The molecular characterization of interaction between Fusarium circinatum and Pinus patula

Abstract

The main objective of this thesis was the elucidation of the host-pathogen interaction between Pinus patula and Fusarium circinatum. This was accomplished by studying differential gene expression at the molecular level. Therefore, the first chapter reports the use of PCR-based methods in gene discovery and transcriptome analysis. The use of these techniques in the identification of novel transcripts in host-pathogen interactions addressed. These examples illustrate the differences and strong features of each technique. Chitinases are linked to defence responses in plants. In chapter tw0, the induction of chitinases in P. patula was assessed at both the protein and genetic level. Western blot analysis and enzyme activity assays indicate that chitinase enzyme is not detected a part of the defence response by P. patula after infection by F. circinatum. This was further confirmed by the lack of significant induction of two Pinus chitinase genes, LP6 and PSCHI4, as determined by RT-PCR analysis. Partial DNA sequence homologues for the LP6 and PSCH14 genes were determined and compared with a variety of plant chitinases. The low levels of detectable chitinase induction in P. patula might explain the high levels of susceptibility to the pitch canker fungus observed in seedlings of this tree. Pinus patula, the most widely planted species in South Africa, is highly susceptible to infection by F. circinatum. In chapter three, suppression subtractive hybridisation was used to elucidate the changes taking place at the molecular level early on in this interaction. Most of the identified transcripts shared homology to both biotic and abiotic stress in plants. The induction of one fragment, displaying homology to phytocyanin proteins, as followed through RT-PCR. Induction levels for this fragment differed significantly between less and more susceptible plants. Although most of the sequences isolated in this study can be Iiked to stress, most have not been linked with specific plant-pathogen interactions. This raises questions in regard to the function of these genes in host-pathogen interactions. Further research identify the function of these sequences in the defence response will be needed. These sequences can also be tested against a family of Pinus trees to ascertain if they will be useful in marker assisted selection. A molecular analysis of culture degeneration and pathogenicity of F. circinatum was attempted in chapter four. In this chapter, the differential induction of transcripts in F. circinatum was determined against several other Fusarium spp. Several of he identified fragments shared homology with stress related proteins. One transcript shared homology to a polyketide synthase, FUM5, that could be linked to fumonisin production in other Fusarium spp. ELISA detected no fumonisin production, although the FUM5 transcripts were detected. The identification of all the transcripts could provide a basis for more intensive gene discovery studies in F. circinatum and other Fusarium spp. The induction of these sequences in different isolates needs to be studied to prove their function in F. circinatum. This study also complements several other studies that studied the morphological characteristics of culture degeneration. Resistance gene analogues have been reported from a diverge set of plant species. In chapter five, degenerate PCR amplification was used to isolate TI-NBS-LRR like resistance gene analogues from a range of Pinus species. These sequences w re further characterised through comparative analysis with previously reported Pinus resistance gene analogues. Through motif analysis, several of the known conserved motifs found in NBS domains were identified and conservation with other plant NBS motifs is indicated. The P-Ioop and GLPL motifs displayed a high level of conservation on amino acid level with other plant NBS motifs. However, slight differences in several of the conserved regions were observed when the Pinus analogues were compared with Arabidopsis thaliana. The identification of differences between angiosperm and gymnosperm NBS sequences indicates that design of new degenerate probes and primers for the isolation of more ancient NBS sequences is needed. Further, phylogenetic and structural analyses of these sequences will also aid in understanding the relationship between angiosperm and gymnosperm NBS sequences. The knowledge gained from such a study will highlight the similarities between angiosperm and gymnosperm defence responses. This study represents the first detailed report on RGA in Pinus.