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Most resistance genes thus far isolated from wheat have a very limited number of functional alleles, with the exception of the powdery mildew PM3 resistance locus. Here we report the isolation of most of the alleles at wheat stem rust resistance gene locus SR9, representing the largest multi-allelic rust resistance locus in common wheat. The seven previously reported resistance alleles (Sr9a, Sr9b, Sr9d, Sr9e, Sr9f, Sr9g, and Sr9h) at the locus were characterised using a synergistic strategy. Loss-of-function mutants and/or transgenic complementation were used to confirm Sr9b, two haplotypes of Sr9e (Sr9e_h1, Sr9e_h2), Sr9g, and Sr9h. Each allele encodes a highly related nucleotide-binding site leucine-rich repeat (NB-LRR) type immune receptor, containing a previously unreported motif at their N termini and an unusual long LRR domain, that confers resistance to a unique spectrum of isolates of the wheat stem rust pathogen. The only SR9 protein effective against stem rust pathogen race TTKSK (Ug99), SR9H, differed from SR9B by a single amino acid. SR9B and SR9G resistance proteins were also distinguished by only a single amino acid. The SR9 allelic series found in the B subgenome are orthologs of wheat stem rust resistance gene Sr21 located in the A subgenome with around 85% identity in protein sequences.

Breeding wheat with durable resistance to the fungal pathogen Puccinia graminis f. sp. tritici (Pgt), a major threat to cereal production, is challenging due to the rapid evolution of pathogen virulence. Increased durability and broad-spectrum resistance can be achieved by introducing more than one resistance gene, but combining numerous unlinked genes by breeding is laborious. Here we generate polygenic Pgt resistance by introducing a transgene cassette of five resistance genes into bread wheat as a single locus and show that at least four of the five genes are functional. These wheat lines are resistant to aggressive and highly virulent Pgt isolates from around the world and show very high levels of resistance in the field. The simple monogenic inheritance of this multigene locus greatly simplifies its use in breeding. However, a new Pgt isolate with virulence to several genes at this locus suggests gene stacks will need strategic deployment to maintain their effectiveness. Combining fungal-resistance genes into a single cassette enables the generation of highly resistant wheat lines.

In the last 20 years, severe wheat stem rust outbreaks have been recorded in Africa, Europe, and Central Asia. This previously well controlled disease, caused by the fungus Puccinia graminis f. sp. tritici, has reemerged as a major threat to wheat cultivation. The stem rust (Sr) resistance gene Sr22 encodes a nucleotide-binding and leucine-rich repeat receptor which confers resistance to the highly virulent African stem rust isolate Ug99. Here, we show that the Sr22 gene is conserved among grasses in the Triticeae and Poeae lineages. Triticeae species contain syntenic loci with single-copy orthologs of Sr22 on chromosome 7, except Hordeum vulgare, which has experienced major expansions and rearrangements at the locus. We also describe 14 Sr22 sequence variants obtained from both Triticum boeoticum and the domesticated form of this species, T. monococcum, which have been postulated to encode both functional and nonfunctional Sr22 alleles. The nucleotide sequence analysis of these alleles identified historical sequence exchange resulting from recombination or gene conversion, including breakpoints within codons, which expanded the coding potential at these positions by introduction of nonsynonymous substitutions. Three Sr22 alleles were transformed into wheat cultivar Fielder and two postulated resistant alleles from Schomburgk (hexaploid wheat introgressed with T. boeoticum segment carrying Sr22) and T. monococcum accession PI190945, respectively, conferred resistance to P. graminis f. sp. tritici race TTKSK, thereby unequivocally confirming Sr22 effectiveness against Ug99. The third allele from accession PI573523, previously believed to confer susceptibility, was confirmed as nonfunctional against Australian P. graminis f. sp. tritici race 98-1,2,3,5,6. [Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Key message: Stem rust resistance genes, SrRL5271 and Sr672.1 as well as SrCPI110651, from Aegilops tauschii, the diploid D genome progenitor of wheat, are sequence variants of Sr46 differing by 1–2 nucleotides leading to non-synonymous amino acid substitutions. Abstract: The Aegilops tauschii (wheat D-genome progenitor) accessions RL 5271 and CPI110672 were identified as resistant to multiple races (including the Ug99) of the wheat stem rust pathogen Puccinia graminis f. sp. tritici (Pgt). This study was conducted to identify the stem rust resistance (Sr) gene(s) in both accessions. Genetic analysis of the resistance in RL 5271 identified a single dominant allele (SrRL5271) controlling resistance, whereas resistance segregated at two loci (SR672.1 and SR672.2) for a cross of CPI110672. Bulked segregant analysis placed SrRL5271 and Sr672.1 in a region on chromosome arm 2DS that encodes Sr46. Molecular marker screening, mapping and genomic sequence analysis demonstrated SrRL5271 and Sr672.1 are alleles of Sr46. The amino acid sequence of SrRL5271 and Sr672.1 is identical but differs from Sr46 (hereafter referred to as Sr46_h1 by following the gene nomenclature in wheat) by a single amino acid (N763K) and is thus designated Sr46_h2. Screening of a panel of Ae. tauschii accessions identified an additional allelic variant that differed from Sr46_h2 by a different amino acid (A648V) and was designated Sr46_h3. By contrast, the protein encoded by the susceptible allele of Ae. tauschii accession AL8/78 differed from these resistance proteins by 54 amino acid substitutions (94% nucleotide sequence gene identity). Cloning and complementation tests of the three resistance haplotypes confirmed their resistance to Pgt race 98-1,2,3,5,6 and partial resistance to Pgt race TTRTF in bread wheat. The three Sr46 haplotypes, with no virulent races detected yet, represent a valuable source for improving stem resistance in wheat.

