Elucidation of defence response mechanisms in pearl millet

Abstract

Pearl millet is a staple food source for millions of African families living in semi-arid regions of the continent. Yet, despite its importance and ability to provide consistent yields, very little research and resources have been directed towards understanding mechanisms governing this crop’s resilience to biotic and abiotic stresses. The research outlined in this thesis therefore aimed to elucidate defence response mechanisms in pearl millet, a non-model cereal crop. This was accomplished through the construction and characterisation of a pearl millet defence response cDNA library, which was subsequently utilised in large scale gene expression studies to profile pearl millet’s response to the defence signalling compounds nitric oxide (NO), methyl jasmonate (MeJA) and salicylic acid (SA), and to the biotrophic rust fungus Puccinia substriata var. indica. A pearl millet cDNA library was constructed by treating pearl millet plants with the defence elicitors chitin and flagellin, and by wounding the plants. Suppression subtractive hybridisation (SSH) was employed to enrich the library for defence response transcripts. In order to characterise the cDNA libraries, a quantitative cDNA microarray-based screening method was developed that enabled identification of false positive transcripts, as well as clones that represented rare or abundant transcripts. Based on this screening method, a number of clones were selected for sequence analysis, and their identity ascertained through homology searches with previously sequenced genes. This revealed a number of genes known to play important roles during pathogen attack. The pearl millet SSH defence response library, consisting of 1920 cDNAs either up- or down regulated in defence response, was spotted onto a glass slide microarray and used in transcript profiling studies to examine pearl millet’s response to the defence signalling molecules NO, MeJA and SA. Whilst only 45 cDNAs responded significantly to NO treatment, 279 and 224 cDNAs responded to MeJA and SA sprays, respectively. Closer examination of MeJA and SA responsive genes revealed that many of the induced transcripts were common to both signalling pathways, demonstrating that a substantial network of regulatory interactions exists between the salicylate and jasmonate pathways, which were previously believed to act in an antagonistic manner. Pathology studies indicated that pretreatment of pearl millet with SA conferred resistance to a virulent isolate of P. substriata var. indica, whereas MeJA application did not significantly reduce subsequent infection levels. Transcript profiling of a susceptible pearl millet line in response to virulent rust infection revealed that genes common to both the jasmonate and salicylate pathways were induced, suggesting that the plant adopts elements from a number of defence signalling pathways in an attempt to ward off infection by the virulent rust fungus. However, in view of results obtained from pearl millet defence signalling molecule pretreatments, it is probably genes that are significantly induced in response to SA, but to a lesser extent by MeJA that actually confer resistance to an avirulent rust isolate. Treatment of pearl millet plants with an avirulent P. substriata strain and subsequent microarray analysis would answer this hypothesis by revealing whether an incompatible reaction elicits more elements of the salicylate defence response pathway.