Arbuscular mycorrhizal fungi (AMF) form a mutually beneficial symbiotic relationship with a majority of land plants through an exchange of nutrients. Despite the importance of AM symbiosis in agricultural and ecological settings, relatively little is known about how the fungal symbiont actively promotes symbiosis. To overcome a host’s immune response, plant pathogens secrete effector proteins that modify a host to suppress an immune response. Few effectors have been identified in AMF, as bioinformatics methods have failed to accurately predict their sequences. To successfully colonize a plant, AMF form structures called arbuscules within plant root cortical cells. Arbuscules are a primary site of nutrient exchange during AMF symbiosis. This work is built on the hypothesis that AMF produce effector proteins to promote symbiosis, and that arbuscules are a site of effector secretion. Using Rhizophagus irregularis, Glomus versiforme and Medicago truncatula this work applies a proteomics-based approach using a new biotechnology to identify fungal proteins secreted by AMF. This novel approach using proteomics and proximity labelling to identify proteins by mass spectrometry is the first time this system has been used to study the plant-AMF relationship. In this work, mass spectrometry identifies a total of 24 R. irregularis proteins and two G. versiforme proteins that are candidate effectors involved in the plant-AMF symbiosis.

Late Blight disease, caused by Phytophthora infestans, is the most significant threat to potato production world-wide. Identifying and deploying more durable host resistance to P. infestans is a promising way forward to sustain the production of potato. To achieve this goal, it is important to seek key pathogen components that are essential for infection and which, upon detection by the host, trigger a resistance response. One such potential key pathogen molecule is the RXLR-containing effector Avr3a. Avr3a is highly up-regulated during infection and is also required for P. infestans pathogenicity. To date, all P. infestans isolates studied contain Avr3a alleles E80M103 and/or K80I103. However, a study of Avr3a diversity in the Toluca Valley, Mexico, has identified additional alleles such as K80I103L139, K80I103H133, E80M103H133 and E80M103G124. Functional studies of these alleles were conducted as part of this thesis, which also include the Avr3a paralogs Pex147-2 and Pex147-3. By examining the amino acid changes in relation to the established protein structure, it was determined that all alterations within the Avr3a variants occur at surface exposed amino acids. The change R124G that leads to Avr3aEMG is located in the a-helix loop 3 and the changes Q133H and M139L (Avr3aKIH, Avr3aEMH and Avr3aKIL) locate to a helix 4. Whereas amino acid substitutions in PEX147-3 only affect surface exposed residues, amino acid changes that occur in PEX147-2 involves a ‘buried’ amino acid that is key to structure and stability. Indeed, with the exception of PEX147-2, all Avr3a variants and PEX147-3 are stable upon transient expression in planta and in yeast cells. In terms of host recognition, the protein products of the Avr3a alleles derived from Avr3aKI are recognised by the cognate host resistance gene product R3a whereas those derived from Avr3aEM evade recognition. Similarly, PEX147-3 is recognised by R3a but PEX147-2 is not. In addition to host recognition, virulence functions of these alleles and paralogs have been elucidated. INF1 and AVR4/CF-4 induced cell death responses, which are dependent on the host defence protein CMPG1, are suppressed by Avr3aKI, Avr3aKIH, Avr3aKIL, Avr3aEM and Avr3aEMG but not by Avr3aEMH. All Avr3a variants interact with and stabilise the host E3 ubiquitin ligase CMPG1 to various degrees in planta and this interaction was found to be weakest for Avr3aEMH. Interestingly, PEX147-3, which did not interact with or stabilise CMPG1, could only suppress INF1 cell death but not CF-4/AVR4 elicited responses. A P. infestans isolate, CS12, which was stably silenced for Avr3aEM expression and subsequently shown to be compromised in virulence on the normally susceptible host Nicotiana benthamiana, was used for in planta complementation studies. As shown previously, upon transient expression in planta prior to infection with C12, Avr3aKI and Avr3aEM successfully restore pathogenicity. Similar levels of virulence re-establishment were only observed for Avr3KI derived alleles Avr3aKIH and Avr3aKIL but not for alleles derived from Avr3aEM. This study concludes that Avr3aEM is currently the only form of the essential effector that is fully functional and evades recognition by the known resistance gene product R3a. This functionality is the likely reason that 70% of all studied isolates in the Toluca Valley are homozygous for Avr3aEM. This form of the effector is therefore a suitable target for identifying more durable resistances.

