Dissection of a rapidly evolving wheat resistance gene cluster by long-read genome sequencing accelerated the cloning of Pm69.

Gene cloning in repeat-rich polyploid genomes remains challenging. Here, we describe a strategy for overcoming major bottlenecks in cloning of the powdery mildew resistance gene (R-gene) Pm69 derived from tetraploid wild emmer wheat. A conventional positional cloning approach was not effective owing to suppressed recombination. Chromosome sorting was compromised by insufficient purity. A Pm69 physical map, constructed by assembling Oxford Nanopore Technology (ONT) long-read genome sequences, revealed a rapidly evolving nucleotide-binding leucine-rich repeat (NLR) R-gene cluster with structural variations. A single candidate NLR was identified by anchoring RNA sequencing reads from susceptible mutants to ONT contigs and was validated by virus-induced gene silencing. Pm69 is likely a newly evolved NLR and was discovered in only one location across the wild emmer wheat distribution range in Israel. Pm69 was successfully introgressed into cultivated wheat, and a diagnostic molecular marker was used to accelerate its deployment and pyramiding with other R-genes.

The AvrPm3-Pm3 effector-NLR interactions control both race-specific resistance and host-specificity of cereal mildews on wheat.

The wheat Pm3 resistance gene against the powdery mildew pathogen occurs as an allelic series encoding functionally different immune receptors which induce resistance upon recognition of isolate-specific avirulence (AVR) effectors from the pathogen. Here, we describe the identification of five effector proteins from the mildew pathogens of wheat, rye, and the wild grass Dactylis glomerata, specifically recognized by the PM3B, PM3C and PM3D receptors. Together with the earlier identified AVRPM3A2/F2, the recognized AVRs of PM3B/C, (AVRPM3B2/C2), and PM3D (AVRPM3D3) belong to a large group of proteins with low sequence homology but predicted structural similarities. AvrPm3b2/c2 and AvrPm3d3 are conserved in all tested isolates of wheat and rye mildew, and non-host infection assays demonstrate that Pm3b, Pm3c, and Pm3d are also restricting the growth of rye mildew on wheat. Furthermore, divergent AVR homologues from non-adapted rye and Dactylis mildews are recognized by PM3B, PM3C, or PM3D, demonstrating their involvement in host specificity.

Distinct domains of the AVRPM3A2/F2 avirulence protein from wheat powdery mildew are involved in immune receptor recognition and putative effector function.

Recognition of the AVRPM3A2/F2 avirulence protein from powdery mildew by the wheat PM3A/F immune receptor induces a hypersensitive response after co-expression in Nicotiana benthamiana. The molecular determinants of this interaction and how they shape natural AvrPm3a2/f2 allelic diversity are unknown. We sequenced the AvrPm3a2/f2 gene in a worldwide collection of 272 mildew isolates. Using the natural polymorphisms of AvrPm3a2/f2 as well as sequence information from related gene family members, we tested 85 single-residue-altered AVRPM3A2/F2 variants with PM3A, PM3F and PM3FL456P/Y458H (modified for improved signaling) in Nicotiana benthamiana for effects on recognition. An intact AvrPm3a2/f2 gene was found in all analyzed isolates and the protein variant recognized by PM3A/F occurred globally at high frequencies. Single-residue alterations in AVRPM3A2/F2 mostly disrupted, but occasionally enhanced, the recognition response by PM3A, PM3F and PM3FL456P/Y458H . Residues enhancing hypersensitive responses constituted a protein domain separate from both naturally occurring polymorphisms and positively selected residues of the gene family. These results demonstrate the utility of using gene family sequence diversity to screen residues for their role in recognition. This approach identified a putative interaction surface in AVRPM3A2/F2 not polymorphic in natural alleles. We conclude that molecular mechanisms besides recognition drive AvrPm3a2/f2 diversification.

Wild emmer genome architecture and diversity elucidate wheat evolution and domestication.

