RESUMEN
Antimicrobial peptides are ancient mediators of the innate defenses of all species of life. These small lytic peptides are being exploited to genetically engineer disease-resistant crop plants. It is anticipated that certain (combinations of) potent antimicrobial peptides will provide agronomically relevant levels of disease control and should contribute to more sustainable agricultural practices.
Asunto(s)
Productos Agrícolas/genética , Ingeniería Genética , Enfermedades de las Plantas/genética , Productos Agrícolas/microbiologíaRESUMEN
Genetic analysis of plant-pathogen interactions has demonstrated that resistance to infection is often determined by the interaction of dominant plant resistance (R) genes and dominant pathogen-encoded avirulence (Avr) genes. It was postulated that R genes encode receptors for Avr determinants. A large number of R genes and their cognate Avr genes have now been analyzed at the molecular level. R gene loci are extremely polymorphic, particularly in sequences encoding amino acids of the leucine-rich repeat motif. A major challenge is to determine how Avr perception by R proteins triggers the plant defense response. Mutational analysis has identified several genes required for the function of specific R proteins. Here we report the identification of Rcr3, a tomato gene required specifically for Cf-2-mediated resistance. We propose that Avr products interact with host proteins to promote disease, and that R proteins "guard" these host components and initiate Avr-dependent plant defense responses.
Asunto(s)
Genes de Plantas , Solanum lycopersicum/genética , Solanum lycopersicum/microbiología , Evolución Biológica , Mapeo CromosómicoRESUMEN
To identify genes from the obligatory biotrophic oomycete Peronospora parasitica that are expressed during infection in Arabidopsis thaliana we employed cDNA-amplified fragment length polymorphism (AFLP) display. cDNA-AFLP fragments from infected and non-infected leaves were separated in parallel by gel electrophoresis and displayed by autoradiography. Most differential gene fragments were derived from P. parasitica.
Asunto(s)
Arabidopsis/microbiología , Genes Fúngicos , Técnicas de Amplificación de Ácido Nucleico , Oomicetos/genética , Secuencia de Bases , Cartilla de ADN , ADN Complementario , Regulación Fúngica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia MolecularRESUMEN
Arabidopsis RPP5 is a member of a large class of pathogen resistance genes encoding nucleotide-binding sites and leucine-rich repeat domains. Yeast two-hybrid analysis showed that RPP5 specifically interacts with At-RSH1, an Arabidopsis RelA/SpoT homolog. In Escherichia coli, RelA and SpoT determine the level of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), which are the effector nucleotides of the bacterial stringent response. Functional analysis in E. coli and in Streptomyces coelicolor A3 (2) showed that At-RSH1 confers phenotypes associated with (p)ppGpp synthesis. We characterized two additional Arabidopsis RelA/SpoT homologs, At-RSH2 and At-RSH3. At-RSH genes may regulate a rapid plant (p)ppGpp-mediated response to pathogens and other stresses.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/metabolismo , Guanosina Pentafosfato/metabolismo , Ligasas/metabolismo , Proteínas de Plantas/metabolismo , Transducción de Señal , Arabidopsis/enzimología , Arabidopsis/genética , Escherichia coli/metabolismo , Guanosina Pentafosfato/genética , Ligasas/genética , Datos de Secuencia Molecular , Mutación , Fenotipo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Streptomyces/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Abstract Molecular genetic approaches were adopted in the model crucifer, Arabidopsis thaliana, to unravel components of RPP5- and RPP1-mediated disease resistance to the oomycete pathogen, Peronospora parasitica. The products of RPP5 and three genes comprising the RPP1 complex locus belong to a major subclass of nucleotide-binding/leucine-rich repeat (NB-LRR) resistance (R) protein that has amino-terminal homology to the cytoplasmic domains of Drosophila and mammalian Toll and interleukin-1 family receptors (the so called 'TIR' domain). Similarities in the domain architecture of these proteins and animal regulators of programmed cell death have also been observed. Mutational screens revealed a number of genes that are required for RPP5-conditioned resistance. Among these are EDS1 and PAD4. Both EDS1 and PAD4 precede the function of salicylic acid-mediated plant responses. The EDS1 and PAD4 genes were cloned and found to encode proteins with similarity to the catalytic site of eukaryotic lipases, suggesting that they may function by hydrolysing a lipid-based substrate.
