173 research outputs found
The Cladosporium fulvum - tomato interaction : physiological and molecular aspects of pathogenesis
No full textIn this thesis research on the physiological and molecular aspects of pathogenesis in the interaction between tomato and Cladosporium fulvum Cooke (syn. Fulvia fulva [Cooke] Cif) is described. This plant-fungus interaction is envisaged to be based on a gene-for-gene relationship. Incompatible interactions (plants are resistant) between certain races of C. fulvum and tomato are thought to result from a specific interaction between products of fungal avirulence genes (racespecific elicitors) and products of corresponding resistance genes (cultivar-specific receptors) that are present in the host. After the elicitor has bound to the receptor, host defense genes are activated. A major feature of the activation of host defense is the accumulation of several pathogenesis-related (PR) proteins. Generally these proteins, which also accumulate in several other plant species, are of low molecular weight, accumulate in the apoplast, are highly resistant to proteolytic cleavage and have extreme iso-electric points. In compatible interactions (plants are susceptible) presumably no molecular recognition of the fungus occurs, resulting in colonization of the apoplastic space between the leaf mesophyll cells.In chapter 2 the purification of a fungal protein (designated P1, molecular mass 14 kD), specific for compatible C. fulvum -tomato interactions is described. Polyclonal antibodies were raised and the protein was shown to be only present in apoplastic fluid isolated from compatible C. fulvum -tomato interactions. Immunolocalization experiments revealed that in compatible interactions the protein was present in the electron-dense matrix between the walls of leaf mesophyll cells and fungal hyphae (chapter 6). Probably P1 plays a role in the establishment or maintenance of basic compatibility and can be regarded as a basic pathogenicity factor.In compatible interactions the fungus is able to hydrolyze the translocation sugar sucrose to glucose and fructose, which in turn are converted into the polyol mannitol by mannitol dehydrogenase (MTLDH) (chapter 3). During the colonization process of the intercellular spaces of the tomato leaves, increasing amounts of mannitol present in the apoplastic fluid coincided with increasing levels of MTLDH activity. The fungal metabolite mannitol cannot be utilized by the plant and possibly functions as a carbohydrate reserve for the fungus. In incompatible interactions no functional nutritional relationship between host and fungus is established and consequently no mannitol accumulation was observed.Chapter 4 describes the partial purification of a race-specific elicitor, the putative product of avirulence gene 4 ( avr4 ) of C. fulvum. The race-specific elicitor precipitated in 60% (v/v) acetone, migrated on high pH, native gels and bound to an anion-exchange column at pH 9.0. The elicitor preparation induced a hypersensitive response and accumulation of PR proteins in near-isogenic line Cf4 of tomato (carrying resistance gene 4), indicating that active host defense is triggered by recognition of a race-specific elicitor by the plant.In incompatible interactions between tomato and C. fulvum the inhibition of fungal growth coincides with a substantial accumulation of PR proteins in the apoplast of the tomato leaf (chapter 5). Two abundantly occurring PR proteins of 35 kD and 26 kD in molecular mass were purified and were shown to have 1,3-β-glucanase and chitinase activity, respectively. Fungal walls that partly consist of 1,3-β-glucans and chitin, can be affected by these hydrolytic enzymes. With polyclonal antibodies that were raised against the purified enzymes one additional 1,3-β-glucanase (33 kD) and three additional chitinases (27, 30 and 32 kD) were detected in apoplastic fluids or homogenates of tomato leaves after inoculation with C.fulvum. Upon inoculation with C.fulvum apoplastic chitinase and 1,3-β-glucanase activities increased more rapidly in incompatible interactions than in compatible ones, indicating that these hydrolytic enzymes might play a role in active host defense.Immunolocalization experiments revealed that in incompatible tomato- C.fulvum interactions 1,3-β-glucanases and chitinases accumulated in intercellular spaces, cytoplasm and electron-dense material that was present in the vacuoles of leaf mesophyll cells (chapter 6). Often 1,3-β-glucanases and chitinases were found to be associated with the electron-dense outer layer of the fungal cell wall. In compatible interactions no localized accumulation of 1,3-β-glucanases and chitinases was observed.In addition to the rapid induction and accumulation of 1,3-β-glucanases and chitinases in incompatible tomato- C.fulvum interactions, a substantial accumulation of PR proteins of about 15 kD in molecular mass occurred. It was shown that in apoplastic fluids isolated from induced tomato leaves three basic PR proteins are present that migrate similarly to the earlier characterized tomato PR protein P14 on SDS-polyacrylamide gels (chapter 7). Two proteins, designated P4 and P6, molecular mass 15.5 kD, isoelectric points (pI) 10.9 and 10.7, respectively, appeared to be serologically related to each other and to the tobacco PR-1 proteins. The third protein, designated P2, molecular mass 15 kD, pI 10.4, was found to be serologically related to PR-R from tobacco. The biological function of P2, P4 and P6 is still unknown.In chapter 8 the characterization of messenger RNA (mRNA) for P6, the most abundant isomer of P14, is described. The mRNA contains an open reading frame of 477 nucleotides, encoding a protein of 159 amino acids, with an N-terminal signal peptide of 24 amino acids. Synthesis of P6 is regulated at the transcriptional level. In the incompatible interaction Cf4/race 5 there was a much faster accumulation of the P6 mRNA than in the compatible one (Cf5/race 5). There are probably two to four genes present in the genome of tomato that encode P14-like proteins
Regulation and activation of SOBIR1-containing receptor complexes involved in plant immune signalling
No full textThe tomato cell surface receptor-like protein (RLP) Cf-4 confers resistance to Avr4-secreting strains of the fungus Cladosporium fulvum, which causes tomato leaf mould disease. Cf-4 constitutively interacts with the receptor-like kinase (RLK) SOBIR1 and interacts with the RLK BAK1 upon recognition of Avr4 by Cf-4. Formation of the Cf-4/SOBIR1/BAK1 complex is proposed to trigger phosphorylation of the intracellular kinase domains of SOBIR1 and BAK1, which subsequently activate downstream signalling resulting in plant immunity. However, components involved in regulating the Avr4-induced formation and subsequent activation of the Cf-4/SOBIR1/BAK1 complex remain unknown. In addition, downstream components required for Cf-4 signalling are largely unknown. The work described in this thesis is aimed at gaining more insight into the molecular mechanisms of Cf-4 signalling. Furthermore, the application of the gained knowledge to genetically engineer plant immunity is pursued. Additionally, as SOBIR1, which is functionally required for all RLPs mediating immunity, also interacts with the RLP CLAVATA2 (CLV2) involved in development, the significance of the CLV2/SOBIR1 complex is investigated
