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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.

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.

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.

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.

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.

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.

L6 is a nucleotide binding site-leucine rich repeat (NBS-LRR) gene that confers race-specific resistance in flax (Linum usitatissimum) to strains of flax rust (Melampsora lini) that carry avirulence alleles of the AvrL567 gene but not to rust strains that carry only the virulence allele. Several mutant and recombinant forms of L6 were made that altered either the methionine-histidine-aspartate (MHD) motif conserved in the NBS domain of resistance proteins or exchanged the short domain C-terminal to the LRR region that is highly variable among L allele products. In transgenic flax some of these alleles are autoactive; they cause a gene dosage-dependent dwarf phenotype and constitutive expression of genes that are markers for the plant defense response. Their effects and penetrance ranged from extreme to mild in their degree of plant stunting, survival, and reproduction. Dwarf plants were also resistant to flax rust strains virulent to wild-type L6 plants, and this nonspecific resistance was associated with a hypersensitive response (HR) at the site of rust infection. The strongest autoactive allele, expressed in Arabidopsis from an ethanol-inducible promoter, gave rise to plant death dependent on the enhanced disease susceptibility 1 (EDS1) gene, which indicates that the mutant flax (Linaceae) L6 gene can signal cell death through a defined disease-resistance pathway in a different plant family (Brassicaceae).

Resistance May Not Be Futile Recently, Ug99, a particularly devastating strain of wheat stem rust fungus, has emerged, which could potentially threaten food security. Now, two genes have been cloned that offer resistance to Ug99. Saintenac et al. (p. 783 , published online 27 June) cloned Sr35 from Triticum monococcum , a diploid wheat species not often cultivated. Periyannan et al. (p. 786 , published online 27 June) cloned Sr33 from Aegilops tauschii , a diploid wild grass that contributed to the hexaploid genome of cultivated wheat. The genes both encode proteins that show features typical of other disease resistance proteins and offer opportunities to slow the pace of Ug99 progression.

• Aims This Botanical Briefing attempts to summarize what is currently known about the molecular bases of disease resistance in cereal species and suggests future research directions. • Scope An increasing number of resistance (R) genes have been isolated from rice, maize, wheat and barley that encode both structurally related and unique proteins. This R protein diversity may be attributable to the different modus operandi employed by pathogen species in some cases, but it is also a consequence of multiple defence strategies being employed against phytopathogens. Mutational analysis of barley has identified additional genes required for activation of an R gene-mediated defence response upon pathogen infection. In some instances very closely related barley R proteins require different proteins for defence activation, demonstrating that, within a single plant species, multiple resistance signalling pathways and different resistance strategies have evolved to confer protection against a single pathogen species. Despite the apparent diversity of cereal resistance mechanisms, some of the additional molecules required for R protein function are conserved amongst cereal and dicotyledonous species and even other eukaryotic species. Thus the derivation of functional homologues and interacting partner proteins from other species is contributing to the understanding of resistance signalling in cereals. The potential and limit of utilizing the rice genome sequence for further R gene isolation from cereal species is also considered, as are the new biotechnological possibilities for disease control arising from R gene isolation. • Conclusions Molecular analyses in cereals have further highlighted the complexity of plant–pathogen co-evolution and have shown that numerous active and passive defence strategies are employed by plants against phytopathogens. Many advances in understanding the molecular basis of disease resistance in cereals have focused on monogenic resistance traits. Future research targets are likely to include less experimentally tractable, durable polygenic resistances and nonhost resistance mechanisms.

Genome sequences reveal that a deluge of DNA from organelles has constantly been bombarding the nucleus since the origin of organelles. Recent experiments have shown that DNA is transferred from organelles to the nucleus at frequencies that were previously unimaginable. Endosymbiotic gene transfer is a ubiquitous, continuing and natural process that pervades nuclear DNA dynamics. This relentless influx of organelle DNA has abolished organelle autonomy and increased nuclear complexity.

