RESUMO
Plant nucleotide-binding leucine-rich repeat (NLRs) immune receptors directly or indirectly recognize pathogen-secreted effector molecules to initiate plant defense. Recognition of multiple pathogens by a single NLR is rare and usually occurs via monitoring for changes to host proteins; few characterized NLRs have been shown to recognize multiple effectors. The barley (Hordeum vulgare) NLR gene Mildew locus a (Mla) has undergone functional diversification, and the proteins encoded by different Mla alleles recognize host-adapted isolates of barley powdery mildew (Blumeria graminis f. sp. hordei [Bgh]). Here, we show that Mla3 also confers resistance to the rice blast fungus Magnaporthe oryzae in a dosage-dependent manner. Using a forward genetic screen, we discovered that the recognized effector from M. oryzae is Pathogenicity toward Weeping Lovegrass 2 (Pwl2), a host range determinant factor that prevents M. oryzae from infecting weeping lovegrass (Eragrostis curvula). Mla3 has therefore convergently evolved the capacity to recognize effectors from diverse pathogens.
Assuntos
Ascomicetos , Eragrostis , Hordeum , Magnaporthe , Virulência/genética , Hordeum/genética , Eragrostis/metabolismo , Plantas/metabolismo , Especificidade de Hospedeiro , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
In the evolution of land plants, the plant immune system has experienced expansion in immune receptor and signaling pathways. Lineage-specific expansions have been observed in diverse gene families that are potentially involved in immunity but lack causal association. Here, we show that Rps8-mediated resistance in barley to the pathogen Puccinia striiformis f. sp. tritici (wheat stripe rust) is conferred by a genetic module: Pur1 and Exo70FX12, which are together necessary and sufficient. Pur1 encodes a leucine-rich repeat receptor kinase and is the ortholog of rice Xa21, and Exo70FX12 belongs to the Poales-specific Exo70FX clade. The Exo70FX clade emerged after the divergence of the Bromeliaceae and Poaceae and comprises from 2 to 75 members in sequenced grasses. These results demonstrate the requirement of a lineage-specific Exo70FX12 in Pur1-mediated immunity and suggest that the Exo70FX clade may have evolved a specialized role in receptor kinase signaling.
RESUMO
The wild relatives and progenitors of wheat have been widely used as sources of disease resistance (R) genes. Molecular identification and characterization of these R genes facilitates their manipulation and tracking in breeding programmes. Here, we develop a reference-quality genome assembly of the wild diploid wheat relative Aegilops sharonensis and use positional mapping, mutagenesis, RNA-Seq and transgenesis to identify the stem rust resistance gene Sr62, which has also been transferred to common wheat. This gene encodes a tandem kinase, homologues of which exist across multiple taxa in the plant kingdom. Stable Sr62 transgenic wheat lines show high levels of resistance against diverse isolates of the stem rust pathogen, highlighting the utility of Sr62 for deployment as part of a polygenic stack to maximize the durability of stem rust resistance.
Assuntos
Aegilops , Basidiomycota , Aegilops/genética , Basidiomycota/genética , Resistência à Doença/genética , Genes de Plantas/genética , Melhoramento Vegetal , Doenças das Plantas/genética , Triticum/genéticaRESUMO
KEY MESSAGE: Understanding the molecular network, including protein-protein interactions, of VRS5 provide new routes towards the identification of other key regulators of plant architecture in barley. The TCP transcriptional regulator TEOSINTE BRANCHED 1 (TB1) is a key regulator of plant architecture. In barley, an important cereal crop, HvTB1 (also referred to as VULGARE SIX-ROWED spike (VRS) 5), inhibits the outgrowth of side shoots, or tillers, and grains. Despite its key role in barley development, there is limited knowledge on the molecular network that is utilized by VRS5. In this work, we performed protein-protein interaction studies of VRS5. Our analysis shows that VRS5 potentially interacts with a diverse set of proteins, including other class II TCP's, NF-Y TF, but also chromatin remodelers. Zooming in on the interaction capacity of VRS5 with other TCP TFs shows that VRS5 preferably interacts with other class II TCP TFs in the TB1 clade. Induced mutagenesis through CRISPR-Cas of one of the putative VRS5 interactors, HvTB2 (also referred to as COMPOSITUM 1 and BRANCHED AND INDETERMINATE SPIKELET 1), resulted in plants that have lost their characteristic unbranched spike architecture. More specifically, hvtb2 mutants exhibited branches arising at the main spike, suggesting that HvTB2 acts as inhibitor of branching. Our protein-protein interaction studies of VRS5 resulted in the identification of HvTB2 as putative interactor of VRS5, another key regulator of spike architecture in barley. The study presented here provides a first step to underpin the protein-protein interactome of VRS5 and to identify other, yet unknown, key regulators of barley plant architecture.