Stripe rust resistance gene YrAet672 from Aegilops tauschii accession CPI110672 encodes a nucleotide-binding and leucine-rich repeat domain containing protein similar to YrAS2388 and both these members were haplotypes of Yr28. New sources of host resistance are required to counter the continued emergence of new pathotypes of the wheat stripe rust pathogen Puccinia striiformis Westend. f. sp. tritici Erikss. (Pst). Here, we show that CPI110672, an Aegilops tauschii accession from Turkmenistan, carries a single Pst resistance gene, YrAet672, that is effective against multiple Pst pathotypes, including the four predominant Pst lineages present in Australia. The YRAet672 locus was fine mapped to the short arm of chromosome 4D, and a nucleotide-binding and leucine-rich repeat gene was identified at the locus. A transgene encoding the YrAet672 genomic sequence, but lacking a copy of a duplicated sequence present in the 3' UTR, was transformed into wheat cultivar Fielder and Avocet S. This transgene conferred a weak resistance response, suggesting that the duplicated 3' UTR region was essential for function. Subsequent analyses demonstrated that YrAet672 is the same as two other Pst resistance genes described in Ae. tauschii, namely YrAS2388 and Yr28. They were identified as haplotypes encoding identical protein sequences but are polymorphic in non-translated regions of the gene. Suppression of resistance conferred by YrAet672 and Yr28 in synthetic hexaploid wheat lines (AABBDD) involving Langdon (AABB) as the tetraploid parent was associated with a reduction in transcript accumulation.

Wheat plants are infected by diverse pathogens of economic significance. They include biotrophic pathogens like mildews and rusts that require living plant cells to proliferate. By contrast necrotrophic pathogens that cause diseases such as tan spot, Septoria nodurum blotch and spot blotch require dead or dying cells to acquire nutrients. Pioneering studies in the flax plant-flax rust pathosystem led to the ‘gene-for-gene’ hypothesis which posits that a resistance gene product in the host plant recognizes a corresponding pathogen gene product, resulting in disease resistance. In contrast, necrotrophic wheat pathosystems have an ‘inverse gene-for-gene’ system whereby recognition of a necrotrophic fungal product by a dominant host gene product causes disease susceptibility, and the lack of recognition of this pathogen molecule leads to resistance. More than 300 resistance/susceptibility genes have been identified genetically in wheat and of those cloned the majority encode nucleotide binding, leucine rich repeat immune receptors. Other resistance gene types are also present in wheat, in particular adult plant resistance genes. Advances in mutational genomics and the wheat pan-genome are accelerating causative disease resistance/susceptibility gene discovery. This has enabled multiple disease resistance genes to be engineered as a transgenic gene stack for developing more durable disease resistance in wheat.

Host resistance is considered the most effective means to control plant diseases; however, individually deployed resistance genes are often rapidly overcome by pathogen adaptation. Combining multiple effective resistance genes is the optimal approach to durable resistance, but the lack of functional markers for resistance genes has hampered implementation. Leaf rust, caused by Puccinia hordei, is an economically significant disease of barley, but only a few major Resistance genes to P. hordei (Rph) have been cloned. In this study, gene Rph3 was isolated by positional cloning and confirmed by mutational analysis and transgenic complementation. The Rph3 gene, which originated from wild barley and was first introgressed into cultivated Egyptian germplasm, encodes a unique transmembrane resistance protein that differs from all known plant disease resistance proteins at the amino acid sequence level. Genetic profiles of diverse accessions indicated limited genetic diversity in Rph3 in domesticated germplasm, and higher diversity in wild barley from the Eastern Mediterranean region. Expression profiling using P. hordei isolates with contrasting pathogenicity for the Rph3 host locus showed that the Rph3 gene was expressed only in interactions with Rph3-avirulent isolates, a phenomenon also observed for transcription activator-like effector-dependent genes known as executors conferring resistance to Xanthomonas spp. Like the known transmembrane executors such as Bs3 and Xa7 heterologous expression of Rph3 in N. benthamiana induced a cell death response. Given that Rph3 shares several features with executor genes, it seems likely that P. hordei contains effectors similar to the transcription activator-like effectors that target host executor genes. The isolation of Rph3 highlights convergent evolutionary processes in diverse plant-pathogen interaction systems, where similar defence mechanisms evolved independently in monocots and dicots and provide evidence for executor genes in the Triticeae tribe.

