No description available.

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.

No description available.

published_or_final_version / Ecology and Biodiversity / Doctoral / Doctor of Philosophy

Many oomycetes, such as the notorious potato late blight pathogen Phytophthora infestans, have devastating effects on crops. Recent findings have implied that eukaryotic plant pathogens deliver effector proteins inside host cells to facilitate colonization by modulating plant defences. Although the translocation mechanisms remain unknown, in oomycetes this process depends on a short conserved amino acid sequence located near the signal peptide of many secreted proteins. This sequence, termed the RxLR motif, is strikingly similar to the core RxLxE/D/Q host cell targeting-signal that is found in virulence proteins from the malaria parasite Plasmodium falciparum. Common infection strategies have been described for these divergent pathogens, albeit one is a plant pathogen while the other infects human cells. In this thesis, stable transformation of P. infestans, combined with a validated assay based on the intracellular recognition of the RxLR effector, A vr3a, was used to study the specificity of effector translocation in oomycetes and to demonstrate the functional similarity between translocation motifs from Plasmodium and two distantly related oomycetes. While accumulating evidence shows that RxLR effectors are delivered into the host cell, the subcellular targeting of these proteins is still unclear. Throughout this PhD project, the difficulty of visualizing translocated effectors during infection was tackled by seeking alternative approaches allowing the detection of fluorescently- tagged effectors once delivered into the plant cell. A further key research question addressed in this thesis was whether the mechanism of translocation required pathogen-encoded proteins or a pathogen- induced environment. Purified fluorescent protein fusions of A vr3a were used to demonstrate that this plant pathogen effector may hold the intrinsic ability to traverse the plasma membrane of animal cells.