The Cladosporium fulvum-tomato interaction: elicitor proteins and their perception
No full textThe gene-for-gene concept postulates that for every dominant gene determining resistance in the host plant, there is a corresponding dominant gene conditioning avirulence in the pathogen. The simplest way to explain the biochemical basis of this concept is direct interaction between an elicitor protein, which is encoded by an avirulence ( Avr ) gene of the pathogen, and a receptor protein, which is encoded by the matching resistance ( R ) gene of the host. Perception of the elicitor protein by the host plant subsequently leads to the activation of defence responses, often including a hypersensitive response (HR).The research described in this thesis is focussed on the characterisation of elicitor proteins of the fungus Cladosporium fulvum and the analysis of their perception by resistant tomato plants. A striking feature of all elicitor proteins of C. fulvum is that their mature form contains an even number of cysteine residues. These cysteine residues are thought to be involved in disulfide bridges, which are essential for proper conformation and stability of the elicitors. Mutational analysis of elicitor proteins ECP1, ECP2 and ECP5, however, revealed that the role of (the even number) of cysteine residues is more complex than anticipated, as not all cysteine residues appeared to be critical for the HR-inducing activity of the elicitor proteins (Chapter 2).During colonisation of the apoplastic space of tomato leaves, C. fulvum secretes elicitor proteins into the apoplast. All Cf genes, mediating resistance to particular races of C. fulvum , are predicted to encode extracytoplasmic, membrane-anchored glycoproteins that contain many leucine-rich repeats (LRRs). LRR domains are thought to be involved in protein-protein interactions. The extracellular localisation of the LRR region of the Cf proteins is consistent with a direct, extracellular perception of the corresponding elicitor proteins. To validate this hypothesis, binding studies were performed between avirulence protein AVR9 and the matching resistance protein Cf-9. Although extensive studies were performed in a multidisciplinary collaboration to prove a direct interaction between the two proteins, no specific binding between AVR9 and Cf-9 could be detected (Chapter 3). This implies that the simplest interpretation of the gene-for-gene concept, involving direct interaction of a pathogen-derived elicitor with a matching resistance gene product, does not hold for the Avr9 / Cf-9 gene pair and that at least a third component is involved in the perception of AVR9 by Cf-9.Also for Avr2/Cf-2 -mediated resistance a third component, Rcr3, seems to be involved. To allow dissection of the biochemical mechanism of perception of avirulence protein AVR2, we set out to clone Avr2 (Chapter 4). Avr2 cDNA was cloned based on the specific HR-inducing activity of the encoded protein in Cf2 tomato plants. Like the other Avr genes of C. fulvum , Avr2 encodes a small, secreted protein with an even number of cysteine residues. Analysis of strains of C. fulvum that are virulent on Cf2 tomato lines revealed various mutations in the Avr2 ORF that all result in the production of a truncated AVR2 protein. Interestingly, an additional modification was discovered, involving the insertion of a LINE-like element (a retrotransposable element), Cfl1, in the Avr2 ORF. Cfl1 is the first LINE-like element identified in C. fulvum and provides the first example of loss of avirulence of a plant pathogen due to insertion of a retrotransposable element in an Avr gene. Analysis of two different rcr3-mutant Cf2 tomato plants revealed that their ability to respond to AVR2 with a HR correlates with their degree of resistance to AVR2-producing strains of C. fulvum . These data support a role for Rcr3 in the perception of AVR2 by Cf-2.Direct perception of elicitor proteins by R proteins has been the prevailing working hypothesis to explain the biochemical basis of the gene-for-gene concept for years. The results of the research that is described in this thesis, however, do not support this hypothesis. Also for most other gene-for-gene relationships studied so far, experimental evidence appears to be more consistent with indirect perception of an AVR protein by an R protein (Chapter 5). Indirect perception implies that, beside the AVR and the R protein, at least a third component is required to induce defence responses. For several gene-for-gene relationships the nature of the putative third component is known. Although each of these components are suggested to be involved in basal defence mechanisms, their nature appears to be diverse. Hence, we argue that, although some elicitors might be directly perceived by the matching R protein, for most gene-for-gene relationships elicitor perception will turn out to be more complex
The Cf-4 en Cf-9 resistance proteins of tomato : molecular aspects of specificity and elicitor perception
No full textTo feed the increasing world population, agricultural production needs continuous improvement. Especially protection of crops from disastrous diseases is crucial. The interaction between the pathogenic fungus Cladosporium fulvum and its host, tomato, serves as a model system for plant-pathogen interactions. Some tomato plants carry resistance ( R ) genes that confer recognition of fungal strains carrying complementary avirulence ( Avr ) genes. A number of these R genes have been cloned, as well as their complementary Avr genes. The aim of the research described in this thesis was to examine how R gene products confer recognition of fungal strains carrying the matching Avr genes. Profound understanding of the molecular basis of this interaction might help us to improve the protection of other crop plants against economically important diseases.Chapter 1 introduces the state of the art on interaction between Cladosporium fulvum and tomato at the time the research described in this thesis was initiated. C. fulvum is a specialised, biotrophic pathogen, causing tomato leaf mold. The fungus infects tomato leaves by entering stomata at the lower side of the leaf. The infection will proceed if no resistance R genes of the plant match any of