The re-emergence of stem rust on wheat in Europe and Africa is reinforcing the ongoing need for durable resistance gene deployment. Here, we isolate from wheat, Sr26 and Sr61, with both genes independently introduced as alien chromosome introgressions from tall wheat grass (Thinopyrum ponticum). Mutational genomics and targeted exome capture identify Sr26 and Sr61 as separate single genes that encode unrelated (34.8%) nucleotide binding site leucine rich repeat proteins. Sr26 and Sr61 are each validated by transgenic complementation using endogenous and/or heterologous promoter sequences. Sr61 orthologs are absent from current Thinopyrum elongatum and wheat pan genome sequences, contrasting with Sr26 where homologues are present. Using gene-specific markers, we validate the presence of both genes on a single recombinant alien segment developed in wheat. The co-location of these genes on a small non-recombinogenic segment simplifies their deployment as a gene stack and potentially enhances their resistance durability., The tall wheat grass-derived stem rust resistance genes Sr26 and Sr61 are among a few ones that are effective to all current dominant races of stem rust, including Ug99. Here, the authors show that the two genes are present in a small non-recombinogenic segment but encode two unrelated NLR proteins.

Stem rust caused by the fungus Puccinia graminis f. sp. tritici (Pgt) is a devastating disease of the global staple crop wheat. Although this disease was largely controlled in the latter half of the twentieth century, new virulent strains of Pgt, such as Ug99, have recently evolved

A stripe rust resistance QTL in durum wheat maps near the bread wheat Yr80 locus with the latter reduced to 15 candidate genes. Some wheat adult plant resistance (APR) genes provide partial resistance in the later stages of plant development to rust diseases and are an important component in protecting wheat crops from these fungal pathogens. These genes provide protection in both bread wheat and durum wheat. Here, we have mapped APR to wheat stripe rust, caused by the fungal pathogen Puccinia striiformis f. sp. tritici, in a cross between durum cultivars Stewart and Bansi. Two resistance QTLs derived from the Stewart parent were identified in multi-generational field trials. One QTL is located on chromosome 1BL and maps to the previously identified Yr29/Lr46/Sr58/Pm39 multi-pathogen APR locus. The second locus, located on chromosome 3BL, maps near the recently described bread wheat APR gene, Yr80. Fine mapping in durum and bread wheat families shows that the durum 3BL locus and Yr80 are closely located, with the later APR gene reduced to 15 candidate genes present in the Chinese Spring genome sequence. Distorted segregation of the durum 3BL region was observed with the Stewart locus preferentially transmitted through pollen when compared with the equivalent Bansi region.

Triticum monococcumssp.monococcumhas useful traits for bread wheat improvement. The synthesis ofTriticum turgidum–T. monococcumamphiploids is an essential step for transferring genes fromT. monococcuminto bread wheat. In this study, 264 wide hybridization combinations were done by crossing 60T. turgidumlines belonging to five subspecies with 83T. monococcumaccessions. Without embryo rescue and hormone treatment, from the 10,810 florets pollinated, 1983 seeds were obtained, with a mean crossability of 18.34% (range 0–89.29%). Many hybrid seeds (90.73%, 923/1017) could germinate and produce plants. A total of 56 new amphiploids (AABBAmAm) were produced by colchicine treatment ofT. turgidum×T. monococcumF1hybrids. The chromosome constitution of amphiploids was characterized by fluorescencein situhybridization using oligonucleotides probes with different chromosome and sub-chromosome specificities. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis indicated that theGlu-A1m-b,Glu-A1m-c,Glu-A1m-dandGlu-A1m-hproteins ofT. monococcumwere expressed in some amphiploids. Despite resistance reduction in several cases, 45 out of 56 amphiploids exhibited resistance to the current predominant Chinese stripe rust races at both the seedling and adult plant stage. These novel amphiploids provide new germplasm for the potential improvement of bread wheat quality and stripe rust resistance.

Nonhost resistance (NHR)protects plants from a large and diverse array of potential phytopathogens. Each phytopathogen can parasitise some plant species, but most plant species are nonhosts that are innately immune due to a series of physical, chemical and inducible defenses these nonadapted pathogens cannot overcome. New evidence supports the NHR paradigm that posits the inability of potential pathogens to colonise nonhost plants is frequently due to molecular incompatibility between pathogen virulence factors and plant cellular targets. While NHR is durable, it is not insurmountable. Environmental changes can facilitate pathogen host jumps or alternatively result in new encounters between previously isolated plant species and pathogens. Climate change is predicted to substantially alter the current distribution of plants and their pathogens which could result in parasitism of new plant species.

