Disulfide bond structure of the AVR9 elicitor of the fungal tomato pathogen Cladosporium fulvum: Evidence for a cystine knot

Disease resistance in plants is commonly activated by the product of an avi rulence (Avr) gene of a pathogen after interaction with the product of a ma tching resistance (R) gene in the host. In susceptible plants, Avr products might function as virulence or pathogenicity factors. The AVR9 elicitor fr om the fungus Cladosporium fulvum induces defense responses in tomato plant s carrying the Cf-9 resistance gene. This 28-residue beta -sheet AVR9 pepti de contains three disulfide bridges, which were identified in this study as Cys2-Cys16, Cys6-Cys19, and Cys12-Cys26. For this purpose, AVR9 was partia lly reduced, and the thiol groups of newly formed cysteines were modified t o prevent reactions with disulfides. After HPLC purification, the partially reduced peptides were sequenced to determine the positions of the modified cysteines, which originated from the reduced disulfide bridge(s). All step s involving molecules with free thiol groups were performed at low pH to su ppress disulfide scrambling. For that reason, cysteine modification by N-et hylmaleimide was preferred over modification by iodoacetamide, Upon (partia l) reduction of native AVR9, the Cys2-Cys16 bridge opened selectively. The resulting molecule was further reduced to two one-bridge intermediates, whi ch were subsequently completely reduced. The (partially) reduced cysteine-m odified AVR9 species showed little or no necrosis-inducing activity, demons trating the importance of the disulfide bridges for biological activity. Ba sed on peptide length and cysteine spacing, it was previously suggested tha t AVR9 isa cystine-knotted peptide. Now, we have proven that the bridging p attern of AVR9 is indeed identical to that of cystine-knotted peptides. Mor eover, NMR data obtained for AVR9 show that it is structurally closely rela ted to the cystine-knotted carboxypeptidase inhibitor. However, AVR9 does n ot show any carboxypeptidase inhibiting activity, indicating that the cysti ne-knot fold is a commonly occurring motif with varying biological function s.