the Avr genes of the fungus. However, the plant recognises the fungus when it carries an R gene that matches an Avr gene present in the fungus. This recognition results in the induction of plant defence responses, including a rapid death of cells surrounding the infection site, called the hypersensitive response (HR). Further fungal growth is prohibited by these defence responses. During its lifecycle on susceptible plants, C. fulvum is restricted to the extracellular space of the tomato leaves and secretes many proteins that potentially play a role in virulence. Also the elicitor proteins encoded by the Avr9 and Avr4 are secreted. Injection of these proteins is sufficient to trigger HR in tomato plants carrying Cf-9 and Cf-4 resistance genes, respectively. Both AVR9 and AVR4 are small, stable, cysteine-rich proteins. The complementary Cf-9 and Cf-4 genes encode highly similar, membrane-anchored, receptor-like proteins with extracytoplasmic leucine-rich repeats (LRRs) and a short cytoplasmic tail. Differences between Cf-9 and Cf-4 proteins are located in the N-terminal half, predominantly in amino acid residues at putative solvent-exposed positions of the LRRs, which is thought to form the 'recognition surface' of these proteins.To examine the role of the various domains of Cf proteins in perception of AVR proteins of C. fulvum in more detail, a functional, transient expression system was developed for the Cf-4 and Cf-9 resistance genes ( chapter 2 ). This expression system is based on infiltration of tobacco leaves with Agrobacterium strains that carry Cf genes on the T-DNA of binary plasmids (agroinfiltration). The AVR proteins are delivered either by injection, agroinfiltration, Potato Virus X-mediated expression or by using Avr -transgenic tobacco plants. This chapter also describes differences between Avr9/Cf-9 - and Avr4/Cf-4 -induced necrosis, which are mainly due to a difference in Avr gene activity upon expression in the plant. Finally, it is shown that the signal transduction pathway leading to HR is conserved in solanaceous plants, but likely not in non-solanaceous plant species. An exception is the non-solanaceous plant lettuce, in which the Avr4/Cf-4 gene pair is functional. The agroinfiltration assay is an excellent expression system to study the effect of mutations in Cf genes. In chapter 3 , agroinfiltration was used to determine specificity determinants in Cf proteins by exchanging domains between Cf-4 and Cf-9 and subsequently examining the effect of these mutations on specificity of perception of AVR proteins. Cf-4 differs from Cf-9 at 67 amino acid positions and also contains three deletions. Significantly, Cf-4 lacks two LRRs compared to Cf-9, which appears essential for Cf-4 function. The two additional LRRs in Cf-9 are required for Cf-9 function. Specificity determinants in Cf-4 reside not only in the LRR domain but also in the B-domain. In contrast, specificity determinants in Cf-9 reside entirely in the LRR domain and are likely scattered throughout this domain. The specificity determinants in the LRRs of Cf-4 cluster in a few adjacent LRRs and reside in only three amino acid residues at putative solvent-exposed positions. Thus, most of the 67 amino acids that vary between Cf-4 and Cf-9 appear not to be required for specificity, but probably serve as a source to generate new specificities. To learn more about specificity determinants of Cf-9 proteins occurring in natural populations, we examined the molecular variation of Cf-9 in Lycopersicon pimpinellifolium (Lp) , from which the Cf-9 locus has been introgressed into cultivated tomato ( chapter 4 ). It appears that AVR9 recognition occurs frequently throughout the Lp population. In addition to Cf-9 , a second gene, designated 9DC , confers AVR9 recognition in Lp . Compared to Cf-9 , 9DC is more polymorphic, occurs more frequently and is more widely spread throughout the Lp population, suggesting that 9DC is older than Cf-9 . The second half of the 9DC gene is nearly identical to the second half of Cf-9 , whereas the first half is nearly identical to Hcr9-9D , a Cf homolog adjacent to Cf-9 at the Cf-9 locus. This suggests that Cf-9 has evolved by intragenic recombination between 9DC and another Cf homolog. The fact that 9DC and Cf-9 proteins both confer recognition of AVR9 but differ in 61 amino acid residues shows that Hcr9 proteins can be highly variable, without affecting their recognitional specificity.After having examined their specificity determinants, we subsequently focused on the cellular location of Cf proteins. The presence of a dilysine motif in the G-domain of Cf-9 ( KK RY) suggests that the protein resides in the endoplasmic reticulum (ER) instead of the plasma membrane (PM). Previously, two conflicting reports on the subcellular location of Cf-9 were published. One report showed that Cf-9 accumulates in the ER and is absent in the plasma membrane, whereas the other showed that Cf-9 resides in the plasma membrane. In chapter 5 we have mutated the dilysine motif and show that the mutant Cf-9 protein remains functional in AVR9 recognition and mediation of HR. The data presented in this chapter, in combination with the two previous reports on Cf-9 localisation, can be explained by assuming that proteins that interact with Cf-9 mask the dilysine motif. This theory suggests that functional Cf-9 protein resides in small quantities in the plasma membrane, where it mediates recognition of the extracellular AVR9 protein as a component of a receptor complex. AVR9 recognition in tomato plants carrying Cf-9 most likely involves the high-affinity binding site (HABS) for AVR9 that was identified in plasma membranes. However, the HABS is not encoded by Cf-9 because it is also present in tomato plants that lack Cf-9 and in many other plant species. As it is likely that both the HABS and the Cf-9 protein reside in the plasma membrane and may be present in the same receptor complex, it is essential to isolate the HABS in order to get more insight in the molecular mechanism of AVR9 perception. In chapter 6 , a procedure is described that allows solubilisation of the HABS without affecting its AVR9-binding activity. Of the 19 detergents that were tested, only octyl glucoside appeared to be suitable for solubilisation of the HABS. Removal of the detergent is crucial in this procedure, as it interferes with AVR9 binding. The described procedure may become an essential tool to study the AVR9 receptor complex at the biochemical level. In the final chapter ( chapter 7 ), the experimental data presented in the previous chapters are discussed. In addition to AVR9/Cf-9 there are many other examples of gene-for-gene interactions where no direct interaction was found between R and Avr gene products. In many cases, there are indications for the involvement of an additional host protein, which may represent the virulence target of the Avr protein. The prevalence of R proteins that 'guard' virulence targets can be explained by natural selection for R genes that are maintained in the plant population through 'trench-warfare', resulting in recognition events that cannot be circumvented by the pathogen without taking a virulence penalty. The 'guard' hypothesis significantly changes the focus of current research to the role of virulence targets of Avr proteins, and might explain absence of functionality of R genes in heterologous plant species, despite the fact that they belong to conserved gene families.</font
