The Cladosporium fulvum - tomato interaction : physiological and molecular aspects of pathogenesis

In 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

An Isoform of the Eukaryotic Translation Elongation Factor 1A (eEF1a) Acts as a Pro-Viral Factor Required for Tomato Spotted Wilt Virus Disease in <i>Nicotiana benthamiana</i>

The tripartite genome of the negative-stranded RNA virus Tomato spotted wilt orthotospovirus (TSWV) is assembled, together with two viral proteins, the nucleocapsid protein and the RNA-dependent RNA polymerase, into infectious ribonucleoprotein complexes (RNPs). These two viral proteins are, together, essential for viral replication and transcription, yet our knowledge on the host factors supporting these two processes remains limited. To fill this knowledge gap, the protein composition of viral RNPs collected from TSWV-infected Nicotiana benthamiana plants, and of those collected from a reconstituted TSWV replicon system in the yeast Saccharomyces cerevisiae, was analysed. RNPs obtained from infected plant material were enriched for plant proteins implicated in (i) sugar and phosphate transport and (ii) responses to cellular stress. In contrast, the yeast-derived viral RNPs primarily contained proteins implicated in RNA processing and ribosome biogenesis. The latter suggests that, in yeast, the translational machinery is recruited to these viral RNPs. To examine whether one of these cellular proteins is important for a TSWV infection, the corresponding N. benthamiana genes were targeted for virus-induced gene silencing, and these plants were subsequently challenged with TSWV. This approach revealed four host factors that are important for systemic spread of TSWV and disease symptom development

The EDS1–PAD4–ADR1 node mediates Arabidopsis pattern-triggered immunity

Plants deploy cell-surface and intracellular leucine rich-repeat domain (LRR) immune receptors to detect pathogens1. LRR receptor kinases and LRR receptor proteins at the plasma membrane recognize microorganism-derived molecules to elicit pattern-triggered immunity (PTI), whereas nucleotide-binding LRR proteins detect microbial effectors inside cells to confer effector-triggered immunity (ETI). Although PTI and ETI are initiated in different host cell compartments, they rely on the transcriptional activation of similar sets of genes2, suggesting pathway convergence upstream of nuclear events. Here we report that PTI triggered by the Arabidopsis LRR receptor protein RLP23 requires signalling-competent dimers of the lipase-like proteins EDS1 and PAD4, and of ADR1 family helper nucleotide-binding LRRs, which are all components of ETI. The cell-surface LRR receptor kinase SOBIR1 links RLP23 with EDS1, PAD4 and ADR1 proteins, suggesting the formation of supramolecular complexes containing PTI receptors and transducers at the inner side of the plasma membrane. We detected similar evolutionary patterns in LRR receptor protein and nucleotide-binding LRR genes across Arabidopsis accessions; overall higher levels of variation in LRR receptor proteins than in LRR receptor kinases are consistent with distinct roles of these two receptor families in plant immunity. We propose that the EDS1–PAD4–ADR1 node is a convergence point for defence signalling cascades, activated by both surface-resident and intracellular LRR receptors, in conferring pathogen immunity

Isolation of apoplastic fluid from leaf tissue by the vacuum infiltration-centrifugation technique

Upon 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

The diverse roles of NB-LRR proteins in plants

Plant innate immunity relies on specialised immune receptors that can detect and defend against a wide variety of microbes. The first group of receptors comprises the transmembrane pathogen- or pattern-recognition receptors (PRRs), which respond to slowly evolving pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs). The second group of immune receptors is formed by the polymorphic disease resistance (R) proteins that detect microbe-derived effector proteins. Most R proteins are members of the nucleotide binding leucine-rich repeat (NB-LRR) class. Although this class comprises one of the biggest protein families in plants, relatively few have been functionally characterised to date. The question rises whether all NB-LRRs function as immune receptors, or that they might have alternative functions. The answer is: yes, they do have alternative functions that are different from the immune receptor function. This review summarises the current knowledge about non-immune receptor signal transduction functions of NB-LRRs in plants

Plant resistance genes : their structure, function and evolution

Plants 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

Avirulence proteins of plant pathogens: determinants of victory and defeat

The simplest way to explain the biochemical basis of the gene-for-gene concept is by direct interaction between a pathogen-derived avirulence (Avr) gene product and a receptor protein, which is encoded by the matching resistance (R) gene of the host plant. The number of R genes for which the matching Avr gene has been cloned is increasing. The number of host-pathogen relationships, however, for which a direct interaction between R and Avr gene products could be proven is still very limited. This observation suggests that in various host-pathogen relationships no physical interaction between R and Avr proteins occurs, and that perception of AVR proteins by their matching R gene products is indirect. Indirect perception implies that at least a third component is required. The 'Guard hypothesis' proposes that this third component could be the virulence target of an AVR protein. Binding of the AVR protein to its virulence target is perceived by the matching R protein, which is 'guarding' the virulence target. An intriguing aspect of the 'Guard hypothesis' is that the Avr gene product causes avirulence of the pathogen through interaction with its virulence target in the plant. This would mean that, although AVR proteins are generally thought to be bifunctional (avirulence as well as virulence factors), this dual function might be based on a single biochemical event. This review focuses on the way AVR proteins are perceived by their matching R gene products. The various components that determine the outcome of the interaction will be discussed, with an emphasis on the dual function of AVR proteins