A quantitative real-time PCR method for in planta monitoring of Phytophthora infestans growth.

AIMS: To establish a reliable and rapid protocol to simultaneously obtain high quality DNA from an infected host plant and the infecting pathogen. To develop an accurate and sensitive low-cost assay for the quantification and in planta monitoring of Phytophthora infestans growth. METHODS AND RESULTS: In this study, we describe a SYBR Green-based quantitative real-time PCR (qPCR) method for the quantification of P. infestans. The method is based on a simultaneous plant-pathogen DNA purification followed by a qPCR in which the relative quantification of pathogen and plant DNA is performed. Besides assuring an accurate quantification, the use of a plant gene provides a reliable indicator of sample quality, allowing the exclusion of inappropriate samples. By applying this methodology, we were able to detect P. infestans in potato leaf and tuber tissue before the first symptoms of the disease were observed and to monitor the in planta growth of the pathogen for 6 days. CONCLUSIONS: This is a reliable low-cost assay that provides rapid, accurate and sensitive quantification of the late blight pathogen, allowing the in planta monitoring of P. infestans growth. SIGNIFICANCE AND IMPACT OF THE STUDY: The quantitative nature of the assay described in this study may be useful in plant breeding programmes and basic research. The method is appropriate for the comparison of cultivars with different, and even subtle, degrees of pathogen resistance and in the screening of new anti-oomycete compounds. The method can be easily adapted to tomato and the model plant Nicotiana benthamiana.

Arginine kinase of the flagellate protozoa Trypanosoma cruzi. Regulation of its expression and catalytic activity.

In epimastigotes of Trypanosoma cruzi, the etiological agent of Chagas' disease, arginine kinase activity increased continuously during the exponential phase of growth. A correlation between growth rate, enzyme-specific activity and enzyme protein was observed. Arginine kinase-specific activity, expressed as a function of enzyme protein, remains roughly constant up to 18 days of culture. In the whole range of the culture time mRNA levels showed minor changes indicating that the enzyme activity is post-transcriptionally regulated. Arginine kinase could be proposed as a modulator of energetic reserves under starvation stress condition.

MRNA encoding a putative RNA helicase of the DEAD-box gene family is up-regulated in trypomastigotes of Trypanosoma cruzi.

Differential display of mRNAs from Trypanosoma cruzi epimastigote and metacyclic trypomastigote stages showed several mRNA species differing in their expression level. The cDNA corresponding to one of these mRNAs was used as a probe in Northern blots and identified a RNA product of 2.6 kb with an expression level eight or more times higher in trypomastigotes than in epimastigotes. This probe was also used to screen a genomic library of T. cruzi CL Brener clone prepared in lambda FIX. A clone of about 15 kb was selected that, after partial sequencing, revealed an open reading frame of 688 amino acids encoding a deduced protein with similarity to RNA helicases of the DEAD-box gene family. The presence of the eight conserved motifs characteristic of the DEAD protein family was observed in the T. cruzi sequence, indicating that it corresponds to a putative RNA helicase gene, which we named HelTc. Southern blot analysis indicated that HelTc is a single-copy gene. Pulsed-field gel electrophoresis separation of chromosomes of several isolates of T. cruzi showed that this gene was localized in one or two chromosomal bands.

Immuno and cytochemical localization of Trypanosoma cruzi nitric oxide synthase

The localization and subcellular distribution of Trypanosoma cruzi nitric oxide synthase was investigated in epimastigote cells by immunocytochemistry at electron and light microscope level, using a polyclonal antibody to neuronal nitric oxide synthase, and also, at light microscope level, by the nicotinamide adenine dinucleotide phosphate-diaphorase histochemical reaction. The immunoreactivity was ultrastructurally localized by electron microscopy in the inner surface of cell membranes and in free cytosolic clusters in the body, flagellum and apical extreme. Light microscopy showed that immunoprecipitates, specific for the Trypanosoma cruzi nitric oxide synthase, co-localized with the formazan precipitates generated by the diaphorase reaction in the same areas identified by electron microscopy. These results, taken together with previous finding from our laboratory could help to explain the involvement of the nitric oxide transduction pathway in T. cruzi epimastigote motility.

