Validation of an in vitro system to trigger changes in the gene expression of effectors of Sclerotinia sclerotiorum.

AIMS: Sclerotinia sclerotiorum, the causal agent of white mold, can infect several host species, including economically important crops. In this study, we propose and validate a new in vitro system able to mimic the conditions of interaction with the host and promote the induction of S. sclerotiorum effectors. METHODS AND RESULTS: For culture media production, we selected three plant species, common bean (Phaseolus vulgaris L, cv. Requinte.), maize (Zea mays, cv. BRS1030) and beggarticks (Bidens pilosa). To validate this system as an in vitro inducer of effectors, the qRT-PCR technique was used to investigate the expression profile of some S. sclerotiorum effector genes in each growth medium at different times after inoculation. CONCLUSION: The results obtained in this study provide a validation of a new method to study S. sclerotiorum during mimetic interaction with different hosts. Although leaf extract does not fully represent the plant environment, the presence of plant components in the culture medium seems to induce effector genes, mimicking in planta conditions. The use of MEVM is simpler than in planta growth, bypasses problems such as the amount of mycelium produced, as well as contamination of host cells during transcriptomic and proteomic analyses. SIGNIFICANCE AND IMPACT OF THE STUDY: We have devised MEVM media as a model mimicking the interaction of S. sclerotiorum and its hosts and used it to evaluate in vitro expression of effectors normally expressed only in planta.

In silico characterization of 1,2-diacylglycerol cholinephosphotransferase and lysophospha-tidylcholine acyltransferase genes in Glycine max L. Merrill.

The enzymes 1,2-diacylglycerol cholinephosphotrans-ferase (CPT) and lysophosphatidylcholine acyltransferase (LPCAT) are important in lipid metabolism in soybean seeds. Thus, understand-ing the genes that encode these enzymes may enable their modification and aid the improvement of soybean oil quality. In soybean, the genes encoding these enzymes have not been completely described; there-fore, this study aimed to identify, characterize, and analyze the in silico expression of these genes in soybean. We identified two gene models encoding CPT and two gene models encoding LPCAT, one of which presented an alternative transcript. The sequences were positioned on the physical map of soybean and the promoter regions were analyzed. Cis-elements responsible for seed-specific expression and responses to biotic and abiotic stresses were identified. Virtual expression analysis of the gene models for CPT and LPCAT indicated that these genes are expressed under different stress conditions, in somatic embryos during differentiation, in immature seeds, root tissues, and calli. Putative ami-no acid sequences revealed the presence of transmembrane domains, and analysis of the cellular localization of these enzymes revealed they are located in the endoplasmic reticulum.

Morphoagronomic characterization and genetic diversity of a common bean RIL mapping population derived from the cross Rudá x AND 277.

Recombinant inbred lines (RILs) are a valuable resource for building genetic linkage maps. The presence of genetic variability in the RILs is essential for detecting associations between molecular markers and loci controlling agronomic traits of interest. The main goal of this study was to quantify the genetic diversity of a common bean RIL population derived from a cross between Rudá (Mesoamerican gene pool) and AND 277 (Andean gene pool). This population was developed by the single seed descent method from 500 F2 plants until the F10 generation. Seven quantitative traits were evaluated in the field in 393 RILs, the parental lines, and five control cultivars. The plants were grown using a randomized block design with additional controls and three replicates. Significant differences were observed among the RILs for all evaluated traits (P < 0.01). A comparison of the RILs and parental lines showed significant differences (P < 0.01) for the number of days to flowering (DFL) and to harvest (DH), productivity (PROD) and mass of 100 beans (M100); however, there were no significant differences for plant architecture, degree of seed flatness, or seed shape. These results indicate the occurrence of additive x additive epistatic interactions for DFL, DH, PROD, and M100. The 393 RILs were shown to fall into 10 clusters using Tocher's method. This RIL population clearly contained genetic variability for the evaluated traits, and this variability will be crucial for future studies involving genetic mapping and quantitative trait locus identification and analysis.

