RESUMEN
In this review, we highlight studies in which whole-genome sequencing, comparative genomics, and population genomics have provided unprecedented insights into past and ongoing pathogen evolution. These include new understandings of the adaptive evolution of secretion systems and their effectors. We focus on Xanthomonas pathosystems that have seen intensive study and improved our understanding of pathogen emergence and evolution, particularly in the context of host specialization: citrus canker, bacterial blight of rice, and bacterial spot of tomato and pepper. Across pathosystems, pathogens appear to follow a pattern of bursts of evolution and diversification that impact host adaptation. There remains a need for studies on the mechanisms of host range evolution and genetic exchange among closely related but differentially host-specialized species and to start moving beyond the study of specific strain and host cultivar pairwise interactions to thinking about these pathosystems in a community context.
Asunto(s)
Enfermedades de las Plantas , Xanthomonas , Xanthomonas/genética , Xanthomonas/patogenicidad , Xanthomonas/fisiología , Enfermedades de las Plantas/microbiología , Evolución Biológica , Interacciones Huésped-Patógeno , Especificidad del Huésped , Genoma Bacteriano/genética , Oryza/microbiología , Citrus/microbiología , Capsicum/microbiología , Genómica , Evolución MolecularRESUMEN
Bacterial-fungal interactions are pervasive in the rhizosphere. While an increasing number of endohyphal bacteria have been identified, little is known about their ecology and impact on the associated fungal hosts and the surrounding environment. In this study, we characterized the genome of an Enterobacter sp. Crenshaw (En-Cren), which was isolated from the generalist fungal pathogen Rhizoctonia solani, and examined the genetic potential of the bacterium with regard to the phenotypic traits associated with the fungus. Overall, the En-Cren genome size was typical for members of the genus and was capable of free-living growth. The genome was 4.6 MB in size, and no plasmids were detected. Several prophage regions and genomic islands were identified that harbor unique genes in comparison with phylogenetically closely related Enterobacter spp. Type VI secretion system and cyanate assimilation genes were identified from the bacterium, while some common heavy metal resistance genes were absent. En-Cren contains the key genes for indole-3-acetic acid (IAA) and phenylacetic acid (PAA) biosynthesis, and produces IAA and PAA in vitro, which may impact the ecology or pathogenicity of the fungal pathogen in vivo. En-Cren was observed to move along hyphae of R. solani and on other basidiomycetes and ascomycetes in culture. The bacterial flagellum is essential for hyphal movement, while other pathways and genes may also be involved.IMPORTANCEThe genome characterization and comparative genomics analysis of Enterobacter sp. Crenshaw provided the foundation and resources for a better understanding of the ecology and evolution of this endohyphal bacteria in the rhizosphere. The ability to produce indole-3-acetic acid and phenylacetic acid may provide new angles to study the impact of phytohormones during the plant-pathogen interactions. The hitchhiking behavior of the bacterium on a diverse group of fungi, while inhibiting the growth of some others, revealed new areas of bacterial-fungal signaling and interaction, which have yet to be explored.
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Enterobacter , Hifa , Enterobacter/genética , Enterobacter/metabolismo , Hifa/metabolismo , Fenilacetatos/metabolismo , Rhizoctonia/genéticaRESUMEN
Goss's wilt, caused by the Gram-positive actinobacterium Clavibacter nebraskensis, is an important bacterial disease of maize. The molecular and genetic mechanisms of resistance to the bacterium, or, in general, Gram-positive bacteria causing plant diseases, remain poorly understood. Here, we examined the genetic basis of Goss's wilt through differential gene expression, standard genome-wide association mapping (GWAS), extreme phenotype (XP) GWAS using highly resistant (R) and highly susceptible (S) lines, and quantitative trait locus (QTL) mapping using 3 bi-parental populations, identifying 11 disease association loci. Three loci were validated using near-isogenic lines or recombinant inbred lines. Our analysis indicates that Goss's wilt resistance is highly complex and major resistance genes are not commonly present. RNA sequencing of samples separately pooled from R and S lines with or without bacterial inoculation was performed, enabling identification of common and differential gene responses in R and S lines. Based on expression, in both R and S lines, the photosynthesis pathway was silenced upon infection, while stress-responsive pathways and phytohormone pathways, namely, abscisic acid, auxin, ethylene, jasmonate, and gibberellin, were markedly activated. In addition, 65 genes showed differential responses (up- or down-regulated) to infection in R and S lines. Combining genetic mapping and transcriptional data, individual candidate genes conferring Goss's wilt resistance were identified. Collectively, aspects of the genetic architecture of Goss's wilt resistance were revealed, providing foundational data for mechanistic studies.
