Ancient variation of the AvrPm17 gene in powdery mildew limits the effectiveness of the introgressed rye Pm17 resistance gene in wheat.

Introgressions of chromosomal segments from related species into wheat are important sources of resistance against fungal diseases. The durability and effectiveness of introgressed resistance genes upon agricultural deployment is highly variable-a phenomenon that remains poorly understood, as the corresponding fungal avirulence genes are largely unknown. Until its breakdown, the Pm17 resistance gene introgressed from rye to wheat provided broad resistance against powdery mildew (Blumeria graminis). Here, we used quantitative trait locus (QTL) mapping to identify the corresponding wheat mildew avirulence effector AvrPm17. It is encoded by two paralogous genes that exhibit signatures of reoccurring gene conversion events and are members of a mildew sublineage specific effector cluster. Extensive haplovariant mining in wheat mildew and related sublineages identified several ancient virulent AvrPm17 variants that were present as standing genetic variation in wheat powdery mildew prior to the Pm17 introgression, thereby paving the way for the rapid breakdown of the Pm17 resistance. QTL mapping in mildew identified a second genetic component likely corresponding to an additional resistance gene present on the 1AL.1RS translocation carrying Pm17. This gene remained previously undetected due to suppressed recombination within the introgressed rye chromosomal segment. We conclude that the initial effectiveness of 1AL.1RS was based on simultaneous introgression of two genetically linked resistance genes. Our results demonstrate the relevance of pathogen-based genetic approaches to disentangling complex resistance loci in wheat. We propose that identification and monitoring of avirulence gene diversity in pathogen populations become an integral part of introgression breeding to ensure effective and durable resistance in wheat.

High-copy transposons from a pathogen give rise to a conserved sRNA family with a novel host immunity target.

Small RNAs (sRNAs) are involved in gene silencing in multiple ways including through cross-kingdom transfers from parasites to their hosts. Little is known about the evolutionary mechanisms enabling eukaryotic microbes to evolve functional mimics of host small regulatory RNAs. Here, we describe the identification and functional characterization of SINE_sRNA1, a sRNA family derived from highly abundant SINE retrotransposons in the genome of the wheat powdery mildew pathogen. SINE_sRNA1 is encoded by a sequence motif that is conserved in multiple SINE families and corresponds to a functional plant miRNA mimic targeting Tae_AP1, a wheat gene encoding an aspartic protease only found in monocots. Tae_AP1 has a novel function enhancing both pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) thus contributing to the cross activation of plant defenses. We conclude that SINE_sRNA1 and Tae_AP1 are functional innovations suggesting the contribution of transposons to the evolutionary arms race between a parasite and its host.

Transcriptomic analysis identifies candidate genes for Aphanomyces root rot disease resistance in pea.

BACKGROUND: Aphanomyces euteiches is a soil-borne oomycete that causes root rot in pea and other legume species. Symptoms of Aphanomyces root rot (ARR) include root discoloration and wilting, leading to significant yield losses in pea production. Resistance to ARR is known to be polygenic but the roles of single genes in the pea immune response are still poorly understood. This study uses transcriptomics to elucidate the immune response of two pea genotypes varying in their levels of resistance to A. euteiches. RESULTS: In this study, we inoculated roots of the pea (P. sativum L.) genotypes 'Linnea' (susceptible) and 'PI180693' (resistant) with two different A. euteiches strains varying in levels of virulence. The roots were harvested at 6 h post-inoculation (hpi), 20 hpi and 48 hpi, followed by differential gene expression analysis. Our results showed a time- and genotype-dependent immune response towards A. euteiches infection, involving several WRKY and MYB-like transcription factors, along with genes associated with jasmonic acid (JA) and abscisic acid (ABA) signaling. By cross-referencing with genes segregating with partial resistance to ARR, we identified 39 candidate disease resistance genes at the later stage of infection. Among the genes solely upregulated in the resistant genotype 'PI180693', Psat7g091800.1 was polymorphic between the pea genotypes and encoded a Leucine-rich repeat receptor-like kinase reminiscent of the Arabidopsis thaliana FLAGELLIN-SENSITIVE 2 receptor. CONCLUSIONS: This study provides new insights into the gene expression dynamics controlling the immune response of resistant and susceptible pea genotypes to A. euteiches infection. We present a set of 39 candidate disease resistance genes for ARR in pea, including the putative immune receptor Psat7g091800.1, for future functional validation.

