Phylogenetic relationship of Phytophthora cryptogea Pethybr. & Laff and P. drechsleri Tucker.

The phylogeny and taxonomy of Phytophthora cryptogea and Phytophthora drechsleri has long been a matter of controversy. To re-evaluate this, a worldwide collection of 117 isolates assigned to either P. cryptogea, P. drechsleri or their sister taxon, Phytophthora erythroseptica were assessed for morphological, physiological (pathological, cultural, temperature relations, mating) and molecular traits. Multiple gene phylogenetic analysis was performed on DNA sequences of nuclear (internal transcribed spacers (ITS), ß-tubulin, translation elongation factor 1α, elicitin) and mitochondrial (cytochrome c oxidase subunit I) genes. Congruence was observed between the different phylogenetic data sets and established that P. drechsleri and P. cryptogea are distinct species. Isolates of P. drechsleri form a monophyletic grouping with low levels of intraspecific diversity whereas P. cryptogea is more variable. Three distinct phylogenetic groups were noted within P. cryptogea with an intermediate group providing strong evidence for introgression of previously isolated lineages. This evidence suggests that P. cryptogea is an operational taxonomic unit and should remain a single species. Of all the morphological and physiological traits only growth rate at higher temperatures reliably discriminated isolates of P. drechsleri and P. cryptogea. As a homothallic taxon, P. erythroseptica, considered the cause of potato pink rot, is clearly different in mating behaviour from the other two species. Pathogenicity, however, was not a reliable characteristic as all isolates of the three species formed pink rot in potato tubers. The phylogenetic evidence suggests P. erythroseptica has evolved from P. cryptogea more recently than the split from the most recent common ancestor of all three species. However, more data and more isolates of authentic P. erythroseptica are needed to fully evaluate the taxonomic position of this species.

Cellular and molecular characterization of Phytophthora parasitica appressorium-mediated penetration.

Data on plant pathogenic oomycetes are scarce and little is known about the early events leading to the onset of infection. The aim of this work was to analyze the penetration process of the soil-borne plant pathogen Phytophthora parasitica, which has a wide host range. Here, we performed a cytological analysis of the colonization of the first plant cell and developed an inoculation assay for characterizing the entire penetration process through cellular and molecular analyses. We showed that P. parasitica infects roots by producing a specialized structure, the appressorium. We produced the first cDNA library for the penetrating stage of a Phytophthora species and showed it to be highly enriched in pathogenicity-related sequences. These included coding sequences for many cell-degrading enzymes, effectors such as RXLR-containing proteins and proteins involved in protection against plant defense responses. Characterization of the appressorium cDNA library and identification of genes overrepresented early in P. parasitica infection provided us with an unprecedented opportunity to decipher the molecular mechanisms involved in penetration of the plant cells during the initiation of infection by a soil-borne oomycete.

Mediation of elicitin activity on tobacco is assumed by elicitin-sterol complexes.

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.

Elicitin genes expressed in vitro by certain tobacco isolates of Phytophthora parasitica are down regulated during compatible interactions.

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.

Crystallization and preliminary X-ray studies of oligandrin, a sterol-carrier elicitor from Pythium oligandrum.

Oligandrin is a 10 kDa acidic protein produced by the fungus micromycete Pythium oligandrum and is a member of the alpha-elicitin group, with sterol- and lipid-carrier properties. Oligandrin has been crystallized at 290 K using PEG 4000 as a precipitant. A cholesterol complex was obtained under the same conditions. The space group of the crystals at low temperature (100 K) is C222, with unit-cell parameters a = 94.0, b = 171.1, c = 55.3 A. Four molecules are present in the asymmetric unit. Data from the free and cholesterol-complexed forms were recorded at synchrotron sources to resolutions of 2.4 (uncomplexed) and 1.9 A (complexed), respectively.

A family of repeated DNA in the genome of the oomycete plant pathogen Phytophthora cryptogea.

