Inhibition of photosynthesis and modification of the wheat leaf proteome by Ptr ToxB: A host‐specific toxin from the fungal pathogen Pyrenophora tritici‐repentis

Tan spot, caused by Pyrenophora tritici‐repentis, is an important foliar disease of wheat. The fungus produces the host‐specific, chlorosis‐inducing toxin Ptr ToxB. To better understand toxin action, we examined the effects of Ptr ToxB on sensitive wheat. Photosynthesis, as measured by infrared gas analysis, declined significantly within 12 h of toxin treatment, prior to the development of chlorosis at 48–72 h. Analysis by 2‐DE revealed a total of 102 protein spots with significantly altered intensities 12–36 h after toxin treatment, of which 66 were more abundant and 36 were less abundant than in the buffer‐treated control. The identities of 47 of these spots were established by MS/MS, and included proteins involved in the light reactions of photosynthesis, the Calvin cycle, and the stress/defense response. Based on the declines in photosynthesis and the identities of the differentially abundant proteins, we hypothesize that Ptr ToxB causes a rapid disruption in the photosynthetic processes of sensitive wheat, leading to the generation of ROS and oxidative stress. Although the photoprotective and repair mechanisms of the host appear to initially still be functional, they are probably overwhelmed by the continued production of ROS, leading to chlorophyll photooxidation and the development of chlorosis.     

Conformational changes and reaction of clostridial glycosylating toxins

The crystal structures of the catalytic fragments of ‘lethal toxin’ from Clostridium sordellii and of ‘α-toxin’ from Clostridium novyi have been established. Almost half of the residues follow the chain fold of the glycosyl-transferase type A family of enzymes; the other half forms large α-helical protrusions that are likely to confer specificity for the respective targeted subgroup of Rho proteins in the cell. In the crystal, the active center of α-toxin contained no substrates and was disassembled, whereas that of lethal toxin, which was ligated with the donor substrate UDP-glucose and cofactor Mn^2 +, was catalytically competent. Surprisingly, the structure of lethal toxin with Ca^2 + (instead of Mn^2 +) at the cofactor position showed a bound donor substrate with a disassembled active center, indicating that the strictly octahedral coordination sphere of Mn^2 + is indispensable to the integrity of the enzyme. The homologous structures of α-toxin without substrate, distorted lethal toxin with Ca^2 + plus donor, active lethal toxin with Mn^2 + plus donor and the homologous Clostridium difficile toxin B with a hydrolyzed donor have been lined up to show the geometry of several reaction steps. Interestingly, the structural refinement of one of the three crystallographically independent molecules of Ca^2 +-ligated lethal toxin resulted in the glucosyl half-chair conformation expected for glycosyl-transferases that retain the anomeric configuration at the C1″ atom. A superposition of six acceptor substrates bound to homologous enzymes yielded the position of the nucleophilic acceptor atom with a deviation of < 1 Å. The resulting donor-acceptor geometry suggests that the reaction runs as a circular electron transfer in a six-membered ring, which involves the deprotonation of the nucleophile by the β-phosphoryl group of the donor substrate UDP-glucose.     

A proteomic evaluation of Pyrenophora tritici‐repentis,causal agent of tan spot of wheat,reveals major differences between virulent and avirulent isolates

Pyrenophora tritici‐repentis causes tan spot, an important foliar disease of wheat. The fungus produces multiple host‐specific toxins, including Ptr ToxB, a chlorosis‐inducing protein encoded by the ToxB gene. A homolog of ToxB is also found in avirulent isolates of the fungus. In order to improve understanding of the role of this homolog and evaluate the general pathogenic ability of P. tritici‐repentis, we compared the proteomes of avirulent race 4 and virulent race 5 isolates of the pathogen. Western blotting analysis revealed the presence of Ptr ToxB in spore germination and culture fluids of race 5 but not race 4. A comprehensive proteome‐level comparison by 2‐DE indicated 133 differentially abundant proteins in the secretome (29 proteins) and mycelium (104 proteins) of races 4 and 5, of which 63 were identified by MS/MS. A number of the proteins found to be up‐regulated in race 5 have been implicated in microbial virulence in other pathosystems, and included the secreted enzymes α‐mannosidase and exo‐β‐1,3‐glucanase, heat‐shock and BiP proteins, and various metabolic enzymes. These proteome‐level differences suggest a reduced general pathogenic ability in race 4 of P. tritici‐repentis, irrespective of toxin production. Such differences may reflect an adaptation to a saprophytic habit.