Uptake of the <em>Fusarium</em> Effector Avr2 by Tomato Is Not a Cell Autonomous Event

Pathogens secrete effector proteins to manipulate the host for their own proliferation. Currently it is unclear whether the uptake of effector proteins from extracellular spaces is a host autonomous process. We study this process using the Avr2 effector protein from Fusarium oxysporum f. sp. lycopersici (Fol). Avr2 is an important virulence factor that is secreted into the xylem sap of tomato following infection. Besides that, it is also an avirulence factor triggering immune responses in plants carrying the I-2 resistance gene. Recognition of Avr2 by I-2 occurs inside the plant nucleus. Here, we show that pathogenicity of an Avr2 knockout Fusarium (FolΔAvr2) strain is fully complemented on transgenic tomato lines that express either a secreted (Avr2) or cytosolic Avr2 (ΔspAvr2) protein, indicating that Avr2 exerts its virulence functions inside the host cells. Furthermore, our data imply that secreted Avr2 is taken up from the extracellular spaces in the presence of the fungus. Grafting studies were performed in which scions of I-2 tomato plants were grafted onto either a ΔspAvr2 or on an Avr2 rootstock. Although the Avr2 protein could readily be detected in the xylem sap of the grafted plant tissues, no I-2-mediated immune responses were induced suggesting that I-2-expressing tomato cells cannot autonomously take up the effector protein from the xylem sap. Additionally, ΔspAvr2 and Avr2 plants were crossed with I-2 plants. Whereas ΔspAvr2/I-2 F1 plants showed a constitutive immune response, immunity was not triggered in the Avr2/I-2 plants confirming that Avr2 is not autonomously taken up from the extracellular spaces to trigger I-2. Intriguingly, infiltration of Agrobacterium tumefaciens in leaves of Avr2/I-2 plants triggered I-2 mediated cell death, which indicates that Agrobacterium triggers effector uptake. To test whether, besides Fol, effector uptake could also be induced by other fungal pathogens the ΔspAvr2 and Avr2 transgenic lines were inoculated with Verticillium dahliae. Whereas ΔspAvr2 plants became hyper-susceptible to infection, no difference in disease development was found in the Avr2 plants as compared to wild-type plants. These data suggest that effector uptake is not a host autonomous process and that Fol and A. tumefaciens, but not V. dahliae, facilitate Avr2 uptake by tomato cells from extracellular spaces

Functional Analysis of Cladosponum fulvum Effector Catalog

Onlangs is de DNA-sequentie van het genoom van Cladosporium fulvum bepaald. Het voornaamste doel daarvan is de identificatie en karakterisering van nieuwe effectors

Pre-advies natte bossen; verdroging, verzuring en eutrofiëring van natte bossen in Nederland: effecten en herstelmaatregelen

In dit rapport wordt een pre-advies gegeven voor herstelmaatregelen in natte bosecosystemen in het kader van het OBN, het Overlevingsplan Bos en Natuur (LNV, 1996) . Doel is het ontwikkelen van een reeks van algemeen toepasbare maatregelen gericht op het herstel van de effecten van verzuring, vermesting (beide als gevolg van luchtverontreiniging) en verdroging, in natte bossen met hoofdfunctie natuur. Een tweede doel is het inventariseren van de mogelijkheden en gevolgen van vernatting van multifunctioneel bos.Advies in het kader van OB

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

Specific members of the TOPLESS family are susceptibility genes for Fusarium wilt in tomato and Arabidopsis

Vascular wilt diseases caused by Fusarium oxysporum are a major threat to many agriculturally important crops. Genetic resistance is rare and inevitably overcome by the emergence of new races. To identify potentially durable and non-race-specific genetic resistance against Fusarium wilt diseases, we set out to identify effector targets in tomato that mediate susceptibility to the fungus. For this purpose, we used the SIX8 effector protein, an important and conserved virulence factor present in many pathogenic F. oxysporum isolates. Using protein pull-downs and yeast two-hybrid assays, SIX8 was found to interact specifically with two members of the tomato TOPLESS family: TPL1 and TPL2. Loss-of-function mutations in TPL1 strongly reduced disease susceptibility to Fusarium wilt and a tpl1;tpl2 double mutant exerted an even higher level of resistance. Similarly, Arabidopsis tpl;tpr1 mutants became significantly less diseased upon F. oxysporum inoculation as compared to wildtype plants. We conclude that TPLs encode susceptibility genes whose mutation can confer resistance to F. oxysporum

Synergistic interaction between the type III secretion system of the endophytic bacterium <i>Pantoea agglomerans</i> DAPP-PG 734 and the virulence of the causal agent of olive knot <i>Pseudomonas savastanoi pv. savastanoi</i> DAPP-PG 722