SOBIR1-containing immune complexes at the plant cell surface: partners and signalling
No full textWe can learn from nature, by studying the mechanisms by which plants defend themselves against pathogens. This knowledge can be applied to improve crops in a sustainable fashion. Plants are sessile organisms with multiple layers of defence against pathogens. A prominent first layer of defence, which is similar to the innate immune system of mammalian cells, is provided by transmembrane (TM)-receptors, which are present at the plant cell surface. Upon pathogen invasion of the extracellular (apoplastic) space, TM-receptors mediate the recognition of pathogen- and/or host derived invasion patterns (IPs) in the extracellular space using their extracellular domain (ECD), which often consists of leucine-rich repeats (LRRs). Next to the ECD, TM-receptors have a single pass TM domain, and an intracellular domain. TM-receptors can be receptor-like kinases (LRR-RLKs), which contain an intracellular kinase domain to enable cytoplasmic signalling, or receptor-like proteins (LRR-RLPs), which contain only a small cytoplasmic tail and no signalling domain. LRR-RLKs and LRR-RLPs are here further referred to as RLKs and RLPs. The regulatory RLK Suppressor Of BIR1-1/Evershed (SOBIR1/EVR, further referred to as SOBIR1) was recently found to constitutively interact with RLPs, and thereby support RLP accumulation as a kind of scaffold protein. Additionally, SOBIR1 was proposed to provide a signalling domain to RLPs, to result in bimolecular RLKs. RLKs, as well as bimolecular RLP/SOBIR1 complexes, both recruit the co-receptor Brassinosteroid-Insensitive 1 - Associated Kinase 1/Somatic Embryogenesis Receptor Kinase 3 (BAK1/SERK3, further referred to as BAK1) upon ligand recognition by the primary ligand receptor. This BAK1 recruitment is thought to activate the TM-receptor complex for downstream signalling. Cultivated tomato (Solanum lycopersicum, Sl) possesses cell surface receptors, introgressed from wild tomato varieties, that provide resistance to the biotrophic leaf pathogen Cladosporium fulvum. These so-called Cf proteins mediate recognition of secreted effectors (also known as avirulence factors (Avrs)) of the pathogen in the apoplast. These Cf proteins are RLPs, and have recently been shown to constitutively interact with the RLK SOBIR1. The work in this thesis was initiated to elucidate the nature of the signalling steps that take place downstream of RLP/SOBIR1 bimolecular RLKs. To this aim, the signalling events that take place upon activation of the Cf-4/SOBIR1 complex by the matching C. fulvum effector Avr4 were studied. In Chapter 1, the plant innate immune system is introduced, and an overview of the current knowledge is given. The main focus lays on cell surface receptor complexes with an ECD consisting of LRRs, on their regulation, and on the immune responses that they trigger. Moreover, the tomato – C. fulvum pathosystem is introduced, with emphasis on the Cf-4/Avr4 gene-for-gene pair. SOBIR1 is required for RLP-mediated resistance to a wide range of pathogens, and it is hypothesized that SOBIR1 is targeted by effector proteins of pathogens to suppress host defence responses. In Chapter 2 it is shown that AvrPto, an effector of the bacterial pathogen Pseudomonas syringae pv. tomato DC3000, interacts with SOBIR1 from Arabidopsis thaliana (At, further referred to as Arabidopsis) and with various Solanaceous SOBIR1 orthologues. This interaction is independent of SOBIR1 kinase activity. Interestingly, AvrPto suppresses AtSOBIR1-induced constitutive immunity, which is observed as cell death (the hypersensitive response (HR)) upon overexpression of AtSOBIR1 in N. benthamiana and tobacco. Additionally, AvrPto compromises the Avr4-triggered HR in Cf-4-transgenic N. benthamiana, without affecting Cf-4/SOBIR1/BAK1 complex formation. These results demonstrate that the bacterial effector AvrPto targets the regulatory RLK SOBIR1, and thereby compromises SOBIR1-mediated defence responses. In Chapter 3 it is shown that kinase activity of SOBIR1 is not essential for its scaffold function, as the kinase-dead mutant SlSOBIR1D473N also stabilizes Cf-4, similar to wild-type SOBIR1. However, kinase activity of SOBIR1 is crucial for downstream immune signalling, and therefore it is hypothesised that SOBIR1 transphosphorylates downstream signalling partners to initiate the activation of defence responses. Phosphorylation of signalling partners is an important molecular switch in various cellular processes, including plant defence. It was observed that AtSOBIR1, which constitutively activates immune responses upon its overexpression in N. benthamiana and tobacco, is highly phosphorylated in planta. Moreover, next to the required kinase activity of SOBIR1, kinase-active BAK1 is also essential for AtSOBIR1-induced constitutive immunity and for the phosphorylation of AtSOBIR1. Furthermore, the activation of a defence response upon perception of Avr4 by Cf-4 depends on signalling-competent BAK1. These results, in addition to observations described in literature about other RLK signalling partners, suggest that SOBIR1 likely first transphosphorylates BAK1 upon its recruitment to the ligand-activated RLP/SOBIR1 complex, after which activated BAK1 transphosphorylates SOBIR1 to subsequently together initiate downstream signalling for immunity. Phosphorylation of SOBIR1 appears to be important for its role in signalling for defence and Chapter 4 elaborates on the findings described in Chapter 3. In Chapter 4, amino acids of the kinase domain of SOBIR1 that could potentially be phosphorylated upon pathogen recognition are identified, and the role of these potential phosphorylation sites in signalling for defence is analysed. Mutational analyses and three-dimensional modelling showed that the threonine (Thr, T) residues T519, T523, and T529, which are all highly conserved in the activation segment of the kinase domain of SOBIR1, are important residues for the role of SOBIR1 in immune signalling. Phosphorylation of these sites likely locks SOBIR1 in an active conformation by controlling the conformation of the activation