RESUMEN
In Arabidopsis ecotype Landsberg erecta (Ler), RPP5 confers resistance to the pathogen Peronospora parasitica. RPP5 is part of a clustered multigene family encoding nucleotide binding-leucine-rich repeat (LRR) proteins. We compared 95 kb of DNA sequence carrying the Ler RPP5 haplotype with the corresponding 90 kb of Arabidopsis ecotype Columbia (Col-0). Relative to the remainder of the genome, the Ler and Col-0 RPP5 haplotypes exhibit remarkable intraspecific polymorphism. The RPP5 gene family probably evolved by extensive recombination between LRRs from an RPP5-like progenitor that carried only eight LRRs. Most members have variable LRR configurations and encode different numbers of LRRs. Although many members carry retroelement insertions or frameshift mutations, codon usage analysis suggests that regions of the genes have been subject to purifying or diversifying selection, indicating that these genes were, or are, functional. The RPP5 haplotypes thus carry dynamic gene clusters with the potential to adapt rapidly to novel pathogen variants by gene duplication and modification of recognition capacity. We propose that the extremely high level of polymorphism at this complex resistance locus is maintained by frequency-dependent selection.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/genética , Arabidopsis/microbiología , Variación Genética , Proteínas de Plantas/genética , Polimorfismo Genético , Secuencia de Aminoácidos , Secuencia Conservada , Evolución Molecular , Hongos/patogenicidad , Haplotipos , Inmunidad Innata , Datos de Secuencia Molecular , Familia de Multigenes , Filogenia , Enfermedades de las Plantas , Proteínas de Plantas/química , Alineación de Secuencia , Homología de Secuencia de AminoácidoAsunto(s)
Bacterias/genética , Bacterias/patogenicidad , Modelos Biológicos , Proteínas de Plantas/genética , Plantas/genética , Plantas/microbiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Repetidas Ricas en Leucina , Proteínas de Plantas/metabolismo , Plantas/virología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas/genética , Proteínas/metabolismo , Virus/genética , Virus/patogenicidadAsunto(s)
Apoptosis/genética , Proteínas de Caenorhabditis elegans , Proteínas de Plantas/genética , Proteínas/genética , Secuencia de Aminoácidos , Animales , Apoptosis/fisiología , Factor Apoptótico 1 Activador de Proteasas , Sitios de Unión/genética , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/fisiología , Secuencia Conservada , Proteínas del Helminto/genética , Proteínas del Helminto/fisiología , Humanos , Datos de Secuencia Molecular , Proteínas de Plantas/fisiología , Proteínas/fisiología , Transducción de SeñalRESUMEN
Plant disease resistance genes operate at the earliest steps of pathogen perception. The Arabidopsis RPP5 gene specifying resistance to the downy mildew pathogen Peronospora parasitica was positionally cloned. It encodes a protein that possesses a putative nucleotide binding site and leucine-rich repeats, and its product exhibits striking structural similarity to the plant resistance gene products N and L6. Like N and L6, the RPP5 N-terminal domain resembles the cytoplasmic domains of the Drosophila Toll and mammalian interleukin-1 transmembrane receptors. In contrast to N and L6, which produce predicted truncated products by alternative splicing, RPP5 appears to express only a single transcript corresponding to the full-length protein. However, a truncated form structurally similar to those of N and L6 is encoded by one or more other members of the RPP5 gene family that are tightly clustered on chromosome 4. The organization of repeated units within the leucine-rich repeats encoded by the wild-type RPP5 gene and an RPP5 mutant allele provides molecular evidence for the heightened capacity of this domain to evolve novel configurations and potentially new disease resistance specificities.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/inmunología , Proteínas de Drosophila , Proteínas de Insectos/genética , Glicoproteínas de Membrana/genética , Enfermedades de las Plantas/genética , Proteínas de Plantas/genética , Receptores de Superficie Celular , Receptores de Interleucina-1/genética , Alelos , Secuencia de Aminoácidos , Animales , Arabidopsis/genética , Secuencia de Bases , Mapeo Cromosómico , ADN de Plantas/química , ADN de Plantas/aislamiento & purificación , Drosophila , Hongos/patogenicidad , Genes de Plantas , Inmunidad Innata/genética , Proteínas de Insectos/química , Glicoproteínas de Membrana/química , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Proteínas de Plantas/química , Estructura Secundaria de Proteína , Receptores de Interleucina-1/química , Alineación de Secuencia , Receptores Toll-LikeRESUMEN
The Ac/Ds transposon system from maize was used for insertional mutagenesis in tomato. Marker genes were employed for the selection of plants carrying a total of 471 unique Ds elements. Three mutants were obtained with Ds insertions closely linked to recessive mutations: feebly (fb), yellow jim (yj) and dopey (dp). The fb seedlings produced high anthocyanin levels, developed into small fragile plants, and were insensitive to the herbicide phosphinothricin. The yj plants had yellow leaves as a result of reduced levels of chlorophyll. The dp mutants completely or partially lacked inflorescences. The fb and yj loci were genetically linked to the Ds donor site on chromosome 3. Reactivation of the Ds element in the fb mutants by crosses with an Ac-containing line resulted in restoration of the wild-type phenotypes. Plant DNA fragments flanking both sides of the Ds element in the fb mutant were isolated by the inverse polymerase chain reaction. Molecular analysis showed that phenotypic reversions of fb were correlated with excisions of Ds. DNA sequence analysis of Fb reversion alleles showed the characteristic Ds footprints. Northern and cDNA sequence analysis indicated that transcription of the FEEBLY (FB) gene was impeded by the insertion of Ds in an intron. Comparison of the predicted amino acid sequence of the FB protein with other database sequences indicated that FB is a novel gene.
