Substitutions of Two Amino Acids in the Nucleotide-Binding Site Domain of a Resistance Protein Enhance the Hypersensitive Response and Enlarge the PM3F Resistance Spectrum in Wheat

Abstract

Proteins with nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains are major components of the plant immune system. They usually mediate resistance against a subgroup of races of a specific pathogen. For the allelic series of the wheat powdery mildew resistance gene Pm3, alleles with a broad and a narrow resistance spectrum have been described. Here, we show that a broad Pm3 spectrum range correlates with a fast and intense hypersensitive response (HR) in a Nicotiana transient-expression system and this activity can be attributed to two particular amino acids in the ARC2 subdomain of the NBS. The combined substitution of these amino acids in narrow-spectrum PM3 proteins enhances their capacity to induce an HR in Nicotiana benthamiana, and we demonstrate that these substitutions also enlarge the resistance spectrum of the Pm3f allele in wheat. Finally, using Bph14, we show that the region carrying the relevant amino acids also plays a role in the HR regulation of another coiled-coil NBS-LRR resistance protein. These results highlight the importance of an optimized NBS-'molecular switch' for the conversion of initial pathogen perception by the LRR into resistance-protein activation, and we describe a possible approach to extend the effectiveness of resistance genes via minimal targeted modifications in the NBS domain.

Multiple Avirulence Loci and Allele-Specific Effector Recognition Control thePm3Race-Specific Resistance of Wheat to Powdery Mildew

“…Consistent with previous studies using autoactive PM3 proteins (Stirnweis et al, 2014a(Stirnweis et al, , 2014b, transient expression of AvrPm3 a2/f2 with Pm3a, Pm3f, or Pm3f L456P/Y458H in N. benthamiana led to cell death. In wheat, transient expression of AvrPm3 a2/f2 with Pm3a led to a significant reduction in the number of cells accumulating the GUS reporter, as did the autoactive Pm3a HR construct, likely because of an activation of Pm3a by AvrPm3 a2/f2 resulting in cell death.…”

“…Brunner et al (2010) previously demonstrated that the two alleles Pm3f and Pm3a have characteristic resistance spectra: Based on the observation that all the isolates recognized by Pm3f were also recognized by Pm3a, but not vice versa, they concluded that Pm3a is a stronger form of Pm3f. Later, Stirnweis et al (2014a) showed that a substitution of only two amino acids in the NBS domain of PM3F (PM3F L456P/Y458H ) was sufficient to broaden Pm3f-mediated resistance to potentially confer the same race specificity as Pm3a, thus substantiating the hypothesis that Pm3a is a stronger version of Pm3f. Therefore, to test for interaction with the resistance gene, Pu_3 and Pu_7 were cloned from the avirulent parent 96224 (Pu_3 avr and Pu_7 avr ) and the virulent parent 94202 (Pu_7 vir ).…”

“…Molecular cloning into Gateway-compatible entry vector was performed using the pENTR/D-TOPO Cloning Kit (Life Technologies) according to the manufacturer. Site-directed mutagenesis and recombination to the binary vector pIPKb004 were performed as previously described by Stirnweis et al (2014a). Primers used for experimental gene annotation and molecular cloning are listed in Supplemental Table 1.…”

“…However, although L6 recognizes more alleles of AvrL567, the subset of alleles recognized by L5 is not encompassed within the subset recognized by L6 (Ravensdale et al, 2012). Stirnweis et al (2014a) subsequently found that two amino acid changes within the Pm3 ARC2 subdomain of the NBS are sufficient to extend the resistance spectrum of the weaker Pm3f allele to that of the stronger Pm3a allele, providing evidence for the concept that specificity of R proteins is the sum of avirulence protein recognition plus activation of the resistance protein (Steinbrenner et al, 2015). This translates into a quantitative variation in the level of virulence in natural isolates, as described by Brunner et al (2010), when they calculated the haustorium index of natural isolates during transient expression assays of several Pm3 alleles.…”

“…Given the unique evolutionary history of the recently diverged allelic series of the Pm3 resistance gene , combined with the evidence of complex interallelic interactions determining race specificity (Brunner et al, 2010;Stirnweis et al, 2014aStirnweis et al, , 2014b, the AvrPm3-Pm3 interaction provides a unique system to study the genetic and molecular mechanisms controlling specificity on the host as well as the pathogen side. In addition, evidence of recent evolution of wheat powdery mildew toward host specificity that is associated with a strong expansion of the effector repertoire (Wicker et al, 2013) provides a unique system to study host-pathogen coevolution in light of the Avr-R interaction.…”