Transformation and gene-disruption in the apple-pathogen, Neonectria ditissima.

BACKGROUND: Apple production in Sweden and elsewhere is being threatened by the fungus, Neonectria ditissima, which causes a disease known as European canker. The disease can cause extensive damage and the removal of diseased wood and heavily infected trees can be laborious and expensive. Currently, there is no way to eradicate the fungus from infected trees and our knowledge of the infection process is limited. Thus, to target and modify genes efficiently, the genetic transformation technique developed for N. ditissima back in 2003 was modified. RESULTS: The original protocol from 2003 was upgraded to use enzymes currently available in the market for making protoplasts. The protoplasts were viable, able to uptake foreign DNA, and able to regenerate back into a mycelial colony, either as targeted gene-disruption mutants or as ectopic mutants expressing the green fluorescent protein (GFP). CONCLUSIONS: A new genetic transformation protocol has been established and the inclusion of hydroxyurea in the buffer during the protoplast-generation step greatly increased the creation of knockout mutants via homologous recombination. Pathogenicity assays using the GFP-mutants showed that the mutants were able to infect the host and cause disease.

Single residues in the LRR domain of the wheat PM3A immune receptor can control the strength and the spectrum of the immune response.

The development of improved plant nucleotide-binding, leucine-rich repeat (LRR) immune receptors (NLRs) has mostly been based on random mutagenesis or on structural information available for specific receptors complexed with the recognized pathogen effector. Here, we use a targeted mutagenesis approach based on the natural diversity of the Pm3 powdery mildew resistance alleles present in different wheat (Triticum aestivum) genotypes. In order to understand the functional importance of the amino acid polymorphisms between the active immune receptor PM3A and the inactive ancestral variant PM3CS, we exchanged polymorphic regions and residues in the LRR domain of PM3A with the corresponding segments of PM3CS. These novel variants were functionally tested for recognition of the corresponding AVRPM3A2/F2 avirulence protein in Nicotiana benthamiana. We identified polymorphic residues in four regions of PM3A that enhance the immune response, but also residues that reduce it or result in complete loss of function. We found that the identified critical residues in PM3A modify its activation threshold towards different protein variants of AVRPM3A2/F2 . PM3A variants with a lowered threshold gave a stronger overall response and gained an extended recognition spectrum. One of these variant proteins with a single amino acid change was stably transformed into wheat, where it conferred race-specific resistance to mildew. This is a proof of concept that improved PM3A variants with an enlarged recognition spectrum can be engineered based on natural diversity by exchanging single or multiple residues that modulate resistance function.

Domestication of High-Copy Transposons Underlays the Wheat Small RNA Response to an Obligate Pathogen.

Plant genomes have evolved several evolutionary mechanisms to tolerate and make use of transposable elements (TEs). Of these, transposon domestication into cis-regulatory and microRNA (miRNA) sequences is proposed to contribute to abiotic/biotic stress adaptation in plants. The wheat genome is derived at 85% from TEs, and contains thousands of miniature inverted-repeat transposable elements (MITEs), whose sequences are particularly prone for domestication into miRNA precursors. In this study, we investigate the contribution of TEs to the wheat small RNA immune response to the lineage-specific, obligate powdery mildew pathogen. We show that MITEs of the Mariner superfamily contribute the largest diversity of miRNAs to the wheat immune response. In particular, MITE precursors of miRNAs are wide-spread over the wheat genome, and highly conserved copies are found in the Lr34 and QPm.tut-4A mildew resistance loci. Our work suggests that transposon domestication is an important evolutionary force driving miRNA functional innovation in wheat immunity.

Characterization of the Resistance to Powdery Mildew and Leaf Rust Carried by the Bread Wheat Cultivar Victo.