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Transcriptoma , Zea mays , Zea mays/genética , Zea mays/microbiología , Estudio de Asociación del Genoma Completo , Mapeo Cromosómico , Secuencia de Bases , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Resistencia a la Enfermedad/genéticaRESUMEN
Understanding gene regulatory networks is essential to elucidate developmental processes and environmental responses. Here, we studied regulation of a maize (Zea mays) transcription factor gene using designer transcription activator-like effectors (dTALes), which are synthetic Type III TALes of the bacterial genus Xanthomonas and serve as inducers of disease susceptibility gene transcription in host cells. The maize pathogen Xanthomonas vasicola pv. vasculorum was used to introduce 2 independent dTALes into maize cells to induced expression of the gene glossy3 (gl3), which encodes a MYB transcription factor involved in biosynthesis of cuticular wax. RNA-seq analysis of leaf samples identified, in addition to gl3, 146 genes altered in expression by the 2 dTALes. Nine of the 10 genes known to be involved in cuticular wax biosynthesis were upregulated by at least 1 of the 2 dTALes. A gene previously unknown to be associated with gl3, Zm00001d017418, which encodes aldehyde dehydrogenase, was also expressed in a dTALe-dependent manner. A chemically induced mutant and a CRISPR-Cas9 mutant of Zm00001d017418 both exhibited glossy leaf phenotypes, indicating that Zm00001d017418 is involved in biosynthesis of cuticular waxes. Bacterial protein delivery of dTALes proved to be a straightforward and practical approach for the analysis and discovery of pathway-specific genes in maize.
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Factores de Transcripción , Zea mays , Zea mays/genética , Zea mays/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional , Bacterias/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Ceras/metabolismoRESUMEN
Phytopathogenic bacteria play important roles in plant productivity, and developments in gene editing have potential for enhancing the genetic tools for the identification of critical genes in the pathogenesis process. CRISPR-based genome editing variants have been developed for a wide range of applications in eukaryotes and prokaryotes. However, the unique mechanisms of different hosts restrict the wide adaptation for specific applications. Here, CRISPR-dCas9 (dead Cas9) and nCas9 (Cas9 nickase) deaminase vectors were developed for a broad range of phytopathogenic bacteria. A gene for a dCas9 or nCas9, cytosine deaminase CDA1, and glycosylase inhibitor fusion protein (cytosine base editor, or CBE) was applied to base editing under the control of different promoters. Results showed that the RecA promoter led to nearly 100% modification of the target region. When residing on the broad host range plasmid pHM1, CBERecAp is efficient in creating base edits in strains of Xanthomonas, Pseudomonas, Erwinia and Agrobacterium. CBE based on nCas9 extended the editing window and produced a significantly higher editing rate in Pseudomonas. Strains with nonsynonymous mutations in test genes displayed expected phenotypes. By multiplexing guide RNA genes, the vectors can modify up to four genes in a single round of editing. Whole-genome sequencing of base-edited isolates of Xanthomonas oryzae pv. oryzae revealed guide RNA-independent off-target mutations. Further modifications of the CBE, using a CDA1 variant (CBERecAp-A) reduced off-target effects, providing an improved editing tool for a broad group of phytopathogenic bacteria.
