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ABSTRACT: Genome sequencing combined with high-throughput functional analyses has proved vital in our quest to understand oomycete-plant interactions. With the identification of effector molecules from Phytophthora spp. we can now embark on dissecting the mechanisms by which effectors modulate host processes and thus ensure parasite fitness. One of the key limitations, however, is to genetically modify Phytophthora and assess gene function during parasitism. Here, we describe a straightforward protocol that allows rapid transformation of Phytophthora capsici, an emerging model in oomycete biology. P. capsici is a broad host range pathogen that can infect a wide variety of plants under lab conditions making it a suitable model for detailed studies on oomycete-host interactions. This protocol relies on electroporation-assisted uptake of DNA in to motile zoospores and allows the rapid identification and characterization of genetically stable transformants.
Methods in molecular biology (Clifton, N.J.) 01/2011; 712:129-35.
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ABSTRACT: Pathogens use specialized secretion systems and targeting signals to translocate effector proteins inside host cells, a process that is essential for promoting disease and parasitism. However, the amino acid sequences that determine host delivery of eukaryotic pathogen effectors remain mostly unknown. The Crinkler (CRN) proteins of oomycete plant pathogens, such as the Irish potato famine organism Phytophthora infestans, are modular proteins with predicted secretion signals and conserved N-terminal sequence motifs. Here, we provide direct evidence that CRN N termini mediate protein transport into plant cells. CRN host translocation requires a conserved motif that is present in all examined plant pathogenic oomycetes, including the phylogenetically divergent species Aphanomyces euteiches that does not form haustoria, specialized infection structures that have been implicated previously in delivery of effectors. Several distinct CRN C termini localized to plant nuclei and, in the case of CRN8, required nuclear accumulation to induce plant cell death. These results reveal a large family of ubiquitous oomycete effector proteins that target the host nucleus. Oomycetes appear to have acquired the ability to translocate effector proteins inside plant cells relatively early in their evolution and before the emergence of haustoria. Finally, this work further implicates the host nucleus as an important cellular compartment where the fate of plant-microbe interactions is determined.
Proceedings of the National Academy of Sciences 10/2010; 107(40):17421-6. · 9.68 Impact Factor
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Séverine Lacombe,
Alejandra Rougon-Cardoso,
Emma Sherwood,
Nemo Peeters,
Douglas Dahlbeck,
H Peter van Esse, Matthew Smoker,
Ghanasyam Rallapalli,
Bart P H J Thomma,
Brian Staskawicz,
Jonathan D G Jones,
Cyril Zipfel
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ABSTRACT: Plant diseases cause massive losses in agriculture. Increasing the natural defenses of plants may reduce the impact of phytopathogens on agricultural productivity. Pattern-recognition receptors (PRRs) detect microbes by recognizing conserved pathogen-associated molecular patterns (PAMPs). Although the overall importance of PAMP-triggered immunity for plant defense is established, it has not been used to confer disease resistance in crops. We report that activity of a PRR is retained after its transfer between two plant families. Expression of EFR (ref. 4), a PRR from the cruciferous plant Arabidopsis thaliana, confers responsiveness to bacterial elongation factor Tu in the solanaceous plants Nicotiana benthamiana and tomato (Solanum lycopersicum), making them more resistant to a range of phytopathogenic bacteria from different genera. Our results in controlled laboratory conditions suggest that heterologous expression of PAMP recognition systems could be used to engineer broad-spectrum disease resistance to important bacterial pathogens, potentially enabling more durable and sustainable resistance in the field.
Nature Biotechnology 03/2010; 28(4):365-9. · 29.50 Impact Factor
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ABSTRACT: To identify proteins involved in tomato Cf-9 resistance protein function, a yeast two-hybrid screen was undertaken using the cytoplasmic C-terminus of Cf-9 as bait. A thioredoxin-homologous clone, interacting specifically with Cf-9, was identified and called CITRX (Cf-9-interacting thioredoxin). Virus-induced gene silencing (VIGS) of CITRX resulted in an accelerated Cf-9/Avr9-triggered hypersensitive response in both tomato and Nicotiana benthamiana, accompanied by enhanced accumulation of reactive oxygen species, alteration of protein kinase activity and induction of defence-related genes. VIGS of CITRX also conferred increased resistance to the fungal pathogen Cladosporium fulvum in the otherwise susceptible Cf0 tomato. CITRX acts as a negative regulator of the cell death and defence responses induced through Cf-9, but not Cf-2. Recognition of the Cf-9 C-terminus by CITRX is necessary and sufficient for this negative regulation. This is the first study that implicates thioredoxin activity in the regulation of plant disease resistance.
The EMBO Journal 06/2004; 23(10):2156-65. · 9.20 Impact Factor
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ABSTRACT: Little is known of how plant disease resistance (R) proteins recognize pathogens and activate plant defenses. Rcr3 is specifically required for the function of Cf-2, a Lycopersicon pimpinellifolium gene bred into cultivated tomato (Lycopersicon esculentum) for resistance to Cladosporium fulvum. Rcr3 encodes a secreted papain-like cysteine endoprotease. Genetic analysis shows Rcr3 is allelic to the L. pimpinellifolium Ne gene, which suppresses the Cf-2-dependent autonecrosis conditioned by its L. esculentum allele, ne (necrosis). Rcr3 alleles from these two species encode proteins that differ by only seven amino acids. Possible roles of Rcr3 in Cf-2-dependent defense and autonecrosis are discussed.
Science 05/2002; 296(5568):744-7. · 31.20 Impact Factor