Assuntos
Hordeum , Grão Comestível/metabolismo , Regulação da Expressão Gênica de Plantas , Hordeum/genética , Hordeum/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Zea mays/metabolismoRESUMO
Crop losses caused by plant pathogens are a primary threat to stable food production. Stripe rust (Puccinia striiformis) is a fungal pathogen of cereal crops that causes significant, persistent yield loss. Stripe rust exhibits host species specificity, with lineages that have adapted to infect wheat and barley. While wheat stripe rust and barley stripe rust are commonly restricted to their corresponding hosts, the genes underlying this host specificity remain unknown. Here, we show that three resistance genes, Rps6, Rps7, and Rps8, contribute to immunity in barley to wheat stripe rust. Rps7 cosegregates with barley powdery mildew resistance at the Mla locus. Using transgenic complementation of different Mla alleles, we confirm allele-specific recognition of wheat stripe rust by Mla. Our results show that major resistance genes contribute to the host species specificity of wheat stripe rust on barley and that a shared genetic architecture underlies resistance to the adapted pathogen barley powdery mildew and non-adapted pathogen wheat stripe rust.
Assuntos
Hordeum/imunologia , Especificidade de Hospedeiro , Imunidade Vegetal , Proteínas de Plantas/imunologia , Adaptação Fisiológica , Alelos , Produtos Agrícolas/genética , Grão Comestível , Melhoramento Vegetal , Doenças das Plantas/imunologia , Puccinia , Receptores Imunológicos , Proteínas Ribossômicas , TriticumRESUMO
Sequence assembly of large and repeat-rich plant genomes has been challenging, requiring substantial computational resources and often several complementary sequence assembly and genome mapping approaches. The recent development of fast and accurate long-read sequencing by circular consensus sequencing (CCS) on the PacBio platform may greatly increase the scope of plant pan-genome projects. Here, we compare current long-read sequencing platforms regarding their ability to rapidly generate contiguous sequence assemblies in pan-genome studies of barley (Hordeum vulgare). Most long-read assemblies are clearly superior to the current barley reference sequence based on short-reads. Assemblies derived from accurate long reads excel in most metrics, but the CCS approach was the most cost-effective strategy for assembling tens of barley genomes. A downsampling analysis indicated that 20-fold CCS coverage can yield very good sequence assemblies, while even five-fold CCS data may capture the complete sequence of most genes. We present an updated reference genome assembly for barley with near-complete representation of the repeat-rich intergenic space. Long-read assembly can underpin the construction of accurate and complete sequences of multiple genomes of a species to build pan-genome infrastructures in Triticeae crops and their wild relatives.
Assuntos
Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Hordeum/genética , Biologia Computacional/métodos , DNA Intergênico , Genoma de Planta , Anotação de Sequência Molecular , Retroelementos , Análise de Sequência de DNA , Sequências Repetidas TerminaisRESUMO
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.