The majority of verified plant disease resistance genes isolated to date are of the NBS-LRR class, encoding proteins with a predicted nucleotide binding site (NBS) and a leucine-rich repeat (LRR) region. We took advantage of the sequence conservation in the NBS motif to clone, by PCR, gene fragments from barley representing putative disease resistance genes of this class. Over 30 different resistance gene analogs (RGAs) were isolated from the barley cultivar Regatta. These were grouped into 13 classes based on DNA sequence similarity. Actively transcribed genes were identified from all classes but one, and cDNA clones were isolated to derive the complete NBS-LRR protein sequences. Some of the NBS-LRR genes exhibited variation with respect to whether and where particular introns were spliced, as well as frequent premature polyadenylation. DNA sequences related to the majority of the barley RGAs were identified in the recently expanded public rice genomic sequence database, indicating that the rice sequence can be used to extract a large proportion of the RGAs from barley and other cereals. Using a combination of RFLP and PCR marker techniques, representatives of all barley RGA gene classes were mapped in the barley genome, to all chromosomes except 4H. A number of the RGA loci map in the vicinity of known disease resistance loci, and the association between RGA S-120 and the nematode resistance locus Ha2 on chromosome 2H was further tested by co-segregation analysis. Most of the RGA sequences reported here have not been described previously, and represent a useful resource as candidates or molecular markers for disease resistance genes in barley and other cereals.

Gene transfer from the chloroplast to the nucleus has occurred over evolutionary time1. Functional gene establishment in the nucleus is rare, but DNA transfer without functionality is presumably more frequent. Here, we measured directly the transfer rate of chloroplast DNA (cpDNA) into the nucleus of tobacco plants (Nicotiana tabacum). To visualize this process, a nucleus-specific neomycin phosphotransferase gene (neoSTLS2) was integrated into the chloroplast genome, and the transfer of cpDNA to the nucleus was detected by screening for kanamycin-resistant seedlings in progeny. A screen for kanamycin-resistant seedlings was conducted with about 250,000 progeny produced by fertilization of wild-type females with pollen from plants containing cp-neoSTLS2. Sixteen plants of independent origin were identified and their progenies showed stable inheritance of neoSTLS2, characteristic of nuclear genes. Thus, we provide a quantitative estimate of one transposition event in about 16,000 pollen grains for the frequency of transfer of cpDNA to the nucleus. In addition to its evident role in organellar evolution, transposition of cpDNA to the nucleus in tobacco occurs at a rate that must have significant consequences for existing nuclear genes.

Expression of the fis1 gene from flax (Linum usitatissimum) is induced by a compatible rust (Melampsora lini) infection. Infection of transgenic plants containing a β-glucuronidase (GUS) reporter gene under the control of the fis1 promoter showed that induction is highly localized to those leaf mesophyll cells within and immediately surrounding rust infection sites. The level of induction reflects the extent of fungal growth. In a strong resistance reaction, such as the hypersensitive fleck mediated by the L6resistance gene, there is very little fungal growth and a microscopic level of GUS expression. Partially resistant flax leaves show levels of GUS expression that were intermediate to the level observed in the fully susceptible infection. Sequence and deletion analysis using both transient Agrobacterium tumefaciens expression and stable transformation assays have shown that the rust-induciblefis1 promoter is contained within a 580-bp fragment. Homologs of fis1 were identified in expressed sequence tag databases of a range of plant species including dicots, monocots, and a gymnosperm. Homologous genes isolated from maize (Zea mays; mis1), barley (Hordeum vulgare; bis1), wheat (Triticum aestivum; wis1), and Arabidopsis encode proteins that are highly similar (76%–82%) to the FIS1 protein. The Arabidopsis homologue has been reported to encode a Δ1-pyrroline-5-carboxylate dehydrogenase that is involved in the catabolism of proline to glutamate. RNA-blot analysis showed that mis1 in maize and the bis1homolog in barley are both up-regulated by a compatible infection with the corresponding species-specific rust. The rust-induced genes homologous to fis1 are present in many plants. The promoters of these genes have potential roles for the engineering of synthetic rust resistance genes by targeting transgene expression to the sites of rust infection.

