NEW YORK (GenomeWeb) – Two new studies have spelled out which genetic mutations are behind the ever-evolving virulence observed in Puccinia graminis f. sp. tritici (Pgt), a fungus known for causing stem rust disease in wheat.
The research teams, both reporting in Science today, focused on Pgt genetic features — specifically avirulence genes — that help the stem rust pest dodge resistant wheat lines that contain immune response-triggering effector proteins such as Sr35 or Sr50, which were introduced through selective breeding. An infamous form of virulent Pgt called Ug99 was first identified in Uganda in the late 1990s, leading to crop losses in Africa, the Middle East, and beyond.
For one of the papers, a Kansas State University-led team used RNA sequencing and whole-genome sequencing to find versions of a fungal gene that leads to virulence, even in wheat plants containing the Sr35 resistance gene.
Starting from Pgt-sensitive and -resistant wheat lines, the researchers tracked early stages of infection microscopically, uncovering hints that a fungal protein secreted early in this process is likely recognized by the Sr35 wheat resistance protein. From there, they used transcriptome and genome sequencing to assess and compare natural Pgt isolates and isolates that had been chemically mutagenized with ethylmethane sulfonate, including 15 Pgt isolates that remained virulent in wheat when Sr35 was present.
In each of the Sr35-resistant isolates, the investigators identified mutations in the same Pgt gene. And through a series of follow-up experiments, they found that the Sr35 wheat resistance protein normally recognizes this secreted fungal protein, dubbed AvrSr35, to prompt plant immunity.
The stem rust fungus appeared to circumvent this process when it produced versions of AvrSr35 containing mobile element insertions that altered AvrSr35 in ways that blocked this interaction, letting Pgt slip past Sr35-based defenses despite elevated expression in the wheat plant leaves over time.
When the researchers re-sequenced a dozen isolates of Pgt that remained virulent in Sr35-positive wheat and 15 that did not, they uncovered two clusters: one with an intact AvrSr35 gene that prompted wheat immune responses and another housing several Pgt isolates with a premature AvrSr35 stop codon created with a miniature inverted transposable element.
Although the Ug99 isolates tested for that study of Pgt did not contain the AvrSr35 mutation, the team's gene expression and co-immunoprecipitation experiments in a tobacco-related Nicotiana plant species suggested Ug-susceptible Sr35 gene-containing wheat carries an Sr35 mutation that allows AvrSr35 to go undetected.
"The discovery of AvrSr35 provides a new tool for Pgt surveillance, identification of host susceptibility targets, and characterization of the molecular determinants of immunity in wheat," senior author Eduard Akhunov, a plant pathology researcher at Kansas State University, and his colleagues wrote.
In a related study, researchers from the University of Sydney, the Commonwealth Scientific and Industrial Research Organization (CSIRO), and elsewhere described another Pgt virulence protein called AvrSr50 that interacts with the rust resistance protein Sr50. For that study, they sequenced a Pgt isolate known as Pgt279, along with a related mutant strain, Pgt632, capable of producing virulent infections in wheat that has the Sr50 resistance protein.
From the roughly 1.1 million single base variants and small insertions or deletions detected in these isolates relative to a Pgt reference sequence, the team narrowed in loss-of-heterozygosity in a region spanning two-dozen genes in the virulent strain. Through a series of follow-up experiments, including efforts to over-express candidate fungal virulence genes in Nicotiana plant models, the investigators identified AvrSr50 and demonstrated that it could infect plants lacking the Sr50 resistance protein.
Their fungal genome sequence data further suggested that a virulent version of AvrSr50 that can infect Sr50-expressing plants contains sequence insertions, while their subsequent analyses provided clues to the origin and expression patterns of virulent AvrSr50 alleles.
"Virulence alleles of AvrSr50 have arisen through DNA insertion and sequence divergence, and our data provide molecular evidence that in addition to sexual recombination, somatic exchange can play a role in the emergence of new virulence traits in Pgt," CSIRO's Peter Dodds and University of Sydney researcher Robert Park, senior authors of that study, and their colleagues wrote.
In a statement, Park noted that the study's findings will make it possible to do DNA testing on stem rust fungi to find wheat crops infected with forms of Pgt that are prone to overcoming Sr50-mediated rust resistance.
For their part, KSU's Akhunov and his colleagues wrote that "[t]he identification of AvrSr35 and AvrSr50 provides valuable tools for molecular surveillance and early detection of virulent fungal pathogen races, which can inform the deployment of resistance genes to prevent epidemics."