loop. Phosphorylation of these amino acids likely stimulates the interaction of T523 and T529 with the arginine (Arg, R) residue and the catalytic aspartic acid (Asp, D) residue of the ‘RD’ motif, respectively. Moreover, phosphorylation on T522, and the tyrosine (Tyr, Y) residues 532 and 538, which are also highly conserved, is likely generating substrate specificity and differential affinity for interacting partners. Co-immunoprecipitation of Cf-4-associated SOBIR1, through a pull-down of Cf-4 in the resting state and in the Avr4-activated state from N. benthamiana:Cf-4-eGFP plants, and subsequent analysis via mass spectrometry (MS), did not identify differential phosphorylation of the SOBIR1 kinase domain. However, in planta overexpression of AtSOBIR1-eGFP, followed by its immunoprecipitation and analysis via MS, revealed that AtSOBIR1 is phosphorylated on several serine (Ser, S) and Thr residues of its kinase domain, including T519. It is concluded that specific phosphorylation of the kinase domain of SOBIR1 likely enables this regulatory RLK to specifically switch on immune signalling downstream of RLPs. Directly downstream of RLKs, plants employ receptor-like cytoplasmic kinases (RLCKs) to signal for defence. Botrytis-Induced Kinase 1 (BIK1) is a central RLCK that signals downstream of several RLKs, including Flagellin-Sensing 2 (FLS2) in Arabidopsis. In Chapter 5, BIK1 is shown to be also important for defence signalling downstream of RLP/SOBIR1-containing complexes, as AtBIK1 was found to interact with AtSOBIR1, as well as with SlSOBIR1. Moreover, overexpression of the closest Solanaceous BIK1 orthologues enhanced the HR triggered by Cf-4 upon recognition of Avr4. On the contrary, overexpression of AtBIK1 appeared to suppress the Cf-4/Avr4-triggered HR. Although a silencing screen of a broad set of N. benthamiana BIK1-homologues did not point to a clear Solanaceous RLCK involved in the Cf-4/SOBIR1-mediated HR, a split-luciferase assay showed the interaction between several tomato BIK1-homologues and SlSOBIR1 and AtFLS2. Furthermore, the tomato RLCK Tomato Protein Kinase 1b (SlTPK1b) was shown to specifically interact with SlSOBIR1. Together, these data suggest that RLCKs play a role in signalling downstream of RLP/SOBIR1 complexes. TM-receptors need to be tightly controlled and regulated to ensure the triggering of a robust defence signal, and at the same time prevent false activation of defence responses. Physical separation of receptors and their co-receptors on the PM by negative regulators helps to keep signalling for defence in check, and thereby retain plant homeostasis concerning growth and development on the one hand and immunity on the other hand. The BAK1-Interacting RLK (BIR) protein family of Arabidopsis is a group of RLKs that negatively regulate immunity by interfering with the hetero-dimerization of the co-receptor BAK1 with ligand-binding immune-receptors. In Chapter 6, it is shown that the BIR protein family is conserved in Solanaceous plants. Gene silencing, overexpression, and protein-protein interaction studies show that the Solanaceous BIR1 orthologues are, similar to AtBIR1, negative regulators of cell death, and that BIR1 might suppress the Avr4-triggered HR in tomato containing Cf-4. Additionally, BIR2 orthologues of tomato and N. benthamiana seem not to be involved in modulating the Cf-4/Avr4-triggered HR. From these results it is concluded that SlBIR1 might be a negative regulator of SOBIR1-mediated defence, possibly through its interaction with SlBAK1, whereas SlBIR2 appears not to be involved in the regulation of Cf-4/SOBIR1-mediated defence. This leads to the hypothesis that BIR1 and BIR2 each likely interact with a different pool of BAK1 present at the plasma membrane, and that each pool is probably involved in a different defence pathway. Chapter 7 summarizes and discusses the major findings described in this thesis. The results of this thesis, together with other recent fundamental discoveries describing plant-microbe interactions on a molecular level, support a refinement of the invasion model that was developed to describe plant-microbe interactions. All TM-receptors with an LRR ECD recruit BAK1 upon their activation by the matching ligand, leading to transphosphorylation events and the initiation of downstream defence responses. These defence responses can be either mild or strong. Strikingly, there are RLP/SOBIR1 complexes that signal for a strong defence response including an HR, and also RLP/SOBIR1 complexes that signal for basal defence responses. All RLP/SOBIR1 complexes tested to far require BAK1 recruitment, and probably transphosphorylation of the kinase domains of SOBIR1 and BAK1 to initiate defence signalling. Therefore, as all signalling initiated by TM-receptors seems to be similar on the molecular level, this supports a spatial division rather than a division based on the intensity of the generated defence responses, which was up till now mostly used. In the ‘spatial invasion model’, IPs are therefore proposed to be classified as either extracellular IPs (ExIPs) or intracellular IPs (InIPs). As a consequence, recognition of ExIPs by TM-receptors leads to extracellularly-triggered immunity (ExTI), and recognition of InIPs by cytoplasmic receptors (which are mostly nucleotide-binding leucine-rich repeat receptors, NB-LRRs) leads to intracellularly-triggered immunity (InTI). Using this spatial dichotomy, the spatial invasion model facilitates a broadly including, but clearly distinguishing nomenclature to describe plant-microbe interactions.</p
Cf-dependent early defence responses induced by avirulence proteins of the tomato pathogen : Cladosporium fulvum
No full textThe outcome of a plant-pathogen interaction is determined by both the presence of resistance ( R ) genes in the plant and matching avirulence ( Avr ) genes in the pathogen. According to the gene-for-gene concept, for a dominant R gene in the host plant resistant to a specific strain of a pathogen, a corresponding dominant Avr gene exists in that strain of the pathogen. R gene-mediated recognition of an Avr gene product triggers a signal transduction cascade, eventually resulting in a hypersensitive response (HR). This HR consists of a collapse of plant cells at the primary site of infection, resulting in an arrest of growth of the pathogen. As it is impractical to study defense signaling responses in whole plants, often cell suspensions are used for this purpose. Directly after treatment of cell suspensions with elicitors, activation of signal transduction processes, such as ion fluxes over the plasma membrane (detectable as alkalization of the extra cellular medium), phospholipid signaling and protein phosphorylation occur. Also reactive oxygen species, which are thought to play a role both in defense signaling