A Melampsora lini β-tubulin gene and flanking sequence has been isolated and characterised. The gene contains 8 introns, is expressed in germinating urediospores and vegetative mycelia and encodes a 448 amino acid protein that is similar to other fungal β-tubulin genes. Evidence for a second β-tubulin gene within the M. lini genome is also presented. The predicted M. lini β-tubulin protein was compared with 34 other fungal β-tubulin proteins in a phylogenetic analysis and, in general, good agreement was observed between the derived β-tubulin based phylogeny and the current taxonomic status of these species. However, a second β-tubulin protein found in five fungal species is atypical when compared to other fungal β-tubulin proteins. Analysis of intron positions within 33 fungal β-tubulin genes showed that intron location was generally characteristic of the fungal class, with euascomycete fungi showing particularly striking conservation of intron position, although intron number was variable and intron sequences showed little conservation.

Rp1 is a complex rust resistance locus of maize. The HRp1-D haplotype is composed of Rp1-D and eight paralogues, seven of which also code for predicted nucleotide binding site-leucine rich repeat (NBS-LRR) proteins similar to the Rp1-D gene. The paralogues are polymorphic (DNA identities 91-97%), especially in the C-terminal LRR domain. The remaining family member encodes a truncated protein that has no LRR domain. Seven of the nine family members, including the truncated gene, are transcribed. Sequence comparisons between paralogues provide evidence for past recombination events between paralogues and diversifying selection, particularly in the C-terminal half of the LRR domain. Variants selected for complete or partial loss of Rp1-D resistance can be explained by unequal crossing over that occurred mostly within coding regions. The Rp1-D gene is altered or lost in all variants, the recombination breakpoints occur throughout the genes, and most recombinant events (9/14 examined) involved the same untranscribed paralogue with the Rp1-D gene. One recombinant with a complete LRR from Rp1-D, but the aminoterminal portion from another homologue, conferred the Rp1-D specificity but with a reduced level of resistance.

A number of agronomically important grasses (sorghum, wheat, panicum, sugar cane, oats, rice and barley) are shown to contain sequences homologous to rp1, a maize gene that confers race-specific resistance to the rust fungus Puccinia sorghi. Mapping of rp1-related sequences in barley identified three unlinked loci on chromosomes 1HL, 3HL and 7HS. The locus located on chromosome 7HS comprises a small gene family of at least four members, two of which were isolated and are predicted to encode nucleotide binding site-leucine-rich repeat (NBS-LRR) proteins that are respectively 58% and 60% identical to the maize rp1 protein. Evidence of positive selection for sequence diversification acting upon these two barley genes was observed; however, diversifying selection was restricted to the carboxy terminal half of the LRR domain. One of these rp1 homologous genes cosegregated with the barley Rpg1 stem rust resistance gene amongst 148 members of the Steptoe × Morex double haploid mapping family. Three other unrelated resistance gene-like sequences, potentially encoding NBS-LRR proteins, are also shown to be linked to the Rpg1 locus but not cosegregating with the gene.

The L6 rust resistance gene from flax generates at least four transcript classes by alternative splicing of the third intron. The most abundant transcript class encodes a resistance protein containing domains that include nucleotide binding site motifs and a leucine-rich repeat region (NBS-LRR). The remaining three transcript classes encode truncated products which lack most of the C-terminal part of the protein containing the leucine-rich region (LRR). The four transcript classes occur in all plant organs examined and no induction of L6 expression was observed following infection of resistant plants with an avirulent rust strain expressing the corresponding A-L6 avirulence gene. Flax plants transgenic for an intronless L6 gene, incapable of encoding truncated resistance proteins by alternative splicing, expressed L6 resistance indistinguishable from that of the wild-type gene. Therefore, a definitive role for alternative transcripts and their predicted truncated products could not be assigned in the flax/flax rust system.

