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 ( K A , D H, and D S ) 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.