Use of practice tests with immediate feedback in an undergraduate molecular biology course.

Multiple studies have shown that testing contributes to learning at all educational levels. In this observational classroom study, we report the use of a learning tool developed for a Genetics and Molecular Biology course at the college level. An interactive set of practice exams that included 136 multiple choice questions (MCQ) or matching queries was developed in the open-source Moodle platform. All MCQ questions contained four answer choices and configured for immediate feedback upon answering. Feedback consisted of providing the right answer and a short explanation of the learning objective examined. The interactive material was tested and refined for several semesters. Usefulness of this tool was assessed in two distinct settings: (1) during a face-to-face semester (Fall 2019) by comparing the grades in a final departmental exam between students who used the tool and those who did not, and (2) during an online semester (Fall 2020) by analyzing the grades in the first and last attempts on study sessions and students' performance in monthly exams. We found that when solving practice tests, students obtained a significantly higher scores in the last attempt compared with their first attempt, and that students who used the material performed better than those who did not. In all cases, answering the practice exams was optional, but students made full use of them preferentially during the online semester. This classroom research exemplifies the documented effectiveness of practice tests enhanced with feedback in biological sciences education through an open-source learning platform.

An online course on applied biochemistry and molecular biology through case-based learning.

An undergraduate online course on Applied Biochemistry and Molecular Biology was developed through different formats of case study that included lecture, class discussion, small-groups discussion, and individual work. Cases covering health, biotechnology, agriculture, and other issues were developed or adapted from the literature to reach the desired learning goals. Multiple web resources were employed for information integration that were presented and discussed in the synchronous sessions and assignments. Formative and summative assessment was achieved through multiple-choice questionnaires, exams, and homework assignments.

In Search of Resistance Against Fusarium Ear Rot: Ferulic Acid Contents in Maize Pericarp Are Associated With Antifungal Activity and Inhibition of Fumonisin Production.

Fusarium verticillioides is a fungal pathogen of maize that causes seedling blight, stem rot, and Fusarium ear rot. Fungal infestation of maize kernels and ears affects grain quality from the ensuing mycotoxin buildup. Among the mycotoxins produced by F. verticillioides, fumonisins accumulate to high levels in Fusarium-infected maize kernels, fumonisin B1 (FB1) being the most abundant in naturally infected maize. Achieving resistance to Fusarium ear rot has been challenging, as various environmental factors facilitate fungal infection. Among the maize grain components that contribute to resistance to F. verticillioides infection, the pericarp is the first barrier faced by the fungus and thus plays a key role. Phenolic acids are major constituents of maize pericarp, of which ferulic acid (FA) is the predominant molecular species. In this work, we explored the relationship between FA levels, fungal infection, and FB1 production in 51 maize genotypes and whether the antioxidant activity of FA might play a role. We confirmed that FA is a major component of the seed pericarp, whose levels as bound FA varied between 4.5 and 26.3 mg/g across maize genotypes. We selected two pools of five maize varieties, with contrasting FA contents: low FA (LFA; 6.14 ± 0.40 mg/g) and high FA (HFA; 15.49 ± 1.31 mg/g). In vitro, HFA extracts inhibited fungal growth with effects comparable to FA concentrations in the 0.25-0.50 mM range. We also established a kernel assay to study F. verticillioides colonization and FB1 production in the LFA and HFA genotypes. Fungal colonization was significantly lower in HFA genotypes relative to LFA genotypes, based on ergosterol levels. Moreover, FB1 production was also inhibited in the HFA genotypes. Importantly, the antioxidant activity of maize pericarp extracts was associated with FA contents, with HFA extracts exhibiting a greater antioxidant activity than LFA extracts. Overall, our results highlight the role of FA and its antioxidant activity on resistance to Fusarium ear rot and provide the basis of a phenotypic trait that can be deployed for breeding selection.

Characterization of the Exo-Metabolome of the Emergent Phytopathogen Fusarium kuroshium sp. nov., a Causal Agent of Fusarium Dieback.