The M rust resistance gene from flax was cloned after two separate approaches, an analysis of spontaneous M mutants with an L6 gene-derived DNA probe and tagging with the maize transposon Activator, independently identified the same gene. The gene encodes a protein of the nucleotide binding site leucine-rich repeat class and is related (86% nucleotide identity) to the unlinked L6 rust resistance gene. In contrast to the L locus, which contains a single gene with multiple alleles, approximately 15 related genes occur at the complex M locus, with only one encoding the M resistance specificity. The M protein contains two direct repeats of 147 and 149 amino acids in the C-terminal part of the leucine-rich region. Three mutant alleles of M encoding a product containing a single repeat unit of 154 amino acids were isolated. The mutant DNA sequences probably occurred by unequal intragenic exchange in the coding region of the repeats. The recombinant alleles lost M resistance and gained no detectable new resistance specificity.

Background Hairpin RNA (hpRNA) transgenes can be effective at inducing RNA silencing and have been exploited as a powerful tool for gene function analysis in many organisms. However, in fungi, expression of hairpin RNA transcripts can induce post-transcriptional gene silencing, but in some species can also lead to transcriptional gene silencing, suggesting a more complex interplay of the two pathways at least in some fungi. Because many fungal species are important pathogens, RNA silencing is a powerful technique to understand gene function, particularly when gene knockouts are difficult to obtain. We investigated whether the plant pathogenic fungus Fusarium oxysporum possesses a functional gene silencing machinery and whether hairpin RNA transcripts can be employed to effectively induce gene silencing. Results Here we show that, in the phytopathogenic fungus F. oxysporum, hpRNA transgenes targeting either a β-glucuronidase (Gus) reporter transgene (hpGus) or the endogenous gene Frp1 (hpFrp) did not induce significant silencing of the target genes. Expression analysis suggested that the hpRNA transgenes are prone to transcriptional inactivation, resulting in low levels of hpRNA and siRNA production. However, the hpGus RNA can be efficiently transcribed by promoters acquired either by recombination with a pre-existing, actively transcribed Gus transgene or by fortuitous integration near an endogenous gene promoter allowing siRNA production. These siRNAs effectively induced silencing of a target Gus transgene, which in turn appeared to also induce secondary siRNA production. Furthermore, our results suggested that hpRNA transcripts without poly(A) tails are efficiently processed into siRNAs to induce gene silencing. A convergent promoter transgene, designed to express poly(A)-minus sense and antisense Gus RNAs, without an inverted-repeat DNA structure, induced consistent Gus silencing in F. oxysporum. Conclusions These results indicate that F. oxysporum possesses functional RNA silencing machineries for siRNA production and target mRNA cleavage, but hpRNA transgenes may induce transcriptional self-silencing due to its inverted-repeat structure. Our results suggest that F. oxysporum possesses a similar gene silencing pathway to other fungi like fission yeast, and indicate a need for developing more effective RNA silencing technology for gene function studies in this fungal pathogen.

Eukaryotic cells originated when an ancestor of the nucleated cell engulfed bacterial endosymbionts that gradually evolved into the mitochondrion and the chloroplast. Soon after these endosymbiotic events, thousands of ancestral prokaryotic genes were functionally transferred from the endosymbionts to the nucleus. This process of functional gene relocation, now rare in eukaryotes, continues in angiosperms. In this article, we show that the chloroplastic acetyl-CoA carboxylase subunit (accD) gene that is present in the plastome of most angiosperms has been functionally relocated to the nucleus in the Campanulaceae. Surprisingly, the nucleus-encoded accD transcript is considerably smaller than the plastidic version, consisting of little more than the carboxylase domain of the plastidic accD gene fused to a coding region encoding a plastid targeting peptide. We verified experimentally the presence of a chloroplastic transit peptide by showing that the product of the nuclear accD fused to green fluorescent protein was imported in the chloroplasts. The nuclear gene regulatory elements that enabled the erstwhile plastidic gene to become functional in the nuclear genome were identified, and the evolution of the intronic and exonic sequences in the nucleus is described. Relocation and truncation of the accD gene is a remarkable example of the processes underpinning endosymbiotic evolution.