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Sistemas CRISPR-Cas , Citosina , Citosina/metabolismo , Edición Génica/métodos , Bacterias/genética , Bacterias/metabolismo , ARNRESUMEN
Transcription activator-like effectors (TALEs) are bacterial proteins that are injected into the eukaryotic nucleus to act as transcriptional factors and function as key virulence factors of the phytopathogen Xanthomonas. TALEs are translocated into plant host cells via the type III secretion system and induce the expression of host susceptibility (S) genes to facilitate disease. The unique modular DNA binding domains of TALEs comprise an array of nearly identical direct repeats that enable binding to DNA targets based on the recognition of a single nucleotide target per repeat. The very nature of TALE structure and function permits the proliferation of TALE genes and evolutionary adaptations in the host to counter TALE function, making the TALE-host interaction the most dynamic story in effector biology. The TALE genes appear to be a relatively young effector gene family, with a presence in all virulent members of some species and absent in others. Genome sequencing has revealed many TALE genes throughout the xanthomonads, and relatively few have been associated with a cognate S gene. Several species, including Xanthomonas oryzae pv. oryzae and X. citri pv. citri, have near absolute requirement for TALE gene function, while the genes appear to be just now entering the disease interactions with new fitness contributions to the pathogens of tomato and pepper among others. Deciphering the simple and effective DNA binding mechanism also has led to the development of DNA manipulation tools in fields of gene editing and transgenic research. In the three decades since their discovery, TALE research remains at the forefront of the study of bacterial evolution, plant-pathogen interactions, and synthetic biology. We also discuss critical questions that remain to be addressed regarding TALEs.
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Oryza , Xanthomonas , Efectores Tipo Activadores de la Transcripción/genética , Efectores Tipo Activadores de la Transcripción/metabolismo , Enfermedades de las Plantas/microbiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Factores de Virulencia , Xanthomonas/genética , Oryza/microbiologíaRESUMEN
Increasing populations and temperatures are expected to escalate food demands beyond production capacities, and the development of maize lines with better performance under heat stress is desirable. Here, we report that constitutive ectopic expression of a heterologous glutaredoxin S17 from Arabidopsis thaliana (AtGRXS17) can provide thermotolerance in maize through enhanced chaperone activity and modulation of heat stress-associated gene expression. The thermotolerant maize lines had increased protection against protein damage and yielded a sixfold increase in grain production in comparison to the non-transgenic counterparts under heat stress field conditions. The maize lines also displayed thermotolerance in the reproductive stages, resulting in improved pollen germination and the higher fidelity of fertilized ovules under heat stress conditions. Our results present a robust and simple strategy for meeting rising yield demands in maize and, possibly, other crop species in a warming global environment.
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Arabidopsis , Termotolerancia , Arabidopsis/genética , Grano Comestible/genética , Oxidación-Reducción , Termotolerancia/genética , Zea mays/genéticaRESUMEN
BACKGROUND: The maize inbred line A188 is an attractive model for elucidation of gene function and improvement due to its high embryogenic capacity and many contrasting traits to the first maize reference genome, B73, and other elite lines. The lack of a genome assembly of A188 limits its use as a model for functional studies. RESULTS: Here, we present a chromosome-level genome assembly of A188 using long reads and optical maps. Comparison of A188 with B73 using both whole-genome alignments and read depths from sequencing reads identify approximately 1.1 Gb of syntenic sequences as well as extensive structural variation, including a 1.8-Mb duplication containing the Gametophyte factor1 locus for unilateral cross-incompatibility, and six inversions of 0.7 Mb or greater. Increased copy number of carotenoid cleavage dioxygenase 1 (ccd1) in A188 is associated with elevated expression during seed development. High ccd1 expression in seeds together with low expression of yellow endosperm 1 (y1) reduces carotenoid accumulation, accounting for the white seed phenotype of A188. Furthermore, transcriptome and epigenome analyses reveal enhanced expression of defense pathways and altered DNA methylation patterns of the embryonic callus. CONCLUSIONS: The A188 genome assembly provides a high-resolution sequence for a complex genome species and a foundational resource for analyses of genome variation and gene function in maize. The genome, in comparison to B73, contains extensive intra-species structural variations and other genetic differences. Expression and network analyses identify discrete profiles for embryonic callus and other tissues.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Proteínas de Plantas/genética , Carácter Cuantitativo Heredable , Zea mays/genética , Secuencia de Bases , Mapeo Cromosómico , Metilación de ADN , Dioxigenasas/genética , Dioxigenasas/metabolismo , Endospermo/genética , Endospermo/metabolismo , Variación Genética , Endogamia , Proteínas de Plantas/clasificación , Proteínas de Plantas/metabolismo , Alineación de Secuencia , Zea mays/clasificación , Zea mays/metabolismoRESUMEN