Una de las opciones para el control de la enfermedad más devastadora del cultivo de papa (Solanum tuberosum), el tizón tardío, producido por Phytophthora infestans, es el desarrollo de variedades resistentes a este patógeno, mediante la transferencia directa de genes de resistencia R por ingeniería genética. En el siguiente trabajo de investigación, se usó el gen RB de Solanum bulbocastanum, que otorga un amplio espectro de resistencia a razas de P. infestans. Para dicho fin, se transformó genéticamente vía Agrobacterium tumefaciens la variedad susceptible de papa Desiree (Solanum tuberosum) con el vector binario pCIP68 que contiene el gen RB. Como resultado, se obtuvieron 19 plantas transformadas con el gen RB, confirmadas por la prueba de resistencia a kanamicina y por la prueba de reacción en cadena de la polimerasa (PCR). Las 19 plantas transgénicas fueron sometidas a infección en invernadero bajo condiciones de bioseguridad con el aislamiento POX067 de P. infestans perteneciente al linaje clonal EC-1 que es dominante en el Perú. Tres de las 19 plantas ([RB]54, [RB]56 y [RB]70) presentaron un alto nivel de resistencia al aislamiento POX067 de P. infestans.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2018-08-22T19:15:30Z No. of bitstreams: 1 2013 - Evandro Silva Pereira Costa.pdf: 2748177 bytes, checksum: 3632d387d4cb90bea88d42726fae79ca (MD5) / Made available in DSpace on 2018-08-22T19:15:30Z (GMT). No. of bitstreams: 1 2013 - Evandro Silva Pereira Costa.pdf: 2748177 bytes, checksum: 3632d387d4cb90bea88d42726fae79ca (MD5) Previous issue date: 2013-12-18 / CAPES / The present work had the objective to characterize 59 accessions of tomato of the cherry group regarding its agronomic characteristics and resistance to the late blight and to select those more adapted to the organic agriculture. As standards were used: Carolina, Perinha ?gua Branca, Pending Yashi, Joanna and the hybrids Super Sweet, Sweet Million and Mascot F1. The experiments were conducted under field conditions in the Horticulture Sector of the Federal Rural University of Rio de Janeiro (UFRRJ) from June 2010 to November 2013 during which eight trials were carried out. In the different trials, productivity, number of total fruits, disease progression, morphological characteristics (coloration, shape, number of locules), physical (longitudinal and equatorial diameter), and physicochemical characteristics of fruits (total soluble solids - TSS , titratable total acidity - TTA, pH, TSS / TTA ratio). It was also evaluated the accumulation of dry mass of the stem, leaves and fruits, content and content of nitrogen (N), potassium (K) and phosphorus (P) in the respective organs. It was observed a great genetic variability between the accessions as to the physical and physical-chemical morphological attributes. Promising accesses were selected for use in breeding programs and with great productive potential and potential for cultivation in organic systems. Among these, we highlight the ENAS 1040, ENAS 1037, ENAS 1031 and ENAS 1026 accessions for cultivation in the spring / summer period and the accesses ENAS 1228, ENAS 1214, ENAS 1227 and ENAS 1220 by the highest total soluble solids (?Brix). The accessions ENAS 1121, EAS 1013, ENAS 1143 and ENAS 1029 were distinguished by the production of fruits with differentiated formats and the accesses ENAS 1007, ENAS 1008, ENAS 1037, ENAS 1033, ENAS 1017, ENAS 1036, ENAS 1062 and ENAS 1029 by coloring of the fruits. The accessions ENAS 1227 and 1026 stood out by the partial resistance to the late blight, equivalent to the standards 'Carolina' and 'Perinha ?gua Branca'. The accessions ENAS 1227, ENAS 1216, ENAS 1153 and ENAS 1060 were the ones that stood out the most regarding the resistance to the late blight and productivity. / O presente trabalho teve como objetivo caracterizar 59 acessos de tomateiro do grupo cereja quanto ?s suas caracter?sticas agron?micas e resist?ncia ? requeima e selecionar aqueles mais adaptados ? agricultura org?nica. Como padr?es foram utilizadas: Carolina, Perinha ?gua Branca, Pendente Yashi, Joanna e os h?bridos Super Sweet, Sweet Million e Mascot F1. Os experimentos foram conduzidos em condi??es de campo, no Setor de Horticultura da Universidade Federal Rural do Rio de Janeiro (UFRRJ) no per?odo de junho de 2010 a novembro de 2013 durante o qual realizaram-se oito ensaios. Nos diferentes ensaios, avaliaram-se produtividade, n?mero de frutos totais, progresso da requeima, caracter?sticas morfol?gicas (colora??o, formato, n?mero de l?culos), f?sicas (di?metro longitudinal e equatorial), e f?sico-qu?mica dos frutos (s?lidos sol?veis totais - SST, acidez total titulav?l - ATT, pH, rela??o SST/ATT). Avaliou-se, ainda, ac?mulo de massa seca do caule, folhas e frutos, teor e conte?do de nitrog?nio (N), pot?ssio (K) e f?sforo (P) nos respectivos ?rg?os. Observou-se grande variabilidade gen?tica entre os acessos quanto aos atributos morfol?gicos f?sicos e f?sico-qu?micos. Selecionaram-se acessos promissores para uso em programas de melhoramento e com grande potencial produtivo e com potencial para cultivo em sistemas org?nicos. Dentre estes, destacam-se os acessos ENAS 1040, ENAS 1037, ENAS 1031 e ENAS 1026 para cultivo no per?odo de primavera/ver?o e os acessos ENAS 1228, ENAS 1214, ENAS 1227 e ENAS 1220 pelos maiores teores de s?lidos sol?veis totais (?Brix). Os acessos ENAS 1121, EANAS 1013, ENAS 1143 e ENAS 1029 destacaram-se pela produ??o de frutos com formatos diferenciados e os acessos ENAS 1007, ENAS 1008, ENAS 1037, ENAS 1033, ENAS 1017, ENAS 1036, ENAS 1062 e ENAS 1029 pela colora??o dos frutos. Os acessos ENAS 1227 e 1026 destacaram-se pela resist?ncia parcial ? requeima, equivalente ? dos padr?es ?Carolina? e ?Perinha ?gua Branca?. Os acessos ENAS 1227, ENAS 1216, ENAS 1153 e ENAS 1060 foram os que mais se destacaram quanto a resist?ncia ? requeima e produtividade.