Phytopathogens have a limited range of host plant species that they can successfully parasitise ie. that they are adapted for. Infection of plants by nonadapted pathogens often results in an active resistance response that is relatively poorly characterised because phenotypic variation in this response often does not exist within a plant species, or is too subtle for genetic dissection. In addition, complex polygenic inheritance often underlies these resistance phenotypes and mutagenesis often does not impact upon this resistance, presumably due to genetic or mechanistic redundancy. Here it is demonstrated that phenotypic differences in the resistance response of Brachypodium distachyon to the nonadapted wheat stripe rust pathogen Puccinia striiformis f. sp. tritici (Pst) are genetically tractable and simply inherited. Two dominant loci were identified on B. distachyon chromosome 4 that each reduce attempted Pst colonisation compared with sib and parent lines without these loci. One locus (Yrr1) is effective against diverse Australian Pst isolates and present in two B. distachyon mapping families as a conserved region that was reduced to 5 candidate genes by fine mapping. A second locus, Yrr2, shows Pst race-specificity and encodes a disease resistance gene family typically associated with host plant resistance. These data indicate that some components of resistance to nonadapted pathogens are genetically tractable in some instances and may mechanistically overlap with host plant resistance to avirulent adapted pathogens., Author summary Plant pathogens are specialists and can colonise only a limited number of plant species (hosts). Pathogen infection of a plant that is not a host of the disease often results in an active plant defense response. This poorly characterised defense response is durable as phytopathogens rarely successfully colonise new hosts. The ability to transfer this resistance to host plants would be highly beneficial in protecting crops from disease. However, this resistance has been difficult to genetically dissect as all members of a plant species are resistant. Here we show that some accessions of the model grass Brachypodium distachyon show some variation in their ability to suppress colonisation by the wheat stripe rust pathogen Puccinia striiformis. Brachypodium is not a host species of wheat stripe rust disease and no accessions are fully susceptible, however, some allow more pathogen growth than others. We have exploited these relatively subtle phenotypic differences to genetically dissect this difference in resistance and identified two Brachypodium loci that contribute increased resistance to the nonadapted wheat stripe rust pathogen.

SummaryIn the last 20 years, stem rust caused by the fungus Puccinia graminis f. sp. tritici (Pgt), has re-emerged as a major threat to wheat and barley cultivation in Africa and Europe. In contrast to wheat with 82 designated stem rust (Sr) resistance genes, barley’s genetic variation for stem rust resistance is very narrow with only seven resistance genes genetically identified. Of these, only one locus consisting of two genes is effective against Ug99, a strain of Pgt which emerged in Uganda in 1999 and has since spread to much of East Africa and parts of the Middle East. The objective of this study was to assess the functionality, in barley, of cloned wheat Sr genes effective against Ug99. Sr22, Sr33, Sr35 and Sr45 were transformed into barley cv. Golden Promise using Agrobacterium-mediated transformation. All four genes were found to confer effective stem rust resistance. The barley transgenics remained susceptible to the barley leaf rust pathogen Puccinia hordei, indicating that the resistance conferred by these wheat Sr genes was specific for Pgt. Cloned Sr genes from wheat are therefore a potential source of resistance against wheat stem rust in barley.

Oat crown rust caused by Puccinia coronata f. sp. avenae is the most destructive foliar disease of cultivated oat. Characterization of genetic factors controlling resistance responses to Puccinia coronata f. sp. avenae in non-host species could provide new resources for developing disease protection strategies in oat. We examined symptom development and fungal colonization levels of a collection of Brachypodium distachyon and B. hybridum accessions infected with three North American P. coronata f. sp. avenae isolates. Our results demonstrated that colonization phenotypes are dependent on both host and pathogen genotypes, indicating a role for race-specific responses in these interactions. These responses were independent of the accumulation of reactive oxygen species. Expression analysis of several defense-related genes suggested that salicylic acid and ethylene-mediated signaling, but not jasmonic acid are components of resistance reaction to P. coronata f. sp. avenae. Our findings provide the basis to conduct a genetic inheritance study to examine if effector-triggered immunity contributes to non-host resistance to P. coronata f. sp. avenae in Brachypodium species.

