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.

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.

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.

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.

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.

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.

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.

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.