Trypanosoma cruzi arginine kinase characterization and cloning. A novel energetic pathway in protozoan parasites.

This work contains the first description of a guanidino kinase in a flagellar unicellular parasite. The enzyme phosphorylates L-arginine and was characterized in preparations from Trypanosoma cruzi, the ethiological agent of Chagas' disease. The activity requires ATP and a divalent cation. Under standard assay conditions (1 mM L-arginine), the presence of 5-fold higher concentrations of canavanine or histidine produced a greater than 50% enzyme inhibition. The base sequence of this enzyme revealed an open reading frame of 357 amino acids and a molecular weight of 40,201. The amino acid sequence shows all of the characteristic consensus blocks of the ATP:guanidino phosphotransferase family and a putative "actinin-type" actin-binding domain. The highest amino acid identities of the T. cruzi sequence, about 70%, were with arginine kinases from Arthropoda. Southern and chromosome blots revealed that the kinase is encoded by a single-copy gene. Moreover, Northern blot analysis showed an mRNA subpopulation of about 2.0 kilobases, and Western blotting of T. cruzi-soluble polypeptides revealed a 40-kDa band. The finding in the parasite of a phosphagen and its biosynthetic pathway, which are totally different from those in mammalian host tissues, points out this arginine kinase as a possible chemotherapy target for Chagas' disease.

Immuno and cytochemical localization of Trypanosoma cruzi nitric oxide synthase.

The localization and subcellular distribution of Trypanosoma cruzi nitric oxide synthase was investigated in epimastigote cells by immunocytochemistry at electron and light microscope level, using a polyclonal antibody to neuronal nitric oxide synthase, and also, at light microscope level, by the nicotinamide adenine dinucleotide phosphate-diaphorase histochemical reaction. The immunoreactivity was ultrastructurally localized by electron microscopy in the inner surface of cell membranes and in free cytosolic clusters in the body, flagellum and apical extreme. Light microscopy showed that immunoprecipitates, specific for the Trypanosoma cruzi nitric oxide synthase, co-localized with the formazan precipitates generated by the diaphorase reaction in the same areas identified by electron microscopy. These results, taken together with previous finding from our laboratory could help to explain the involvement of the nitric oxide transduction pathway in T. cruzi epimastigote motility.

L-arginine uptake and L-phosphoarginine synthesis in Trypanosoma cruzi.

A very specific L-arginine transporter showing high affinity has been characterized in Trypanosoma cruzi epimastigotes. Uptake was found to be dependent on L-arginine concentration and it was saturable. Values for maximum velocity and Km ranged between 48.1-57.5 pmol.min-1 per 3 x 10(7) cells and between 4.2-5.5 microM, respectively. The calculated activation energy and Q10 were 31.1 KJ.mol-1, and 1.7, respectively. Uptake velocity significantly increased when cells were preincubated in the absence of L-arginine. Cells retained the labeled amino acid independently of the presence or absence of exogenous L-arginine. The specificity of L-arginine uptake was demonstrated by competition assays in the presence of 80-fold molar excess of natural amino acids and several L-arginine derivatives. The highest levels of inhibition were caused by L-homoarginine, D-arginine, L-canavanine, L-ornithine, and L-citrulline. L-arginine uptake by T. cruzi epimastigotes was not affected by the presence of potassium or sodium ions in the incubation mixture or by pH changes in the range between 5.5-8.5. The major product of L-arginine uptake was characterized as phosphoarginine. Moreover, arginine kinase activity was detected in soluble extracts from T. cruzi epimastigotes.

Factors from Trypanosoma cruzi interacting with AP-1 sequences.