Wheat (Triticum spp.) is one of the founder crops that likely drove the Neolithic transition to sedentary agrarian societies in the Fertile Crescent more than 10,000 years ago. Identifying genetic modifications underlying wheat's domestication requires knowledge about the genome of its allo-tetraploid progenitor, wild emmer (T. turgidum ssp. dicoccoides). We report a 10.1-gigabase assembly of the 14 chromosomes of wild tetraploid wheat, as well as analyses of gene content, genome architecture, and genetic diversity. With this fully assembled polyploid wheat genome, we identified the causal mutations in Brittle Rachis 1 (TtBtr1) genes controlling shattering, a key domestication trait. A study of genomic diversity among wild and domesticated accessions revealed genomic regions bearing the signature of selection under domestication. This reference assembly will serve as a resource for accelerating the genome-assisted improvement of modern wheat varieties.

AvrPm2 encodes an RNase-like avirulence effector which is conserved in the two different specialized forms of wheat and rye powdery mildew fungus.

There is a large diversity of genetically defined resistance genes in bread wheat against the powdery mildew pathogen Blumeria graminis (B. g.) f. sp. tritici. Many confer race-specific resistance to this pathogen, but until now only the mildew avirulence gene AvrPm3a2/f2 that is recognized by Pm3a/f was known molecularly. We performed map-based cloning and genome-wide association studies to isolate a candidate for the mildew avirulence gene AvrPm2. We then used transient expression assays in Nicotiana benthamiana to demonstrate specific and strong recognition of AvrPm2 by Pm2. The virulent AvrPm2 allele arose from a conserved 12 kb deletion, while there is no protein sequence diversity in the gene pool of avirulent B. g. tritici isolates. We found one polymorphic AvrPm2 allele in B. g. triticale and one orthologue in B. g. secalis and both are recognized by Pm2. AvrPm2 belongs to a small gene family encoding structurally conserved RNase-like effectors, including Avra13 from B. g. hordei, the cognate Avr of the barley resistance gene Mla13. These results demonstrate the conservation of functional avirulence genes in two cereal powdery mildews specialized on different hosts, thus providing a possible explanation for successful introgression of resistance genes from rye or other grass relatives to wheat.

Hybridization of powdery mildew strains gives rise to pathogens on novel agricultural crop species.

Throughout the history of agriculture, many new crop species (polyploids or artificial hybrids) have been introduced to diversify products or to increase yield. However, little is known about how these new crops influence the evolution of new pathogens and diseases. Triticale is an artificial hybrid of wheat and rye, and it was resistant to the fungal pathogen powdery mildew (Blumeria graminis) until 2001 (refs. 1,2,3). We sequenced and compared the genomes of 46 powdery mildew isolates covering several formae speciales. We found that B. graminis f. sp. triticale, which grows on triticale and wheat, is a hybrid between wheat powdery mildew (B. graminis f. sp. tritici) and mildew specialized on rye (B. graminis f. sp. secalis). Our data show that the hybrid of the two mildews specialized on two different hosts can infect the hybrid plant species originating from those two hosts. We conclude that hybridization between mildews specialized on different species is a mechanism of adaptation to new crops introduced by agriculture.

Multiple Avirulence Loci and Allele-Specific Effector Recognition Control the Pm3 Race-Specific Resistance of Wheat to Powdery Mildew.

In cereals, several mildew resistance genes occur as large allelic series; for example, in wheat (Triticum aestivum and Triticum turgidum), 17 functional Pm3 alleles confer agronomically important race-specific resistance to powdery mildew (Blumeria graminis). The molecular basis of race specificity has been characterized in wheat, but little is known about the corresponding avirulence genes in powdery mildew. Here, we dissected the genetics of avirulence for six Pm3 alleles and found that three major Avr loci affect avirulence, with a common locus_1 involved in all AvrPm3-Pm3 interactions. We cloned the effector gene AvrPm3(a2/f2) from locus_2, which is recognized by the Pm3a and Pm3f alleles. Induction of a Pm3 allele-dependent hypersensitive response in transient assays in Nicotiana benthamiana and in wheat demonstrated specificity. Gene expression analysis of Bcg1 (encoded by locus_1) and AvrPm3 (a2/f2) revealed significant differences between isolates, indicating that in addition to protein polymorphisms, expression levels play a role in avirulence. We propose a model for race specificity involving three components: an allele-specific avirulence effector, a resistance gene allele, and a pathogen-encoded suppressor of avirulence. Thus, whereas a genetically simple allelic series controls specificity in the plant host, recognition on the pathogen side is more complex, allowing flexible evolutionary responses and adaptation to resistance genes.