Fusarium kuroshium is the fungal symbiont associated with the ambrosia beetle Euwallacea kuroshio, a plague complex that attacks avocado, among other hosts, causing a disease named Fusarium dieback (FD). However, the contribution of F. kuroshium to the establishment of this disease remains unknown. To advance the understanding of F. kuroshium pathogenicity, we profiled its exo-metabolome through metabolomics tools based on accurate mass spectrometry. We found that F. kuroshium can produce several key metabolites with phytotoxicity properties and other compounds with unknown functions. Among the metabolites identified in the fungal exo-metabolome, fusaric acid (FA) was further studied due to its phytotoxicity and relevance as a virulence factor. We tested both FA and organic extracts from F. kuroshium at various dilutions in avocado foliar tissue and found that they caused necrosis and chlorosis, resembling symptoms similar to those observed in FD. This study reports for first-time insights regarding F. kuroshium associated with its virulence, which could lead to the potential development of diagnostic and management tools of FD disease and provides a basis for understanding the interaction of F. kuroshium with its host plants.

Maize Thymidine Kinase Activity Is Present throughout Plant Development and Its Heterologous Expression Confers Tolerance to an Organellar DNA-Damaging Agent.

Thymidine kinase 1 (TK1) phosphorylates thymidine nucleosides to generate thymidine monophosphate. This reaction belongs to the pyrimidine salvage route that is phylogenetically conserved. In the model plant Arabidopsis thaliana, TK activity contributes to maintain nuclear and organellar genome integrity by providing deoxythymidine-triphosphate (dTTP) for DNA synthesis. Arabidopsis has two TK1 genes (TK1a and TK1b) and double mutants show an albino phenotype and develop poorly. In contrast, maize (Zea mays L.) has a single TK1 (ZmTK1) gene and mutant plants are albino and display reduced genome copy number in chloroplasts. We studied the role of ZmTK1 during development and genotoxic stress response by assessing its activity at different developmental stages and by complementing Arabidopsis tk1 mutants. We found that ZmTK1 transcripts and activity are present during germination and throughout maize development. We show that ZmTK1 translocation to chloroplasts depends on a 72-amino-acid N-signal and its plastid localization is consistent with its ability to complement Arabidopsis tk1b mutants which are hypersensitive to ciprofloxacin (CIP), a genotoxic agent to organellar DNA. Also, ZmTK1 partly complemented the Arabidopsis double mutant plants during development. Our results contribute to the understanding of TK1 function in monocot species as an organellar enzyme for genome replication and repair.

Sphingolipid Effects on the Plasma Membrane Produced by Addition of Fumonisin B1 to Maize Embryos.

Fumonisin B1 is a mycotoxin produced by Fusarium verticillioides that modifies the membrane properties from animal cells and inhibits complex sphingolipids synthesis through the inhibition of ceramide synthase. The aim of this work was to determine the effect of Fumonisin B1 on the plant plasma membrane when the mycotoxin was added to germinating maize embryos. Fumonisin B1 addition to the embryos diminished plasma membrane fluidity, increased electrolyte leakage, caused a 7-fold increase of sphinganine and a small decrease in glucosylceramide in the plasma membrane, without affecting phytosphingosine levels or fatty acid composition. A 20%-30% inhibition of the plasma membrane H+-ATPase activity was observed when embryos were germinated in the presence of the mycotoxin. Such inhibition was only associated to the decrease in glucosylceramide and the addition of exogenous ceramide to the embryos relieved the inhibition of Fumonisin B1. These results indicate that exposure of the maize embryos for 24 h to Fumonisin B1 allowed the mycotoxin to target ceramide synthase at the endoplasmic reticulum, eliciting an imbalance of endogenous sphingolipids. The latter disrupted membrane properties and inhibited the plasma membrane H+-ATPase activity. Altogether, these results illustrate the mode of action of the pathogen and a plant defense strategy.

The Protective Effect of Trichoderma asperellum on Tomato Plants against Fusarium oxysporum and Botrytis cinerea Diseases Involves Inhibition of Reactive Oxygen Species Production.