Interaction between factors from Trypanosoma cruzi extracts and AP-1 sequences was studied by electrophoretic mobility shift assays. Using a double-stranded probe carrying the AP-1 sequence from the SV40 promoter, three specific complexes designated A, B, and C were detected. Complexes A and C were formed when using single-stranded probes. The relative amount of complex B, specific for double-stranded DNA, increased as a function of probe length. Complexes were stabilized by cross-linking with UVC irradiation and resolved on denaturing SDS-PAGE. Complex A generated bands of 60- and 39 kDa; complex B produced two bands of 46- and 43 kDa; and complex C generated one band of 43 kDa. The AP-1 binding activity was much higher in purified nuclear preparations than in soluble fractions, and was detected in crude extracts from the three forms of the parasite. The binding signal, however, was much stronger in amastigote and trypomastigote than in the epimastigote forms. Specific binding was increased by oxidative stress. Antibodies raised against peptides corresponding to conserved domains of mammalian c-Jun and c-Fos detected bands of 40- and 60 kDa, respectively, in a nuclear epimastigote preparation.

Partial characterization of guanylyl cyclase activity in calf thyroid.

The aim of the present study was to perform the partial characterization of the enzyme guanylyl cyclase (GC) in bovine thyroid. The results obtained showed the presence of two types of GC: one is soluble and comprises around 79% of total activity, while the other is particulate. Treatment with 1% Triton X-100 increased both activities. When the kinetics of the enzyme was analyzed, using the complex MnGTP as a substrate, the results showed a Michaelis type kinetics for the soluble enzyme, with a Km of 0.037 mM, whereas the particulate GC showed a positive allosteric behavior with a S0.5 of 0.214 mM and a Hill coefficient of 1.9, indicating that the enzyme has at least two binding sites for the substrate. When the influence of different Mn2+ concentrations was studied, a positive allosteric behavior for the soluble GC was found, with a S0.5 of 1.2 mM and a Hill coefficient of 2.2. The kinetics of the particulate enzyme under similar conditions was of Michaelis type, with a Km of 0.752 mM. Although the enzyme is highly dependent on Mn2+, it was of interest to investigate the possible effects of other divalent cations, such as Ca2+ and Mg2+. The replacement of Mn2+ for Mg2+ caused a complete disappearance of the particulate enzyme activity, while the soluble activity decreased by 85%. Addition of Ca2+ had no effect on either GC. However, with suboptimum. concentrations of Mn2+, high Ca2+ concentration caused an increase in soluble activity, but it comprised only 20% of maximum activity with optimum Mn2+ concentrations. With the particulate enzyme a slight but significant inhibition was observed.

Entamoeba histolytica: signaling through G proteins.

The intracellular signaling pathways of Entamoeba histolytica are largely unknown. Although the expression of guanine nucleotide binding proteins (G proteins) is expected from functional studies, their biochemical characterization remains elusive in this protozoan. Using a combination of biochemical and immunological studies, we provide strong evidence for the presence of a Gs protein in amoeba. Our results strengthen our understanding of the signal transduction mechanisms in E. histolytica as potential sites of a new therapeutic strategy.

Control of Trypanosoma cruzi epimastigote motility through the nitric oxide pathway.

Trypanosoma cruzi epimastigote motility can be enhanced by addition of L-arginine, to the culture. This effect is blocked by N-methyl-L-arginine, a competitive inhibitor of the nitric oxide synthase. N-methyl-D-aspartate and L-glutamate, two agonists of the NMDA/L-glutamate receptor, also enhanced motility. This stimulation is blocked by MK-801 a noncompetitive antagonist of the NMDA receptor. In addition, sodium nitroprusside, a guanylyl cyclase stimulator and 8-Br-cyclic GMP, and analog of cyclic GMP, also stimulated epimastigote motility. It is suggested that an increase of intracellular cyclic GMP levels mediated by nitric oxide may be responsible for the increase in epimastigote motility.

Signal transduction mechanisms in Trypanosoma cruzi.

Trypanosoma cruzi, the etiological agent of Chagas disease, is an adequate model for studies on the evolution of signal transduction pathways. These pathways involve molecular entities such as membrane receptors, transduction G proteins, protein kinases and second messengers (Ca(2+), cyclic AMP, cyclic GMP, nitric oxide). In this article, Mirtha M. Flawiá, María T. Téllez-Iñón and Héctor N. Torres describe the studies performed on T. cruzi transduction pathways and their role in the control of metacyclogenesis and cell motility.