Soybean rust resistance sources and inheritance in the common bean (Phaseolus vulgaris L.).

Soybean rust (SBR), caused by the fungus Phakopsora pachyrhizi, has been reported in common bean (Phaseolus vulgaris L.) cultivars and elite lines that were infected under controlled and natural field conditions in South Africa, the United States, Argentina, and Brazil. Although SBR is currently not a top priority problem for the common bean crop, many bean breeders are concerned about this disease because of the high severity and virulence diversity of P. pachyrhizi and its broad host range. In this study, a set of 44 P. vulgaris genotypes were tested for resistance to P. pachyrhizi; these genotypes included resistance sources to several fungal common bean diseases, carioca-, black- and red-seeded Brazilian cultivars, and elite lines that were developed by the main common bean breeding programs in Brazil. Twenty-four SBR resistance sources were identified. They presented the reddish-brown (RB) lesion type, characterizing resistance reactions. In addition to the RB lesion type, the PI181996 line presented the lowest disease severity mean score, considering its associated standard error value. For this reason, it was crossed with susceptible lines to study the inheritance of resistance. The results support the hypothesis that resistance to SBR in PI181996 is monogenic and dominant. We propose that this SBR resistance gene, the first to be identified and characterized in common bean, might be designated as Pkp-1.

Development of a SCAR marker for the analysis of B chromosome presence in Partamona helleri (Hymenoptera, Apidae).

Chromosomes in hymenopteran insects cannot currently be analysed in adult individuals. The only available cytogenetic techniques need to be performed in larvae. Here we develop and implement a SCAR (Sequence Characterized Amplified Region) marker, associated with B chromosomes in the bee Partamona helleri, which has proven to be very useful to reveal B chromosome presence in adults from natural populations. The marker was tested in ten different colonies simultaneously analysed by both molecular (ten adults per colony) and cytogenetic (20 larvae per colony) techniques. The presence of the SCAR marker always showed the same pattern as B chromosome presence: both were present or absent in all individuals from a same colony, or both were present in only part of the individuals from a same colony. This molecular marker is thus a useful tool for analysing new aspects of this B chromosome system such as B frequency and geographical distribution, B transmission, or B effects in adult individuals.

A RAPD marker associated with B chromosomes in Partamona helleri (Hymenoptera, Apidae).

The hymenopteran Partamona helleri is found in southwestern Brazil in the Mata Atlântica from the north of the state of Santa Catarina until the south of Bahia. This work shows that P. helleri can carry up to four B chromosomes per individual. In order to obtain more information about P. helleri B chromosomes, the RAPD technique was used to detect DNA fragments associated with these chromosomes. The results showed that the RAPD technique is useful to detect specific sequences associated with B chromosomes. One RAPD marker was identified, cloned and used as probe in a DNA blot analysis. This RAPD marker hybridized with sequences present only in individuals containing B chromosomes.

Characterization of alpha-galactosidases from germinating soybean seed and their use for hydrolysis of oligosaccharides.

Raffinose oligosaccharides (RO) are the major factors responsible for flatulence following ingestion of soybean derived products. Removal of RO from seeds or soymilk would then have a positive impact on the acceptance of soy-based foods. Enzymic hydrolysis of the RO is accomplished by alpha-galactosidase. While the content of RO decreases during seed germination, the activity of alpha-galactosidase increases substantially. Two alpha-galactosidases were isolated from germinating seeds by partition in an aqueous two-phase system followed by ion-exchange and affinity chromatography. One of the enzyme preparations (P1) showed a single protein with M(r) of 33 kDa, and the second (P2) had two proteins with M(r) of 31 and 33 kDa. Maximal activities against the synthetic substrate rho-nitrophenyl-alpha-D-galactopyranoside (rhoNPGal) were detected at pH 5.0-5.5 and 45-50 degrees C. Both enzymes were fairly stable at 40 degrees C, but lost most of their activities after 30 min at 50 degrees C. The K(m) values for hydrolysis of rhoNPGal by the P1 and P2 enzymes were 1.55 and 0.76 mM, respectively. The K(m) values determined for hydrolysis of raffinose and melibiose by the P2 enzyme were 5.53 and 5.34 mM, respectively and galactose was a competitive inhibitor (K(i)=0.65 mM). To different extents, both enzymes were sensitive to inhibition by galactose, melibiose, CuSO(4), and SDS. Sucrose and beta-mercaptoethanol showed discrete inhibitory effects on both enzymes.

