Molecular characterisation of Eucalyptus grandis PGIP

Abstract

Coniothyrium zuluense is the causal agent of a serious Eucalyptus stem canker disease in South Africa (Wingfield et al., 1997). Eucalypts are the most important hardwood plantations in the world, and in South Africa these hardwoods occupy approximately 1.5 million hectares of plantation area, an area that is soon to be increased by an additional 600 000 hectares. As exotics, Eucalyptus plantations are constantly exposed to infection by fungal pathogens such as C. zuluense, which by secreting cell-¬wall degrading enzymes contribute to the degradation of plant cell walls and subsequent reduction and in the quality of timber produced. This ultimately affects the South African paper, pulp and timber industries. Selection of resistant clones through traditional breeding methods is the most common method currently employed in overcoming the problem of fungal infection. The genetic manipulation of Eucalyptus trees for enhanced resistance to fungal diseases is an alternative to the time-consuming and tedious approach of conventional breeding. The identification of several antifungal proteins, particularly polygalacturonase-inhibiting proteins (PGIPs) from various plant species including Eucalyptus, lead to the hypothesis that over-expression of these proteins could potentially reduce pathogen attack. However, prior to the expression of PGIPs in plants, isolation and molecular characterization of these genes are required. The aims of this study were therefore (l) to clone and characterize the complete Eucalyptus grandis pgip gene, (2) to transform Nicotiana tabacum (tobacco) plants with the E. grandis pgip gene and (3) to test for inhibition of C. zuluense PGs by PGIPs extracted from transgenic tobacco plants. This forms the first step towards the generation of E. grandis clones that are more disease tolerant. A review of the role of fungal endopolygalacturonases and polygalacturonase¬inhibitors in plant-pathogen interactions are presented in chapter I. Strategies employed to isolate and characterize pgip genes from a range of plant species are highlighted and the importance ofPGIPs in disease resistance is discussed. In chapter 2, the molecular cloning and characterization of the E. grandis pgip gene is discussed. The work presented in this chapter is a follow up on work previously conducted by Chimwamurombe (2001). Previously, a partial Eucalyptus pgip gene sequence was obtained with the use of degenerate oligonucleotide primers. In this study, the complete Eucalyptus pgip gene was obtained through the employment of genome walking strategies. Transformation of Nicotiana tabacum cv LA Burley plants with the Eucalyptus pgip gene and the molecular characterization of transgenic tobacco plants is discussed in chapter 3. The transformation and expression of foreign genes in tobacco plants is a well-established protocol, making tobacco the most appropriate candidate plant for assessing the functionality of the plant transformation construct. The production of endopolygalacturonases from virulent C. zuluense isolates and the subsequent PGIP assays conducted to determine levels of PG inhibition are included in this chapter. This thesis consists of three independent chapters representing studies on the molecular characterization of an E. grandis pgip gene and focusing on the potential for inhibition of PGs produced by C. zuluense by Eucalyptus PGIP extracted from transgenic tobacco plants. Repetition of certain aspects in the individual chapters has been unavoidable and the thesis is presented following a uniform style.