Science Collected Works - Theses
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ItemSelf-incompatibility in Raphanus raphanistrum (Wild Radish) : Insights and Applications(University of Melbourne, 2013)Wild radish (Raphanus raphanistrum) is a self-incompatible (SI) winter annual and is a major crop weed in Australia, being present in all Australian states and territories except the Northern Territory. Control of wild radish costs an estimated A$210 million per annum and the species has recently overtaken ryegrass as the worst weed to control in Western Australia (WA), with 93% of the WA populations resistant to one or more herbicides. There is thus an urgent need for herbicide-independent control measures that can target wild radish reproduction. This thesis aims to evaluate the potential of the self-incompatibility (SI) system of R. raphanistrum as a reproductive target and uses two approaches to activate the SI response in a constitutive manner: the first uses SP11 (S-locus protein 11) molecules and the second uses stigmatic ROS (reactive oxygen species) inducers. Chapter 1 is a literature review and surveys current understanding of the SI response, with a particular focus on the Brassicaceae which are the emphasis of this thesis. The SI response is controlled by a multiallellic locus, the S-locus, and pollen rejection or acceptance is determined by the interaction of the products of two tightly-linked S-locus genes: SP11, encoding a pollen-expressed protein ligand and SRK, encoding a receptor kinase expressed by the stigma. Binding of SP11 to its cognate SRK causes an allele-specific activation of the SI response resulting in rejection of self-pollen. SP11 thus has the potential to inhibit reproduction in R. raphanistrum in a specific and non-toxic manner. However, to act as a reproductive blocker, the allele-specific action of SP11 requires that the number of S-alleles present in the Australian R. raphanistrum populations to be low - information that is currently lacking. The chapter concludes with a statement of the aims of this thesis. Chapter 2 describes the identification and characterisation of S-alleles in a wild R. raphanistrum population (ML8) from WA (Western Australia). A diallel was used to demonstrate the presence of a robust SI system and compatibility relationships in the population were determined. SRK S-domain and kinase domain sequences were amplified from genomic DNA: in particular, S-domains for three S-alleles were isolated and physically linked to their respective kinase domains. SP11 sequences were amplified from floral bud RNA and their relationship to SRKs determined. Based on SRK kinase domain sequences and a theoretical estimation, a total of 30 S-alleles was predicted to be present in the entire Australian R. raphanistrum population - a number that is considered low and renders the SP11 molecule a viable reproductive blocker candidate. Chapter 3 reports further work on characterizing the gene products of SP11 and SRK, with particular emphasis on the interaction between these two SI determinants. Recombinant SP11 produced in E. coli was able to prevent the germination of otherwise compatible pollen on R. raphanistrum stigmas in an allele-specific manner. A recombinant version of SRK that was missing the kinase domain (eSRK) was transiently expressed in tobacco (Nicotiana benthamiana) leaves and the hypervariable sub-domain (HVR) of eSRK was produced in E. coli. However, none of the recombinant eSRK or HVR interacted with SP11 in pull-down and dot-blot assays. Chapter 4 describes the effect of ROS on the SI response in R. raphanistrum. Chemicals known to induce or inhibit ROS production were used to manipulate ROS levels in the stigma. Treatment with sodium pyruvate, ascorbic acid and menandione bisulphite led to >8-fold increase in ROS stigmatic levels. Stigmas with elevated ROS levels were able to reject compatible pollen in a manner that appeared identical to the rejection of incompatible pollen. The reasons why increased stigmatic ROS levels should lead to pollen rejection are discussed. This thesis concludes with a summary of key experimental findings in light of current understanding of SI and suggests potential avenues for further research. A discussion of an integrated weed management approach incorporating reproductive blockers described in this thesis with existing control strategies is also presented.