Assuntos
Basidiomycota/patogenicidade , Brachypodium/genética , Resistência à Doença/genética , Especificidade de Hospedeiro , Doenças das Plantas/genética , Brachypodium/imunologia , Brachypodium/microbiologia , Mapeamento Cromossômico , Hordeum/microbiologia , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Locos de Características Quantitativas/genética , Triticum/microbiologiaRESUMO
KEY MESSAGE: We identified two novel wheat stem rust resistance genes, Sr-1644-1Sh and Sr-1644-5Sh in Aegilops sharonensis that are effective against widely virulent African races of the wheat stem rust pathogen. Stem rust is one of the most important diseases of wheat in the world. When single stem rust resistance (Sr) genes are deployed in wheat, they are often rapidly overcome by the pathogen. To this end, we initiated a search for novel sources of resistance in diverse wheat relatives and identified the wild goatgrass species Aegilops sharonesis (Sharon goatgrass) as a rich reservoir of resistance to wheat stem rust. The objectives of this study were to discover and map novel Sr genes in Ae. sharonensis and to explore the possibility of identifying new Sr genes by genome-wide association study (GWAS). We developed two biparental populations between resistant and susceptible accessions of Ae. sharonensis and performed QTL and linkage analysis. In an F6 recombinant inbred line and an F2 population, two genes were identified that mapped to the short arm of chromosome 1Ssh, designated as Sr-1644-1Sh, and the long arm of chromosome 5Ssh, designated as Sr-1644-5Sh. The gene Sr-1644-1Sh confers a high level of resistance to race TTKSK (a member of the Ug99 race group), while the gene Sr-1644-5Sh conditions strong resistance to TRTTF, another widely virulent race found in Yemen. Additionally, GWAS was conducted on 125 diverse Ae. sharonensis accessions for stem rust resistance. The gene Sr-1644-1Sh was detected by GWAS, while Sr-1644-5Sh was not detected, indicating that the effectiveness of GWAS might be affected by marker density, population structure, low allele frequency and other factors.
Assuntos
Resistência à Doença/genética , Genes de Plantas , Doenças das Plantas/genética , Poaceae/genética , Basidiomycota , Mapeamento Cromossômico , Estudos de Associação Genética , Ligação Genética , Modelos Lineares , Desequilíbrio de Ligação , Modelos Genéticos , Fenótipo , Doenças das Plantas/microbiologia , Poaceae/microbiologia , Locos de Características QuantitativasRESUMO
The domestication of plants is underscored by the selection of agriculturally favorable developmental traits, including flowering time, which resulted in the creation of varieties with altered growth habits. Research into the pathways underlying these growth habits in cereals has highlighted the role of three main flowering regulators: VERNALIZATION1 (VRN1), VRN2, and FLOWERING LOCUS T (FT). Previous reverse genetic studies suggested that the roles of VRN1 and FT are conserved in Brachypodium distachyon yet identified considerable ambiguity surrounding the role of VRN2 To investigate the natural diversity governing flowering time pathways in a nondomesticated grass, the reference B. distachyon accession Bd21 was crossed with the vernalization-dependent accession ABR6. Resequencing of ABR6 allowed the creation of a single-nucleotide polymorphism-based genetic map at the F4 stage of the mapping population. Flowering time was evaluated in F4:5 families in five environmental conditions, and three major loci were found to govern flowering time. Interestingly, two of these loci colocalize with the B. distachyon homologs of the major flowering pathway genes VRN2 and FT, whereas no linkage was observed at VRN1 Characterization of these candidates identified sequence and expression variation between the two parental genotypes, which may explain the contrasting growth habits. However, the identification of additional quantitative trait loci suggests that greater complexity underlies flowering time in this nondomesticated system. Studying the interaction of these regulators in B. distachyon provides insights into the evolutionary context of flowering time regulation in the Poaceae as well as elucidates the way humans have utilized the natural variation present in grasses to create modern temperate cereals.
Assuntos
Brachypodium/genética , Flores/genética , Flores/fisiologia , Variação Genética , Locos de Características Quantitativas/genética , Mapeamento Cromossômico , Cruzamentos Genéticos , Ecótipo , Regulação da Expressão Gênica de Plantas , Ligação Genética , Genótipo , Geografia , Fenótipo , Folhas de Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de TempoRESUMO
Wild relatives of domesticated crop species harbor multiple, diverse, disease resistance (R) genes that could be used to engineer sustainable disease control. However, breeding R genes into crop lines often requires long breeding timelines of 5-15 years to break linkage between R genes and deleterious alleles (linkage drag). Further, when R genes are bred one at a time into crop lines, the protection that they confer is often overcome within a few seasons by pathogen evolution. If several cloned R genes were available, it would be possible to pyramid R genes in a crop, which might provide more durable resistance. We describe a three-step method (MutRenSeq)-that combines chemical mutagenesis with exome capture and sequencing for rapid R gene cloning. We applied MutRenSeq to clone stem rust resistance genes Sr22 and Sr45 from hexaploid bread wheat. MutRenSeq can be applied to other commercially relevant crops and their relatives, including, for example, pea, bean, barley, oat, rye, rice and maize.