Induction of Trypanosoma cruzi metacyclogenesis in the gut of the hematophagous insect vector, Rhodnius prolixus, by hemoglobin and peptides carrying alpha D-globin sequences.

Trypanosoma cruzi, a protozoan responsible for the American trypanosomiasis (Chagas disease), multiplies and differentiates in the gut of triatomine insect vectors. The effects of hemoglobin and synthetic peptides carrying alpha D-globin fragments on both the growth and the transformation of T. cruzi epimastigotes (noninfective) into metacyclic trypmastigotes (infective forms) were studied. This differentiation in the insect's gut is expressed when hemoglobin and synthetic peptides corresponding to residues 30-49 and 35-73 of the alpha D-globin were added to the plasma diet. However, synthetic peptide 41-73 does not induce differentiation of epimastigotes even in the presence of the two former synthetic peptides. Thus, these data delineate an unusual molecular mechanism which modulates the dynamics of transformation of epimastigotes into metacyclic trypomastigotes in the triatomine vector's gut.

The nitric oxide transduction pathway in Trypanosoma cruzi.

A nitric oxide synthase was partially purified from soluble extracts of Trypanosoma cruzi epimastigote forms. The conversion of L-arginine to citrulline by this enzyme activity required NADPH and was blocked by EGTA. The reaction was activated by Ca2+, calmodulin, tetrahydrobiopterin, and FAD, and inhibited by N omega-methyl-L-arginine. L-Glutamate and N-methyl-D-aspartate stimulated in vivo conversion of L-arginine to citrulline by epimastigote cells. These stimulations could be blocked by EGTA, MK-801, and ketamine and enhanced by glycine. A sodium nitroprusside-activated guanylyl cyclase activity was detected in cell-free, soluble preparations of T. cruzi epimastigotes. L-Glutamate, N-methyl-D-aspartate, and sodium nitroprusside increased epimastigote cyclic GMP levels. MK-801 bound specifically to T. cruzi epimastigote cells. This binding was competed by ketamine and enhanced by glycine or L-serine. Evidence thus indicates that in T. cruzi epimastigotes, L-glutamate controls cyclic GMP levels through a pathway mediated by nitric oxide.

Adenylyl cyclase and G-proteins in Phytomonas.

Phytomonas sp. membranes have an adenylyl cyclase activity which is greater in the presence of Mn2+ than with Mg2+. The Mg2+ and Mn2+ activity ratio varies from one membrane preparation to another, suggesting that the adenylyl cyclase has a variable activation state. A[35S]GTP-gamma-S-binding activity with a Kd of 171 nM was detected in Phytomonas membranes. Incubation of these membranes with activated cholera or pertussis toxin and [adenylate 23P]NAD+ led to incorporation of radioactivity into bands of about 40-44 kDa. Crude membranes were electrophoresed on SDS-polyacrylamide gels and analyzed, by Western blotting, with the 9188 anti-alpha[s] antibody and the AS/7 antibody (anti-alpha[i], anti-alpha[i1], and anti-alpha[i2]. These procedures resulted in the identification of polypeptides of approximately 40-44 kDa. Phytomonas adenylyl cyclase could be activated by treatment of membrane preparations with cholera toxin, in the presence of NAD+, while similar treatment with pertussis toxin did not affect this enzyme activity. These studies indicate that in Phytomonas, adenylyl cyclase activity is coupled to an unknown receptor entity through G alpha[s] proteins.

Stimulation of Trypanosoma cruzi adenylyl cyclase by an alpha D-globin fragment from Triatoma hindgut: effect on differentiation of epimastigote to trypomastigote forms.