Leaf rust and powdery mildew are two important foliar diseases in wheat. A recombinant inbred line (RIL) population, obtained by crossing two bread wheat cultivars ('Victo' and 'Spada'), was evaluated for resistance to the two pathogens at seedling stage. Upon developing a genetic map of 8726 SNP loci, linkage analysis identified three resistance Quantitative Trait Loci (QTLs), with 'Victo' contributing the resistant alleles to all loci. One major QTL (QPm.gb-7A) was detected in response to Blumeria graminis on chromosome 7A, which explained 90% of phenotypic variation (PV). The co-positional relationship with known powdery mildew (Pm) resistance loci suggested that a new source of resistance was identified in T. aestivum. Two QTLs were detected in response to Puccinia triticina: a major gene on chromosome 5D (QLr.gb-5D), explaining a total PV of about 59%, and a minor QTL on chromosome 2B (QLr.gb-2B). A positional relationship was observed between the QLr.gb-5D with the known Lr1 gene, but polymorphisms were found between the cloned Lr1 and the corresponding 'Victo' allele, suggesting that QLr.gb-5D could represent a new functional Lr1 allele. Lastly, upon anchoring the QTL on the T. aestivum reference genome, candidate genes were hypothesized on the basis of gene annotation and in silico gene expression analysis.

A chromosome-scale genome assembly reveals a highly dynamic effector repertoire of wheat powdery mildew.

Blumeria graminis f. sp. tritici (B.g. tritici) is the causal agent of the wheat powdery mildew disease. The highly fragmented B.g. tritici genome available so far has prevented a systematic analysis of effector genes that are known to be involved in host adaptation. To study the diversity and evolution of effector genes we produced a chromosome-scale assembly of the B.g. tritici genome. The genome assembly and annotation was achieved by combining long-read sequencing with high-density genetic mapping, bacterial artificial chromosome fingerprinting and transcriptomics. We found that the 166.6 Mb B.g. tritici genome encodes 844 candidate effector genes, over 40% more than previously reported. Candidate effector genes have characteristic local genomic organization such as gene clustering and enrichment for recombination-active regions and certain transposable element families. A large group of 412 candidate effector genes shows high plasticity in terms of copy number variation in a global set of 36 isolates and of transcription levels. Our data suggest that copy number variation and transcriptional flexibility are the main drivers for adaptation in B.g. tritici. The high repeat content may play a role in providing a genomic environment that allows rapid evolution of effector genes with selection as the driving force.

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.

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.

Impact of biotic and abiotic factors on the expression of fungal effector-encoding genes in axenic growth conditions.

In phytopathogenic fungi, the expression of hundreds of small secreted protein (SSP)-encoding genes is induced upon primary infection of plants while no or a low level of expression is observed during vegetative growth. In some species such as Leptosphaeria maculans, this coordinated in-planta upregulation of SSP-encoding genes expression relies on an epigenetic control but the signals triggering gene expression in-planta are unknown. In the present study, biotic and abiotic factors that may relieve suppression of SSP-encoding gene expression during axenic growth of L. maculans were investigated. Some abiotic factors (temperature, pH) could have a limited effect on SSP gene expression. In contrast, two types of cellular stresses induced by antibiotics (cycloheximide, phleomycin) activated strongly the transcription of SSP genes. A transcriptomic analysis to cycloheximide exposure revealed that biological processes such as ribosome biosynthesis and rRNA processing were induced whereas important metabolic pathways such as glycogen and nitrogen metabolism, glycolysis and tricarboxylic acid cycle activity were down-regulated. A quantitatively different expression of SSP-encoding genes compared to plant infection was also detected. Interestingly, the same physico-chemical parameters as those identified here for L. maculans effectors were identified to regulate positively or negatively the expression of bacterial effectors. This suggests that apoplastic phytopathogens may react to similar physiological parameters for regulation of their effector genes.

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.

Genetic and molecular characterization of a locus involved in avirulence of Blumeria graminis f. sp. tritici on wheat Pm3 resistance alleles.

