RESUMO
Plant-parasitic nematodes secrete effectors that manipulate plant cell morphology and physiology to achieve host invasion and establish permanent feeding sites. Effectors from the highly expanded SPRYSEC (SPRY domain with a signal peptide for secretion) family in potato cyst nematodes have been implicated in activation and suppression of plant immunity, but the mechanisms underlying these activities remain largely unexplored. To study the host mechanisms used by SPRYSEC effectors, we identified plant targets of GpRbp-1 from the potato cyst nematode Globodera pallida. Here, we show that GpRbp-1 interacts in yeast and in planta with a functional potato homologue of the Homology to E6-AP C-Terminus (HECT)-type ubiquitin E3 ligase UPL3, which is located in the nucleus. Potato lines lacking StUPL3 are not available, but the Arabidopsis mutant upl3-5 displaying a reduced UPL3 expression showed a consistently small but not significant decrease in susceptibility to cyst nematodes. We observed a major impact on the root transcriptome by the lower levels of AtUPL3 in the upl3-5 mutant, but surprisingly only in association with infections by cyst nematodes. To our knowledge, this is the first example that a HECT-type ubiquitin E3 ligase is targeted by a pathogen effector and that a member of this class of proteins specifically regulates gene expression under biotic stress conditions. Together, our data suggest that GpRbp-1 targets a specific component of the plant ubiquitination machinery to manipulate the stress response in host cells.
Assuntos
Regulação da Expressão Gênica de Plantas , Proteínas de Helminto/metabolismo , Solanum tuberosum/parasitologia , Tylenchoidea/patogenicidade , Ubiquitina-Proteína Ligases/metabolismo , Animais , Arabidopsis/parasitologia , Proteínas de Arabidopsis/metabolismo , Domínio B30.2-SPRY , Ligases/metabolismo , Proteínas Nucleares/metabolismo , UbiquitinaçãoRESUMO
Plants have evolved a limited repertoire of NB-LRR disease resistance (R) genes to protect themselves against myriad pathogens. This limitation is thought to be counterbalanced by the rapid evolution of NB-LRR proteins, as only a few sequence changes have been shown to be sufficient to alter resistance specificities toward novel strains of a pathogen. However, little is known about the flexibility of NB-LRR R genes to switch resistance specificities between phylogenetically unrelated pathogens. To investigate this, we created domain swaps between the close homologs Gpa2 and Rx1, which confer resistance in potato (Solanum tuberosum) to the cyst nematode Globodera pallida and Potato virus X, respectively. The genetic fusion of the CC-NB-ARC of Gpa2 with the LRR of Rx1 (Gpa2CN/Rx1L) results in autoactivity, but lowering the protein levels restored its specific activation response, including extreme resistance to Potato virus X in potato shoots. The reciprocal chimera (Rx1CN/Gpa2L) shows a loss-of-function phenotype, but exchange of the first three LRRs of Gpa2 by the corresponding region of Rx1 was sufficient to regain a wild-type resistance response to G. pallida in the roots. These data demonstrate that exchanging the recognition moiety in the LRR is sufficient to convert extreme virus resistance in the leaves into mild nematode resistance in the roots, and vice versa. In addition, we show that the CC-NB-ARC can operate independently of the recognition specificities defined by the LRR domain, either aboveground or belowground. These data show the versatility of NB-LRR genes to generate resistance to unrelated pathogens with completely different lifestyles and routes of invasion.
Assuntos
Resistência à Doença/genética , Doenças das Plantas/imunologia , Proteínas de Plantas/metabolismo , Potexvirus/fisiologia , Solanum tuberosum/genética , Tylenchoidea/fisiologia , Animais , Proteínas de Repetições Ricas em Leucina , Mutação com Perda de Função , Fenótipo , Doenças das Plantas/parasitologia , Doenças das Plantas/virologia , Folhas de Planta/genética , Folhas de Planta/imunologia , Folhas de Planta/parasitologia , Folhas de Planta/virologia , Proteínas de Plantas/genética , Raízes de Plantas/genética , Raízes de Plantas/imunologia , Raízes de Plantas/parasitologia , Raízes de Plantas/virologia , Brotos de Planta/genética , Brotos de Planta/imunologia , Brotos de Planta/parasitologia , Brotos de Planta/virologia , Domínios Proteicos , Proteínas/genética , Proteínas/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Proteínas Recombinantes de Fusão , Solanum tuberosum/imunologia , Solanum tuberosum/parasitologia , Solanum tuberosum/virologiaRESUMO
BACKGROUND: The yellow potato cyst nematode, Globodera rostochiensis, is a devastating plant pathogen of global economic importance. This biotrophic parasite secretes effectors from pharyngeal glands, some of which were acquired by horizontal gene transfer, to manipulate host processes and promote parasitism. G. rostochiensis is classified into pathotypes with different plant resistance-breaking phenotypes. RESULTS: We generate a high quality genome assembly for G. rostochiensis pathotype Ro1, identify putative effectors and horizontal gene transfer events, map gene expression through the life cycle focusing on key parasitic transitions and sequence the genomes of eight populations including four additional pathotypes to identify variation. Horizontal gene transfer contributes 3.5 % of the predicted genes, of which approximately 8.5 % are deployed as effectors. Over one-third of all effector genes are clustered in 21 putative 'effector islands' in the genome. We identify a dorsal gland promoter element motif (termed DOG Box) present upstream in representatives from 26 out of 28 dorsal gland effector families, and predict a putative effector superset associated with this motif. We validate gland cell expression in two novel genes by in situ hybridisation and catalogue dorsal gland promoter element-containing effectors from available cyst nematode genomes. Comparison of effector diversity between pathotypes highlights correlation with plant resistance-breaking. CONCLUSIONS: These G. rostochiensis genome resources will facilitate major advances in understanding nematode plant-parasitism. Dorsal gland promoter element-containing effectors are at the front line of the evolutionary arms race between plant and parasite and the ability to predict gland cell expression a priori promises rapid advances in understanding their roles and mechanisms of action.
