Chile (Capsicum annuum) plants transformed with the RB gene from Solanum bulbocastanum are resistant to Phytophthora capsici.

Phytophthora capsici is a soil borne pathogen, and is among the most destructive pathogens for Capsicum annuum (chile). P. capsici is known to cause diseases on all parts of the chile plants. Therefore, it requires independent resistance genes to control disease symptoms that are induced by each of the P. capsici strains. This requirement of multiple resistance genes to confer resistance to P. capsici, in chile makes breeding for resistance a daunting pursuit. Against this backdrop, a genetic engineering approach would be to introduce a broad host resistance gene into chile in order to protect it from different races of P. capsici. Notably, a broad host resistance gene RB from Solanum bulbocastanum has been shown to confer resistance to P. infestans in both S. tuberosum and S. lycopersicum. We agroinfiltrated the RB gene into the leaves of susceptible chile plants, demonstrating that the gene is also capable of lending resistance to P. capsici in chile. We introduced the RB gene into chile by developing an Agrobacterium tumefaciens mediated transformation system. The integration of the RB gene into the genome of the primary transformants and its subsequent transfer to the F1 generation was confirmed by genomic PCR using primers specific for the RB gene. A 3:1 ratio for the presence and absence of the RB gene was observed in the F1 progeny. In addition to showing resistance to P. capsici in a leaf inoculation experiment, about 30% of the F1 progeny also exhibited resistance to root inoculation. Our data, when taken together, suggests that the RB gene from S. bulbocastanum confers resistance against P. capsici in C. annuum, thereby demonstrating that the RB gene has an even broader host range than reported in the literature-both in terms of the host and the pathogen.

An intragenic approach to confer glyphosate resistance in chile (Capsicum annuum) by introducing an in vitro mutagenized chile EPSPS gene encoding for a glyphosate resistant EPSPS protein.

Chile pepper (Capsicum annuum) is an important high valued crop worldwide, and when grown on a large scale has problems with weeds. One important herbicide used is glyphosate. Glyphosate inactivates the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme in the synthesis of aromatic amino acids. A transgenic approach towards making glyphosate resistant plants, entails introducing copies of a gene encoding for glyphosate-resistant EPSPS enzyme into the plant. The main objective of our work was to use an intragenic approach to confer resistance to glyphosate in chile which would require using only chile genes for transformation including the selectable marker. Tobacco was used as the transgenic system to identify different gene constructs that would allow for the development of the intragenic system for chile, since chile transformation is inefficient. An EPSPS gene was isolated from chile and mutagenized to introduce substitutions that are known to make the encoded enzyme resistant to glyphosate. The promoter for EPSPS gene was isolated from chile and the mutagenized chile EPSPS cDNA was engineered behind both the CaMV35S promoter and the EPSPS promoter. The leaves from the transformants were checked for resistance to glyphosate using a cut leaf assay. In tobacco, though both gene constructs exhibited some degree of resistance to glyphosate, the construct with the CaMV35S promoter was more effective and as such chile was transformed with this gene construct. The chile transformants showed resistance to low concentrations of glyphosate. Furthermore, preliminary studies showed that the mutated EPSPS gene driven by the CaMV35S promoter could be used as a selectable marker for transformation. We have shown that an intragenic approach can be used to confer glyphosate-resistance in chile. However, we need a stronger chile promoter and a mutated chile gene that encodes for a more glyphosate resistant EPSPS protein.