The intracellular bacterial pathogen Legionella pneumophila is the etiological agent of Legionnaires’ disease, a severe pneumonia; it has also served as a valuable tool in studying host-pathogen interactions. The study of L. pneumophila pathogenesis has led to the discovery of novel biochemical and enzymatic mechanisms and a better understanding of host cell immune responses and signaling. L. pneumophila replicates within eukaryotic cells through the use of a type IV secretion system and over 330 effector proteins injected into the host cell. Only approximately 10% of these effectors have been characterized, but regardless of the small fraction, the complexity of L. pneumophila infection is clear. A good demonstration of this complexity is the large number of effector activities the bacteria uses to manipulate the small GTPase involved in ER to Golgi trafficking, Rab1. Six different effectors with eight separate activities modulate the activity of Rab1 to aid in the replication of the bacteria. We recently discovered that the protein SetA is yet another effector targeting Rab1. SetA glucosylates Rab1 using a canonical DxD motif and the glucose moiety interferes with both GTP hydrolysis and guanosine nucleotide dissociation inhibitor (GDI) binding. Based on our findings, the role of SetA is likely to aid in maintaining a pool of free Rab1, increasing availability for use by other L. pneumophila effectors. Another example of the complexity of L. pneumophila pathogenesis is the use of metaeffectors. Metaeffectors are effectors that regulate other effectors, both being produced by L. pneumophila. Three mechanisms of metaeffector regulation have been identified: 1) removal of a modification on host proteins placed by the cognate effector, 2) direct modification of the cognate effector or 3) direct binding to the cognate effector. Through the use of Size Exclusion Chromatography (SEC), binding assays with purified proteins and bacterial two-hybrid analysis, we found the mechanism of regulation for the SidI metaeffector Lpg2505 to be inactivation through direct binding. Atypical of previously identified effector characteristics, the binding of SidI by Lpg2505 occurs within the bacterial cell prior to translocation. The expression pattern of both effectors in L. pneumophila in addition to the other findings suggest a temporal role for Lpg2505 activity in which inactivation of SidI occurs after sufficient bacterial replication has occurred.<br>

Magnaporthe grisea is a notorious pathogenic fungus that causes rice blast disease worldwide. Proteins secreted by the fungus are likely candidates for being effectors that are potentially recognized by determinants of resistance or susceptibility in host plants. However, knowledge of the role of secreted proteins of M. grisea is still limited. In this study, I identified 29 proteins that were secreted into culture filtrates from M. grisea strains expressing candidate proteins. I confirmed secretion of these proteins and tested them for elicitor activity on plants. Among them, I studied two groups: cell wall degrading enzymes (CWDEs) and small cysteine-rich proteins. Cysteine-rich proteins have been shown in other systems to function as elicitors. Initially, I expressed and purified proteins in M. grisea to obtain proteins by a homologous expression system. Although this was effective for a number of proteins, the need for greater amounts of protein led me to express several proteins in the Pichia pastoris system. Several candidate proteins were purified and found to induce symptoms on rice and maize. Hypothetical proteins MG10424.4 and MG09998.4 were both found to have elicitor activity. Lipase MG07016.4 did not induce response of plants and we concluded that the lipase activity of MG07016.4 does not function as an elicitor. I also purified a small cysteine-rich protein, which belongs to the group of cluster 180 proteins in M. grisea, MG10732.4 from P. pastoris. It is able to cause yellowing symptoms and hydrogen peroxide production in plants and it might contain elicitor activity.

No description available.

No description available.

No description available.

