Phosphorylation and proteome dynamics in pathogen-resistant tomato plants

Microbial plant pathogens impose a continuous threat on global food production. Similar to disease resistance in mammals, an innate immune system allows plants to recognise pathogens and swiftly activate defence. For the work described in this thesis, the interaction between tomato and the extracellular fungal pathogen Cladosporium fulvum serves as a model system to study host resistance and susceptibility in plant-pathogen interactions. Resistance to C. fulvum in tomato plants follows the gene-for-gene hypothesis, which requires the presence of a Cf resistance gene in tomato and presence of the cognate avirulence gene (Avr) in C. fulvum. Upon perception of the Avr by a tomato plant, a typical hypersensitive response (HR) is induced that renders the plant resistant to C. fulvum. In the years preceding this thesis work, most research was focussed on understanding which Avrs are produced by C. fulvum and how these Avrs are actually perceived by resistant plants (Chapter 1). The goal of the work described in this thesis is to reveal downstream signalling cascades triggered upon Avr perception. Therefore, the HR was studied by using a model system in which the Cf-4 protein of tomato and the Avr4 protein from C. fulvum were simultaneously expressed in tomato seedlings. Since the Cf-4/Avr-induced responses are inhibited at 33°C and high humidity, these Cf-4/Avr4 seedlings initiate a synchronized and reproducible HR after incubation at 33°C and a subsequent shift to 20°C, which allows studying downstream responses. To prevent pathogen proliferation in the resistant plant, defence signalling cascades need to be activated extremely fast upon pathogen recognition. Therefore, many downstream signalling cascades depend on post-translational modifications (PTMs) that allow a rapid, reversible, controlled and highly specific transduction of perceived signals. An overview of the various types of PTMs and their role in the resistance response of plants to pathogens is provided in Chapter 2. In addition, examples are provided of successful pathogens that manipulate PTMs. Protein phosphorylation seems to play an important role in the Cf-4/Avr4-triggered HR, since Avr4 perception leads to the specific activation of at least three mitogen-activated protein kinases, LeMPK1, -2 and -3, which requires phosphorylation by an upstream kinase (Chapter 3). Each of these three kinases seems to have a different role in downstream defence signalling, since the kinases were shown to have different phosphorylation specificities and therefore most likely have different downstream target substrates. Furthermore, these kinases appear to play a different role with regard to HR and full resistance to C. fulvum in tomato (Chapter 3). Since protein phosphorylation was shown to play an important role in Cf-4/Avr4-induced defence signalling, the phosphoproteome of Cf-4/Avr4 and control seedlings after HR initiation was studied using a new approach (Chapter 4). This approach led to the identification of 50 phosphoproteins, most of which have not been described in tomato before. Quantification revealed 13 phosphoproteins with an altered abundance in the Cf-4/Avr4 seedlings as compared to the control, which implies HR-induced differential phosphorylation of these proteins. Phosphorylation-mediated regulation of the activity of these proteins pointed to a swift decrease in photosynthetic activity upon HR-initiation, which was confirmed by experiments in which the actual efficiency of the photosynthesis in the Cf-4/Avr4 seedlings was determined upon induction of the HR. Furthermore, a shift from aerobic to anaerobic respiration, which possibly results from oxygen depletion caused by a massive oxidative burst consuming large amounts of oxygen, seems to take place upon initiation of the HR. Finally, differential phosphorylation of the four cytoplasmic isoforms of the Hsp90 chaperone protein was observed, suggesting that they play distinct roles during defence signalling (Chapter 4). In addition to the HR, other associated defence responses are initiated upon recognition of C. fulvum. One of these responses is the secretion of defence-related proteins into the apoplast, which is the environment where C. fulvum operates. Therefore, the dynamics of the apoplastic proteome of resistant, Cf-4-expressing plants and susceptible tomato plants lacking Cf-4, were studied after inoculation with a strain of C. fulvum that secretes Avr4 (Chapter 5). Analysis of the apoplastic proteome revealed a slow accumulation of defence proteins in the apoplast of susceptible plants, which is most likely the result of perception of general elicitors of C. fulvum by tomato. In resistant plants, the same set of proteins accumulates in the apoplast, but this occurs much faster and to higher levels. The accelerated response is caused by the Cf-4/Avr4-initiated HR that also leads to cell death. The HR, in combination with the accelerated protein secretion, renders the plants resistant to C. fulvum. In addition, in susceptible plants C. fulvum seems to specifically downregulate genes encoding cell wall proteins of which the accumulation possibly hampers nutrient and water uptake and thereby proliferation of the pathogen in the tomato apoplast. Possibly, an effector of C. fulvum targets a receptor for general elicitors, thereby suppressing transcription of these genes (Chapter 5). Most data described in this thesis have been obtained from Cf-4/Avr4 seedlings in which the HR can be inhibited by incubating the plants at 33°C. The present data suggest that this temperature-sensitivity occurs at the site of signal perception. Possibly, cytoplasmic Hsp90 stabilizes R protein complexes localized at the plasma membrane. Upon high temperature stress, an increased demand for Hsp90 occurs in the cells to stabilize unfolding proteins that play a role in basal cellular processes, which could lead to the release and subsequent degradation of R protein complexes, rendering defence signalling temperature-sensitive (Chapter 6). The temperature-sensitivity of the Cf-4/Avr4-initiated HR provides a very clean and reproducible tool to study the HR, in the absence of the fungus that produces the Avr. Furthermore, the data described in this thesis provide evidence that the Cf-4/Avr4 seedlings recover from the temperature stress before the specific Cf-4/Avr4-triggered HR is initiated. The possibility to separate the events directly associated with the HR from the full resistance response of the plant to the invading fungus, provides new insight into the complexity of plant defence responses and their specific suppression upon successful colonization by C. fulvum (Chapter 6). Comparison of the defence response to other processes that occur in the cell underlines that resistance and HR execution cannot be seen as an independent and separate process in resistant plants that have recognized a pathogen. On the contrary, signalling cascades seem to depend on similar components and on cascades that possibly converge, eventually leading to a similar response (Chapter 6). Finally, an up to date model for the Cf-4/Avr4-triggered HR and resistance is proposed, based on data that have been published before and the results obtained with the research described in this thesis (Chapter 6). <br/