Assuntos
Genoma de Protozoário , Doenças das Plantas/parasitologia , Solanum tuberosum/parasitologia , Tylenchoidea/genética , Tylenchoidea/patogenicidade , Animais , Elementos Facilitadores Genéticos , Perfilação da Expressão Gênica , Transferência Genética Horizontal , Ilhas Genômicas , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Estágios do Ciclo de Vida , Motivos de Nucleotídeos , Matrizes de Pontuação de Posição Específica , Sítios de Splice de RNA , Splicing de RNA , Transcriptoma , Tylenchoidea/crescimento & desenvolvimento , Virulência/genéticaRESUMO
The Grp1 locus confers broad-spectrum resistance to the potato cyst nematode species Globodera pallida and Globodera rostochiensis and is located in the GP21-GP179 interval on the short arm of chromosome V of potato. A high-resolution map has been developed using the diploid mapping population RHAM026, comprising 1,536 genotypes. The flanking markers GP21 and GP179 have been used to screen the 1,536 genotypes for recombination events. Interval mapping of the resistances to G. pallida Pa2 and G. rostochiensis Ro5 resulted in two nearly identical LOD graphs with the highest LOD score just north of marker TG432. Detailed analysis of the 44 recombinant genotypes showed that G. pallida and G. rostochiensis resistance could not be separated and map to the same location between marker SPUD838 and TG432. It is suggested that the quantitative resistance to both nematode species at the Grp1 locus is mediated by one or more tightly linked R genes that might belong to the NBS-LRR class.
Assuntos
Cromossomos de Plantas , Genes de Plantas , Imunidade Inata/genética , Doenças das Plantas/parasitologia , Solanum tuberosum , Tylenchoidea/patogenicidade , Animais , Mapeamento Cromossômico , Marcadores Genéticos , Genótipo , Escore Lod , Solanum tuberosum/genética , Solanum tuberosum/parasitologiaRESUMO
Plant-parasitic cyst nematodes secrete cell wall modifying proteins during their invasion of host plants. In this study, we used a monoclonal antibody to immunopurify and to sequence the N terminus of the most abundant cellulases in stylet secretions of preparasitic juveniles of Globodera rostochiensis. The N-terminal amino acid sequence perfectly matched the sequence of an expressed sequence tag of two nearly identical genes, named Gr-eng3 and Gr-eng4, which show relatively low similarity with the previously identified Gr-eng1 and Gr-eng2 (i.e., 62% similarity and 42% identity). The recombinantly produced proteins from Gr-eng3 and Gr-eng4 demonstrated specific activity on carboxymethylcellulose, indicating that these genes encode active cellulases. To date, the cellulases in cyst nematodes are comprised of three possible domain structure variants with different types of ancillary domains at the C terminus of the glycosyl hydrolase family 5 (GHF5) domain. We used Bayesian inference to show that the phylogeny of the GHF5 domain based on currently available data suggest that the extant nematode cellulases arose through reshuffling of the GHF5 domain with different types of ancillary domains as relatively independent units. Knocking-down Gr-eng3 and Gr-eng4 using RNA interference resulted in a reduction of nematode infectivity by 57%. Our observations show that the reduced infectivity of the nematodes can be attributed to poor penetration of the host's root system at the onset of parasitism.
Assuntos
Celulases/genética , Celulases/metabolismo , Nematoides/enzimologia , Sequência de Aminoácidos , Animais , Celulases/química , Dados de Sequência Molecular , Filogenia , Doenças das Plantas/parasitologia , Proteína D-Aspartato-L-Isoaspartato Metiltransferase , Solanum tuberosum/parasitologiaRESUMO
Southern analysis showed that Gr-EXPB1, a functional expansin from the potato cyst nematode Globodera rostochiensis, is member of a multigene family, and EST data suggest expansins to be present in other plant parasitic nematodes as well. Homology modeling predicted that Gr-EXPB1 domain 1 (D1) has a flat beta-barrel structure with surface-exposed aromatic rings, whereas the 3D structure of Gr-EXPB1-D2 was remarkably similar to plant expansins. Gr-EXPB1 shows highest sequence similarity to two extracellular proteins from saprophytic soil-inhabiting Actinobacteria, and includes a bacterial type II carbohydrate-binding module. These results support the hypothesis that a number of pathogenicity factors of cyst nematodes is of procaryotic origin and were acquired by horizontal gene transfer.