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Title: Discovery and analysis of candidate effector genes in the parasitic plant, Striga hermonthica
Author: Bradley, James
ISNI:       0000 0004 8504 8571
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2019
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Striga hermonthica (Del.) Benth. is an obligate out-crossing parasitic weed that parasitises the roots of staple cereal crops throughout Sub-Saharan Africa, including maize, sorghum, millets and upland rice. S. hermonthica infection reduces crop yields by 20–100 % and has a huge impact on food security for millions of subsistence farmers. In order to form and maintain a successful attachment, the germinated Striga seed radicle must penetrate the host root, whilst evading or suppressing the host plant's immune system. To achieve this, parasitic plants, like other plant-associated organisms such as fungi and nematodes, deploy virulence-related proteins termed 'effectors' that interact with host biology. However, very little is known about the identity of such proteins from parasitic plants. The accessibility of high-throughput sequencing technology and the recent availability of a S. hermonthica genome sequence now facilitate genome-wide approaches for effector discovery. The aim of this thesis was to integrate genomic, transcriptomic and bioinformatic approaches to discover genes encoding effector candidates (ECs) in S. hermonthica, and to develop ways to functionally validate these ECs. A pipeline to computationally predict the S. hermonthica secretome was developed. From this secretome, suites of putative ECs were identified based on a series of structural and functional characteristics of effector proteins. This analysis revealed ECs with diverse functions including those related to cell wall modification, protease activity (e.g. cysteine, aspartyl and subtilase proteases) and/or oxidoreductase activity. Furthermore, some ECs were found that shared homology with effectors identified from other plant parasites. An experimental population genomics approach was also used to identify ECs from S. hermonthica. Here, hundreds of S. hermonthica individuals growing on a very susceptible rice variety (NERICA 7) or a largely resistant rice variety (NERICA 17), were collected and sequenced. The sequenced reads were aligned to the S. hermonthica reference genome to identify single nucleotide polymorphisms and obtain measures of allele frequency differences between the S. hermonthica grown on the two host varieties that differed in their level of resistance/susceptibility. This analysis identified 39 ECs with statistically different allele frequencies associated with the ability of some S. hermonthica individuals to successfully parasitise NERICA 17. These genes encoded proteins involved in cell wall modification, protease, kinase and lipase activities, and a range of other functions. In addition, this analysis revealed an extremely genetically polymorphic S. hermonthica population, strongly suggesting S. hermonthica has a high evolutionary potential to overcome host resistance; which could have profound implications on the development of crop varieties with durable resistance. S. hermonthica attachments were also collected from the roots of NERICA 7 or NERICA 17 host plants at 2, 4, and 7 days post infection to profile gene expression changes in S. hermonthica over time. The differentially expressed genes (DEGs) identified during this time-course included a significant proportion of putative secreted proteins. In addition, some DEGs were identified as ECs by the population genomics approach. Among the genes that showed very strong changes in expression over the time-course were those encoding putative secreted peroxidases, PAR1-like proteins, cytokinin dehydrogenase enzymes, aspartyl and subtilase proteases, and leucine rich repeat-containing proteins. To functionally validate these as bona fide effectors, it was desirable to genetically transform S. hermonthica plants. A recently reported method using magnetic nanoparticles as carriers of DNA was trialed in an attempt to transform S. hermonthica pollen, which could then be used to generate stably transformed seed. This approach led to the transformation of S. hermonthica pollen but the efficiency was very low and resulted in a loss of pollen viability. An alternative way to study EC function, using Agroinfiltration to transiently express ECs in the leaves of Nicotiana spp., was also developed to test whether five ECs identified from the secretome of S. hermonthica would elicit a cell death phenotype. These ECs shared sequence similarity with the Ave1 effector from Verticillium dahliae that had been shown to trigger cell death in the leaves of Nicotiana spp., although no such response was found for these particular ECs from S. hermonthica.
Supervisor: Scholes, Julie ; Butlin, Roger ; Chaudhuri, Roy Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available