No description available.

Aphids are economically important insect pests, which feed on phloem sap using stylets. Aphids cause significant losses of crop yield, through draining plant resources and vectoring over 275 plant viruses. In plant-pathogen interactions, basal plant defense involving pathogen associated molecular pattern (PAMP) triggered immunity (PTI) and effector triggered immunity (ETI) effectively fends off the majority of plant pathogens. I aimed to discover whether these mechanisms are also involved in the plant response to aphids. I found that elicitors present within aphids can evoke PTI/ETI defense responses. In Arabidopsis thaliana, perception of aphid elicitors requires the Leucine-Rich Repeat Receptor- Like Kinase (LRR-RLK) BAK1, which is required for multiple PTI responses via interaction with other RLKs. I identified two RLKs which may detect aphid elicitors and provide specificity to aphid detection. Successful aphid colonization of plants is thought to involve the suppression of PTI and ETI via effectors, leading to effector-triggered susceptibility (ETS). I investigated a Myzus persicae effector, Mp10, and found that it was required for success on Arabidopsis and could block immune signalling. A plant target for Mp10 was identified via a yeast twohybrid screen. Further investigations suggest that the Mp10 target has previously unknown roles in immune receptor trafficking. Mp10 induces ETI-like responses when expressed in plants, which I found were not dependent upon Mp10 effector action or salicylic acid. A yeast two-hybrid screen of candidate aphid effectors revealed interactions with plant resistance proteins, which may play a role in the aphid-plant interaction. Aphid effector proteins were also found to interact with each other, suggesting a role in the regulation of effector action and delivery into plants. Taken together, the research described in this thesis has elucidated the roles of PTI, ETS and ETI in insect-plant interactions and identified specific plant and aphid proteins that are involved in these.

No description available.

Modern agriculture relies on chemical herbicides to control weedy species that compete with crops. In the UK, an estimated 80 % of cropland is infested with the weed species black-grass (Alopecurus myosuroides) that has evolved resistance to multiple herbicides with different modes-of-action. Studies in resistant black-grass identified a phi (F) class glutathione transferase, AmGSTF1, which was constitutively expressed. Heterologous expression of AmGSTF1 in a transgenic host plant granted a multiple herbicide resistant (MHR) phenotype and it was found that the enzyme induced the activities of endogenous detoxification enzymes as well as catalytically detoxifying damaging hydroperoxides in vitro, which can form as a downstream consequence of herbicide treatment. In the current work, AmGSTF1 mutants have been derived and exploited to better understand the function of AmGSTF1 in eliciting MHR. Using a catalytically-retarded mutant, it is shown that the enzyme elicited MHR without requiring catalysis. Instead, the mutant induced the activities of endogenous detoxification enzymes. Another mutant, lacking a cysteinyl residue (Cys120), has demonstrated that Cys120 plays a key role in the interaction of AmGSTF1 with xenobiotics. In particular, Cys120 can be alkylated and inhibited by 4-chloro-7-nitro-benzoxadiazole (NBD-Cl), a compound that can reverse MHR when sprayed on black-grass plants. Enzyme inhibition and alkylation studies found that AmGSTF1 could be alkylated by other chemicals but that this did not induce notable inhibition of the protein. The cysteinyl mutant also induced MHR in a transgenic host plant by inducing the activities of endogenous detoxification enzymes. The properties of AmGSTF1 orthologues from annual rye-grass (Lolium rigidum) and maize (Zea mays) were also explored and found to display very similar functional properties as AmGSTF1. Transcriptome profiling demonstrated that AmGSTF1 did not induce changes in host plant biochemistry by perturbing gene expression. These studies have therefore demonstrated a central regulatory role for GSTF1 enzymes in co-ordinating MHR associated with manipulating host detoxification pathways and challenges the scientific dogma that glutathione transferases require catalytic activity to elicit herbicide resistance.