Trichoderma species are fungi widely employed as plant-growth-promoting agents and for biological control. Several commercial and laboratory-made solid formulations for mass production of Trichoderma have been reported. In this study, we evaluated a solid kaolin-based formulation to promote the absortion/retention of Trichoderma asperellum in the substrate for growing tomato plants. The unique implementation of this solid formulation resulted in an increased growth of the tomato plants, both in roots and shoots after 40 days of its application. Plants were challenged with two fungal pathogens, Fusarium oxysporum and Botrytis cinerea, and pretreatment with T. asperellum resulted in less severe wilting and stunting symptoms than non-treated plants. Treatment with T. asperellum formulation inhibited Reactive Oxygen Species (ROS) production in response to the pathogens in comparison to plants that were only challenged with both pathogens. These results suggest that decrease in ROS levels contribute to the protective effects exerted by T. asperellum in tomato.

Role of pyrimidine salvage pathway in the maintenance of organellar and nuclear genome integrity.

Nucleotide biosynthesis proceeds through a de novo pathway and a salvage route. In the salvage route, free bases and/or nucleosides are recycled to generate the corresponding nucleotides. Thymidine kinase (TK) is the first enzyme in the salvage pathway to recycle thymidine nucleosides as it phosphorylates thymidine to yield thymidine monophosphate. The Arabidopsis genome contains two TK genes -TK1a and TK1b- that show similar expression patterns during development. In this work, we studied the respective roles of the two genes during early development and in response to genotoxic agents targeting the organellar or the nuclear genome. We found that the pyrimidine salvage pathway is crucial for chloroplast development and genome replication, as well as for the maintenance of its integrity, and is thus likely to play a crucial role during the transition from heterotrophy to autotrophy after germination. Interestingly, defects in TK activity could be partially compensated by supplementation of the medium with sugar, and this effect resulted from both the availability of a carbon source and the activation of the nucleotide de novo synthesis pathway, providing evidence for a compensation mechanism between two routes of nucleotide biosynthesis that depend on nutrient availability. Finally, we found differential roles of the TK1a and TK1b genes during the plant response to genotoxic stress, suggesting that different pools of nucleotides exist within the cells and are required to respond to different types of DNA damage. Altogether, our results highlight the importance of the pyrimidine salvage pathway, both during plant development and in response to genotoxic stress.

Long-term response on growth, antioxidant enzymes, and secondary metabolites in salicylic acid pre-treated Uncaria tomentosa microplants.

OBJECTIVE: To obtain micro propagated Uncaria tomentosa plantlets with enhanced secondary metabolites production, long-term responses to salicylic acid (SA) pre-treatments at 1 and 100 µM were evaluated after propagation of the plantlets in a SA-free medium. RESULTS: SA pre-treatments of single node cuttings OF U. tomentosa produced long-term responses in microplants grown for 75 days in a SA-free medium. Reduction in survival rate, root formation, and stem elongation were observed only with 100 µM SA pre-treatments with respect to the control (0 + DMSO).Both pre-treatments enhanced H2O2 and inhibited superoxide dismutase and catalase activities, while guaiacol peroxidase was increased only with 1 µM SA. Also, both pre-treatments increased total monoterpenoid oxindole alkaloids by ca. 55 % (16.5 mg g(-1) DW), including isopteropodine, speciophylline, mitraphylline, isomitraphylline, rhynchopylline, and isorhynchopylline; and flavonoids by ca. 21 % (914 µg g(-1) DW), whereas phenolic compounds were increased 80 % (599 µg g(-1) DW) at 1 µM and 8.2 % (359 µg g(-1) DW) at 100 µM SA. CONCLUSION: Pre-treatment with 1 µM SA of U.tomentosa microplants preserved the survival rate and increased oxindole alkaloids, flavonoids, and phenolic compounds in correlation with H2O2 and peroxidase activity enhancements, offering biotechnological advantages over non-treated microplants.

Disruption of sphingolipid biosynthesis in Nicotiana benthamiana activates salicylic acid-dependent responses and compromises resistance to Alternaria alternata f. sp. lycopersici.