Assuntos
Clonagem Molecular/métodos , Resistência à Doença/genética , Genes de Plantas/genética , Mutagênese Sítio-Dirigida/métodos , Doenças das Plantas/genética , Plantas/genética , Melhoramento Genético/métodos , Doenças das Plantas/prevenção & controle , Plantas Geneticamente Modificadas/genética , Análise de Sequência de DNA/métodosRESUMO
Nonhost resistance is often conceptualized as a qualitative separation from host resistance. Classification into these two states is generally facile, as they fail to fully describe the range of states that exist in the transition from host to nonhost. This poses a problem when studying pathosystems that cannot be classified as either host or nonhost due to their intermediate status relative to these two extremes. In this study, we investigate the efficacy of the Poaceae-stripe rust (Puccinia striiformis Westend.) interaction for describing the host-nonhost landscape. First, using barley (Hordeum vulgare L.) and Brachypodium distachyon (L.) P. Beauv. We observed that macroscopic symptoms of chlorosis and leaf browning were associated with hyphal colonization by P. striiformis f. sp. tritici, respectively. This prompted us to adapt a protocol for visualizing fungal structures into a phenotypic assay that estimates the percent of leaf colonized. Use of this assay in intermediate host and intermediate nonhost systems found the frequency of infection decreases with evolutionary divergence from the host species. Similarly, we observed that the pathogen's ability to complete its life cycle decreased faster than its ability to colonize leaf tissue, with no incidence of pustules observed in the intermediate nonhost system and significantly reduced pustule formation in the intermediate host system as compared to the host system, barley-P. striiformis f. sp. hordei. By leveraging the stripe rust pathosystem in conjunction with macroscopic and microscopic phenotypic assays, we now hope to dissect the genetic architecture of intermediate host and intermediate nonhost resistance using structured populations in barley and B. distachyon.
RESUMO
Aegilops sharonensis Eig (Sharon goatgrass) is a wild diploid relative of wheat within the Sitopsis section of Aegilops. This species represents an untapped reservoir of genetic diversity for traits of agronomic importance, especially as a source of novel disease resistance. To gain a foothold in this genetic resource, we sequenced the cDNA from leaf tissue of two geographically distinct Ae. sharonensis accessions (1644 and 2232) using the 454 Life Sciences platform. We compared the results of two different assembly programs using different parameter sets to generate 13 distinct assemblies in an attempt to maximize representation of the gene space in de novo transcriptome assembly. The most sensitive assembly (71,029 contigs; N50 674 nts) retrieved 18,684 unique best reciprocal BLAST hits (BRBH) against six previously characterised grass proteomes while the most specific assembly (30,609 contigs; N50 815 nts) retrieved 15,687 BRBH. We combined these two assemblies into a set of 62,243 non-redundant sequences and identified 139 belonging to plant disease resistance genes of the nucleotide binding leucine-rich repeat class. Based on the non-redundant sequences, we predicted 37,743 single nucleotide polymorphisms (SNP), equivalent to one per 1,142 bp. We estimated the level of heterozygosity as 1.6% in accession 1644 and 30.1% in 2232. The Ae. sharonensis leaf transcriptome provides a rich source of sequence and SNPs for this wild wheat relative. These sequences can be used with existing monocot genome sequences and EST sequence collections (e.g. barley, Brachypodium, wheat, rice, maize and Sorghum) to assist with genetic and physical mapping and candidate gene identification in Ae. sharonensis. These resources provide an initial framework to further build on and characterise the genetic and genomic structure of Ae. sharonensis.