Drought stress is a major constraint in global maize production, causing almost 30-90% of the yield loss depending upon growth stage and the degree and duration of the stress. Here, we report that ectopic expression of Arabidopsis glutaredoxin S17 (AtGRXS17) in field grown maize conferred tolerance to drought stress during the reproductive stage, which is the most drought sensitive stage for seed set and, consequently, grain yield. AtGRXS17-expressing maize lines displayed higher seed set in the field, resulting in 2-fold and 1.5-fold increase in yield in comparison to the non-transgenic plants when challenged with drought stress at the tasseling and silking/pollination stages, respectively. AtGRXS17-expressing lines showed higher relative water content, higher chlorophyll content, and less hydrogen peroxide accumulation than wild-type (WT) control plants under drought conditions. AtGRXS17-expressing lines also exhibited at least 2-fold more pollen germination than WT plants under drought stress. Compared to the transgenic maize, WT controls accumulated higher amount of proline, indicating that WT plants were more stressed over the same period. The results present a robust and simple strategy for meeting rising yield demands in maize under water limiting conditions.
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Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Estrés Fisiológico/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sequías , Expresión Génica Ectópica/genética , Grano Comestible/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Tolerancia a la Sal/genética , Estrés Fisiológico/fisiología , Termotolerancia/genética , Zea mays/genéticaRESUMEN
Many plant disease resistance (R) genes function specifically in reaction to the presence of cognate effectors from a pathogen. Xanthomonas oryzae pathovar oryzae (Xoo) uses transcription activator-like effectors (TALes) to target specific rice genes for expression, thereby promoting host susceptibility to bacterial blight. Here, we report the molecular characterization of Xa7, the cognate R gene to the TALes AvrXa7 and PthXo3, which target the rice major susceptibility gene SWEET14. Xa7 was mapped to a unique 74-kb region. Gene expression analysis of the region revealed a candidate gene that contained a putative AvrXa7 effector binding element (EBE) in its promoter and encoded a 113-amino-acid peptide of unknown function. Genome editing at the Xa7 locus rendered the plants susceptible to avrXa7-carrying Xoo strains. Both AvrXa7 and PthXo3 activated a GUS reporter gene fused with the EBE-containing Xa7 promoter in Nicotiana benthamiana. The EBE of Xa7 is a close mimic of the EBE of SWEET14 for TALe-induced disease susceptibility. Ectopic expression of Xa7 triggers cell death in N. benthamiana. Xa7 is prevalent in indica rice accessions from 3000 rice genomes. Xa7 appears to be an adaptation that protects against pathogen exploitation of SWEET14 and disease susceptibility.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Genes prv , Oryza/genética , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/genética , Xanthomonas/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Resistencia a la Enfermedad/genética , Oryza/metabolismo , Oryza/microbiología , Fitomejoramiento , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Alineación de Secuencia , Xanthomonas/genéticaRESUMEN
Aphids secrete proteins from their stylets that evidence indicates function similar to pathogen effectors for virulence. Here, we describe two small candidate effector gene families of the pea aphid, Acyrthosiphon pisum, that share highly conserved secretory signal peptide coding regions and divergent non-secretory coding sequences derived from miniature exons. The KQY candidate effector family contains eleven members with additional isoforms, generated by alternative splicing. Pairwise comparisons indicate possible four unique KQY families based on coding regions without the secretory signal region. KQY1a, a representative of the family, is encoded by a 968 bp mRNA and a gene that spans 45.7 kbp of the genome. The locus consists of 37 exons, 33 of which are 15 bp or smaller. Additional KQY members, as well as members of the KHI family, share similar features. Differential expression analyses indicate that the genes are expressed preferentially in salivary glands. Proteomic analysis on salivary glands and saliva revealed 11 KQY members in salivary proteins, and KQY1a was detected in an artificial diet solution after aphid feeding. A single KQY locus and two KHI loci were identified in Myzus persicae, the peach aphid. Of the genes that can be anchored to chromosomes, loci are mostly scattered throughout the genome, except a two-gene region (KQY4/KQY6). We propose that the KQY family expanded in A. pisum through combinatorial assemblies of a common secretory signal cassette and novel coding regions, followed by classical gene duplication and divergence.