The L6 rust resistance gene from flax was cloned after tagging with the maize transposable element Activator. The gene is predicted to encode two products of 1294 and 705 amino acids that result from alternatively spliced transcripts. The longer product is similar to the products of two other plant disease resistance genes, the tobacco mosaic virus resistance gene N of tobacco and the bacterial resistance gene RPS2 of Arabidopsis. The similarity involves the presence of a nucleotide (ATP/GTP) binding site and several other amino acid motifs of unknown function in the N-terminal half of the polypeptides and a leucine-rich region in the C-terminal half. The truncated product of L6, which lacks most of the leucine-rich C-terminal region, is similar to the truncated product that is predicted from an alternative transcript of the N gene. The L6, N, and RPS2 genes, which control resistance to three widely different pathogen types, are the foundation of a class of plant disease resistance genes that can be referred to as nucleotide binding site/leucine-rich repeat resistance genes.

Nuclear genomes of eukaryotes are bombarded by a continuous deluge of organellar DNA which contributes significantly to eukaryote evolution. Here, we present a new PCR-based method that allows the specific amplification of nuclear integrants of organellar DNA (norgs) by exploiting recent deletions present in organellar genome sequences. We have used this method to amplify nuclear integrants of plastid DNA (nupts) from the nuclear genomes of several nicotiana species and to study the evolutionary forces acting upon these sequences. The role of nupts in endosymbiotic evolution and the different genetic factors influencing the time available for a chloroplastic gene to be functionally relocated in the nucleus are discussed.

The nuclear genome of eukaryotes contains large amounts of cytoplasmic organelle DNA (nuclear integrants of organelle DNA [norgs]). The recent sequencing of many mitochondrial and chloroplast genomes has enabled investigation of the potential role of norgs in endosymbiotic evolution. In this article, we describe a new polymerase chain reaction-based method that allows the identification and evolutionary study of recent and older norgs in a range of eukaryotes. We tested this method in the genus Nicotiana and obtained sequences from seven nuclear integrants of plastid DNA (nupts) totaling 25 kb in length. These nupts were estimated to have been transferred 0.033 to 5.81 million years ago. The spectrum of mutations present in the potential protein-coding sequences compared with the noncoding sequences of each nupt revealed that nupts evolve in a nuclear-specific manner and are under neutral evolution. Indels were more frequent in noncoding regions than in potential coding sequences of former chloroplastic DNA, most probably due to the presence of a higher number of homopolymeric sequences. Unexpectedly, some potential protein-coding sequences within the nupts still contained intact open reading frames for up to 5.81 million years. These results suggest that chloroplast genes transferred to the nucleus have in some cases several millions of years to acquire nuclear regulatory elements and become functional. The different factors influencing this time frame and the potential role of nupts in endosymbiotic gene transfer are discussed.

The process of activation tagging in plants involves the random distribution of plant regulatory sequences throughout the genome. The insertion of a regulatory sequence in the vicinity of an endogenous gene can alter the transcriptional pattern of this gene resulting in a mutant phenotype that arises from excess functional gene product. Activation tagging has been undertaken extensively in a number of dicot plants and also in rice. This has been achieved primarily by high-throughput plant transformation using T-DNA sequences that encode regulatory elements. Apart from rice, most cereals do not have a suitably efficient transformation system for high-throughput transformation. In this article, we detail an activation tagging system in barley that exploits the mobility of the maize Ac/Ds transposable element system to distribute a highly expressed promoter throughout the barley genome. The advantage of this approach in this species is that a relatively small number of primary transgenics are required to generate an activation tagging population. Insertion of this transposable element into genes can also generate insertional inactivation mutants enabling both gene overexpression and gene knockout mutants to be identified in the same population.