and in direct defense against the pathogen, are produced after AVR perception. The interaction between Cladosporium fulvum and tomato is a well-studied plant-pathogen interaction that obeys the gene-for-gene concept. From this pathosystem several resistance ( Cf ) and Avr genes have been cloned, from which the matching gene pairs Cf-4/Avr4 and Cf-9/Avr9 are the best studied. Although many efforts were undertaken to study defense signaling in cell suspensions derived from Cf- carrying tomato plants, they were not responsive to the matching AVRs. Therefore, Cf4 +-and Cf-9 +- tobacco cell suspensions, which are responsive to the matching AVR protein, were used to study Cf/Avr- mediated defense signaling. In Chapter 2, defense responses in Cf-9 +- tobacco leaves and Cf-9 +- tobacco cell suspensions induced by both wild-type (WT-AVR9) and mutant AVR9 analogues were studied. Upon injection into leaves of both tomato MM-Cf9 and Cf-9 +- tobacco leaves, the mutant AVR9 peptides R08K, F10A and F21A showed higher, lower and no necrosis-inducing activity, respectively, as compared to WT-AVR9. Similar relative activities were found for these 4 peptides when assayed in Cf-9 +- tobacco cell suspensions. R08K showed a stronger, whereas F10A and F21A showed a lower oxidative burst-inducing activity as compared to WT-AVR9. In a medium alkalization assay equal activities were observed for R08K and WT-AVR9, whereas F10A showed a lower activity and no medium alkalization activity at all was observed for F21A. Surprisingly, the oxidative burst was induced at peptide concentrations that were 100 times lower as compared to those inducing medium alkalization. Concentrations inducing a full medium alkalization response are similar to peptide concentrations that induce necrosis in leaves of Cf-9- carrying tomato or tobacco plants. Treatment of Cf-9 +- tobacco cell suspensions with WT-AVR9 resulted in the activation of a MAP kinase, whereas F21A activated the MAP kinase only to a small extent. WT-AVR9 also induced massive cell death at 18 hr after addition to Cf-9 +- tobacco cell suspensions, whereas tobacco cells not expressing Cf-9 remained viable, illustrating the specificity of this response. In Chapter 3 we have shown that, upon AVR4 treatment of Cf-4 +-tobacco cells, levels of the second messenger phosphatidic acid (PA) increased dramatically. This response occurred within 2 min after addition of AVR4 and was highly specific. The PA conversion product diacylglycerol pyrophosphate (DGPP) accumulated between 4-8 minutes after addition of AVR4. Whether DGPP is a second messenger in its own right or serves as a negative regulator for PA signaling is still unclear. A differential labeling strategy showed that AVR4-induced PA accumulation resulted predominantly from the conversion of diacylglycerol (DAG) into PA by diacylglycerol kinase (DGK). DAG can be generated during signaling events by the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) by phospholipase C (PLC). Pretreatment of Cf-4 +- tobacco cells with the PLC inhibitors neomycin and U73122 blocked AVR4-induced PA accumulation, indicating that PA is indeed generated via PLC activity. The AVR4-induced oxidative burst was blocked by the NADPH oxidase inhibitor diphenylene iodonium (DPI), whereas it did not block AVR4-induced PA accumulation. Conversely, the PLC inhibitor U73122 blocked both AVR4-induced PA accumulation and oxidative burst in a dose-dependent way. Treatment with a synthetic, water-soluble PA derivative induced a small, and transient oxidative burst in Cf-4 +- tobacco cells. These data demonstrate the importance of phospholipid signaling in the AVR4-induced oxidative burst. Additional studies showed that AVR4-induced medium alkalization and MAP kinase activation were also blocked by PLC inhibitors, suggesting that these responses are also PLC-dependent. During the experiments described in Chapter 2 it became clear that AVR9-induced defense responses are temperature sensitive. The temperature-sensitivity of Cf/Avr- mediated defense responses is studied in Chapter 4. Injection of AVR4 or AVR9 in leaves of tobacco and tomato plants carrying Cf - 4 or Cf-9, respectively, resulted in necrosis in the injected area at 20°C, whereas at 33°C this response was suppressed. At 20°C, tomato seedlings expressing both a Cf gene and matching Avr gene germinate but develop systemic HR after unfolding of the cotyledons and subsequently die. These seedlings could be rescued at 33°C but rapidly died after transfer to 20°C. Gel blot analysis of RNA isolated at different time points after transfer of the rescued Cf/Avr seedlings to 20°C, revealed a controlled induction of expression of various typical defense-related genes. This synchronized onset of HR provides an excellent basis for the identification of novel, HR-related genes by cDNA-AFLP analysis. In cell suspensions we found that both the AVR4- and AVR9-induced medium alkalization response is slowly suppressed at 33°C, but quickly recovers upon transfer to 15°C. For AVR4- and AVR9-induced medium alkalization, differences in the kinetics of the suppression of this response at an elevated temperature were demonstrated. The high affinity binding site for AVR9 is thought to be the AVR9 receptor, which is involved in the initiation of Cf-9/Avr9- mediated defense responses. It was shown that binding of AVR9 to microsomal fractions isolated from cell suspensions incubated at 33°C was decreased by 80%, as compared to microsomal fractions isolated from cell suspensions incubated at 20°C. The decrease of AVR9 binding was caused by a decrease in amount of binding sites rather than by a decrease in the affinity of the binding site for AVR9, providing a molecular basis for the temperature sensitivity of Cf-9/Avr9- mediated defense. In Chapter 5 the results described in this thesis are discussed and some additional unpublished data are included. A model for AVR-induced signaling in the C. fulvum/ tomato interaction is presented. In addition, new techniques, future experiments and perspectives to further unravel Cf/Avr- mediated signaling are discussed
Isolation of apoplastic fluid from leaf tissue by the vacuum infiltration-centrifugation technique
No full textUpon infection of plants by pathogens, at least at the early stages of infection, the interaction between the two organisms occurs in the apoplast. To study the molecular basis of host susceptibility vs. resistance on the one hand, and pathogen virulence vs. avirulence on the other, the identification of extracellular compounds such as pathogen effectors that determine the outcome of the interaction is essential. Here, I describe the vacuum infiltration-centrifugation technique, which is an extremely simple and straightforward method to explore one of the most important battlefields of a plant–pathogen interaction; the apoplast