We have identified a family of repetitive sequences, called PCISs (Phytophthora Cryptogea Insertion Sequences), in the genome of Phytophthora cryptogea. They vary greatly in size (in a 100-1200 bp range) and appear to represent 3' terminal fragments of a larger element. Two subfamilies were characterised on the basis of nucleotide sequences. PCISs exhibit insertion polymorphism, as well as a very low sequence divergence. Most copies are flanked by terminal direct repeats, suggesting that their spreading involves insertion events rather than rearrangements. PCISs are found in very few copies in Phytophthora cinnamomi. The spreading of these sequences within the genus Phytophthora is discussed.

Are elicitins cryptograms in plant-Oomycete communications?

Stimulation of plant natural defenses is an important challenge in phytoprotection prospects. In that context, elicitins, which are small proteins secreted by Phytophthora and Pythium species, have been shown to induce a hypersensitive-like reaction in tobacco plants. Moreover, these plants become resistant to their pathogens, and thus this interaction constitutes an excellent model to investigate the signaling pathways leading to plant resistance. However, most plants are not reactive to elicitins, although they possess the functional signaling pathways involved in tobacco responses to elicitin. The understanding of factors involved in this reactivity is needed to develop agronomic applications. In this review, it is proposed that elicitins could interact with regulating cell wall proteins before they reach the plasma membrane. Consequently, the plant reactivity or nonreactivity status could result from the equilibrium reached during this interaction. The possibility of overexpressing the elicitins directly from genomic DNA in Pichia pastoris allows site-directed mutagenesis experiments and structure/function studies. The recent discovery of the sterol carrier activity of elicitins brings a new insight on their molecular activity. This constitutes a crucial property, since the formation of a sterol-elicitin complex is required to trigger the biological responses of tobacco cells and plants. Only the elicitins loaded with a sterol are able to bind to their plasmalemma receptor, which is assumed to be an allosteric calcium channel. Moreover, Phytophthora and Pythium do not synthesize the sterols required for their growth and their fructification, and elicitins may act as shuttles trapping the sterols from the host plants. Sequence analysis of elicitin genes from several Phytophthora species sheds unexpected light on the phylogenetic relationships among the genus, and suggests that the expression of elicitins is under tight regulatory control. Finally, general involvement of these lipid transfer proteins in the biology of Pythiaceae, and in plant defense responses, is discussed. A possible scheme for the coevolution between Phytophthora and tobacco plants is approached.

Elicitins, proteinaceous elicitors of plant defense, are a new class of sterol carrier proteins.

Some phytopathogenic fungi within Phytophthora species are unable to synthesize sterols and therefore must pick them up from the membranes of their host-plant, using an unknown mechanism. These pseudo-fungi secrete elicitins which are small hydrophilic cystein-rich proteins. The results show that elicitins studied interact with dehydroergosterol in the same way, but with some time-dependent differences. Elicitins have one binding site with a similar strong affinity for dehydroergosterol. Using a non-steroid hydrophobic fluorescent probe, we showed that phytosterols are able to similarly bind to elicitins. Moreover, elicitins catalyze sterol transfer between phospholipidic artificial membranes. Our results afford the first evidence for a molecular activity of elicitins which appears to be extracellular sterol carrier proteins. This property should contribute to an understanding of the molecular mechanism involved in sterol uptake by Phytophthora. It opens new perspectives concerning the role of such proteins in plant-microorganism interactions, since elicitins trigger defence reactions in plants.

Diversity of Virulence in Phytophthora parasitica on Tobacco, as Reflected by Nuclear RFLPs.

ABSTRACT A worldwide collection of P. parasitica isolates was investigated for the ability to infect tobacco and tomato, as related to elicitin production. Elicitin was produced by all nontobacco isolates, and nonproducing strains all were isolated from tobacco. In addition, producing strains were isolated from tobacco and coexisted with nonproducing (TE ) strains. Elicitin production generally was associated with low virulence on tobacco and frequent pathogenicity on tomato, whereas TE isolates generally were highly virulent and specialized to tobacco. Analysis of both mitochondrial and nuclear DNA restriction fragment length polymorphisms indicated, for the first time, that black shank isolates can be distinguished from other P. parasitica isolates on the basis of genetic criteria. Our results suggest that severe black shank is caused by a limited number of TE strains that have been disseminated by clonal evolution. Mutations in the TE phenotype seem to have arisen independently in several genetic backgrounds and distinct geographic areas. The fortuitous absence of elicitin production has precluded population replacements in areas of intensive tobacco cultivation. Thus, monitoring the loss of elicitin production in developing tobacco areas should be considered in disease management.