Crop improvement is limited by the availability of valuable traits in sexually compatible species. Access to new characters using genetic engineering would be of great value. Barley has been transformed using microprojectile bombardment and by direct gene transfer to protoplasts, but neither method has been able to produce fertile transformants in large numbers with simple transgene integration characteristics. Agrobacterium-mediated transformation was first achieved in 1997, and it has become the method of choice. Using immature embryos of the barley variety Golden Promise as the target organ, the binary vector pWBVec8 containing the intron-interrupted hygromycin resistance gene hph as the selectable marker, and selection of transformed cells on hygromycin, the Agrobacterium method is efficient, and the transgene insertion characteristics are superior to other methods. However, the procedure is strongly genotype dependent. In this report, we describe a transformation protocol giving details of plant culture, embryo isolation and preparation, vector details, Agrobacterium culture, infection methods, subsequent procedures for callus generation and plantlet production, and analysis of transgenic plants.

Plant resistance proteins (R proteins) recognize corresponding pathogen avirulence (Avr) proteins either indirectly through detection of changes in their host protein targets or through direct R–Avr protein interaction. Although indirect recognition imposes selection against Avr effector function, pathogen effector molecules recognized through direct interaction may overcome resistance through sequence diversification rather than loss of function. Here we show that the flax rust fungus AvrL567 genes, whose products are recognized by the L5, L6, and L7 R proteins of flax, are highly diverse, with 12 sequence variants identified from six rust strains. Seven AvrL567 variants derived from Avr alleles induce necrotic responses when expressed in flax plants containing corresponding resistance genes ( R genes), whereas five variants from avr alleles do not. Differences in recognition specificity between AvrL567 variants and evidence for diversifying selection acting on these genes suggest they have been involved in a gene-specific arms race with the corresponding flax R genes. Yeast two-hybrid assays indicate that recognition is based on direct R–Avr protein interaction and recapitulate the interaction specificity observed in planta . Biochemical analysis of Escherichia coli -produced AvrL567 proteins shows that variants that escape recognition nevertheless maintain a conserved structure and stability, suggesting that the amino acid sequence differences directly affect the R–Avr protein interaction. We suggest that direct recognition associated with high genetic diversity at corresponding R and Avr gene loci represents an alternative outcome of plant–pathogen coevolution to indirect recognition associated with simple balanced polymorphisms for functional and nonfunctional R and Avr genes.

Alternative splicing is an important step in controlling gene expression and has been shown to occur for a number of plant disease resistance (R) genes. The specific biological role of alternatively spliced transcripts from most R genes is unknown, yet in two cases it is clear that functional disease resistance cannot be activated without them. We report 12 splice isoforms of the M flax rust resistance gene, a TIR-NBS-LRR class of R gene. Collectively, these isoforms are predicted to encode at least nine different polypeptide products, only one of which is a full length peptide believed to confer functional M gene-specific disease resistance. An additional intron to that previously described was found in the 5' untranslated region. Splicing of this leader intron removes an upstream ORF (muORF) sequence. In some transcripts the leader intron is retained and in this case we predict negligible translation initiation of the full length M gene-encoding ORF. The majority of the alternatively spliced isoforms of M would encode truncated TIR and TIR-NBS containing proteins. Although the role of alternative splicing and the existence and function of the products they encode is still unclear, the complexities of the splicing profile, and the 5' UTR of the M gene, are likely to serve in mechanisms to regulate R protein levels.