Regulation of the avirulence gene Avr9 of the fungal tomato pathogen Cladosporium fulvum
No full textDuring growth of a pathogen in host tissue, pathogenicity genes are usually highly expressed. A detailed understanding of how these pathogenicity genes are regulated is required to gain a better insight in the molecular communication between pathogen and host. Chapter one describes several bacterial and fungal genes, which are envisaged to be involved in pathogenicity and are induced in vitro during growth under nutrient-limiting conditions. Based on the data described in this chapter, we speculate that in plants, pathogens encounter an environment in which nutrients are limiting. Lack of nitrogen might be one of the key factors that induce these pathogenicity genes.The interaction between the fungus Cladosporium fulvum and its only host, tomato, is used as a model system to study plant-pathogen interactions. This interaction is a typical gene-for-gene relationship, that states that for each avirulence ( Avr ) gene in the pathogen there is a corresponding resistance ( R ) gene in the plant. Direct or indirect interaction between the products of Avr and R genes leads to incompatibility.The object of the research performed in this thesis was to obtain a better understanding of the factor(s) involved in regulation of the C. fulvum avirulence gene Avr 9, which is highly expressed in planta during colonisation of the intercellular spaces of tomato leaves. The product of this gene is specifically recognised by tomato plants carrying matching resistance gene Cf-9 . After recognition, the plant mounts a hypersensitive response (HR) that eventually leads to resistance against the fungus.Before the study was initiated it was known that the Avr 9 gene is induced under conditions of nitrogen starvation in vitro . Furthermore, several (TA)GATA sequences were found to be present in the Avr 9 promoter. These sequences had earlier been identified as the binding sites for a wide-domain GATA-type regulator (AREA in Aspergillus nidulans and NIT2 in Neurospora crassa ), involved in nitrogen utilisation. Both observations made it likely to hypothesise that a similar regulator would be involved in induction of Avr 9 expression in C. fulvum and that nitrogen-limitation in the apoplast is the environmental factor that induces Avr 9 expression in planta .Chapter two describes the Avr 9 promoter activity in A. nidulans transformants, containing a single copy of an Avr 9 promoter- uid A (GUS) reporter gene fusion in different are A backgrounds ( are A wild-type , are A minus, are A constitutive), targeted at the arg B locus, following nitrogen starvation. Induction of the Avr 9 promoter was found to be similarly regulated in A. nidulans and C. fulvum , indicating that the AREA protein of A. nidulans is able to induce the Avr 9 promoter and that C. fulvum contains an AREA-like regulator that can bind to (TA)GATA sequences.Chapter three describes a mutational analysis of these (TA)GATA sequences which reveals that two TAGATA-boxes, located most proximal to the start codon, both containing two invertedly orientated TAGATA sequences, are crucial for inducibility of Avr 9 promoter activity in A. nidulans .Mutated Avr 9 promoter fragments which did not show any inducibility in A. nidulans were fused to the Avr 9 coding region and introduced (not targeted) into strains of C. fulvum lacking Avr 9. However, in C. fulvum transformants the Avr 9 gene was induced when they were grown in rich, liquid media, a condition which normally suppresses Avr 9 gene expression. We have no Southern data on the transformants but it could be that multiple integrations have caused the loss of nitrogen-dependent Avr 9 regulation both in vitro and in planta . This result emphasises that for reliable promoter studies in C. fulvum a gene-targeting system is required.The development of such a system for C . fulvum is described in Chapter four . For this purpose, the C. fulvumpyr 1 gene was isolated. The pyr 1 gene codes for the enzyme orotidine-5'-monophosphate decarboxylase, which is involved in the pyrimidine biosynthetic pathway and is considered to be a versatile selection marker for filamentous fungi. The isolation of the C. fulvumpyr 1 gene was based on complementation of an A. nidulanspyr G-minus mutant strain which was simultaneously transformed with digested genomic DNA of C. fulvum containing the wild-type pyr 1 gene and an autonomously-replicating plasmid.C. fulvumpyr 1 + transformants were obtained by introducing a vector, containing the C. fulvumpyr 1 gene with a defined mutation, into a C. fulvum pyr 1-mutant strain. Southern blot analysis of these transformants showed that site-directed integration of this vector at the pyr 1 locus had occurred. Thus, targeting of constructs of interest to the pyr 1 locus of C. fulvum is feasible.Isolation of the are A/ nit -2 homologue of C. fulvum , designated Nrf 1 (for n itrogen r esponse f actor 1), is described in Chapter five . The gene encodes a protein which contains a putative zinc finger DNA-binding domain that is 98% identical to the zinc finger domain present in the AREA and NIT2 proteins. Function equivalence of Nrf 1 to are A was demonstrated by complementation of an A. nidulansare A-minus mutant with Nrf 1. Expression analysis in liquid media revealed that, in contrast to what occurs in wild-type C. fulvum strains, in Nrf 1-deficient strains the Avr 9 gene is not induced under conditions of nitrogen starvation. However, Nrf 1-deficient strains were still avirulent on tomato plants containing the Cf-9 resistance gene, indicating that in planta still sufficient quantities of the AVR9 elicitor are produced. It appears that, although NRF1 is a major regulator of the Avr 9 gene expression, in planta at least one additional positive regulator of Avr 9 gene expression is active.In Chapter six we studied the effect of elevated nitrogen levels on expression of Avr 9 in C. fulvum grown in planta . We observed that tomato plants containing both the Cf-9 gene and elevated levels of nitrate in the apoplast show partial resistance against strains of C. fulvum containing the Avr 9 gene. This implies that the elevated level of nitrate in the apoplast represses Avr 9 expression.In Chapter seven the data obtained in this research project are discussed in relation to other known avirulence genes. It is still unknown why, in their host, pathogens would produce proteins that betray them. A possible role for the AVR9 elicitor as a kind of "survival protein" for the fungus during infection is discussed. Although, it appears that regulation of the Avr 9 gene is associated with nitrogen circuits in C. fulvum , regulation of Avr 9 by NRF1 in vitro and in planta is not similar. The isolation of additional plant factor(s) which are able to induce Avr 9 is a challenge for future research.</p