RNase activity prevents the growth of a fungal pathogen in tobacco leaves and increases upon induction of systemic acquired resistance with elicitin.

The hypersensitive response and systemic acquired resistance (SAR) can be induced in tobacco (Nicotiana tabacum L.) plants by cryptogein, an elicitin secreted by Phytophthora cryptogea. Stem application of cryptogein leads to the establishment of acquired resistance to subsequent leaf infection with Phytophthora parasitica var nicotianae, the agent of the tobacco black shank disease. We have studied early events that occur after the infection and show here that a tobacco gene encoding the extracellular S-like RNase NE is expressed in response to inoculation with the pathogenic fungus. Upon induction of SAR with cryptogein, the accumulation of NE transcripts coincided with a rapid induction of RNase activity and with the increase in the activity of at least two different extracellular RNases. Moreover, exogenous application of RNase activity in the extracellular space of leaves led to a reduction of the fungus development by up to 90%, independently of any cryptogein treatment and in the absence of apparent necrosis. These results indicate that the up-regulation of apoplastic RNase activity after inoculation could contribute to the control of fungal invasion in plants induced to SAR with cryptogein.

Characterization of a gene cluster of Phytophthora cryptogea which codes for elicitins, proteins inducing a hypersensitive-like response in tobacco.

Elicitins, proteinaceous elicitors secreted by Phytophthora spp., act as inducers of a hypersensitive-like response in tobacco during incompatible interactions. We have isolated and cloned sequences encoding cryptogein and related isoforms from P. cryptogea that belong to the elicitin family. The isolation of a genomic clone led to the characterization of four clustered genes. Two of these genes encode distinct elicitins, and two genes would encode, if expressed, a class of highly acidic elicitins which had not been observed so far. Northern blots indicate that elicitin genes are expressed in the fungus grown in vitro, though at different levels. Southern hybridization revealed that elicitins are encoded by a multigene family in several other species of Phytophthora. Moreover, isolates of Phytophthora parasitica var. nicotianae, pathogenic to tobacco, which do not produce elicitins, possess several elicitin-encoding genes. Involvement of elicitins in plant-pathogen interactions is discussed.

Migration of the Fungal Protein Cryptogein within Tobacco Plants.

Cryptogein (CRY), a protein secreted by Phytophthora cryptogea, causes necrosis on tobacco (Nicotiana tabacum) plants at the site of application (the stem or the roots) and also on distant leaves. Autoradiography of plantlets after root absorption of radioiodinated CRY demonstrated a rapid migration of the label to the leaf lamina via the veins. Using an anti-CRY antiserum, a CRY-related antigen was detected in the stem and leaves of CRY-treated plants at a distance from the site of application. This antigen had the same molecular weight as CRY and was detected in the leaves as early as 1 hour after stem treatment, i.e. long before necrosis was detectable. The antigen was also detected in plants inoculated with P. cryptogea. The distant location of the necrosis induced by the fungus or by CRY can be ascribed to the migration of this protein, which is toxic to tobacco cells. It is proposed that CRY, which also elicits defense reactions in tobacco, might contribute to the hypersensitive response of tobacco to P. cryptogea.

Structural and transcriptional characterization of the external spacer of a ribosomal RNA nuclear gene from a higher plant.