The root hemi-parasitic witchweeds Striga hermonthica and S. asiatica are considered the most important biotic constraint to cereal crop production in sub-Saharan Africa (SSA). These parasites infect the staple cereal crops (rice, maize, sorghum and millet) resulting in considerable yield losses. Control of these parasites is very difficult as the Striga seed bank is widespread and damage to the crop occurs long before the parasite emerges above ground. Resistant cultivars are considered to be an effective and affordable component of an integrated Striga management strategy but very few are available to farmers as sources of resistance to Striga are relatively scarce and little is known about the molecular genetic basis of resistance to this parasite. Rice is an economically important cereal crop in SSA that is mostly cultivated by resource-poor farmers. Both cultivated rice species, Oryza sativa (L.) and Oryza glaberrima (Steud.), are grown in Africa. To take advantage of superior traits from each species, AfricaRice Center and partners developed inter-specific rice cultivars called NERICA (NEw RICe for Africa) for rain-fed upland ecosystems. Because of their high yields, even on low nutrient soils where Striga spp. are prevalent, the NERICA cultivars have been widely adopted by farmers. Despite this, very little is known about their resistance to different species and ecotypes of Striga. The aims of this study are to determine how resistant and/or tolerant the upland NERICA cultivars are to different species and ecotypes of Striga under controlled environment and Striga-infested field conditions, to identify whether resistance is broad spectrum or specific to particular ecotypes of Striga and to characterize the phenotype of the resistance at a histological level. Finally using a Chromosome Segment Substitution Line (CSSL) population derived from a cross between an O. glaberrima cultivar MG12 (donor parent) and an O. sativa cultivar Caiapo (recurrent parent), the genetic basis of post-attachment resistance to Striga is investigated. The NERICA rice cultivars showed different susceptibilities to both S. hermonthica and S. asiatica species under controlled environment conditions. Some cultivars showed good broad-spectrum resistance against several Striga ecotypes and species whilst others showed intermediate resistance or were very susceptible. In addition, some cultivars showed resistance to a particular ecotype of Striga but were susceptible to others. The phenotype of a resistant interaction was often characterized by necrosis at the host parasite interface and an inability of the parasite to penetrate the host root endodermis. In general, the most resistant NERICA cultivars grew better than the very susceptible cultivars although even a small number of parasites caused a reduction in above ground host biomass. There was however, genetic variation for tolerance to Striga (the ability to grow and yield well in the presence of Striga) amongst the NERICA cultivars. The NERICA cultivars were also grown in field trials at Kyela in Tanzania (under S. asiatica infestation) and at Mbita Point in Kenya (under S. hermonthica infestation) in 2010 and 2011 to determine the impact of environment on the expression of resistance. The resistance of the NERICA cultivars against S. hermonthica and S. asiatica, in the field, was broadly similar to that observed in the laboratory although there were some exceptions. These results allow us to recommend particular cultivars for Striga-infested regions but they also illustrate the necessity of understanding the genetic basis of resistance to different ecotypes of Striga for breeding of durable resistance (and pyramiding of appropriate resistance genes) in host cultivars adapted to different rice agro-ecosystems in sub-Saharan Africa. Sixty four lines of an inter-specific CSSL population and the parent cultivars MG12 and Caiapo were phenotyped for resistance to S. hermonthica. MG12 showed good resistance to S. hermonthica whilst Caiapo was very susceptible. The CSSLs showed a range of susceptibility to the parasite, however, only two CSSLs showed the same strong resistance phenotype as MG12. Graphical genotyping and a Quantitative Trait Loci (QTL) analysis revealed a large QTL on chromosome 12 (designated STR12.1) which explained at least 80 % of the variation for resistance in the population and suggests that resistance to S. hermonthica (in MG12) is due to one (or a few genes) of major effect. This finding opens the way for the identification of candidate Striga resistance genes (through fine mapping approaches) and their transfer to farmer-preferred cultivars via marker assisted breeding.

The obligately outbreeding root hemiparasite Striga hermonthica (Orobanchaceae)is a serious threat to subsistence agriculture in sub-Saharan Africa. Resistance to this parasite in its crop hosts, such as rice, sorghum and maize, is not common, and the evolution of host adaptations that are able to overcome new sources of resistance is an ever-present risk. Research into host adaptation in S. hermonthica has generally sought to correlate the genetic relationship between Striga individuals with host identity; however, such approaches must be supported by lab-based evidence of host adaptation, otherwise ad hoc field sampling may result in the confounding of host identity with isolation-by-distance. Additionally, genetic variation used to reconstruct relationships is unlikely to provide an insight into relationships at functional loci underlying host adaptation. In this thesis, I use a range of new approaches to investigate several different aspects of parasite adaptation in the S. hermonthica-Sorghum bicolor pathosystem. Host adaptations, or pre-adaptations, are commonly revealed using tests for differential virulence between Striga populations and host genotypes; that is, by demonstrating population-level genotype-by-genotype interactions. Evidence for such interactions was found between three West African populations of S. hermonthica and five sorghum cultivars. These interactions were shown to be strongest at the parasite post-attachment life stage, and to depend on the parasite virulence metric used. Environmental influences on host-parasite interactions were strong and variable between years and sites. Candidate genetic loci for virulence, responding to selection in a micro-evolutionary fashion, were identified by Fst differentiation-based approaches ('outlier analyses') that aim to uncover associations between particular loci and environmental drivers, such as host identity. An AFLP outlier analysis was used on Striga plants parasitising nine sorghum cultivars in a field trial in Burkina Faso. Significant locus-specific differentiation was detected at 14 out of 1275 loci. However, predicted allele frequencies at these loci did not correlate with a field measure of Striga virulence across host-selected sub-populations. Simulation results suggested that the estimated levels of Fst at outlying loci could mean that alleles underlying host adaptation exist at intermediate frequencies in populations. A three-generation pedigree, created from a cross between S. hermonthica individuals from an East African and a West African population, enabled further insights into the genetics of adaptation. Individuals from a pseudo-backcrossed F1 (BCF1) generation, grown on two different sorghum hosts and in axenic culture, indicated significant host-related segregation distortion. Analyses of virulence in the BCF1 also provided strong evidence for epistasis, and for an effect of maternal identity. A second outlier analysis of host adaptation, investigating the East African population used in the pedigree, indicated some correspondence between outlier loci and loci found to be differentially segregating between different hosts in the BCF1 generation, and demonstrated the differing genomic extents of these phenomena. The results accumulated across these experiments provide evidence for a complex, polygenic basis to virulence in S. hermonthica.