Post-translational modification of host proteins in pathogen-triggered defence signalling in plants

Microbial plant pathogens impose a continuous threat to global food production. Similar to animals, an innate immune system allows plants to recognize pathogens and swiftly activate defence. To activate a rapid response, receptor-mediated pathogen perception and subsequent downstream signalling depends on post-translational modification (PTM) of components essential for defence signalling. We discuss different types of PTMs that play a role in mounting plant immunity, which include phosphorylation, glycosylation, ubiquitination, sumoylation, nitrosylation, myristoylation, palmitoylation and glycosylphosphatidylinositol (GPI)-anchoring. PTMs are rapid, reversible, controlled and highly specific, and provide a tool to regulate protein stability, activity and localization. Here, we give an overview of PTMs that modify components essential for defence signalling at the site of signal perception, during secondary messenger production and during signalling in the cytoplasm. In addition, we discuss effectors from pathogens that suppress plant defence responses by interfering with host PTMs

Quantitative phosphoproteomics of tomato mounting a hypersensitive response reveals a swift suppression of photosynthetic activity and a differential role for Hsp90 isoforms

An important mechanism by which plants defend themselves against pathogens is the rapid execution of a hypersensitive response (HR). Tomato plants containing the Cf-4 resistance gene mount an HR that relies on the activation of phosphorylation cascades, when challenged with the Avr4 elicitor secreted by the pathogenic fungus Cladosporium fulvum. Phosphopeptides were isolated from tomato seedlings expressing both Cf-4 and Avr4 using titanium dioxide columns and LC-MS/MS analysis led to the identification of 50 phosphoproteins, most of which have not been described in tomato before. Phosphopeptides were quantified using a label-free approach based on the MS peak areas. We identified 12 phosphopeptides for which the abundance changed upon HR initiation, as compared to control seedlings. Our results suggest that photosynthetic activity is specifically suppressed in a phosphorylation-dependent way during the very early stages of HR development. In addition, phosphopeptides originating from four Hsp90 isoforms exhibited altered abundances in Cf-4/Avr4 seedlings compared to control seedlings, suggesting that the isoforms of this chaperone protein have a different function in defense signaling. We show that label-free relative quantification of the phosphoproteome of complex samples is feasible, allowing extension of our knowledge on the general physiology and defense signaling of plants mounting the HR

Potexvirussen volgen in water, grond en bollen

Inundatie of biofertigatie helpen niet tegen TVX. De virussen PlAMV en TVX verspreiden zich onder meer via onkruid, groenbemsters en grond. Wageningen Universiteit, Vertify en BKD zoeken in de PPS ‘Op weg naar virusvrij en afzetgericht telen’ hoe potexvirussen zich in het veld verspreiden. Virusvrij starten en onkruidvrij telen zijn essentieel, blijkt uit de PPS

System-Wide Hypersensitive Response-Associated Transcriptome and Metabolome Reprogramming in Tomato