A lambda recombinant phage, carrying a radish rDNA fragment spanning the complete external spacer and its borders, has been isolated and characterized by sequencing. The fragment is 2911 bp long and includes 486 bp of the 3' end of the 25S rRNA sequence, 2349 bp of spacer and the first 76 bp of the 5' end of the 18S rRNA sequence. The spacer can be divided into three regions: two unique domains flanking a 830-bp region of repeated sequences. Seven repeats ranging from 80 to 103 bp can be recognized. They are separated by short arrays of 12-21 adenylic residues. Each repeat slightly differs from the others by single-nucleotide changes or short deletions. Examination of single-nucleotide changes common to two units suggests that a duplication arose during the evolution of this sequence. The repeated region was subcloned and used as a probe to demonstrate that it is highly species-specific: in stringent conditions it does not cross-hybridize with the spacer of ribosomal genes from closely related species such as Brassica. Transcription products, starting or finishing within the spacer sequence, were mapped by northern blotting, primer extension and S1 mapping. Two major precursors were identified starting respectively at positions 2095 and 2280. The region surrounding the start at 2095 presents extensive homology with an analogous region in maize, rye, mung bean, Xenopus and tse-tse fly. However, longer transcripts can be detected. Several 3' ends downstream of the 25S terminus were also observed. Taken together these results indicate that rDNA transcription and pre-rRNA processing in plants are more complex than anticipated from previous studies.

Organization and evolution of a higher plant alphoid-like satellite DNA sequence.

Two recombinant plasmids containing, respectively, three and eight tandem repeats of a 177 base-pair (bp) element from radish nuclear DNA have been isolated. These plasmids were used as probes to investigate the organization and the copy number of this element within the genome. This sequence is present in congruent to 0.6 million copies. Restriction analysis provides evidence for sequence heterogeneity and reveals the occurrence of non-overlapping subfamilies. Nine units were sequenced and found to be remarkably conserved. However, sequences in the two clones clearly belong to two distinct subgroups. Our data suggest that these sequences evolved in a concerted manner and that homogenization mechanisms such as gene conversions certainly took place. The 177 bp sequence is made from three 60 bp blocks that are derived from a common ancestor. Exchanges between the three blocks probably occurred before they became fixed as a patchwork of short sequences, the 177 bp element. This unit of 177 bp was then amplified in several steps. The presence of such a repeated sequence can be detected in other Cruciferae when hybridizations are carried out under low stringency conditions. Direct comparison with a previously published mustard satellite DNA sequence indicates a similar organization and a 75% homology. Homology was also found with shorter regions (congruent to 60 bp) of broad bean and corn satellite DNA. Finally, homology was also found with several animal alphoid sequences, suggesting that this family also occurs in the plant genomes.

Post-transcriptional regulation of glyceraldehyde-3-phosphate-dehydrogenase gene expression in rat tissues.

We have isolated and identified cDNA clones containing part of the coding sequence for rat glyceraldehyde-3-phosphate-dehydrogenase (GAPDH, E.C. 1.2.1.12). By using one of these clones as a probe, we have shown that: i) the abundance of GAPDH mRNA is different in various tissues of the adult rat and in good correlation with the abundance of the enzyme; ii) the transcription rates are quite similar in all tissues tested. We therefore conclude that the tissue-specific differential GAPDH gene expression is regulated by adjusting the abundance of its mRNA at the post-transcriptional level.

Complete nucleotide sequence of the messenger RNA coding for chicken muscle glyceraldehyde-3-phosphate dehydrogenase.

The complete nucleotide sequence for chicken glyceraldehyde-3-phosphate dehydrogenase mRNA has been determined, thereby extending the longest such sequence previously reported (Dugaiczyk et al. Biochemistry, 1983, 22, 1605-1613) by 27 nucleotides. The complete mRNA with the exclusion of poly(A) is 1284 nucleotides long and contains 56 nucleotides of 5' non coding sequence and 229 nucleotides of 3' non coding region. Knowledge of the complete sequence allows us to propose secondary structures models which may be of biological significance.

A warning on the use of synthetic DNA primers for initiation of reverse transcription on RNA templates: unexpected initiation at a mismatched nucleotide.

A synthetic 13-mer oligodeoxynucleotide designed to specifically initiate on lactic dehydrogenase (LDH) mRNA was shown to prime with high efficiency the synthesis of DNA complementary to the last 133 nucleotides of mitochondrial 16S ribosomal RNA on a mouse liver total poly(A)+ RNA template. One especially interesting feature of this reaction was that reverse transcriptase has been able to initiate at the 3' terminal C residue of the primer despite the fact that it was facing an A. This observation therefore establishes the ability of reverse transcriptase to initiate directly on a mismatched nucleotide.