Activation and signal transduction of SOBIR1/BAK1-containing immune complexes present at the plant cell surface
No full textPlants have evolved a two-layered innate immune system to cope with invading microbes from the surrounding environment. Receptors present at the plasma membrane, which are either receptor-like kinases (RLKs) or receptor-like proteins (RLPs), form the first layer of the plant innate immune system and play an important role in mediating host resistance against various pathogens. Our research focuses on the interaction between the extracellular pathogenic fungus Cladosporium fulvum and tomato (Solanum lycopersicum, Sl). Previously, we have shown that the cell-surface receptor Cf-4, which is a leucine-rich repeat (LRR)-RLP, specifically recognizes the apoplastic effector Avr4 secreted by C. fulvum. This recognition leads to the resistance of tomato to C. fulvum. In contrast to RLKs, RLPs lack an intracellular kinase domain for downstream signaling. Interestingly, our subsequent research has found SUPPRESSOR OF BIR1-1/EVERSHED (SOBIR1/EVR, further referred to as SOBIR1), which is an LRR-RLK, to constitutively interact with Cf-4 and to thereby provide Cf-4 with a kinase domain. Consistent with LRR-RLK-mediated immune signaling, the Cf-4/SOBIR1 complex recruits the LRR-RLK BRI-ASSOCIATED KINASE 1/SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3 (BAK1/SERK3, further referred to as BAK1) to initiate plant immunity, upon the perception of Avr4 by Cf-4. Notably, BAK1 recruitment was later found to be a general phenomenon upon ligand-mediated activation of the LRR-RLP/SOBIR1 complex. The research described in this thesis was aimed to elucidate the molecular mechanisms behind the activation of the Cf-4/SOBIR1/BAK1 complex and to identify essential receptor-like cytoplasmic kinases (RLCKs) that play a role in immune signaling downstream of the Cf-4/SOBIR1 complex.SOBIR1 is a positive regulator of LRR-RLP-mediated immune signaling and appears to be present throughout the plant kingdom. Overexpression of Arabidopsis (Arabidopsis thaliana, At) SOBIR1 results in constitutive activation of cell death and associated defense responses in both Arabidopsis and the model Solanaceous plant Nicotiana benthamiana (Nb). Nevertheless, no symptoms of auto-immunity were observed when NbSOBIR1, SlSOBIR1, or SlSOBIR1-like was overexpressed in leaves of N. benthamiana plants. To generate the material that can be used to characterize the function of SOBIR1 in planta and to study the fundamentals of plant immunity triggered by Avr4/Cf-4, we employed the CRISPR/Cas9 system to knock out SOBIR1 in N. benthamiana. We show that we successfully knocked out SOBIR1 and its homolog SOBIR1-like both in wild-type N. benthamiana and in N. benthamiana stably expressing the Cf-4 transgene. Strikingly, N. benthamiana sobir1 (/sobir1-like) knock-out plants are non-responsive to the Avr4/Cf-4 combination, and consistently, N. benthamiana:Cf-4 sobir1 (/sobir1-like) knock-out plants are also non-responsive to Avr4.The sobir1 (/sobir1-like) knock-out plants were subsequently implemented for complementation studies, combined with a site-directed mutagenesis screen of putative phosphorylation sites present in SOBIR1. One serine (Ser/S) and four threonine (Thr/T) residues that are present in the activation segment of the kinase domain of SOBIR1 were studied. NbSOBIR1T522, as well as its analogous residues in SlSOBIR1 and SlSOBIR1-like, was identified to be essential for Avr4/Cf-4-induced reactive oxygen species (ROS) accumulation, the activation of a mitogen-activated protein kinase (MAPK) cascade, and the hypersensitive response (HR). Further in vitro phosphorylation assays demonstrated that this particular Thr residue is required for the intrinsic kinase activity of SOBIR1. Additionally, we provide in vitro evidence to support the SOBIR1/BAK1 activation model that was proposed before.Recently, Tyr phosphorylation has been recognized to be a common feature of RLK activation in plants. Therefore, all Tyr residues present in the kinase domain of NbSOBIR1, SlSOBIR1, and SlSOBIR1-like were individually changed into the non-phosphorylatable amino acid phenylalanine (Phe/F) and the obtained mutants were included in the complementation study. We show that NbSOBIR1Y469, as well as its analogous residues in SlSOBIR1 and SlSOBIR1-like, plays a crucial role in Avr4/Cf-4-triggered MAPK activation and the HR, whereas this residue is not essential for the Avr4/Cf-4-induced ROS production or the intrinsic kinase activity of SOBIR1. However, no phosphorylated Tyr residue was detected in the kinase domain of either AtSOBIR1 or NbSOBIR1 by mass spectrometry. Therefore, we propose that SOBIR1 employs this important Tyr residue in its kinase domain to trigger plant immunity by binding a specific substrate, instead of being important in a phosphorylated state.Cell-surface receptors deploy a large number of downstream RLCKs to relay the immune signals from the extracellular space into the plant cells. We show that eight members from the RLCK class VII subfamily 6 (RLCK-VII-6) in N. benthamiana:Cf-4 plants play a pivotal role in regulating the production of ROS that is stimulated by multiple extracellular immunogenic patterns (ExIPs), including the Avr4 protein. Strikingly, these eight members are dispensable for Avr/Cf-4-triggered MAPK activation and the HR. More importantly, despite their different subcellular localization, these eight members appear to function redundantly as positive regulators of the Avr4/Cf-4-induced ROS accumulation. Furthermore, members from the RLCK-VII-7 and -8 subfamilies might also be essential for the Avr4/Cf-4-triggered ROS burst. However, the molecular mechanisms by which the various members from the RLCK-VII-6 and RLCK-VII-7/8 subfamilies regulate the production of ROS in N. benthamiana are not known
Plant resistance genes : their structure, function and evolution
No full textPlants have developed efficient mechanisms to avoid infection or to mount responses that render them resistant upon attack by a pathogen. One of the best-studied defence mechanisms is based on gene-for-gene resistance through which plants, harbouring specific resistance (R) genes, specifically recognise pathogens carrying matching avirulence (Avr) genes. Here a review of the R genes that have been cloned is given. Although in most cases it is not clear how R gene encoded proteins initiate pathways leading to disease resistance, we will show that there are clear parallels with disease prevention in animal systems. Furthermore, some evolutionary mechanisms acting on R genes to create novel recognitional specificities will be discussed
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