Pathogens secrete molecules, termed effectors, to manipulate their host to the benefit of the pathogen. Effectors of plant parasitic nematodes are predicted to have a range of functions such as facilitating invasion, initiation and maintenance of the feeding site, and suppression of host defences. The genome sequence of the potato cyst nematode Globodera pallida was analysed to identify putative effectors. They include: 129 effectors similar to those previously identified from cyst nematodes, 53 cell wall modifying enzymes and 117 novel putative effectors. Only four effectors were common between G. pallida and the root-knot nematode Meloidogyne incognita. These could have a conserved role in plant parasitism. A large SPRY domain containing gene family was identified in G. pallida. It has 299 members, of which 30 are predicted to be secreted and therefore categorised as effectors. Phylogenetic analysis showed that the family is hugely expanded and specific to Globodera species. Fifty-four putative effectors were cloned from G. pallida cDNA. Transgenic lines of Arabidopsis thaliana and Solanum tuberosum L. ‘Désirée’ were produced, to express a range of these effectors and act as tools for functional characterisation. Potato lines that expressed selected effectors were subjected to phenotypic analysis and pathogen susceptibility assays. The largest range of aberrant phenotypes was observed for those plants expressing GpIA7 and GpIVG9. Potato lines expressing GpIA7 showed altered growth phenotypes and an increased susceptibility to Phytophthora infestans CS-12. GpIVG9-expressing potato lines showed accelerated growth, distorted leaves and increased susceptibility to nematode invasion. A more in-depth functional characterisation was conducted on a ubiquitin extension protein effector. The G. pallida ubiquitin extension protein suppressed PAMPtriggered immunity and the C-terminal extension was required for this activity. The outcomes from this work and the tools generated for future experimentation will contribute to elucidating the complex interactions between pathogens and their hosts.

No description available.

No description available.

Behavioural and electrophysiological responses of winged virginoparous Aphis fabae to volatile compounds of faba bean, Vicia faba, were studied and semiochemicals used in host location identified. In olfactometer bioassays, aphids responded positively to V. faba volatiles from an intact plant. This response also occurred when volatiles from an air entrainment sample of a V. faba plant were tested. Coupled gas chromatography-electroantennography revealed the presence of 16 electrophysiologically active volatile compounds in the air entrainment sample and 15 of these were identified as (E)-2-hexenal, (Z)-3-hexen-1-ol, 1-hexanol, benzaldehyde, 6-methyl-5-hepten-2-one, octanal, (Z)-3-hexen-1-yl acetate, (R)-linalool, methyl salicylate, decanal, undecanal, (E)-caryophyllene, (E)-β-farnesene, (S)-germacrene D, and (E,E,)-4,8,12-trimethyl-1,3,7,11-tridecatetraene. A synthetic blend consisting of all identified compounds in the same concentration and ratio as in the air entrainment sample elicited a similar behavioural response from the aphids as the air entrainment sample. Each compound was tested for behavioural activity individually at the same concentration as in the air entrainment sample and subsequently over a range of different doses. It was found that the response to the complete blend was not due to a response to a single compound. Dose response experiments also revealed ten of the compounds elicited negative behavioural responses from aphids. Further behavioural experiments revealed that these responses were context-specific and behavioural activity of individual compounds was different when they were presented alongside other compounds in the blend. It was hypothesised that a blend of host volatiles in a species-specific ratio may be used by A. fabae to recognise its host. To determine whether or not ratios of volatiles could provide a reliable cue to host seeking aphids, intra-specific and diurnal variation of ratios of volatiles emitted were investigated. Although considerable variation in ratios was observed the quantities of some pairs of compounds were positively correlated, indicating a degree of consistency in the ratios.