Improved Selection with Newly Identified RAPD Markers Linked to Resistance Gene to Four Pathotypes of Colletotrichum lindemuthianum in Common Bean.

ABSTRACT Three F(2) populations derived from crosses between the resistant cultivar AB 136 and the susceptible cultivar Michelite (MiA), and one F(2) population derived from a cross between AB 136 and Mexico 222 (MeA), were used to identify markers linked to anthracnose resistance genes present in cultivar AB 136. Primer OPZ04 produced a DNA band (OPZ04(560)) linked in coupling phase to the resistance gene for pathotype 89 (8.5 +/- 0.025 cM) in one population derived from the cross MiA. In the same population, primer OPZ09 produced one band (OPZ09(950)) linked in repulsion phase (20.4 +/- 0.014 cM) to the same resistance gene. The simultaneous use of markers in coupling and in repulsion phases allowed the identification of the three genotypic classes. In the other two populations from cross MiA, OPZ04(560) was linked in coupling phase to resistance genes for pathotypes 73 (2.9 +/- 0.012 cM) and 81 (2.8 +/- 0.017 cM). In population MeA, OPZ04(560) was linked in coupling phase (7.5 +/- 0.033 cM) to resistance to pathotype 64. These data suggest that a single gene or complex locus of linked resistance genes present in cultivar AB 136 confers resistance to all four pathotypes of C. lindemuthianum.

Identification of Random Amplified Polymorphic DNA Markers Linked to the Co-4 Resistance Gene to Colletotrichum lindemuthianum in Common Bean.

ABSTRACT New cultivars of the common bean (Phaseolus vulgaris) with durable resistance to anthracnose can be developed by pyramiding major resistance genes using marker-assisted selection. To this end, it is necessary to identify sources of resistance and molecular markers tightly linked to the resistance genes. The objectives of this work were to study the inheritance of resistance to anthracnose in the cultivar TO (carrying the Co-4 gene), to identify random amplified polymorphic DNA (RAPD) markers linked to Co-4, and to introgress this gene in the cultivar Rudá. Populations F(1), F(2), F(2:3), BC(1)s, and BC(1)r from the cross Rudá x TO were inoculated with race 65 of Colletotrichum lindemuthianum, causal agent of bean anthracnose. The phenotypic ratios (resistant/susceptible) were 3:1 in the F(2) population, 1:1 in the BC(1)s, and 1:0 in the BC(1)r, confirming that resistance to anthracnose in the cultivar TO was monogenic and dominant. Six RAPD markers linked to the Co-4 gene were identified, four in the coupling phase: OPY20(830C) (0.0 centimorgan [cM]), OPC08(900C) (9.7 cM), OPI16(850C) (14.3 cM), and OPJ01(1,380C) (18.1 cM); and two in the repulsion phase: OPB03(1,800T) (3.7 cM) and OPA18(830T) (17.4 cM). OPY20(830C) and OPB03(1,800T), used in association as a codominant pair, allowed the identification of the three genotypic classes with a high degree of confidence. Marker OPY20(830C), which is tightly linked to Co-4, is being used to assist in breeding for resistance to anthracnose.

Urinary protein/creatinine ratio in hypertensive pregnant women.

