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Abstract:

Tomato (Solanum lycopersicum) is one the most important horticultural crops in the world and is of high economic and nutritional value. As a consequence, it is a part of the diets of several cultures from around the world. Besides its economic and socio-cultural value, tomatoes are also ideal model fruit plants for plant research since they possess a diploid genome which has been fully sequenced, have a short generation time, require relatively low maintenance, show resilience to abiotic stresses such as drought, and are easy to transform plant tissues. Since the 2000s, the greenhouse-based commercial cultivation of hydroponically grown tomato plants has been threatened by hairy root disease (HRD). Tomato plants suffering from HRD show increased proliferation of root tissues, preference for vegetative growth over generative growth, dwarfism, loss in apical dominance, and a significant decrease in fruit yields. Rhizogenic agrobacteria belonging to biovar (BV) 1 genomic species were found to be the etiological agents behind HRD. During infection, BV1 rhizogenic agrobacteria were found to transfer a part of their genome to the host plant which was found to subsequently integrate within the hosts genome. The transferred genomic region is known as transfer DNA (T-DNA) which encodes several open reading frames (ORFs) under the control of promoters which permit their expression in plants. These ORFs were found to play diverse roles including inducing cellular dedifferentiation and redifferentiation processes within infected plant cells resulting in their conversion to root tissues. The expression of the ORFs were found to be controlled spatially and temporally and their concerted actions were found to result in symptoms typical of HRD highlighting the complex molecular landscape during HRD rhizogenesis. However, despite their association with plant pathogenesis, BV1 rhizogenic agrobacteria offer several research and economic incentives. For instance, BV1 rhizogenic agrobacteria have been utilized for plant transformation applications to overcome recalcitrance of some plant species towards Agrobacterium-mediated transformation or overcome regeneration bottlenecks. Furthermore, some of the traits associated with HRD such as the increased production of specialized plant metabolites of medicinal/economic importance have resulted in their application to generate hairy root cultures which are propagated in bioreactors for large scale production of secondary metabolites, or in the ornamental plant breeding sector to generate plants with desirable traits such as dwarfism or increased branching. The multifaceted pathogenic lifestyle, research and economic potential of BV1 rhizogenic agrobacteria has resulted in over three decades of research to understand the molecular functions of the enigmatic T-DNA ORFs towards HRD. Despite this, the molecular functions of the T-DNA ORFs remain poorly understood. Therefore, the main objective of this thesis was to develop tools and technologies that would facilitate the study of the tomato-BV1 rhizogenic agrobacteria interaction and unravel the contribution of the T-DNA genes towards HRD. We used the BV1 rhizogenic Agrobacterium strain K599 as the model strain in most studies since (1) it is one of the best-studied BV1 rhizogenic agrobacteria and (2) the T-DNA encodes only 11 ORFs compared to some other strains of root-inducing agrobacteria which can encode up to 18 ORFs and as such represents a minimum T-DNA configuration that is capable of inducing HRD. To unravel the molecular functions of individual T-DNA ORFs towards HRD, we constructed knockout mutant strains of target T-DNA ORFs. We achieved this by developing a CRISPR-mediated cytosine base-editor for Agrobacterium spp. which we implemented to introduce premature stop codons within the T-DNA encoded ORF3, ORF4, RolB, RolC, ORF13, ORF14 and Cucumopine synthase effectively generating functional knockout mutants of the targeted T-DNA ORFs. We also performed whole genome sequencing of the base-edited mutants targeting the rolB, rolC and orf13 ORFs which revealed a low number of off-targets with a preference for -TC motifs containing genomic regions. For rolA, we used allelic exchange to generate a functional knockout mutant strain. As a preliminary study to easily test the virulence and root-inducing potential of the constructed K599 T-DNA mutant strains, we utilized the carrot disc bioassays. The assay revealed that no single T-DNA ORF was absolutely essential for HRD in carrots since all T-DNA mutant agrobacteria strains induced hairy roots to some extent, however, some genes such as rolB were found to be critical for the timely onset of hairy root induction and the knockout of other genes such as orf13 resulted in the appearance of hairy roots at the same time as the wildtype K599 strain. Furthermore, the carrot disk assays also revealed that the knockout of most genes, except for orf3n, resulted in highly decreased symptom severity in comparison to wildtype K599. Lastly, we also used the carrot disk assay to assess the virulence of some wildtype BV1 rhizogenic agrobacteria strains. The constructed orf13 and orf14 functional knockout mutants and wildtype rhizogenic Agrobacterium strain K599 were further selected for performing a comparative transcriptome study on tomato hairy roots and attempt to decipher the functions of orf13 and orf14. We utilized an in vitro tomato composite plant bioassay to generate tomato hairy roots which were harvested and subject to mRNA sequencing. Our comparative transcriptome analyses revealed significant upregulation of C-terminally encoded peptides (CEPs) which were found to partake in plant responses related to regulation of root development and nitrate import during HRD rhizogenesis. Furthermore, we observed an interesting cross-talk between the phytohormones jasmonic acid, ethylene, abscisic acid and auxin which were found to affect the expression of several plant defense related genes belonging to eleven PATHOGENESIS RELATED families. In the comparisons between hairy roots induced by orf13 functional knockout mutant versus K599 and orf14 functional knockout mutant versus K599, 16 and 4 genes were found to be significantly differentially expressed, respectively. We also developed a soil-based tomato HRD disease bioassay which we utilized to assess the susceptibilities of tomato cultivar 'Moneymaker' and the tomato rootstocks, 'Maxifort', 'Optifort' and 'Arnold' without artificial wounding and used it to characterize the virulence patterns of BV1 rhizogenic agrobacteria strains K599, NCPPB4062, ST15.13/012, ST15.13/057 and ST15.13/097 during tomato HRD. Accordingly, we found 'Optifort' and 'Maxifort' to show increased susceptibility to BV1 rhizogenic agrobacteria in comparison to 'Arnold' and 'Moneymaker' and strains K599, ST15.13/057 and ST15.13/012 were found to be virulent strains which correlated with the findings from our carrot bioassay. Furthermore, we observed a differential development of root system architecture in response to rhizogenic agrobacteria which was found to be independent from their effects on root biomass. Taken together, the studies conducted during this thesis have helped set up a suitable platform to carry out agrobacteria research and study the plant-rhizogenic agrobacteria interactions in the context of tomato HRD. The fundamental knowledge gained, and bioassays developed can potentially be harnessed in future resistance breeding programs and testing developed tomato varieties and rootstocks for HRD susceptibility as well as identify strains for plant transformation applications.