecular biology and biotechnology provide opportunities to introduce foreign resistance genes into existing cultivars and to develop new, environmentally friendly products that can protect susceptible bananas from Foc. Better knowledge of the Fusarium wilt pathogen, its diversity, and its mechanisms of pathogenesis will contribute significantly to developing these novel approaches for control of the disease. Molecular information on the pathogenicity of Foc, however, is limited, whereas other formae speciales of F. oxysporum have been better studied. In this thesis, Agrobacterium tumefaciens-mediated transformation of (ATMT) was employed to investigate genes responsible for pathogenicity of Foc to banana. Chapter 1 provides an overview of pathogenicity in F. oxysporum. Pathogenic and non-pathogenic forms of the fungus are first introduced to the reader, and then the biology, epidemiology and etiology of pathogenic forms of F. oxysporum are discussed. The genetic make-up and ability of the Fusarium wilt fungus to cause disease in plants concludes the first part of the review. In recent years, there has been a noted increase in the number of techniques available to study hostpathogen interactions. The second part of the review concentrates on these techniques and their applications in studying pathogenicity of the Fusarium wilt pathogen. In Chapter 2, an ATMT and screening system for Foc was developed. Five A. tumefaciens strains were evaluated for their efficiency to transform Foc with a randomly integrating vector that confers hygromycin B resistance and expression of green fluorescent protein (GFP). A small insertion mutant library of Foc was created, and a subset of transformants was characterized by determining the number of T-DNA inserts present, the location and identity of predicted genes disrupted by T-DNA insertion, and whether transformants of Foc were altered in their virulence against susceptible banana plants. In Chapter 3, the role of a known pathogenicity gene, Frp1, of the tomato pathogen F. oxysporum f. sp. lycopersici (Fol) was investigated in Foc. The first objective was to isolate and characterize the Frp1 gene in Foc, and to compare it to the homologous gene in Fol. A vector containing a modified Fol Frp1 gene was then obtained and used for targeted disruption of the gene in Foc via ATMT. Mutants in which the Frp1 gene was disrupted were then analyzed for GFP expression, culture morphology, and alterations in pathogenicity to banana.