RESUMEN
Xanthomonas spp. encompass a wide range of plant pathogens that use numerous virulence factors for pathogenicity and fitness in plant hosts. In this Review, we examine recent insights into host-pathogen co-evolution, diversity in Xanthomonas populations and host specificity of Xanthomonas spp. that have substantially improved our fundamental understanding of pathogen biology. We emphasize the virulence factors in xanthomonads, such as type III secreted effectors including transcription activator-like effectors, type II secretion systems, diversity resulting in host specificity, evolution of emerging strains, activation of susceptibility genes and strategies of host evasion. We summarize the genomic diversity in several Xanthomonas spp. and implications for disease outbreaks, management strategies and breeding for disease resistance.
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Interacciones Huésped-Patógeno/genética , Enfermedades de las Plantas/microbiología , Factores de Virulencia/genética , Virulencia/genética , Xanthomonas/genética , Xanthomonas/patogenicidad , Plantas/microbiologíaRESUMEN
Xanthomonas vasicola pv. vasculorum is an emerging bacterial plant pathogen that causes bacterial leaf streak on corn. First described in South Africa in 1949, reports of this pathogen have greatly increased in the past years in South America and in the United States. The rapid spread of this disease in North and South America may be due to more favorable environmental conditions, susceptible hosts and/or genomic changes that favored the spread. To understand whether genetic mechanisms exist behind the recent spread of X. vasicola pv. vasculorum, we used comparative genomics to identify gene acquisitions in X. vasicola pv. vasculorum genomes from the United States and Argentina. We sequenced 41 genomes of X. vasicola pv. vasculorum and the related sorghum-infecting X. vasicola pv. holcicola and performed comparative analyses against all available X. vasicola genomes. Time-measured phylogenetic analyses showed that X. vasicola pv. vasculorum strains from the United States and Argentina are closely related and arose from two introductions to North and South America. Gene content comparisons identified clusters of genes enriched in corn X. vasicola pv. vasculorum that showed evidence of horizontal transfer including one cluster corresponding to a prophage found in all X. vasicola pv. vasculorum strains from the United States and Argentina as well as in X. vasicola pv. holcicola strains. In this work, we explore the genomes of an emerging phytopathogen population as a first step toward identifying genetic changes associated with the emergence. The acquisitions identified may contain virulence determinants or other factors associated with the spread of X. vasicola pv. vasculorum in North and South America and will be the subject of future work.
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Xanthomonas , Argentina , Genómica , Filogenia , Enfermedades de las Plantas , Sudáfrica , América del Sur , Estados Unidos , Zea maysRESUMEN
Genome sequences provide genomic maps with a single-base resolution for exploring genetic contents. Sequencing technologies, particularly long reads, have revolutionized genome assemblies for producing highly continuous genome sequences. However, current long-read sequencing technologies generate inaccurate reads that contain many errors. Some errors are retained in assembled sequences, which are typically not completely corrected by using either long reads or more accurate short reads. The issue commonly exists, but few tools are dedicated for computing error rates or determining error locations. In this study, we developed a novel approach, referred to as k-mer abundance difference (KAD), to compare the inferred copy number of each k-mer indicated by short reads and the observed copy number in the assembly. Simple KAD metrics enable to classify k-mers into categories that reflect the quality of the assembly. Specifically, the KAD method can be used to identify base errors and estimate the overall error rate. In addition, sequence insertion and deletion as well as sequence redundancy can also be detected. Collectively, KAD is valuable for quality evaluation of genome assemblies and, potentially, provides a diagnostic tool to aid in precise error correction. KAD software has been developed to facilitate public uses.