The endophytic bacterium Pantoea agglomerans DAPP-PG 734 was previously isolated from olive knots caused by infection with Pseudomonas savastanoi pv. savastanoi DAPP-PG 722. Whole-genome analysis of this P. agglomerans strain revealed the presence of a Hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS). To assess the role of the P. agglomerans T3SS in the interaction with P. savastanoi pv. savastanoi, we generated independent knockout mutants in three Hrp genes of the P. agglomerans DAPP-PG 734 T3SS (hrpJ, hrpN, and hrpY). In contrast to the wildtype control, all three mutants failed to cause a hypersensitive response when infiltrated in tobacco leaves, suggesting that P. agglomerans T3SS is functional and injects effector proteins in plant cells. In contrast to P. savastanoi pv. savastanoi DAPP-PG 722, the wildtype strain P. agglomerans DAPP-PG 734 and its Hrp T3SS mutants did not cause olive knot disease in 1-year-old olive plants. Coinoculation of P. savastanoi pv. savastanoi with P. agglomerans wildtype strains did not significantly change the knot size, while the DAPP-PG 734 hrpY mutant induced a significant decrease in knot size, which could be complemented by providing hrpY on a plasmid. By epifluorescence microscopy and confocal laser scanning microscopy, we found that the localization patterns in knots were nonoverlapping for P. savastanoi pv. savastanoi and P. agglomerans when coinoculated. Our results suggest that suppression of olive plant defences mediated by the Hrp T3SS of P. agglomerans DAPP-PG 734 positively impacts the virulence of P. savastanoi pv. savastanoi DAPP-PG 722

The Genomes of the Fungal Plant Pathogens Cladosporium fulvum and Dothistroma septosporum Reveal Adaptation to Different Hosts and Lifestyles But Also Signatures of Common Ancestry

We sequenced and compared the genomes of the Dothideomycete fungal plant pathogens Cladosporium fulvum (Cfu) (syn. Passalora fulva) and Dothistroma septosporum (Dse) that are closely related phylogenetically, but have different lifestyles and hosts. Although both fungi grow extracellularly in close contact with host mesophyll cells, Cfu is a biotroph infecting tomato, while Dse is a hemibiotroph infecting pine. The genomes of these fungi have a similar set of genes (70% of gene content in both genomes are homologs), but differ significantly in size (Cfu >61.1-Mb; Dse 31.2-Mb), which is mainly due to the difference in repeat content (47.2% in Cfu versus 3.2% in Dse). Recent adaptation to different lifestyles and hosts is suggested by diverged sets of genes. Cfu contains an a-tomatinase gene that we predict might be required for detoxification of tomatine, while this gene is absent in Dse. Many genes encoding secreted proteins are unique to each species and the repeat-rich areas in Cfu are enriched for these species-specific genes. In contrast, conserved genes suggest common host ancestry. Homologs of Cfu effector genes, including Ecp2 and Avr4, are present in Dse and induce a Cf-Ecp2- and Cf-4-mediated hypersensitive response, respectively. Strikingly, genes involved in production of the toxin dothistromin, a likely virulence factor for Dse, are conserved in Cfu, but their expression differs markedly with essentially no expression by Cfu in planta. Likewise, Cfu has a carbohydrate-degrading enzyme catalog that is more similar to that of necrotrophs or hemibiotrophs and a larger pectinolytic gene arsenal than Dse, but many of these genes are not expressed in planta or are pseudogenized. Overall, comparison of their genomes suggests that these closely related plant pathogens had a common ancestral host but since adapted to different hosts and lifestyles by a combination of differentiated gene content, pseudogenization, and gene regulatio

Studies towards the Intrinsic Function of the AVR4 and AVR9 Elicitors of the Fungal Tomato Pathogen Cladosporium fulvum