Multilayered defense responses ensure that plants are hosts to only a few adapted pathogens in the environment. The host range of a plant pathogen depends on its ability to fully overcome plant defense barriers, with failure at any single step sufficient to prevent life cycle completion of the pathogen. Puccinia striiformis, the causal agent of stripe rust (=yellow rust), is an agronomically important obligate biotrophic fungal pathogen of wheat and barley. It is generally unable to complete its life cycle on the non-adapted wild grass species Brachypodium distachyon, but natural variation exists for the degree of hyphal colonization by Puccinia striiformis. Using three B. distachyon mapping populations, we identified genetic loci conferring colonization resistance to wheat-adapted and barley-adapted isolates of P. striiformis. We observed a genetic architecture composed of two major effect QTLs (Yrr1 and Yrr3) restricting the colonization of P. striiformis. Isolate specificity was observed for Yrr1, whereas Yrr3 was effective against all tested P. striiformis isolates. Plant immune receptors of the nucleotide binding, leucine-rich repeat (NB-LRR) encoding gene family are present at the Yrr3 locus, whereas genes of this family were not identified at the Yrr1 locus. While it has been proposed that resistance to adapted and non-adapted pathogens are inherently different, the observation of (1) a simple genetic architecture of colonization resistance, (2) isolate specificity of major and minor effect QTLs, and (3) NB-LRR encoding genes at the Yrr3 locus suggest that factors associated with resistance to adapted pathogens are also critical for non-adapted pathogens., Author summary Plants are constantly exposed to a multitude of potential pathogens but remain immune to most of these due to a multilayered immune system. Pathogens have specialized by adapting to certain host plants and their defense barriers. Most of our understanding of plant-pathogen interactions stems from these highly specialized interactions, because they are characterized by qualitative interactions (resistant or susceptible). It has generally been assumed that the genetic and molecular basis of resistance to non-adapted pathogens is fundamentally different, as either no variation exists in a species (complete immunity) or variation encompasses only early pathogen invasion (colonization), but not full susceptibility. We have studied the interaction between the agronomically important fungal stripe rust pathogen (Puccinia striiformis) of wheat and barley with the wild grass species Brachypodium distachyon. Rust infections consist of two stages: colonization of plant tissues followed by a reproductive phase. We identified natural variation for the degree of P. striiformis colonization in different B. distachyon accessions and dissected the genetic architecture controlling resistance at this infection stage. QTLs conferring resistance possessed several characteristics similar to adapted host systems, indicating that resistance to adapted and non-adapted pathogens are not intrinsically different.

Recent research has indicated that a subset of defense-related genes is downregulated in the Arabidopsis DNA demethylase triple mutant rdd (ros1 dml2 dml3) resulting in increased susceptibility to the fungal pathogen Fusarium oxysporum. In rdd plants these downregulated genes contain hypermethylated transposable element sequences (TE) in their promoters, suggesting that this methylation represses gene expression in the mutant and that these sequences are actively demethylated in wild-type plants to maintain gene expression. In this study, the tissue-specific and pathogen-inducible expression patterns of rdd-downregulated genes were investigated and the individual role of ROS1, DML2, and DML3 demethylases in these spatiotemporal regulation patterns was determined. Large differences in defense gene expression were observed between pathogen-infected and uninfected tissues and between root and shoot tissues in both WT and rdd plants, however, only subtle changes in promoter TE methylation patterns occurred. Therefore, while TE hypermethylation caused decreased gene expression in rdd plants it did not dramatically effect spatiotemporal gene regulation, suggesting that this latter regulation is largely methylation independent. Analysis of ros1-3, dml2-1, and dml3-1 single gene mutant lines showed that promoter TE hypermethylation and defense-related gene repression was predominantly, but not exclusively, due to loss of ROS1 activity. These data demonstrate that DNA demethylation of TE sequences, largely by ROS1, promotes defense-related gene expression but does not control spatiotemporal expression in Arabidopsis. Summary: Ros1-mediated DNA demethylation of promoter transposable elements is essential for activation of defense-related gene expression in response to fungal infection in Arabidopsis thaliana.

Summary The hexaploid wheat (Triticum aestivum) adult plant resistance gene, Lr34/Yr18/Sr57/Pm38/Ltn1, provides broad-spectrum resistance to wheat leaf rust (Lr34), stripe rust (Yr18), stem rust (Sr57) and powdery mildew (Pm38) pathogens, and has remained effective in wheat crops for many decades. The partial resistance provided by this gene is only apparent in adult plants and not effective in field-grown seedlings. Lr34 also causes leaf tip necrosis (Ltn1) in mature adult plant leaves when grown under field conditions. This D genome-encoded bread wheat gene was transferred to tetraploid durum wheat (T. turgidum) cultivar Stewart by transformation. Transgenic durum lines were produced with elevated gene expression levels when compared with the endogenous hexaploid gene. Unlike nontransgenic hexaploid and durum control lines, these transgenic plants showed robust seedling resistance to pathogens causing wheat leaf rust, stripe rust and powdery mildew disease. The effectiveness of seedling resistance against each pathogen correlated with the level of transgene expression. No evidence of accelerated leaf necrosis or up-regulation of senescence gene markers was apparent in these seedlings, suggesting senescence is not required for Lr34 resistance, although leaf tip necrosis occurred in mature plant flag leaves. Several abiotic stress-response genes were up-regulated in these seedlings in the absence of rust infection as previously observed in adult plant flag leaves of hexaploid wheat. Increasing day length significantly increased Lr34 seedling resistance. These data demonstrate that expression of a highly durable, broad-spectrum adult plant resistance gene can be modified to provide seedling resistance in durum wheat.