RESUMEN
Approaches to manipulating disease resistance in plants is expanding exponentially due to advances in our understanding of plant defense mechanisms and new tools for manipulating the plant genome. The application of effective strategies is only limited now by adoption of rapid classical genetic techniques and the acceptance of genetically engineered traits for some problems. The use of genome editing and cis-genetics, where possible, may facilitate applications that otherwise require considerable time or genetic engineering, depending on settling legal definitions of the products. Nonetheless, the variety of approaches to developing disease resistance has never been greater.
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Resistencia a la Enfermedad , Edición Génica , Genoma de Planta , Ingeniería Genética , Humanos , Plantas Modificadas GenéticamenteRESUMEN
Plants are vulnerable to disease through pathogen manipulation of phytohormone levels, which otherwise regulate development, abiotic, and biotic responses. Here, we show that the wheat pathogen Xanthomonas translucens pv. undulosa elevates expression of the host gene encoding 9-cis-epoxycarotenoid dioxygenase (TaNCED-5BS), which catalyzes the rate-limiting step in the biosynthesis of the phytohormone abscisic acid and a component of a major abiotic stress-response pathway, to promote disease susceptibility. Gene induction is mediated by a type III transcription activator-like effector. The induction of TaNCED-5BS results in elevated abscisic acid levels, reduced host transpiration and water loss, enhanced spread of bacteria in infected leaves, and decreased expression of the central defense gene TaNPR1 The results represent an appropriation of host physiology by a bacterial virulence effector.
Asunto(s)
Ácido Abscísico/metabolismo , Enfermedades de las Plantas/microbiología , Reguladores del Crecimiento de las Plantas/biosíntesis , Triticum/microbiología , Xanthomonas/fisiología , Dioxigenasas/genética , Dioxigenasas/metabolismo , Susceptibilidad a Enfermedades , Regulación de la Expresión Génica de las Plantas , Interacciones Huésped-Patógeno , Enfermedades de las Plantas/inmunología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Triticum/genética , Triticum/inmunología , Triticum/metabolismo , Virulencia , Xanthomonas/patogenicidadRESUMEN
Bacterial blight of rice is an important disease in Asia and Africa. The pathogen, Xanthomonas oryzae pv. oryzae (Xoo), secretes one or more of six known transcription-activator-like effectors (TALes) that bind specific promoter sequences and induce, at minimum, one of the three host sucrose transporter genes SWEET11, SWEET13 and SWEET14, the expression of which is required for disease susceptibility. We used CRISPR-Cas9-mediated genome editing to introduce mutations in all three SWEET gene promoters. Editing was further informed by sequence analyses of TALe genes in 63 Xoo strains, which revealed multiple TALe variants for SWEET13 alleles. Mutations were also created in SWEET14, which is also targeted by two TALes from an African Xoo lineage. A total of five promoter mutations were simultaneously introduced into the rice line Kitaake and the elite mega varieties IR64 and Ciherang-Sub1. Paddy trials showed that genome-edited SWEET promoters endow rice lines with robust, broad-spectrum resistance.
