Fusarium oxysporum f.sp vasinfectum (FOV) has merged as a very serious pathogen of cotton, which apparently lacks good sources of endogenous resistance to Fusarium wilt. In contrast, there are several well defined genes in tomato for resistance to the related pathogen Fusarium oxysporum f.sp lycopersici (FOL) and tomato has the best characterised system of plant interaction with Fusarium. The tomato I-2 gene for resistance to FOL has been cloned and found to encode a coiled-coil-nucleotide-binding site- leucine-rich repeat(CC-NBS_LRR) protein similar to proteins encoded by a number of resistance genes to various plant pathogens in various plant species.

Under previous CRDC funding, we engineered the I-2 gene to allow its expression/over expression under the control of a strong constitutive promoter (the 35S from cauliflower mosaic virus) and, in collaboration with CSIRO Plant Industry, attempted to produce 35S:I-2 transgenic cotton. However, we were unable to generate transgenic cotton lines containing 35:SI-2. We interpreted this result to indicate that over expression and/or inappropriate temporal or spatial expressions of the I-2 protein caused inappropriate activation of cotton defence mechanisms leading to cell death. Interestingly, this finding is consistent with the expression of some disease resistance genes in different plant species eg. the flax L6 resistance gene expressed in tobacco causes the constitutive activation of tobacco plant defences.

We continued to test I-2 function in cotton by Agrobacterium-mediated transient expression of the 35S:I-2 gene in cotton leaves. We found that over expression of the I-2 in cotton leaf tissues resulted in necrosis consistent with activation of cotton defences. Again, this finding is consisitent with results that would be expected following over expression of an active resistance gene. More importantly, it indicated that the I-2 was biologically active in cotton.

Given that over expression of I-2 caused inappropriate activation of cotton defence mechanisms, we explored the possibility of using the I-2 promoter in place of the 35S promoter. A glucuronidase (GUS) reporter gene was fused to the I-2 promoter and the fusion construct was expressed in hairy roots of cotton induced by Agrobacterium rhizogenes. Staining for GUS activity showed that the I-2 promoter was active and showed a similar pattern of expression in cotton to that in a tomato ie: in cells adjacent to to the vascular tissues of the root. We therefore made a DNA construct with the I-2 gene driven by its own promoter for use in cotton transformation experiments.

following the conclusion of our previous CRDC funding, we proceeded, in collaboration with the CSIRO Plant Industry, to produce transgenic cotton with the I-2 gene under the control of its own promoter. Twenty two T0 lines were produced, with 9 of these identified as independent lines. In this project, we have analysied the progeny of these primary transformers to determine how many transgenic loci are present. Many of the primary transformants carry multiple I-2 loci. All I-2 lines were screened for their resistance/tolerance towards FOV in glasshouse trials. Whilst the preliminary results from an initial glass house trial were encouraging, later FOV bioassays demostrated that the possession of the I-2 gene appeared to have no effect on resistance/tolerance in cotton against FOV.