A previously reported Agrobacterium tumefaciens transformation system that transformed wheat cultivar Fielder at high efficiency was shown to also transform eight out of nine Triticum aestivum (hexaploid wheat) cultivars tested and two Triticum turgidum (durum wheat) cultivars. Transformation efficiencies of these wheat lines ranged from 1.5 to 51 %. Included amongst this germplasm were elite Australian hexaploid wheat cultivars that are currently in commercial cultivation and two of these cultivars, Gladius and Westonia, were transformed at 32 and 45 % efficiency, respectively. Similar high transformation efficiencies were observed for durum wheat cultivars Kronos (51 %) and Stewart (26 %). This highly efficient transformation system was used to generate transgenic plants in the absence of selection and high heritability of unselected transgenes was observed. Selectable marker free transgenic wheat plants were produced at 3 % efficiency. These data demonstrate highly efficient Agrobacterium transformation of diverse wheat germplasm, including elite cultivars, which enables routine production of selectable marker free transgenics.

Large numbers of candidate effectors from fungal pathogens are being identified through whole-genome sequencing and in planta expression studies. Although Agrobacterium-mediated transient expression has enabled high-throughput functional analysis of effectors in dicot plants, this assay is not effective in cereal leaves. Here, we show that a nonpathogenic Pseudomonas fluorescens engineered to express the type III secretion system (T3SS) of P. syringae and the wheat pathogen Xanthomonas translucens can deliver fusion proteins containing T3SS signals from P. syringae (AvrRpm1) and X. campestris (AvrBs2) avirulence (Avr) proteins, respectively, into wheat leaf cells. A calmodulin-dependent adenylate cyclase reporter protein was delivered effectively into wheat and barley by both bacteria. Absence of any disease symptoms with P. fluorescens makes it more suitable than X. translucens for detecting a hypersensitive response (HR) induced by an effector protein with avirulence activity. We further modified the delivery system by removal of the myristoylation site from the AvrRpm1 fusion to prevent its localization to the plasma membrane which could inhibit recognition of an Avr protein. Delivery of the flax rust AvrM protein by the modified delivery system into transgenic tobacco leaves expressing the corresponding M resistance protein induced a strong HR, indicating that the system is capable of delivering a functional rust Avr protein. In a preliminary screen of effectors from the stem rust fungus Puccinia graminis f. sp. tritici, we identified one effector that induced a host genotype-specific HR in wheat. Thus, the modified AvrRpm1:effector–Pseudomonas fluorescens system is an effective tool for large-scale screening of pathogen effectors for recognition in wheat.

The model temperate grass Brachypodium distachyon is considered a nonhost for wheat rust diseases caused by Puccinia graminis f. sp. tritici, P. triticina, and P. striiformis. Up to 140 Brachypodium accessions were infected with these three rust species, in addition to P. graminis ff. spp. avena and phalaridis. Related B. distachyon lines showed similar cytological nonhost resistance (NHR) phenotypes, and an inverse relationship between P. graminis f. sp. tritici and P. striiformis growth was observed in many lines, with accessions that allowed the most growth of P. graminis f. sp. tritici showing the least P. striiformis development and vice versa. Callose deposition patterns during infection by all three rust species showed similarity to the wheat basal defense response while cell death that resulted in autofluorescence did not appear to be a major component of the defense response. Infection of B. distachyon with P. graminis f. sp. avena and P. graminis f. sp. phalaridis produced much greater colonization, indicating that P. graminis rusts with Poeae hosts show greater ability to infect B. distachyon than those with Triticeae hosts. P. striiformis infection of progeny from two B. distachyon families demonstrated that these NHR phenotypes are highly heritable and appear to be under relatively simple genetic control, making this species a powerful tool for elucidating the molecular basis of NHR to cereal rust pathogens.

Plant phenotypes resistant and susceptible to fungal pathogens are usually scored using qualitative, subjective methods that are based upon disease symptoms or by an estimation of the amount of visible fungal growth. Given that plant resistance genes often confer partial resistance to fungal pathogens, a simple, sensitive, nonsubjective quantitative method for measuring pathogen growth would be highly advantageous. This report describes an in planta quantitative assay for fungal biomass based upon detection of chitin using wheat germ agglutinin conjugated to a fluorophore. Using this assay, the growth of wheat rust pathogens on wheat was assayed and the additivity of several adult plant and seedling resistance genes to Puccinia striiformis, P. graminis, and P. triticina was assayed on both glasshouse- and field-grown material. The assay can discriminate between individual rust pustules on a leaf segment or, alternatively, compare fungal growth on field plots. The quantification of Erysiphe necator (powdery mildew) growth on Vitis vinifera (grapevine) is also demonstrated, with resistant and susceptible cultivars readily distinguished. Given that chitin is a major cell wall component of many plant fungal pathogens, this robust assay will enable simple and accurate measurement of biomass accumulation in many plant–fungus interactions.

