Mapping of avirulence genes in Phytophthora infestans with amplified fragment length polymorphism markers selected by bulked segregant analysis.
(25/452)
In this study we investigated the genetic control of avirulence in the diploid oomycete pathogen Phytophthora infestans, the causal agent of late blight on potato. The dominant avirulence (Avr) genes matched six race-specific resistance genes introgressed in potato from a wild Solanum species. AFLP markers linked to Avr genes were selected by bulked segregant analysis and used to construct two high-density linkage maps, one containing Avr4 (located on linkage group A2-a) and the other containing a cluster of three tightly linked genes, Avr3, Avr10, and Avr11 (located on linkage group VIII). Bulked segregant analysis also resulted in a marker linked to Avr1 and this allowed positioning of Avr1 on linkage group IV. No bulked segregant analysis was performed for Avr2, but linkage to a set of random markers placed Avr2 on linkage group VI. Of the six Avr genes, five were located on the most distal part of the linkage group, possibly close to the telomere. The high-density mapping was initiated to facilitate future positional cloning of P. infestans Avr genes. (+info)
Phytotoxic protein PcF, purification, characterization, and cDNA sequencing of a novel hydroxyproline-containing factor secreted by the strawberry pathogen Phytophthora cactorum.
(26/452)
A novel protein factor, named PcF, has been isolated from the culture filtrate of Phytophthora cactorum strain P381 using a highly sensitive leaf necrosis bioassay with tomato seedlings. Isolated PcF protein alone induced leaf necrosis on its host strawberry plant. The primary structure and cDNA sequence of this novel phytotoxic protein was determined, and BLAST searches of Swiss-Prot, EMBL, and GenBank(TM)/EBI data banks showed that PcF shared no significant homology with other known sequences. The 52-residue PcF protein, which contains a 4-hydroxyproline residue along with three S-S bridges, exhibits a high content of acidic sidechains, accounting for its isoelectric point of 4.4. The molecular mass of isolated PcF is 5,622 +/- 0.5 Da as determined by mass spectrometry and matches that calculated from the deduced amino acid sequence with cDNA sequencing. The cDNA sequence indicates that PcF is first produced as a larger precursor, comprising an additional N-terminal, 21-residue secretory signal peptide. Maturation of this protein involves the hydroxylation of proline 49, a feature that is unique among other known secreted fungal phytopathogenic proteins. (+info)
Elicitin genes expressed in vitro by certain tobacco isolates of Phytophthora parasitica are down regulated during compatible interactions.
(27/452)
Phytophthora spp. secrete proteins called elicitins in vitro that can specifically induce hypersensitive response and systemic acquired resistance in tobacco. In Phytophthora parasitica, the causal agent of black shank, most isolates virulent on tobacco are unable to produce elicitins in vitro. Recently, however, a few elicitin-producing P. parasitica strains virulent on tobacco have been isolated. We investigated the potential diversity of elicitin genes in P. parasitica isolates belonging to different genotypes and with various virulence levels toward tobacco as well as elicitin expression pattern in vitro and in planta. Although elicitins are encoded by a multigene family, parAl is the main elicitin gene expressed. This gene is highly conserved among isolates, regardless of the elicitin production and virulence levels toward tobacco. Moreover, we show that elicitin-producing P. parasitica isolates virulent on tobacco down regulate parAl expression during compatible interactions, whichever host plant is tested. Conversely, one elicitin-producing P. parasitica isolate that is pathogenic on tomato and avirulent on tobacco still expresses parAl in the compatible interaction. Therefore, some P. parasitica isolates may evade tobacco recognition by down regulating parA1 in planta. The in planta down regulation of parA1 may constitute a suitable mechanism for P. parasitica to infect tobacco without deleterious consequences for the pathogen. (+info)
Induction of plant gp91 phox homolog by fungal cell wall, arachidonic acid, and salicylic acid in potato.
(28/452)
The oxidative burst has been suggested to be a primary event responsible for triggering the cascade of defense responses in various plant species against infection with avirulent pathogens or pathogen-derived elicitors. The molecular mechanisms of rapid production of active oxygen species (AOS), however, are not well known. We isolated homologs of gp91 phox, a plasma membrane protein of the neutrophil NADPH oxidase, from a potato cDNA library. Molecular cloning of the cDNA showed that there are two isogenes, designated StrbohA and StrbohB, respectively. The RNA gel blot analyses showed that StrbohA was constitutively expressed at a low level, whereas StrbohB was induced by hyphal wall components (HWC elicitor) from Phytophthora infestans in potato tubers. Treatment of potato tubers with HWC elicitor caused a rapid but weak transient accumulation of H2O2 (phase I), followed by a massive oxidative burst 6 to 9 h after treatment (phase II). Diphenylene iodonium (DPI), an inhibitor of the neutrophil NADPH oxidase, blocked both bursts, whereas pretreatment of the protein synthesis inhibitor cycloheximide with the tuber abolished only the second burst. These results suggest that the expression of StrbohA and StrbohB contributes to phase I and II bursts, respectively. The same is true for arachidonic acid, a lipid component of P. infestans-stimulated biphasic oxidative burst, whereas an endogenous signaling molecule, salicylic acid, only induced a weak phase II burst. Both molecules induced the StrbohB expression, which is in agreement with the second burst. To characterize the signal transduction pathway leading to the oxidative burst, we examined the role of protein phosphorylation in HWC-stimulated StrbohB gene expression. K252a and staurosporine, two protein kinase inhibitors, blocked the transcript accumulation. Two inhibitors of extracellular Ca2+ movement, however, did not abolish the transcript accumulation of StrbohB, suggesting that certain calcium-independent protein kinases are involved in the process of StrbohB gene expression. Additionally, we examined a causal relationship between the oxidative burst and expression of defense genes induced by the HWC elicitor. The transcript accumulation of genes related to sesquiterpenoid phytoalexin synthesis (lubimin and rishitin) and phenylpropanoid pathway was inhibited slightly by the DPI treatment, suggesting that the oxidative burst is not essential to activate these genes. Interestingly, the concomitant presence of DPI with the elicitor resulted in an increase in lubimin accumulation and a decrease in rishitin accumulation. Because it is known that lubimin is metabolized into rishitin via oxylubimin, we propose that AOS mediates the synthesis of rishitin from lubimin. (+info)
Adhesion of Phytophthora palmivora zoospores: electron microscopy of cell attachment and cyst wall fibril formation.