A peptide from hindguts of the Triatoma hematophagous Chagas insect vector activates adenylyl cyclase activity in Trypanosoma cruzi epimastigote membranes and stimulates the in vitro differentiation of epimastigotes to metacyclic trypomastigotes. Hindguts were obtained from insects fed 2 days earlier with chicken blood. Purification was performed by gel filtration and HPLC on C18 and C4 columns. SDS/PAGE of the purified peptide showed a single band of about 10 kDa. The following sequence was determined for the 20 amino-terminal residues of this peptide: H2N-Met-Leu-Thr-Ala-Glu-Asp-Lys-Lys-Leu-Ile-Gln- Gln-Ala-Trp-Glu-Lys-Ala-Ala-Ser-His. This sequence is identical to the amino terminus of chicken alpha D-globin. On a Western blot, the peptide immunoreacted with a polyclonal antibody against chicken globin D. A synthetic peptide corresponding to residues 1-40 of the alpha D-globin amino terminus also stimulated adenylyl cyclase activity and promoted differentiation. This 125I-labeled synthetic peptide bound specifically to T. cruzi epimastigote cells. Activation of epimastigote adenylyl cyclase by the hemoglobin-derived peptide may play an important role in T. cruzi differentiation and consequently in the transmission of Chagas disease.

G-protein from Medicago sativa: functional association to photoreceptors.

G-protein subunits were characterized from Medicago sativa (alfalfa) seedlings. Crude membranes and GTP-Sepharose-purified fractions were electrophoresed on SDS/polyacrylamide gels and analysed by Western blotting with 9193 (anti-alpha common) and AS/7 (anti-alpha t, anti-alpha i1 and anti-alpha i2) polyclonal antibodies. These procedures led to the identification of a specific polypeptide band of about 43 kDa. Another polypeptide reacting with the SW/1 (anti-beta) antibody, of about 37 kDa, was also detected. The 43 kDa polypeptide bound specifically [alpha-32P]GTP by a photoaffinity reaction and was ADP-ribosylated by activated cholera toxin, but not by pertussis toxin. Irradiation of etiolated Medicago sativa protoplast preparations at 660 nm for 1 min produced a maximal increase in the guanosine 5'-[gamma-thio]triphosphate (GTP[35S])-binding rate. After this period of irradiation, the binding rate tended to decrease. The effect of a red-light (660 nm) pulse on the binding rate was reversed when it was immediately followed by a period of far-red (> 730 nm) illumination. These results may suggest that activation of GTP[S]-binding rate was a consequence of conversion of phytochrome Pr into the Ptr form.

An alpha D-globin fragment from Triatoma infestans hindgut stimulates Trypanosoma cruzi adenylyl cyclase and promotes metacyclogenesis.

A peptide from hindguts of the Triatoma infestans, the hematophagous Chagas' insect vector, activates adenylyl cyclase activity in Trypanosoma cruzi epimastigote membranes and stimulates the in vitro differentiation of epimastigotes (proliferative and non-infectious forms) to metacyclic trypomastigotes (non-proliferative and infectious forms). The peptide was purified from hindguts of insects fed two days before with chicken blood. After purification, the peptide showed upon SDS-PAGE a single band of about 10 kDa. The sequence for 20 residues of the amino terminus of this peptide was: H2N-Met-Leu-Thr-Ala-Glu-Asp-Lys-Lys-Leu-Ile-Gln-Gln-Ala-Trp-Glu-Lys-Ala- Ala-Ser-His. This sequence corresponds to the amino terminus of chicken alpha D-globin. A synthetic peptide with the sequence of the 40 amino acids corresponding to the amino terminus of alpha D-globin, also stimulated T. cruzi adenylyl cyclase activity and promoted metacyclogenesis.

Isolation of adenylate cyclase mutants from Rhizobium meliloti deficient in nodulation.

Six Rhizobium meliloti mutants were isolated after Tn5-mediated mutagenesis as resistant to inhibition by a mixture of amino acids (serine, methionine, glycine and leucine). All were defective in adenylate cyclase activity and failed to form nodules in infected roots of Medicago sativa. Furthermore, like other nodulation mutants, they showed altered motility and increased secretion of exopolysaccharides; addition of cAMP to the growth medium abolished some of these phenotypic defects. The possibility that adenylate cyclase participates in the transduction of signals inducing nodulation is discussed.