RESUMO
The type III effector protein AvrPto acts as a virulence factor in susceptible plants lacking a cognate resistance gene but triggers hypersensitive response and disease resistance in tomato plants carrying the Pto gene or in tobacco plants carrying an unknown resistance gene. To assist the characterization of cellular responses caused by AvrPto in the plant, a pathogen-free system was adopted to isolate genes up-regulated 12 h after induced expression of AvrPto. By using subtraction cloning and transgenic tobacco plants expressing avrPto as a transgene, we isolated 125 nonredundant cDNA clones that represent avrPto-response genes (ARG). In addition to genes that are known to be induced by Pto-avrPto recognition, a number of new genes were also isolated. Most of ARG showed a specific induction in tobacco plants challenged with incompatible or nonhost pathogens. The use of an avrPto mutant that selectively eliminated the avrPto recognition in tobacco demonstrated that the ARG were induced in a highly specific manner by the avirulence, instead of the virulence activity of avrPto.
Assuntos
Proteínas de Bactérias/toxicidade , Genes de Plantas , Nicotiana/genética , Nicotiana/microbiologia , Proteínas de Bactérias/genética , DNA Complementar/genética , DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes Bacterianos , Mutação , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Plantas Geneticamente Modificadas , Pseudomonas syringae/genética , Pseudomonas syringae/patogenicidade , Regulação para Cima/efeitos dos fármacos , VirulênciaRESUMO
Pto confers disease resistance to Pseudomonas syringae pv tomato carrying the cognate avrPto gene. Overexpression of Pto under the cauliflower mosaic virus 35S promoter activates spontaneous lesions and confers disease resistance in tomato (Lycopersicon esculentum) plants in the absence of avrPto. Here, we show that these AvrPto-independent defenses require a functional Prf gene. Several Pto-interacting (Pti) proteins are thought to play a role in Pto-mediated defense pathways. To test if interactions with Pti proteins are required for the AvrPto-independent defense responses by Pto overexpression, we isolated several Pto mutants that were unable to interact with one or more Pti proteins, but retained normal interaction with AvrPto. Overexpression of two mutants, Pto(G50S) and Pto(R150S), failed to activate AvrPto-independent defense responses or confer enhanced resistance to the virulent P. s. pv tomato. When introduced into plants carrying 35S::Pto, 35S::Pto(G50S) dominantly suppressed the AvrPto-independent resistance caused by former transgene. 35S::Pto(G50S) also blocked the induction of a number of defense genes by the wild-type 35S::Pto. However, 35S::Pto(G50S) and 35S::Pto(R150S) plants were completely resistant to P. s. pv tomato (avrPto), indicating a normal gene-for-gene resistance. Furthermore, 35S::Pto(G50S) plants exhibited normal induction of defense genes in recognition of avrPto. Thus, the AvrPto-independent defense activation and gene-for-gene resistance mediated by Pto are functionally separable.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Plantas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Bactérias/metabolismo , Expressão Gênica , Imunidade Inata/genética , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Solanum lycopersicum/microbiologia , Mutação , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Pseudomonas/genética , Pseudomonas/crescimento & desenvolvimentoRESUMO
SUMMARY Wheat leaf rust disease, caused by the biotrophic fungus Puccinia triticina, is a result of complex interactions requiring the coordinated activities of the two organisms involved. In an effort to understand the molecular basis of wheat-rust interactions, we isolated and characterized cDNA corresponding to in planta induced fungal genes (PIGs) from susceptible wheat leaves infected with P. triticina by using suppression subtractive hybridization to construct a cDNA library. 350 clones were sequenced, of which 104 were unique. Forty-four cDNA clones encode ribosomal proteins, comprising the single largest category of clones isolated. Twenty-five of these ribosomal protein genes are likely to be of fungal origin, as was suggested by sequence homology. Hybridization of 56 selected non-ribosomal protein clones to rust germling cDNA or genomic DNA probes showed that at least 44 were of fungal origin, demonstrating that the library was highly enriched for fungal cDNA. Differential expression analysis identified 26 non-ribosomal protein genes that were induced in rust-infected leaves. At least 21 of the induced genes were from the rust fungus, indicating that the majority of the induced genes were rust PIGs that are likely to play a role in parasitism. Some of the induced genes share homology to known PIGs or virulence genes in other fungi, suggesting similarities in parasitism among different fungi. Eight clones correspond to novel PIGs that have not been reported in any organism. This paper reports, for the first time, the isolation of P. triticina PIGs and discusses the use of total rust genomic DNA to identify the source of genes.