Proline accumulation and catabolism play significant roles in adaptation to a variety of plant stresses including osmotic stress, drought, temperature, freezing, UV irradiation, heavy metals and pathogen infection. In this study, the gene Delta1 -pyrroline-5-carboxylate dehydrogenase (P5CDH), which catalyzes the second step in the conversion of proline to glutamate, is characterized in a number of cereal species. P5CDH genes from hexaploid wheat, Triticum turgidum (durum wheat), Aegilops tauschii, Triticum monococcum, barley, maize and rice were shown to be conserved in terms of gene structure and sequence, present as a single copy per haploid, non-polyploid genome and located in evolutionarily conserved linkage groups. A wheat cDNA sequence was shown by yeast complementation to encode a functional P5CDH activity. A divergently-transcribed rab7 gene was identified immediately 5' of P5CDH in all grasses examined, except rice. The rab7/P5CDH intergenic region in these species, which presumably encompasses 5' regulatory elements of both genes, showed a distinct pattern of sequence evolution with sequences in juxtaposition to each ORF conserved between barley, wheat, A. tauschii and T. monococcum. More distal 5' sequence in this intergenic region showed a higher rate of divergence, with no homology observed between these regions in the wheat and barley genomes. Maize and rice showed no similarity in regions 5' of P5CDH when compared with wheat, barley, and each other, apart from a 22 bp region of conserved non-coding sequence (CNS) that is similar to a proline response element identified in the promoter of the Arabidopsis proline dehydrogenase gene. A palindromic motif similar to this cereal CNS was also identified 5' of the Arabidopsis AtP5CDH gene showing conservation of this sequence in monocot and dicot lineages.

The maize Rp1-D gene confers race-specific resistance against Puccinia sorghi (common leaf rust) isolates containing a corresponding avrRp1-D avirulence gene. An Rp1-D genomic clone and a similar Rp1-D transgene regulated by the maize ubiquitin promoter were transformed independently into susceptible maize lines and shown to confer Rp1-D resistance, demonstrating that this resistance can be transferred as a single gene. Transfer of these functional transgenes into wheat and barley did not result in novel resistances when these plants were challenged with isolates of wheat stem rust (P. graminis), wheat leaf rust (P. triticina), or barley leaf rust (P. hordei). Regardless of the promoter employed, low levels of gene expression were observed. When constitutive promoters were used for transgene expression, a majority of Rp1-D transcripts were truncated in the nucleotide binding site-encoding region by premature polyadenylation. This aberrant mRNA processing was unrelated to gene function because an inactive version of the gene also generated such transcripts. These data demonstrate that resistance gene transfer between species may not be limited only by divergence of signaling effector molecules and pathogen avirulence ligands, but potentially also by more fundamental gene expression and transcript processing limitations.

Transfer of organelle DNA into the nuclear genome has been significant in eukaryotic evolution, because it appears to be the origin of many nuclear genes. Most studies on organelle DNA transfer have been restricted to evolutionary events but experimental systems recently became available to monitor the process in real time. We designed an experimental screen to detect plastid DNA (ptDNA) transfers to the nucleus in whole plants grown under natural conditions. The resultant genotypes facilitated investigation of the evolutionary mechanisms underlying ptDNA transfer and nuclear integration. Here we report the characterization of nuclear loci formed by integration of newly transferred ptDNA. Large, often multiple, fragments of ptDNA between 6.0 and 22.3 kb in size are incorporated into chromosomes at single Mendelian loci. The lack of chloroplast transcripts of comparable size to the ptDNA integrants suggests that DNA molecules are directly involved in the transfer process. Microhomology (2–5 bp) and rearrangements of ptDNA and nuclear DNA were frequently found near integration sites, suggesting that nonhomologous recombination plays a major role in integration. The mechanisms of ptDNA integration appear similar to those of biolistic transformation of plant cells, but no sequence preference was identified near junctions. This article provides substantial molecular analysis of real-time ptDNA transfer and integration that has resulted from natural processes with no involvement of cell injury, infection, and tissue culture. We highlight the impact of cytoplasmic organellar genome mobility on nuclear genome evolution.