Mutagenesis continues to play an essential role for understanding plant gene function and, in some instances, provides an opportunity for plant improvement. The development of gene editing technologies such as TALENs and zinc fingers has revolutionised the targeted mutation specificity that can now be achieved. The CRISPR/Cas9 system is the most recent addition to gene editing technologies and arguably the simplest requiring only two components; a small guide RNA molecule (sgRNA) and Cas9 endonuclease protein which complex to recognise and cleave a specific 20 bp target site present in a genome. Target specificity is determined by complementary base pairing between the sgRNA and target site sequence enabling highly specific, targeted mutation to be readily engineered. Upon target site cleavage, error-prone endogenous repair mechanisms produce small insertion/deletions at the target site usually resulting in loss of gene function. CRISPR/Cas9 gene editing has been rapidly adopted in plants and successfully undertaken in numerous species including major crop species. Its applications are not restricted to mutagenesis and target site cleavage can be exploited to promote sequence insertion or replacement by recombination. The multiple applications of this technology in plants are described.

In higher plants, DNA transfer from the plastid (chloroplast) genome to the nucleus is a frequent, ongoing process. However, there has been uncertainty over whether this transfer occurs by a direct DNA mechanism or whether RNA intermediates are involved. Previous experiments utilising transplastomic Nicotiana tabacum (tp7 and tp17) enabled the detection of plastid-to-nucleus transfer in real time. To determine whether RNA intermediates are involved in this transfer, transplastomic lines (tpneoACG) were generated containing, in their plastid genomes, a nuclear promoter-driven kanamycin resistance gene (neo) with a start codon that required plastid RNA editing but otherwise identical to tp7 and tp17. Therefore it was expected that kanamycin resistance would only be acquired following RNA-mediated transfer of neo to the nucleus. Screening of tpneoACG progeny revealed several kanamycin-resistant plants, each of which contained the neo gene located in the nucleus. Surprisingly, neo was unedited in all these plants, indicating that neoACG was active in the absence of an edited start codon and suggesting that RNA intermediates were not involved in the transfers. However, analysis of tpneoACG revealed that only a low proportion of transcripts potentially able to mediate neo transfer were edited, thus precluding unequivocal conclusions regarding the role of RNA in plastid-to-nucleus transfer. The low proportion of edited transcripts was found to be due to predominant antisense neo transcripts, rather than to low editing efficiency of the sense transcripts. This study highlights a number of important considerations in the design of experiments utilising plastid RNA editing. The results also suggest that RNA editing sites reduce but do not eliminate functional plastid-to-nucleus gene transfer. This is relevant both in an evolutionary context and in placing RNA editing-dependent genes in the plastid genome as a means of transgene containment.

Activation tagging, as the result of random genomic insertion of either promoter or enhancer sequences, can produce novel, dominant mutations by over-expression of endogenous genes. This powerful genomics tool has been used extensively in dicot species such as Arabidopsis, while rice is the only cereal for which an equivalent system exists. In this study we describe an activation tagging system in barley based upon the maize Ac/Ds transposable element system. A modified Ds element (UbiDs) containing two maize polyubiquitin promoters, transposed in families derived from multiple independent UbiDs transformants and generated new Ds insertion events at frequencies ranging from 0% to 52% per family. The majority of transposed UbiDs elements activated high levels of adjacent flanking sequence transcription. Transposon-mediated expression was detected in all barley cell and tissue types analysed suggesting that this system is applicable to all aspects of plant development and biogenesis. In addition to transcriptional activation, this system is also capable of generating insertional knockout mutants and a UbiDs inactivated allele of the granule bound starch synthase I gene (waxy) was recovered that lead to reduced amylose accumulation. The recovery and analysis of dominant over-expression phenotypes generated by this system will provide a novel approach to understanding gene function in large cereal genomes where gene redundancy may mask conventional loss-of-function mutations.

Plants have developed diverse molecular and cellular mechanisms to cope with a lifetime of exposure to a variety of pathogens. Host transcriptional reprogramming is a central part of plant defense upon pathogen recognition. Recent studies link DNA methylation and demethylation as well as chromatin remodeling by posttranslational histone modifications, including acetylation, methylation, and ubiquitination, to changes in the expression levels of defense genes upon pathogen challenge. Remarkably these inducible defense mechanisms can be primed prior to pathogen attack by epigenetic modifications and this heightened resistance state can be transmitted to subsequent generations by inheritance of these modification patterns. Beside the plant host, epigenetic mechanisms have also been implicated in virulence development of pathogens. This review highlights recent findings and insights into epigenetic mechanisms associated with interactions between plants and pathogens, in particular bacterial and fungal pathogens, and demonstrates the positive role they can have in promoting plant defense.