OBJECTIVES: To determine the correlation between the protein/creatinine ratio and 24-h proteinuria; to estimate the sensitivity and specificity of this ratio for the diagnosis of significant proteinuria; to establish its cutoff point with the best predictive value for the diagnosis of significant proteinuria in patients with systemic arterial hypertension. STUDY DESIGN: A cross-sectional study of 47 hypertensive patients who had been pregnant for 20 weeks or more seen at the Maternity of the University Hospital of Porto Alegre. The studied factor was the protein/creatinine ratio measured in a single random urine sample and the outcome was protein determination in 24-h urine. The level of significance was set at 0.05. RESULTS: The correlation coefficient between the protein/creatinine ratio and 24-h proteinuria was 0.94 when urine was properly collected. A receiver-operator characteristic curve was constructed to determine the sensitivity and specificity of the ratio for the diagnosis of significant proteinuria (> or = 300 mg in 24 h). Specificity and predictive positive value were 100% for a ratio > or = 0.8. The best values for sensitivity, specificity, positive predictive value, and negative predictive value in the diagnosis of proteinuria > or = 300 mg in 24 h were obtained when the protein/creatinine ratio was 0.5 (0.96, 0.96, 0.96, and 0.96, respectively). CONCLUSION: The protein/creatinine ratio measured in a single urine sample taken at random from hypertensive pregnant women showed good sensitivity and specificity for the diagnosis of 24-h proteinuria > or = 300 mg and was strongly correlated with 24-h proteinuria. A ratio of 0.5 mg/mg is predictive of significant proteinuria and can be used for the diagnosis and follow-up of hypertensive pregnant women.

Biochemical profile of amniotic fluid for the assessment of fetal and renal development.

Creatinine plays a key role in the function and maturation of fetal kidneys throughout pregnancy. It is important to identify other markers that may help in the diagnosis of renal dysfunction. Our aim was to determine the profile of and the correlation between biochemical markers to be used to assess renal function and maturation of the fetus in the amniotic fluid during pregnancy and to determine the distribution of normal values for creatinine, N-acetyl-beta-D-glucosaminidase (NAG), beta2-microglobulin, glucose, urea, sodium, potassium, phosphorus, calcium, uric acid, albumin, and osmolality in three gestational age groups. This was a cross-section study that assessed 115 samples of amniotic fluid during three different periods of pregnancy, i.e., 13 to 20, 27 to 34, and 36 to 42 weeks. Concentrations of creatinine, NAG, urea, potassium and uric acid increased during pregnancy (P<0.05). Beta2-microglobulin, glucose, sodium, phosphorus, calcium, and albumin concentration and osmolality decreased (P<0.05), whereas beta2-microglobulin, glucose and uric acid presented significant correlations with gestational age and creatinine, respectively (r>0.6, P<0.05). Urea, potassium and phosphorus showed mild correlations with both (r>0.5, P<0.05). NAG, sodium, albumin and osmolality did not show significant correlations (r<0.5, P<0.05). These tests confirmed the important role of creatinine in terms of correlation with gestational age. beta2-Microglobulin, glucose and uric acid were significant as markers of function and maturation of fetal kidneys, whereas NAG did not demonstrate a useful role for the assessment of renal maturation.

The role of colchicine in prevention of hepatic cirrhosis induced by carbon tetrachloride.

BACKGROUND/AIMS: The present study was undertaken to determine whether colchicine has a beneficial effect in the prevention of hepatic cirrhosis when it is given simultaneously with CCl4. METHODOLOGY: Wistar rats were employed as experimental animals and divided into 6 groups: Group I received saline solution, Group II, saline solution and mineral oil; Group III, colchicine (10 micrograms/100 g) and mineral oil; Group IV, colchicine (10 micrograms/100 g) and CCl4; Group V, colchicine (5 micrograms/100 g) and CCl4; and, Group VI received saline solution and CCl4. The effect of colchicine was evaluated by liver function tests, serum total proteins, electrolytes and histological evaluation. RESULTS: The results demonstrated higher values of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and total bilirubin in groups IV and V when compared with group VI (p < 0.05). No difference between group VI and groups IV and V was observed in histological evaluation, serum total proteins and electrolytes (p < 0.05). CONCLUSIONS: Colchicine, as given in this study, did not have any protective effect in the prevention of cirrhosis induced by carbon tetrachloride.