(29/452)
Zoospores of Phytophthora palmivora adhered to a plastic film surface were examined by electron microscopy. Three stages of adhesion were compared: (1) non-adhesive, unencysted zoospores, (2) adhered incipient cysts, and (3) adhered mature cysts. Thin sections of incipient cysts revealed cells attached to the film surface through the partially discharged contents of the so-called peripheral vesicles; this seems to be the first step in cell adhesion. In mature cysts, the adhesive appeared to have been compacted into an electron-dense deposit binding the cyst wall to the plastic surface. The adhesion zone was also examined in face view after lysing attached incipient cysts with sodium dodecyl sulphate. Cyst wall microfibrils were seen together with an amorphous substance (presumably the adhesive material). The microfibrils were in various stages of formation. Seemingly, adhesion and microfibril formation take place concurrently. The possibility was considered that the material contained in the peripheral vesicles serves in both cell adhesion and microfibril elaboration. (+info)
Mediation of elicitin activity on tobacco is assumed by elicitin-sterol complexes.
(30/452)
Elicitins secreted by phytopathogenic Phytophthora spp. are proteinaceous elicitors of plant defense mechanisms and were demonstrated to load, carry, and transfer sterols between membranes. The link between elicitor and sterol-loading properties was assessed with the use of site-directed mutagenesis of the 47 and 87 cryptogein tyrosine residues, postulated to be involved in sterol binding. Mutated cryptogeins were tested for their ability to load sterols, bind to plasma membrane putative receptors, and trigger biological responses. For each mutated elicitin, the chemical characterization of the corresponding complexes with stigmasterol (1:1 stoichiometry) demonstrated their full functionality. However, these proteins were strongly altered in their sterol-loading efficiency, specific binding to high-affinity sites, and activities on tobacco cells. Ligand replacement experiments strongly suggest that the formation of a sterol-elicitin complex is a requisite step before elicitins fasten to specific binding sites. This was confirmed with the use of two sterol-preloaded elicitins. Both more rapidly displaced labeled cryptogein from its specific binding sites than the unloaded proteins. Moreover, the binding kinetics of elicitins are related to their biological effects, which constitutes the first evidence that binding sites could be the biological receptors. The first event involved in elicitin-mediated cell responses is proposed to be the protein loading with a sterol molecule. (+info)
A pathogen-inducible divinyl ether synthase (CYP74D) from elicitor-treated potato suspension cells.
(31/452)
In elicitor-treated potato cells, 9-lipoxygenase-derived oxylipins accumulate with the divinyl ether colneleic acid as the major metabolite. Here, the identification of a potato cDNA is described, whose predicted amino acid sequence corresponds to divinyl ether synthases, belonging to the recently identified new P450 subfamily CYP74D. The recombinant protein was expressed in Escherichia coli and shown to metabolize 9-hydroperoxy linoleic acid to colneleic acid at pH 6.5. This fatty acid derivative has been implicated in functioning as a plant antimicrobial compound. RNA blot analyses revealed accumulation of divinyl ether synthase transcripts both upon infiltration of potato leaves with Pseudomonas syringae and after infection with Phytophthora infestans. (+info)
High frequency mitotic gene conversion in genetic hybrids of the oomycete Phytophthora sojae.
(32/452)
Microbial populations depend on genetic variation to respond to novel environmental challenges. Plant pathogens are notorious for their ability to overcome pesticides and host resistance genes as a result of genetic changes. We report here that in particular hybrid strains of Phytophthora sojae, an oomycete pathogen of soybean, high frequency mitotic gene conversion rapidly converts heterozygous loci to homozygosity, resulting in heterokaryons containing highly diverse populations of diploid nuclei. In hybrids involving strain P7076, conversion rates of up to 3 x 10(-2) per locus per nucleus per generation were observed. In other hybrids, rates were of the order of 5 x 10(-5). Independent gene conversion was observed within a selected linkage group including loci as close as 0.7 kb apart and in unlinked markers throughout the genome. Gene conversions continued throughout vegetative growth and were stimulated by further sexual reproduction. At many loci, conversion showed extreme disparity, with one allele always being lost, suggesting that conversion was initiated by allele-specific double-stranded breaks. Pedigree analysis indicated that individual loci undergo multiple independent conversions within the nuclei of a vegetative clone and that conversion may be preceded by a heritable "activation" state. (+info)