| Wheat stem rust is an infectious crop disease caused by the fungus Puccinia graminis f.sp.tritici(Pgt)that brings about devastating yield losses in global wheat industry.In 1999,a previously uncharacterized strain of Pgt designated Ug99 was detected in Uganda and found to be unsusceptible to commercial wheat stem rust resistance genes such as the nucleotide-binding,leucine-rich repeat receptor(NLR)Sr31.The increasing threat posed by broad virulence spectrum and continuous spreading of Ug99 prompted discovery of a nearly one hundred wheat stem resistance genes in recent years.Among them,Sr35,the newly identified wheat stem rust resistance gene and NLR from the diploid wheat species Triticum monococcum,has been demonstrated to be capable of recognizing and binding the Pgt avirulence factor35(AvrSr35),thus conferring resistance to the host wheat plant against Ug99 and Ug99-related wheat stem rust strains.Considering that molecular basis for recognition of AvrSr35 by Sr35 is unknown,this paper uses structural biology and biochemical methods to study the mechanisms underlying interaction between Sr35 and AvrSr35 and provide insights into pathon mechanism of Ug99 infection and wheat-related immune responses,which will aid in formulating effective control measures against wheat stem rust.First,the thesis explores the strength of the AvrSr35-Sr35 interaction with GST pull-down assay,gel filtration assay,analytical ultracentrifugation and microscale thermophoresis,determining the dissociation constant of 7.8 mM.Subsequently,the crystal structure of AvrSr35 determined by X-ray crystal diffraction method is thoroughly analyzed.The crystal structure indicates that AvrSr35 exhibits a simple structure composed of 20 α-helices of varying lengths and that two molecules of AvrSr35 bind to form a diagonally symmetrical dimer.In order to investigate the influence of AvrSr35 dimerization on recognition by Sr35,the amino acids D350 and R381 located at the AvrSr35 dimerization interface were mutated to alanine to disrupt the dimerization.Comparison of dissociation constants suggested that affinity of the AvrSr35 mutant for Sr35 is similar to that of wild-type AvrSr35,indicating that the binding of AvrSr35 to Sr35 does not depend on AvrSr35 dimerization.To identify the protein regions by which AvrSr35 binds to Sr35,this paper tested binding affinities between AvrSr35 truncations and Sr35.The results showed that the 171–558 aa core fragment of AvrSr35 is essential for mediating the binding to Sr35,except for the loop composed of 480–505 aa.According to previous studies,upon activation plant NLRs oligomerize and form resistosomes to carry out functions in immunity.Similarly,this paper observed that ATP induces formation of the Sr35 resistosome after AvrSr35-mediated activation of Sr35.Furthermore,by analyzing the density map of the Sr35 resistosome determined using cryo-EM,we found that the Sr35 resistosome is formed by two Sr35-AvrSr35 complex pentamers connected to each other.In addition,amino acid sequence alignment,homology modeling and molecular docking indicated that binding to AvrSr35 causes rearrangements within NOD domain of Sr35 and that insertion of ATP stabilizes the activated conformation of the NOD domain,which in turn induces the oligomerization of Sr35 and assembly of resistosome.In summary,this paper explores the structural basis for activation of the wheat resistance protein Sr35 and its recognition of the Ug99 effector AvrSr35,and proposes a model for the underlying molecular mechanisms. |