Identification of Races of Colletotrichum lindemuthianum with the Aid of PCR-Based Molecular Markers.

Inoculation of a common bean differential series is the usual method for identification of races of Colletotrichum lindemuthianum. This procedure is extremely useful for phytopathological as well as breeding purposes, but it requires strict control of the number of spores and incubation conditions. Furthermore, this method may result in misclassifications of isolates because of the subjectivity of symptom evaluation. We propose the use of DNA-based molecular markers as an auxiliary tool to aid the classification of races of C. lindemuthianum. Specific DNA bands were identified for races 73, 65, and 64 by polymerase chain reaction (PCR) amplification of bulked DNA samples from isolates of these three races with random primers. The presence of these bands was checked on four isolates previously classified by inoculation on a differential series as belonging to races 23, 72, 79, and 585. The molecular procedure showed that two of these isolates had been misclassified, confirming the high potential of the proposed procedure to aid the identification of races of C. lindemuthianum. Amplification products obtained with 44 different primers also allowed the determination of the genetic distances among isolates from races 73, 65, and 64. These data were used to cluster the isolates into three groups that coincide with the ones obtained by inoculation.

Genetic analysis of Melipona quadrifasciata LEP. (Hymenoptera: Apidae, Meliponinae) with RAPD markers.

Melipona quadrifasciata ("mandaçaia") can be subdivided into two subspecies: M. q. anthidioides and M. q. quadrifasciata. In the present study we used RAPD markers to estimate intercolonial genetic variation among 69 colonies of Melipona quadrifasciata. Ten workers per colony were analyzed. The intercolony genetic distances based on RAPD markers ranged from 29.5% (colonies collected in the State of São Paulo vs colonies from the State of Minas Gerais) to 34.2% (São Paulo vs Santa Catarina). These results indicate a high genetic similarity among the colonies analyzed. According to the genetic distances two different groups could be distinguished. The first containing the samples from Santa Catarina region and the second, samples from Paraná, São Paulo, Minas Gerais, and Espírito Santo. Based on the molecular analysis, bees belonging to the different subspecies M. q. quadrifasciata (from Santa Catarina) and M. q. anthidiodes (from the other regions) were distinguished.

Purification and characterization of zein-degrading proteases from endosperm of germinating maize seeds.

We have purified a group of four proteases (molecular mass 26-33 kilodalton) from germinating seeds of maize (Zea mays L. var W64A) using ammonium sulfate and isoelectric precipitations, anion exchange chromatography, and electroelution from preparative nondenaturing polyacrylamide gels. Their appearance in the endosperm of germinating seeds coincides with the onset of zein degradation. We have shown that these proteases degrade zeins dissolved in alcoholic solution as well as aggregated in protein bodies from developing maize kernels. The apparent molecular weights and net negative charges of each of these proteases are very similar. Additionally, they are inhibited by thiol-blocking agents and activated by reducing compounds. These characteristics suggest that they are a group of cysteine proteases involved in the first steps of storage protein degradation.

Fermentation of starch by Klebsiella oxytoca p2, containing plasmids with alpha-amylase and pullulanase genes.

Klebsiella oxytoca P2(pC46), an ethanol-producing recombinant, has been evaluated in fermentation of maltose and starch. The maximum ethanol produced by P2(pC46) was 0.34 g ethanol/g maltose and 0.38, 0.40, or 0.36 g ethanol/g starch in fermentation of 1, 2, or 4% starch, representing 68, 71, and 64% the theoretical yield. The pC46 plasmid transformed to cells of K. oxytoca P2 reduced the ethanol production from maltose and starch. In fermentation of starch after its digestion at 60 degrees C for 24 h, in two-step fermentation, the time for maximum ethanol production was reduced to 12-24 h and the theoretical yield was around 90%. The increase in starch concentration resulted in lower alpha-amylase activity but in higher pullulanase activity. The high activity and thermostability of the amylolytic enzymes from this transformant suggest that it has a potential for amylolytic enzymes source.