<p>The bacterial pathogen <i>Legionella pneumophila</i> modulates host immunity using effectors translocated by its Dot/Icm transporter to facilitate its intracellular replication. A number of these effectors employ diverse mechanisms to interfere with protein ubiquitination, a post-translational modification essential for immunity. Here, we have found that <i>L. pneumophila</i> induces monoubiquitination of the E2 enzyme UBE2N by its Dot/Icm substrate MavC(Lpg2147). Ubiquitination of UBE2N by MavC abolishes its activity in the formation of K63-linked polyubiquitin chains, which dampens NF-kB signaling in the initial phase of bacterial infection. The inhibition of UBE2N activity by MavC creates a conundrum because this E2 enzyme is important in multiple signaling pathways, including some that are important for intracellular <i>L. pneumophila</i> replication. Here we also show that the activity of UBE2N is restored by MvcA(Lpg2148), an ortholog of MavC. MvcA functions to deubiquitinate UBE2N-Ub using the same catalytic triad required for its deamidase activity. Structural analysis of the MvcA-UBE2N-Ub complex reveals a crucial role of the insertion domain in MvcA in substrate recognition. Our findings reveal that two remarkably similar proteins catalyze the forward and reverse reactions to impose temporal regulation of the activity of UBE2N during <i>L. pneumophila</i> infection.</p>

This was a project joint between the University of Dundee and The James Hutton Institute where both parties were interested in further understanding the interactions between plant host proteins and pathogen effector proteins. An objective of this thesis was to determine the host target of the Phytophthora infestans effector PiAVR2 and the means by which this avirulence protein is recognised by the potato resistance protein, R2. Prior to this PhD, forward genetic studies identified three RXLR effector encoding genes within the AVR2 locus. By use of transient co-expression with the resistance gene R2 it was determined which of these genes was PiAVR2. A virulent form of PiAVR2, named PiAVR2-like, was found within isolates of P. infestans. Isolates which only express PiAVR2-like are virulent on potato cultivars expressing R2. Isolates which express both forms, or only the PiAVR2 form, are avirulent on cultivars expressing R2. This suggests that expressing only PiAVR2-like is key to the virulence of the pathogen on R2 expressing cultivars. There are 10 known orthologues of R2 which all recognise PiAVR2. However none can recognise PiAVR2-like. The characterisation of the means by which P. infestans overcomes R2 resistance has provided a strategy, based on identifying R genes that recognise PiAVR2-like, to provide durable late blight disease resistance. It was also discovered that both PiAVR2 and PiAVR2-like physically interact with the same host target proteins, BSL1, BSL2a and BSL2b. The BSLs are part of a family of Kelch repeat containing Ser/Thr phosphatases which function as activators of the brassinosteroid signal transduction pathway. It was shown that silencing of the BSL1 and BSL2a genes within plants results in the attenuation of PiAVR2 recognition by R2. In the case of BSL1 it was further shown that an interaction between R2 and BSL1 only occurs in the presence of PiAVR2. This implies that R2 recognises PiAVR2 by an indirect mechanism, utilising either the Guard or Decoy Hypotheses, and that BSL1 is essential for this recognition. This is the first reported demonstration of indirect recognition of an intracellular eukaryotic plant pathogen effector protein.

Pests and pathogens represent a serious and continuing threat to potato and tomato production worldwide. In this thesis, I have developed a new NB-LRRs probe library accounting for the recent improved annotations of both potato and tomato (Jupe et al., 2013 and Andolfo et al., 2014). The probe library was successfully used to map a late blight resistance in the diploid potato population B3C1HP. Using bulked-segregant resistance gene enrichment and sequencing (RenSeq) analysis in this population, which segregated 1:1 for the phenotype, the resistance was mapped to the lower end of chromosome 9. Furthermore, I developed a novel diagnostic tool, dRenSeq, to screen existing germplasm collection for the presence or absence of known, already characterised disease resistance genes, to prioritise novel resistances for research and breeding. dRenSeq was applied successfully on a set of S. okadae accessions as a proof of concept. The tomato late blight resistance gene Rpi-Ph3 was another focal point in this work, and the use of RenSeq was envisaged to identify Rpi-Ph3. However, another team published the gene (Zhang et al., 2014) and efforts were redirected towards the development of PCR markers to aid marker-assisted selection in breeding programs and to identify the cognate avirulence gene, Avr-Ph3. In addition, the new probe library was assessed in silico to evaluate if it would have enabled the identification of Rpi-Ph3 and homologous sequences. The identification of Avr-Ph3 was established through a large effector screen in an association panel of tomato accessions, co-infiltrations with Rpi-Ph3 in the model Solanaceae plant Nicotiana benthamiana and pathogen assays. The effector screen required the prior establishment of a robust transient expression system in tomato.