Asunto(s)
Elementos Transponibles de ADN/genética , Genes de Plantas/genética , Mutación , Proteínas de Plantas/genética , Solanum lycopersicum/genética , Aminobutiratos/farmacología , Antocianinas/biosíntesis , Secuencia de Bases , Clorofila/análisis , Clonación Molecular/métodos , ADN Complementario/genética , ADN de Plantas/genética , Genes Recesivos , Ligamiento Genético , Herbicidas/farmacología , Solanum lycopersicum/efectos de los fármacos , Solanum lycopersicum/crecimiento & desarrollo , Datos de Secuencia Molecular , Mutagénesis Insercional , ARN de Planta/análisis , Análisis de Secuencia de ADN , Zea mays/genéticaRESUMEN
The fungal pathogen Alternaria alternata f. sp. lycopersici produces host-selective AAL-toxins that cause Alternaria stem canker in tomato. Susceptibility to the disease is based on the relative sensitivity of the host to the AAL-toxins and is controlled by the Asc locus on chromosome 3L. Chemical mutagenesis was employed to study the genetic basis of sensitivity to AAL-toxins and susceptibility to fungal infection. Following the treatment of seeds of a susceptible line with ethyl methanesulphonate (EMS), resistant M2 mutants were obtained. Most plants with induced resistances showed toxin-sensitivity responses that were comparable to those of resistant control lines carrying the Asc locus. In addition, genetic analysis of the mutagenised plants indicated that the mutations occurred at the Asc locus. Furthermore, novel mutants were identified that were insensitive to the AAL-toxins at the seedling stage but toxin-sensitive and susceptible to fungal infection at mature stages. No AAL-toxin-insensitive insertion mutants were identified following a transposon mutagenesis procedure. Molecular mechanisms involved in host defence against A a. lycopersici are discussed.
RESUMEN
The fungal pathogen Alternaria alternata f. sp. lycopersici produces AAL-toxins that function as chemical determinants of the Alternaria stem canker disease in the tomato (Lycopersicon esculentum). In resistant cultivars, the disease is controlled by the Asc locus on chromosome 3. Our aim was to characterize novel sources of resistance to the fungus and of insensitivity to the host-selective AAL-toxins. To that end, the degree of sensitivity of wild tomato species to AAL-toxins was analyzed. Of all members of the genus Lycopersicon, only L. cheesmanii was revealed to be sensitive to AAL-toxins and susceptible to fungal infection. Besides moderately insensitive responses from some species, L. pennellii and L. peruvianum were shown to be highly insensitive to AAL-toxins as well as resistant to the pathogen. Genetic analyses showed that high insensitivity to AAL-toxins from L. pennellii is inherited in tomato as a single complete dominant locus. This is in contrast to the incomplete dominance of insensitivity to AAL-toxins of L. esculentum. Subsequent classical genetics, RFLP mapping and allelic testing indicated that high insensitivity to AAL-toxins from L. pennellii is conferred by a new allele of the Asc locus.
Asunto(s)
Alternaria/patogenicidad , Micotoxinas/toxicidad , Enfermedades de las Plantas/genética , Solanum lycopersicum/genética , Ligamiento Genético , Inmunidad Innata , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
The fungal disease resistance locus Alternaria stem canker (Asc) in tomato has been suggested to encode the enzyme aspartate carbamoyltransferase (ACTase). To test this hypothesis a segment of the tomato ACTase gene was amplified by the polymerase chain reaction (PCR) using degenerate primers. The PCR product obtained was subsequently used to isolate an ACTase cDNA clone. Restriction fragment length polymorphism (RFLP) linkage analysis showed that the ACTase gene and the Asc locus do not cosegregate. RFLP mapping positioned the ACTase gene on chromosome 11, while the Asc locus is located on chromosome 3. These results exclude the possibility that the ACTase protein is encoded by the Asc locus.
Asunto(s)
Alternaria , Aspartato Carbamoiltransferasa/genética , Genes de Plantas/genética , Enfermedades de las Plantas/microbiología , Plantas/enzimología , Alternaria/inmunología , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , ADN de Cadena Simple , Genes de Plantas/inmunología , Ligamiento Genético , Datos de Secuencia Molecular , Plantas/genética , Plantas/microbiología , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
The maize doubleDs element is stably maintained in the tomato genome. Upon the subsequent introduction of Ac into a plant containing doubleDs, disruption of the doubleDs structure and DNA rearrangements at the site of the doubleDs element were observed. No indications were obtained for excision of the complete doubleDs structure. The consequences of transactivation of doubleDs in these experiments are different from those described for transactivation of single Ds elements in tomato. The mechanisms by which such rearrangements could have occurred in tomato are discussed in relation to complex insertions containing doubleDs in maize.