Recognition of the extracellular race-specific elicitor proteins AVR4 and AVR9 produced by the pathogenic fungus Cladosporium fulvum is mediated by the tomato resistance genes Cf-4 and Cf-9 , respectively. Recognition of these elicitors triggers host defense responses resulting in full resistance against the fungus. So far, intrinsic functions have not been identified for these two race-specific elicitors and all other characterized proteineous elicitors of C. fulvum . A short overview of the present state of the knowledge on the role of elicitor proteins in virulence is given in the introduction (chapter 1). In this thesis, we provide details on the molecular structure of both AVR4 and AVR9. Based on the protein structure homologies, known protein motifs were identified in both proteins. Subsequently, we analyzed whether these structural homologies could be translated into functional homologies based on bioassays.To this purpose, the disulfide bonds of AVR4 and AVR9 were elucidated. The chosen approach relied on the reducing agent tris-(2-carboxyethyl)-phosphine (TCEP), which allowed partial reduction of disulfide bonds at acidic pH. After partial reduction, the thiol groups of newly formed cysteines were modified in order to prevent disulfide bond shuffling. The disulfide bond pattern was identified following two different approaches. For AVR9 (chapter 3), the newly formed thiols were blocked by N -ethylmaleimide (NEM) and 4-vinylpyridine (VP). The resulting modified cysteines are compatible with standard protein sequencing protocols making use of the Edman degradation. For AVR4 (chapter 4), partial reduction was achieved by cyanylation of the sulfhydryl groups with 1-cyano-4-diethylamino-pyridinium (CDAP). This modification facilitated specific base-induced cleavage of the peptide bond yielding peptide fragments that could easily be identified by mass spectrometry.The disulfide bonds in the mature AVR9 protein involve Cys2-Cys16, Cys6-Cys19, and Cys12-Cys26, respectively. Cysteine spacing and the disulfide bond pattern of AVR9 are identical to those found in cystine-knotted inhibitor peptides. The cystine knot motif is best described by a "ring" formed by two disulfide bonds and their connecting amino acid residues, which is penetrated by a third disulfide bond. NMR data confirm that AVR9 is structurally most related to the cystine-knotted carboxypeptidase inhibitor (CPI). However, although structurally related to CPI, AVR9 does not show any carboxypeptidase inhibiting activity. Yet, AVR9 could still very well inhibit other plant proteases.Sequence homology revealed that AVR4 contains the invertebrate chitin-binding domain (inv ChBD) (chapter 5). This motif was previously reported to occur in most eukaryotic kingdoms except in plants and fungi. Six cysteine residues are conserved in the inv ChBD, which are interconnected by three disulfide bonds in mature AVR4: Cys11-Cys41, Cys35-Cys80, and Cys57-Cys72. AVR4 contains one additional disulfide bond, Cys21-Cys27 (chapter 4). Tachycitin is the only inv ChBD protein for which the disulfide bond pattern and 3D structure have been reported; the conserved cysteines in tachycitin show an identical disulfide bond pattern to that found in AVR4. Interestingly, AVR4 is the only fungal representative of the inv ChBD family found so far.It could be proven experimentally that AVR4 indeed binds specifically to chitin, but not to other related polysaccharides such as chitosan (chapter 5). Fluorescently labeled AVR4 localizes at chitin present in cell walls of Trichoderma viride and Fusarium solani f.sp. phaseoli . AVR4 can protect these fungi against the deleterious effect of class I plant chitinases (family-19 catalytic domain). Chitin in cell walls of in vitro -grown C. fulvum is not accessible and the fungus does not produce AVR4 under these conditions. However, chitin appeared accessible for AVR4 in cell walls of C. fulvum growing in the intercellular space of tomato where AVR4 is abundantly secreted by the fungus (chapter 5). These results suggest that AVR4 might contribute to the virulence of C. fulvum as it can protect the fungus during infection of tomato against constitutive and induced tomato chitinases.Independent disruption of the three conserved disulfide bonds resulted in protease sensitive isoforms of AVR4. Many strains of C. fulvum virulent on Cf-4 tomato circumvent recognition by single Cys-to-Tyr mutations in the AVR4 protein. However, the identified amino acid mutations only involve two of the three conserved disulfide bonds. Disruption of any of the four disulfide bonds in AVR4 did not result in a complete loss of chitin-binding, although Cys57-Cys72 might contribute to chitin-binding activity. These results indicate that in naturally occurring mutant alleles of avr4, the intrinsic function of AVR4 (chitin-binding ability) remained. Thus, races 4 of C. fulvum circumvent recognition mediated by the Cf-4 resistance gene without losing the correlated virulence function of AVR4 .The two main classes of chitin-binding domains, the invertebrate (CBM14) and the plant ChBD (CBM18), appear to exemplify convergent evolution. The thermodynamic properties ( KA ,DH, andDS ) of AVR4 binding to chito-oligomers with a degree-of-polymerization (DP) of 1 to 6 were compared to those of the plant lectins hevein and Urtica dioica agglutinin(UDA) (chapter 6). AVR4 only interacts with oligomers with DP≥3, while the plant lectins interact with the monomer N-acetyl-glucosamine (GlcNAc). The non-covalent complex between AVR4 and chito-oligomers could specifically be detected with ESI MS (upper limit in the millimolar range). NMR data indicated that the chitin-binding site has a topology similar to that of tachycitin a well-characterized representative of the CBM14 type of ChBD proteins, but that different amino acid residues within the motif are important for the interaction with chito-oligomers.Thus the expression pattern (both timing and local concentration), affinity and localization of AVR4 support a role as an integral cell wall protein forming a protective barrier against plant chitinases.In conclusion, our studies have proved that structural studies of AVR proteins do not only reveal structural homologies with other proteins (AVR9) present in structural databases, but also functional homologies, as has been proven for AVR4. In the future close collaborations between molecular biologists and structural biologist are required to accelerate progress in functional genomics and proteomics