Wheat powdery mildew is caused by the obligate biotrophic fungus Blumeria graminis f. sp. tritici. The allelic series of the wheat Pm3 gene conferring race-specific resistance against powdery mildew has been well characterized functionally, and recently the corresponding avirulence gene AvrPm3a/f triggering the specific recognition by Pm3a and Pm3f alleles was cloned. Here, we describe the genetic and molecular analysis of two additional Blumeria loci involved in the resistance mediated by the Pm3c and Pm3f alleles. We genetically identified the two loci and mapped at high resolution one locus involved in the avirulence towards both Pm3c and Pm3f. The single candidate gene Bcg1 was identified in a physical target interval of 26kb defined by flanking genetic markers. Bcg1 encodes a small secreted protein sharing structural homology with ribonucleases and belongs to a family of clustered putative effector genes under diversifying selection. We found a very good, but not complete, correlation of Bcg1 haplotypes with the phenotypes of natural isolates. Two mutants were generated that were affected in their phenotypes towards Pm3a and Pm3f but did not show any sequence polymorphism in Bcg1. Our results suggest that avirulence to Pm3 in Blumeria is determined by a complex network of genes, in which Bcg1 might have a central role as a modifier of the Pm3/AvrPm3 interactions.

Agrobacterium tumefaciens Gene Transfer: How a Plant Pathogen Hacks the Nuclei of Plant and Nonplant Organisms.

Agrobacterium species are soilborne gram-negative bacteria exhibiting predominantly a saprophytic lifestyle. Only a few of these species are capable of parasitic growth on plants, causing either hairy root or crown gall diseases. The core of the infection strategy of pathogenic Agrobacteria is a genetic transformation of the host cell, via stable integration into the host genome of a DNA fragment called T-DNA. This genetic transformation results in oncogenic reprogramming of the host to the benefit of the pathogen. This unique ability of interkingdom DNA transfer was largely used as a tool for genetic engineering. Thus, the artificial host range of Agrobacterium is continuously expanding and includes plant and nonplant organisms. The increasing availability of genomic tools encouraged genome-wide surveys of T-DNA tagged libraries, and the pattern of T-DNA integration in eukaryotic genomes was studied. Therefore, data have been collected in numerous laboratories to attain a better understanding of T-DNA integration mechanisms and potential biases. This review focuses on the intranuclear mechanisms necessary for proper targeting and stable expression of Agrobacterium oncogenic T-DNA in the host cell. More specifically, the role of genome features and the putative involvement of host's transcriptional machinery in relation to the T-DNA integration and effects on gene expression are discussed. Also, the mechanisms underlying T-DNA integration into specific genome compartments is reviewed, and a theoretical model for T-DNA intranuclear targeting is presented.

Global genomic analyses of wheat powdery mildew reveal association of pathogen spread with historical human migration and trade.

The fungus Blumeria graminis f. sp. tritici causes wheat powdery mildew disease. Here, we study its spread and evolution by analyzing a global sample of 172 mildew genomes. Our analyses show that B.g. tritici emerged in the Fertile Crescent during wheat domestication. After it spread throughout Eurasia, colonization brought it to America, where it hybridized with unknown grass mildew species. Recent trade brought USA strains to Japan, and European strains to China. In both places, they hybridized with local ancestral strains. Thus, although mildew spreads by wind regionally, our results indicate that humans drove its global spread throughout history and that mildew rapidly evolved through hybridization.

Comparative Transcriptome Analysis of Wheat Lines in the Field Reveals Multiple Essential Biochemical Pathways Suppressed by Obligate Pathogens.