The late blight pathogen, Phytophthora infestans, is the most destructive pathogen of its solanaceous hosts potato and tomato. It is a threat to global food security and it is therefore important to understand the cellular and molecular dynamics underlying colonisation of its host plants. This greater understanding will inform strategies to improve host plant resistance. In addition to studying the cell biology of the interaction, it is important to understand the temporal changes in gene expression and regulation during host-pathogen interactions at the earliest infection time points. Previously published transcriptomic studies of P. infestans used two days post infection (dpi) as the earliest sampling time point. Expression of a marker gene (Hmp1) for biotrophy and a selection of effector coding genes has been reported as early as 12 hours post inoculation (hpi), suggesting that infection was initiated before then. Transcriptomic studies of P. infestans have focussed mostly on leaf tissue, and there is still a lack of research on the transcriptome of P. infestans grown in alternative plant tissues such as tubers, or in host cell-free apoplastic fluid. This thesis explores transcriptomic studies of the early, biotrophic stages of potato infection by Phytophthora infestans, which is critical for understanding which genes are involved at what stages of infection development. By using the latest sensitive microarray technology to study the P. infestans transcriptome in an infection time course that remained biotrophic for its duration, a list of 1,707 transcripts of P. infestans were discovered to be differentially expressed. This list included 114 transcripts for RxLR effectors, out of which 26 were detected from 12 hours post infection, including: Avr2, Avr3a, Avrblb1 (ipi01), Avrblb2, and the recently characterised RD2. Also of interest was that transcripts encoding a PAMP (CBEL) detected at 12 hours, were suppressed in the pathogen by 24 hours. Transcripts encoding 55 RxLR effectors were co-expressed (with >95 % correlation coefficient) with the biotrophy marker gene Hmp1, suggesting that these effectors are important throughout the biotrophic stages of infection. QRT-PCR and cell biology data supported the expression of the biotrophy marker gene Hmp1 as early as 12 hours after infection and this was further supported by the co-expression of avirulence genes such as Avr2 and Avr3a. A set of 17 transcripts, including six cytoplasmic effectors (RxLR effectors), as well as a transcript encoding an apoplastic effector (glucanase inhibitor), was found to be infection-specific, supporting the hypothesis that these genes might have roles in establishing biotrophy. By examining pathogen behaviour in tuber tissue, clear cell biology evidence of functional haustoria was found. Gene expression analysis of a selection of leaf infection-related genes suggested that effectors are used to promote infection also in host tuber tissue. However, some cytoplasmic RXLR effector proteins such as PITG_05146 and PITG_15128, which were up-regulated during biotrophic infection of leaf tissue, were not detected during tuber infection, indicating potential differences in pathogenic requirements. A microarray experiment was conducted on in vitro stages of zoospores, and mycelium grown in apoplastic fluid of N. benthamiana, nutrient rich pea broth, and sterile water. This revealed 13,819 transcripts that were differentially expressed between any two conditions. This list included transcripts encoding 322 RxLR effectors, of which avirulence effectors such as Avr2, Avr3a, and RD2 were highly up-regulated during hyphal growth in apoplastic fluid compared to other in vitro stages. This provides evidence that the apoplast contains chemical signals that induce expression of infection-related genes in P. infestans. Curiously, the leaf infection-specific genes identified in Chapter 3 were not expressed when P. infestans was grown in apoplastic fluid, revealing that additional stimuli are required for induction of all necessary pathogen genes during infection. Future research, building upon the findings from this project, should be focused on the following areas: 1) Explore whether haustoria are produced only in order to deliver effectors or if there are other purposes as well, such as nutrient uptake; 2) The continued exploration of differences between genes co-expressed with Hmp1 during leaf infection, tuber infection, and in apoplastic fluid to further dissect the transcriptional regulation of these genes; 3) Identify whether Hmp1-co-expressed genes of unknown function may play a role in haustorium formation; 4) Investigate, using molecular transformation and cell biology, whether secreted proteins co-expressed with Hmp1 are secreted from haustoria; 5) Investigate the role(s) of infection-specific genes in establishing disease. 6) Transcriptomic studies of P. infestans biotrophic infection of tuber tissue to determine the differences in pathogenic adaptation in this tissue type, compared to leaf infection.

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.