Assuntos
Genoma de Planta , Poaceae/genética , Transcriptoma , Triticum/genética , Heterozigoto , Doenças das Plantas/genética , Folhas de Planta/genética , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Retrotransposons' high capacity for mutagenesis is a threat that genomes need to control tightly. Transcriptional gene silencing is a general and highly effective control of retrotransposon expression. Yet, some retrotransposons manage to transpose and proliferate in plant genomes, suggesting that, as shown for plant viruses, retrotransposons can escape silencing. However no evidence of retrotransposon silencing escape has been reported. Here we analyze the silencing control of the tobacco Tnt1 retrotransposon and report that even though constructs driven by the Tnt1 promoter become silenced when stably integrated in tobacco, the endogenous Tnt1 elements remain active. Silencing of Tnt1-containing transgenes correlates with high DNA methylation and the inability to incorporate H2A.Z into their promoters, whereas the endogenous Tnt1 elements remain partially methylated at asymmetrical positions and incorporate H2A.Z upon induction. Our results show that the promoter of Tnt1 is a target of silencing in tobacco, but also that endogenous Tnt1 elements can escape this control and be expressed in their natural host.
Assuntos
Inativação Gênica , Nicotiana/genética , Retroelementos/genética , Cromatina/metabolismo , Metilação de DNA , Epigênese Genética , Regulação da Expressão Gênica , Ordem dos Genes , Histonas/metabolismo , Metiltransferases/metabolismo , Regiões Promotoras Genéticas , Estresse Fisiológico/genética , Nicotiana/metabolismo , Transcrição GênicaRESUMO
Retrotransposons are a major component of eukaryote genomes, being especially abundant in plant genomes. They are frequently found inserted in gene-rich regions and have greatly contributed to the evolution of gene coding capacity and regulation. Retrotransposon insertions can influence the expression of neighboring genes in many ways, such as modifying their promoter or terminator sequences and altering their epigenetic control. Plant retrotransposons are highly regulated and their expression is usually associated with stress situations. While the control of transcription of some plant retrotransposons has been analyzed in some detail, little is known about the transcriptional termination of these elements. Here we show that the transcripts of the tobacco retrotransposon Tnt1 display a high variability of polyadenylation sites, only a fraction of them terminating at the major termination site. We also report on the ability of Tnt1 to extend its transcription into flanking genomic sequences and we analyze a particular case in which Tnt1 transcripts include sequences of an oppositely oriented resistance-like gene. The expression of this gene and the neighboring Tnt1 copy generate transcripts overlapping in more that 800 nucleotides, which could anneal and form dsRNAs and enter into silencing regulatory pathways. Resistance gene loci are usually composed of tandem arrays of resistance-like genes, a number of which contain mutations, including retrotransposon insertions, and are considered as to be pseudogenes. Given that plant retrotransposons are usually regulated by stress, the convergent expression of these resistance-like pseudogenes and the interleaving inducible retrotransposons may contribute to the control of plant responses to stress.
Assuntos
Regulação da Expressão Gênica de Plantas , Genes de Plantas , Nicotiana/genética , Retroelementos/genética , Transcrição Gênica , Sequência de Bases , Proteínas de Fluorescência Verde/genética , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Elementos Reguladores de Transcrição/genética , Sequências Reguladoras de Ácido Nucleico , Retroelementos/fisiologia , Estresse Fisiológico/genética , Sequências Repetidas Terminais/genéticaRESUMO
Post-transcriptional gene silencing (PTGS) is a sequence-specific RNA degradation process conserved in fungi, plants and animals. The trigger of the mechanism is double-stranded RNA derived from transgenic or endogenous loci and formed by intra- or inter-molecular interactions of single-stranded RNAs or the action of RNA-dependent RNA polymerases (RDRs). Double-stranded RNA from various sources is processed by one of the four Dicer-like (DCL) proteins in Arabidopsis, and the resulting short RNAs enter into at least four different pathways, one of which involves the production of trans-acting short interfering RNAs (tasiRNAs). We report here a novel gene (SDE5) that is required for transgene silencing and the production of tasiRNAs. Mutation in SDE5 also results in hyper-susceptibility to cucumber mosaic virus but not turnip mosaic virus. However, like RDR6, SDE5 is not involved in inverted repeat-induced transgene silencing or the biogenesis of microRNAs and 24 nt siRNAs produced by DCL3. Based on these results, we propose that SDE5 acts together with RDR6 in generating double-stranded RNA from specific single-stranded RNAs. As the sequence of SDE5 has sequence features shared by TAP, a human mRNA export factor, we propose that its role could be in the transport of RNA molecules that are converted into a double-stranded form by RDR6.