Mildew and rust are the most devastating cereal pathogens, and in wheat they can cause up to 50% yield loss every year. Wheat lines containing resistance genes are used to effectively control fungal diseases, but the molecular mechanisms underlying the interaction between wheat and its fungal pathogens are poorly understood. Here, we used RNA sequencing (RNA-Seq) to compare the transcriptomic landscape of susceptible and resistant wheat lines to identify genes and pathways that are targeted by obligate biotrophic fungal pathogens. The five lines differed in the expression of thousands of genes under infection as well as control conditions. Generally, mixed infection with powdery mildew and leaf rust resulted in downregulation of numerous genes in susceptible lines. Interestingly, transcriptomic comparison between the nearly isogenic lines Thatcher and Thatcher-Lr34 identified 753 genes that are uniquely downregulated in the susceptible line upon infection. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis, revealed the suppression of six major biochemical pathways, namely nuclear transport, alternative splicing, DNA damage response, ubiquitin-mediated proteolysis, phosphoinositol signaling, and photosynthesis. We conclude that powdery mildew and leaf rust evade the wheat defense system by suppression of programmed cell death (PCD) and responses to cellular damage. Considering the broad range of the induced changes, we propose that the pathogen targets "master regulators" at critical steps in the respective pathways. Identification of these wheat genes targeted by the pathogen could inspire new directions for future wheat breeding.

Cross-Kingdom RNAi of Pathogen Effectors Leads to Quantitative Adult Plant Resistance in Wheat.

Cross-kingdom RNA interference (RNAi) is a biological process allowing plants to transfer small regulatory RNAs to invading pathogens to trigger the silencing of target virulence genes. Transient assays in cereal powdery mildews suggest that silencing of one or two effectors could lead to near loss of virulence, but evidence from stable RNAi lines is lacking. We established transient host-induced gene silencing (HIGS) in wheat, and demonstrate that targeting an essential housekeeping gene in the wheat powdery mildew pathogen (Blumeria graminis f. sp. tritici) results in significant reduction of virulence at an early stage of infection. We generated stable transgenic RNAi wheat lines encoding a HIGS construct simultaneously silencing three B.g. tritici effectors including SvrPm3 a1/f1 , a virulence factor involved in the suppression of the Pm3 powdery mildew resistance gene. We show that all targeted effectors are effectively downregulated by HIGS, resulting in reduced fungal virulence on adult wheat plants. Our findings demonstrate that stable HIGS of effector genes can lead to quantitative gain of resistance without major pleiotropic effects in wheat.

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.

Cereal powdery mildew effectors: a complex toolbox for an obligate pathogen.

Cereal powdery mildews are major pathogens of cultivated monocot crops, and all are obligate biotrophic fungi that can only grow and reproduce on living hosts. This lifestyle is combined with extreme host specialization where every mildew subspecies (referred to as forma specialis) can only infect one plant species. Recently there has been much progress in our understanding of the possible roles effectors play in this complex host-pathogen interaction. Here, we review current knowledge on the origin, evolution, and mode of action of cereal mildew effectors, with a particular focus on recent advances in the identification of bona fide effectors and avirulence effector proteins from wheat and barley powdery mildews.

Non-parent of Origin Expression of Numerous Effector Genes Indicates a Role of Gene Regulation in Host Adaption of the Hybrid Triticale Powdery Mildew Pathogen.

Powdery mildew is an important disease of cereals. It is caused by one species, Blumeria graminis, which is divided into formae speciales each of which is highly specialized to one host. Recently, a new form capable of growing on triticale (B.g. triticale) has emerged through hybridization between wheat and rye mildews (B.g. tritici and B.g. secalis, respectively). In this work, we used RNA sequencing to study the molecular basis of host adaptation in B.g. triticale. We analyzed gene expression in three B.g. tritici isolates, two B.g. secalis isolates and two B.g. triticale isolates and identified a core set of putative effector genes that are highly expressed in all formae speciales. We also found that the genes differentially expressed between isolates of the same form as well as between different formae speciales were enriched in putative effectors. Their coding genes belong to several families including some which contain known members of mildew avirulence (Avr) and suppressor (Svr) genes. Based on these findings we propose that effectors play an important role in host adaptation that is mechanistically based on Avr-Resistance gene-Svr interactions. We also found that gene expression in the B.g. triticale hybrid is mostly conserved with the parent-of-origin, but some genes inherited from B.g. tritici showed a B.g. secalis-like expression. Finally, we identified 11 unambiguous cases of putative effector genes with hybrid-specific, non-parent of origin gene expression, and we propose that they are possible determinants of host specialization in triticale mildew. These data suggest that altered expression of multiple effector genes, in particular Avr and Svr related factors, might play a role in mildew host adaptation based on hybridization.