Manipulation of defence related lignification in wheat
Lignin is a complex phenolic hetropolymer with an established role in structure, support and defence in higher plants. The chemical structure of lignin is as yet undefined but controlled by an enzymatic pathway leading to three monomeric subunits. Lignin accumulates in plants in response to pathogen challenge. A scanning densitrometric assay to detect lignin was developed that was non-invasive, quantitative and quick to perform. The assay was used in conjunction with assessments of phytotoxicity, mycotoxity and pathogen resistance to assess the efficacy of potential biochemical inhibitors of the phenylpropanoid pathway in vivo. With this information, tolerances for biochemical inhibition of the phenylpropanoid pathway were obtained. This allowed further investigation of the basis of genetic and metabolic regulation of one form of one enzyme of the pathway, phenylalanine ammonia lyase, in wheat. Evidence of a potential role for endogenous elicitation in the ligninification pathway was also gained by the use of the assay. Elicitation in terms of the hypersensitive response was also investigated during attempts to purify the fungal elicitor Avr2 using the tovnaXo!Cladosporium fulvum model; however this work was completed by an alternative genetic screen protocol published elsewhere. Control of ligninificiation and the enzymes that produce the polymer is therefore an essential part of the defence response in wheat. This has important implications for genetic modification of the pathway. It was shown in this study that the phenylpropanoid pathway controls one aspect of resistance in wheat and concludes that care must be taken when manipulating the pathway in plants for increased digestibility or ease of pulping. In addition, a separate project was undertaken in order to purify an avirulence protein possessed by the Cladosporiumfulvum fungus. The projects aim was to obtain amino acid sequence(s) of potential interacting proteins that would be used to design primer sequences to provide a genetic sequence of the target avirulence protein Avr2. Although several candidate proteins were obtained and amino acid sequencing attempted; a competing group obtained the genetic sequence of Avr2. The sequence of this clone predicts a protein whose molecular weight and isoelectric point falls within a region of proteins whose isoelectric points and molecular weights show activity in a bioassay for Cf-2 interacting proteins. This data supports the conclusion that the work by Luderer et al (2002) defines the genetic sequence of Avr 2.