Genetic diversity of the ectomycorrhizal fungus Pisolithus tinctorius based on RAPD-PCR analysis.

Twenty Pisolithus tinctorius isolates from different geographic locations and different hosts were characterized by the random amplified polymorphic DNA technique. Thirteen arbitrary primers generated 87 DNA fragments, all of them polymorphic. These data were used to calculate genetic distances among the isolates. The pairwise genetic distances ranged from 1 to 100%, with an average of 58.7%. Cluster analysis based on the amplified fragments grouped the isolates according to their host and geographical origins. Group I contained isolates collected in Brazil and group II those collected in the Northern Hemisphere. In addition to the diversity seen at the molecular level, the isolates also showed host specificity. Greenhouse experiments demonstrated that isolates from the Northern Hemisphere colonized mainly Pinus whereas isolates from Brazil colonized only Eucalyptus. The molecular data suggest that the Pisolithus tinctorius isolates analyzed belong to two distinct groups. The data also suggest new guidelines for future investigations on the taxonomy and systematic of this important fungus species. Furthermore, these results support future experiments aimed at the selection and development of improved isolates of P. tinctorius.

Fructose-1,6-diphosphate: potential protection in cyclosporine-induced renal impairment.

There is evidence that fructose-1,6-diphosphate (FDP) provides protection from hepatic and cardiac toxic-induced damage and ischemic renal insult. To determine if FDP also protects against cyclosporine (CsA)-induced nephrotoxicity, two groups of adult male Wistar rats were studied for whole kidney clearance rates. After two initial control periods, group 1 received only CsA (CsA, n = 8). Group 2 received FDP 350 mg/kg, followed by CsA 50 mg/kg (FDP-CsA, n = 6). In both groups, after a 30-min equilibration period, two additional clearance rates were measured (Post 1 and Post 2). A significant reduction in clearance rates was observed after drug infusion in both groups (approximately 58 and 64% in CsA and FDP-CsA groups, respectively, p < 0.05) with a recovery to control values in the Post 2 period in the FDP-CsA group. These data suggest a protective effect of FDP on CsA-induced renal impairment.

Inheritance pattern of RAPD markers in Melipona quadrifasciata (Hymenoptera: Apidae, Meliponinae).

Melipona quadrifasciata is an important pollinator agent in several regions of Brazil. Data concerning the genetics of this species are scarce in the literature. In this work we used the random amplified polymorphic DNA (RAPD) technique to determine the degree of polymorphism and the inheritance pattern of these molecular markers in this species. Our ultimate goal is to establish tools to be used in the study of the genomic organization of M. quadrifasciata. Genomic DNA from progenies F(1) and BC(1) were assayed with 79 different primers, yielding an average of 6.67 bands and 1.68 polymorphisms per primer. Three types of polymorphisms were detected: band presence/absence, band intensity, and fragment-length polymorphisms. Most of the observed polymorphisms were band presence/absence, typical of RAPD-dominant markers. The number of observed polymorphisms and their segregation in accordance with a Mendelian proportion confirm the importance of this technique for genome analysis of species like M. quadrifasciata that are poorly studied at the genetic level.

Transformation of Penicillium griseoroseum nitrate reductase mutant with the nia gene from Fusarium oxysporum.

A heterologous transformation system for Penicillium griseoroseum has been developed. This system is based on nia, the structural gene from Fusarium oxysporum encoding nitrate reductase. Penicillium griseoroseum niaD mutants have been selected from chlorate-resistant colonies. Among 24 chlorate-resistant colonies analyzed, 2 were confirmed to be niaD mutants. Transformation frequencies of 8 transformants/microgram of DNA were obtained. DNA hybridization analyses of five transformants showed distinct integration patterns of the plasmid and in all of them the integration occurred at tandem arrays. The transformation system established in this work will be useful for genetic studies of the pectinolytic complex genes from P. griseoroseum.