Construction and testing of an endochitinase/ {u03B2}-1,3-glucanase fusion gene for ability to confer fusarium wilt resistance in tomato

Fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici (Fol) is one of the most devastating diseases of tomato. The most effective, practical, and environmentally friendly strategy to control the disease would be to generate resistant tomato cultivars. Such a strategy can be achieved through the introduction of resistance genes or genes that function in defence, such as chitinase and {u03B2}-1,3-glucanasee, into susceptible tomato cultivars. This study reports an attempt to generate transgenic tomato carrying endochitinase (ech42) and {u03B2}-1,3-glucanase (gluc78) genes from the biocontrol fungus Trichoderma atroviride strain PI and test their combined ability to confer resistance to Fusarium wilt. Several steps involving various technically challenging approaches were performed to try to achieve this objective. First, an endochitinase {u03B2}-1,3-glucanase fusion gene was constructed using PCR-driven overlap extension to obtain a transcrip-tional and translational fusion. A modified PCR-driven overlap extension method was developed and it proved efficient in creating the chimeric endochitinase {u03B2}-1,3-glucanase fusion gene. A red fluorescent protein (rfp) reporter gene construct was also generated using this method. Second, Fol race 3 was transformed with a green fluorescent protein (gfp) reporter gene to enable the growth of Fol race 3 to be monitored in tomato roots. Third, transformation of tomato hairy roots and whole tomato transformation were attempted with the endochitinase {u03B2}-1,3-glucanase fusion gene using the rfp gene as a reporter. Fol race 3 was transformed with gfp under the control of the constitutive gpd promoter (gpd promoter-gfp) and the inducible SIX1 (Avr3) promoter (SIX1 promoter-gfp) using Agrobacterium tumefaciens-mediated transformation. The SIX1 promoter-gfp construct would allow the expression of gfp only when Fol is growing within its tomato host [164]. Fol race 3 transformation with gpd promoter-gfp was efficient and transformants were obtained at high frequency. On the other hand, transformation of Fol race 3 with SIX1 promoter-gfp occurred at low frequency and there was little evidence for GFP fluorescence when putative transformants were used to infect tomato roots. The pathogenicity of both transformed lines was not altered towards resistant and susceptible tomato cultivars but the SIX1 promoter-gfp Fol transformants showed reduced avirulence towards the resistant tomato line. The general lack of SIX1 promoter-gfp transformants able to express gfp may be attributed to the lack of 300 bp of the SIX1 promoter region that could contain necessary elements for efficient expression of the SIX1 promoter-gfp construct. To examine this hypothesis, the additional 300 bp of the SIX1 region should be added to the SIX1 promoter-gfp construct and used to transform Fol. The rfp reporter gene was incorporated along with the chimeric gene into the T-DNA of binary vectors to enable screening of transformed hairy roots with the chimeric gene. However, only weak expression of rfp transcript and no expression of chimeric gene transcript was detected following transient expression analysis in Nicotiana benthamiana. Hairy root transformation employing Agrobacterium rhizogenes was performed both in vitro and ex vitro. However, the lack of RFP fluorescence prevented detection of hairy roots transformed with the chimeric gene. Hairy roots may have been transformed but the ocs promoter was probably not strong enough to drive sufficient rfp expression for detection of RFP fluorescence. Replacing the ocs promoter with a stronger promoter like the 35S promoter could have helped to enhance rfp expression. Transformation was also attempted using A, tumefaciens carrying the chimeric gene but it failed to generate any transformed tomato plants. Although the ability of the chimeric endochitinase {u03B2}-1,3-glucanase gene to confer resistance against Fusarium wilt in tomato remained untested, it remains a valuable objective for future research