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Resistencia a la Enfermedad , Proteínas de Transporte de Membrana/genética , Oryza/crecimiento & desarrollo , Efectores Tipo Activadores de la Transcripción/genética , Xanthomonas/patogenicidad , Proteínas Bacterianas/genética , Sistemas CRISPR-Cas , Edición Génica , Regulación de la Expresión Génica de las Plantas , Mutación , Oryza/genética , Oryza/microbiología , Proteínas de Plantas/genética , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Xanthomonas/genéticaRESUMEN
Blight-resistant rice lines are the most effective solution for bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo). Key resistance mechanisms involve SWEET genes as susceptibility factors. Bacterial transcription activator-like (TAL) effectors bind to effector-binding elements (EBEs) in SWEET gene promoters and induce SWEET genes. EBE variants that cannot be recognized by TAL effectors abrogate induction, causing resistance. Here we describe a diagnostic kit to enable analysis of bacterial blight in the field and identification of suitable resistant lines. Specifically, we include a SWEET promoter database, RT-PCR primers for detecting SWEET induction, engineered reporter rice lines to visualize SWEET protein accumulation and knock-out rice lines to identify virulence mechanisms in bacterial isolates. We also developed CRISPR-Cas9 genome-edited Kitaake rice to evaluate the efficacy of EBE mutations in resistance, software to predict the optimal resistance gene set for a specific geographic region, and two resistant 'mega' rice lines that will empower farmers to plant lines that are most likely to resist rice blight.
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Resistencia a la Enfermedad , Proteínas de Transporte de Membrana/genética , Oryza/crecimiento & desarrollo , Efectores Tipo Activadores de la Transcripción/metabolismo , Xanthomonas/patogenicidad , Proteínas Bacterianas/genética , Sitios de Unión , Sistemas CRISPR-Cas , Bases de Datos Genéticas , Edición Génica , Regulación de la Expresión Génica de las Plantas , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Mutación , Oryza/genética , Oryza/microbiología , Proteínas de Plantas/genética , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Xanthomonas/metabolismoRESUMEN
Newly emerged wheat blast disease is a serious threat to global wheat production. Wheat blast is caused by a distinct, exceptionally diverse lineage of the fungus causing rice blast disease. Through sequencing a recent field isolate, we report a reference genome that includes seven core chromosomes and mini-chromosome sequences that harbor effector genes normally found on ends of core chromosomes in other strains. No mini-chromosomes were observed in an early field strain, and at least two from another isolate each contain different effector genes and core chromosome end sequences. The mini-chromosome is enriched in transposons occurring most frequently at core chromosome ends. Additionally, transposons in mini-chromosomes lack the characteristic signature for inactivation by repeat-induced point (RIP) mutation genome defenses. Our results, collectively, indicate that dispensable mini-chromosomes and core chromosomes undergo divergent evolutionary trajectories, and mini-chromosomes and core chromosome ends are coupled as a mobile, fast-evolving effector compartment in the wheat pathogen genome.
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Micosis/genética , Enfermedades de las Plantas/genética , Triticum/genética , Ascomicetos/genética , Cromosomas Fúngicos , Reordenamiento Génico/genética , Genoma Fúngico/genética , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Poaceae/genética , Factores de Transcripción/genéticaRESUMEN
Transcription Activator-Like effectors (TALes) represent the largest family of type III effectors among pathogenic bacteria and play a critical role in the process of infection. Strains of Xanthomonas oryzae pv. oryzae (Xoo) and some strains of other Xanthomonas pathogens contain large numbers of TALe genes. Previous techniques to clone individual or a complement of TALe genes through conventional strategies are inefficient and time-consuming due to multiple genes (up to 29 copies) in a given genome, and technically challenging due to the repetitive sequences (up to 33 nearly identical 102-nucleotide repeats) of individual TALe genes. Thus, only a limited number of TALe genes have been molecularly cloned and characterized, and the functions of most TALe genes remain unknown. Here, we present an easy and efficient cloning technique to clone TALe genes selectively through in vitro homologous recombination and single-strand annealing, and demonstrate the feasibility of this approach with four different Xoo strains. Based on the Gibson assembly strategy, two complementary vectors with scaffolds that can preferentially capture all TALe genes from a pool of genomic fragments were designed. Both vector systems enabled cloning of a full complement of TALe genes from each of four Xoo strains and functional analysis of individual TALes in rice in approximately 1 month compared to 3 months by previously used methods. The results demonstrate a robust tool to advance TALe biology and a potential for broad usage of this approach to clone multiple copies of highly competitive DNA elements in any genome of interest.