The hypersensitive response (HR) is considered to be the hallmark of the resistance response of plants to pathogens. To study HR-associated transcriptome and metabolome reprogramming in tomato (Solanum lycopersicum), we used plants that express both a resistance gene to Cladosporium fulvum and the matching avirulence gene of this pathogen. In these plants, massive reprogramming occurred, and we found that the HR and associated processes are highly energy demanding. Ubiquitin-dependent protein degradation, hydrolysis of sugars, and lipid catabolism are used as alternative sources of amino acids, energy, and carbon skeletons, respectively. We observed strong accumulation of secondary metabolites, such as hydroxycinnamic acid amides. Coregulated expression of WRKY transcription factors and genes known to be involved in the HR, in addition to a strong enrichment of the W-box WRKY-binding motif in the promoter sequences of the coregulated genes, point to WRKYs as the most prominent orchestrators of the HR. Our study has revealed several novel HR-related genes, and reverse genetics tools will allow us to understand the role of each individual component in the HR

System-Wide Hypersensitive Response-Associated Transcriptome and Metabolome Reprogramming in Tomato

The hypersensitive response (HR) is considered to be the hallmark of the resistance response of plants to pathogens. To study HR-associated transcriptome and metabolome reprogramming in tomato (Solanum lycopersicum), we used plants that express both a resistance gene to Cladosporium fulvum and the matching avirulence gene of this pathogen. In these plants, massive reprogramming occurred, and we found that the HR and associated processes are highly energy demanding. Ubiquitin-dependent protein degradation, hydrolysis of sugars, and lipid catabolism are used as alternative sources of amino acids, energy, and carbon skeletons, respectively. We observed strong accumulation of secondary metabolites, such as hydroxycinnamic acid amides. Coregulated expression of WRKY transcription factors and genes known to be involved in the HR, in addition to a strong enrichment of the W-box WRKY-binding motif in the promoter sequences of the coregulated genes, point to WRKYs as the most prominent orchestrators of the HR. Our study has revealed several novel HR-related genes, and reverse genetics tools will allow us to understand the role of each individual component in the HR

Creation of a new genus in the family Secoviridae substantiated by sequence variation of newly identified strawberry latent ringspot

To obtain insight into the sequence diversity of strawberry latent ringspot virus (SLRSV), isolates from collections and diagnostic samples were sequenced by high-throughput sequencing. For five SLRSV isolates, the complete genome sequences were determined, and for 18 other isolates nearly complete genome sequences were determined. The sequence data were analysed in relation to sequences of SLRSV and related virus isolates available in the NCBI GenBank database. The genome sequences were annotated, and sequences of the protease-polymerase (Pro-Pol) region and coat proteins (CPs) (large and small CP together) were used for phylogenetic analysis. The amino acid sequences of the Pro-Pol region were very similar, whereas the nucleotide sequences of this region were more variable. The amino acid sequences of the CPs were less similar, which was corroborated by the results of a serological comparison performed using antisera raised against different isolates of SLRSV. Based on these results, we propose that SLRSV and related unassigned viruses be assigned to a new genus within the family Secoviridae, named “Stralarivirus”. Based on the phylogenetic analysis, this genus should include at least three viruses, i.e., SLRSV-A, SLRSV-B and lychnis mottle virus. The newly generated sequence data provide a basis for designing molecular tests to screen for SLRSV

The novel Cladosporium fulvum lysin motif effector Ecp6 is a virulence factor with orthologues in other fungal species

During tomato leaf colonization, the biotrophic fungus Cladosporium fulvum secretes several effector proteins into the apoplast. Eight effectors have previously been characterized and show no significant homology to each other or to other fungal genes. To discover novel C. fulvum effectors that might play a role in virulence, we utilized two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to visualize proteins secreted during C. fulvum-tomato interactions. Three novel C. fulvum proteins were identified: CfPhiA, Ecp6 and Ecp7. CfPhiA shows homology to proteins found on fungal sporogenous cells called phialides. Ecp6 contains lysin motifs (LysM domains) that are recognized as carbohydrate-binding modules. Ecp7 encodes a small, cysteine-rich protein with no homology to known proteins. Heterologous expression of Ecp6 significantly increased the virulence of the vascular pathogen Fusarium oxysporum on tomato. Furthermore, by RNA interference (RNAi)-mediated gene silencing we demonstrate that Ecp6 is instrumental for C. fulvum virulence on tomato. Hardly any allelic variation was observed in the Ecp6 coding region of a worldwide collection of C. fulvum strains. Although none of the C. fulvum effectors identified so far have obvious orthologues in other organisms, conserved Ecp6 orthologues were identified in various fungal species. Homology-based modelling suggests that the LysM domains of C. fulvum Ecp6 may be involved in chitin binding