Race Ug99 (TTKSK) of Puccinia graminis f. sp. tritici, detected in Uganda in 1998, has been recognized as a serious threat to food security because it possesses combined virulence to a large number of resistance genes found in current widely grown wheat (Triticum aestivum) varieties and germplasm, leading to its potential for rapid spread and evolution. Since its initial detection, variants of the Ug99 lineage of stem rust have been discovered in Eastern and Southern African countries, Yemen, Iran, and Egypt. To date, eight races belonging to the Ug99 lineage are known. Increased pathogen monitoring activities have led to the identification of other races in Africa and Asia with additional virulence to commercially important resistance genes. This has led to localized but severe stem rust epidemics becoming common once again in East Africa due to the breakdown of race-specific resistance gene SrTmp, which was deployed recently in the ‘Digalu’ and ‘Robin’ varieties in Ethiopia and Kenya, respectively. Enhanced research in the last decade under the umbrella of the Borlaug Global Rust Initiative has identified various race-specific resistance genes that can be utilized, preferably in combinations, to develop resistant varieties. Research and development of improved wheat germplasm with complex adult plant resistance (APR) based on multiple slow-rusting genes has also progressed. Once only the Sr2 gene was known to confer slow rusting APR; now, four more genes—Sr55, Sr56, Sr57, and Sr58—have been characterized and additional quantitative trait loci identified. Cloning of some rust resistance genes opens new perspectives on rust control in the future through the development of multiple resistance gene cassettes. However, at present, disease-surveillance-based chemical control, large-scale deployment of new varieties with multiple race-specific genes or adequate levels of APR, and reducing the cultivation of susceptible varieties in rust hot-spot areas remains the best stem rust management strategy.

As there are numerous pathogen species that cause disease and limit yields of crops, such as wheat (Triticum aestivum), single genes that provide resistance to multiple pathogens are valuable in crop improvement. The mechanistic basis of multi-pathogen resistance is largely unknown. Here we use comparative genomics, mutagenesis and transformation to isolate the wheat Lr67 gene, which confers partial resistance to all three wheat rust pathogen species and powdery mildew. The Lr67 resistance gene encodes a predicted hexose transporter (LR67res) that differs from the susceptible form of the same protein (LR67sus) by two amino acids that are conserved in orthologous hexose transporters. Sugar uptake assays show that LR67sus, and related proteins encoded by homeoalleles, function as high-affinity glucose transporters. LR67res exerts a dominant-negative effect through heterodimerization with these functional transporters to reduce glucose uptake. Alterations in hexose transport in infected leaves may explain its ability to reduce the growth of multiple biotrophic pathogen species.

International audience; We identify the wheat stem rust resistance gene Sr50 (using physical mapping, mutation and complementation) as homologous to barley Mla, encoding a coiled-coil nucleotide-binding leucine-rich repeat (CC-NB-LRR) protein. We show that Sr50 confers a unique resistance specificity different from Sr31 and other genes on rye chromosome 1RS, and is effective against the broadly virulent Ug99 race lineage. Extensive haplotype diversity at the rye Sr50 locus holds promise for mining effective resistance genes.

With increasing global food demands in the face of challenging biotic and abiotic pressures on crop production, there is a vital need for good crop improvement strategies. Gene editing and gene targeting using designer nucleases are relatively new, sophisticated approaches that can be used for crop improvement. Designer nucleases are molecules that can be engineered to cleave virtually any endogenous DNA target sequence, making this technology inherently more powerful over current, essentially random mutation strategies. These molecules can also be used to promote targeted DNA insertions and homologous recombination. Further modifications of these molecules can convert them into designer transcription factors that can activate or suppress a gene of choice. Four designer nuclease platforms are currently available: meganucleases, zinc finger nucleases, TALENs and the more recently developed CRISPR/Cas9 system. All four of these systems have been shown to function in crop plants and have been used for site-specific gene targeting and gene editing. Herein, we describe the basis of each designer nuclease platform, highlighting the advantages and disadvantages of each, and give examples of their application in crop improvement.

A gene fis1 from flax (Linum usitatissimum), which is induced in mesophyll cells at the site of rust (Melampsora lini) infection, is also expressed in vascular tissue, particularly in floral structures of healthy plants. This paper reports that the promoter controlling this expression is contained within 282 bp 5' to the coding region and that fis1 gene induction is specifically by the rust pathogen and not by other fungal pathogens or by wounding. The fis1 gene has 73% homology with an Arabidopsis gene which encodes delta-1-pyrroline-5-carboxylate dehydrogenase (P5CDH) which is a part of the proline degradation pathway. Transgenic flax plants that either over-express fis1 or show reduced fis1 expression due to RNA-mediated gene silencing have an unaltered morphology. However, plants with reduced fis1 expression have markedly increased sensitivity to exogenous proline and show alteration in epidermal cell morphology, callose deposition and the production of hydrogen peroxide during proline-induced death. These lines, which show a biologically significant level of fis1 suppression, have an unaltered reaction to either virulent or avirulent rust infections, as do fis1 over-expression lines. These data indicate that the fis1 gene plays a role in proline metabolism and most likely encodes for a P5CDH enzyme. However, the precise role of fis1 and P5C catabolism in the development of rust disease remains unclear.