Sphingolipids play an important role in signal transduction pathways that regulate physiological functions and stress responses in eukaryotes. In plants, recent evidence suggests that their metabolic precursors, the long-chain bases (LCBs) act as bioactive molecules in the immune response. Interestingly, the virulence of two unrelated necrotrophic fungi, Fusarium verticillioides and Alternaria alternata, which are pathogens of maize and tomato plants, respectively, depends on the production of sphinganine-analog mycotoxins (SAMs). These metabolites inhibit de novo synthesis of sphingolipids in their hosts causing accumulation of LCBs, which are key regulators of programmed cell death. Therefore, to gain more insight into the role of sphingolipids in plant immunity against SAM-producing necrotrophic fungi, we disrupted sphingolipid metabolism in Nicotiana benthamiana through virus-induced gene silencing (VIGS) of the serine palmitoyltransfersase (SPT). This enzyme catalyzes the first reaction in LCB synthesis. VIGS of SPT profoundly affected N. benthamiana development as well as LCB composition of sphingolipids. While total levels of phytosphingosine decreased, sphinganine and sphingosine levels increased in SPT-silenced plants, compared with control plants. Plant immunity was also affected as silenced plants accumulated salicylic acid (SA), constitutively expressed the SA-inducible NbPR-1 gene and showed increased susceptibility to the necrotroph A. alternata f. sp. lycopersici. In contrast, expression of NbPR-2 and NbPR-3 genes was delayed in silenced plants upon fungal infection. Our results strongly suggest that LCBs modulate the SA-dependent responses and provide a working model of the potential role of SAMs from necrotrophic fungi to disrupt the plant host response to foster colonization.

Fumonisin B1, a toxin produced by Fusarium verticillioides, modulates maize ß-1,3-glucanase activities involved in defense response.

Fusarium verticillioides is an important pathogen in maize that causes various diseases affecting all stages of plant development worldwide. The fungal pathogen could be seed borne or survive in soil and penetrate the germinating seed. Most F. verticillioides strains produce fumonisins, which are of concern because of their toxicity to animals and possibly humans, and because they enhance virulence against seedlings of some maize genotypes. In this work, we studied the action of fumonisin B1 (FB1) on the activity of maize ß-1,3-glucanases involved in plant defense response. In maize embryos, FB1 induced an acidic isoform while suppressing the activity of two basic isoforms. This acidic isoform was induced also with 2,6-dichloroisonicotinic acid, an analog of salicylic acid. Repression of the basic isoforms suggested a direct interaction of the enzymes with the mycotoxin as in vitro experiments showed that pure FB1 inhibited the basic ß-1,3-glucanases with an IC(50) of 53 µM. When germinating maize embryos were inoculated with F. verticillioides the same dual effect on ß-1,3-glucanase activities that we observed with the pure toxin was reproduced. Similar levels of FB1 were recovered at 24 h germination in maize tissue when they were treated with pure FB1 or inoculated with an FB1-producing strain. These results suggest that ß-1,3-glucanases are a relevant physiological target and their modulation by FB1 might contribute to F. verticillioides colonization.

Fumonisin production by Fusarium verticillioides strains isolated from maize in Mexico and development of a polymerase chain reaction to detect potential toxigenic strains in grains.

Fumonisins are mycotoxins produced by Fusarium verticillioides (Sacc. Nirenberg) in maize (Zea mays L.), a staple crop in Mexico. In this study, we report the isolation and identification of 67 Fusarium strains isolated from maize kernels collected in Northwest and Central Mexico. The strains were characterized regarding fumonisin B(1) production and the presence of the FUM1 gene. F. verticillioides was the predominant species isolated in both geographic regions, but the isolates from Northwest Mexico produced higher levels of fumonisin. A polymerase chain reaction (PCR)-based method, to detect a region of the FUM1 gene involved in fumonisin biosynthesis, was developed and employed to detect mycotoxigenic fungi in pure culture and in contaminated maize. The presence of the FUM1 gene was associated with fumonisin production in most isolates, except seven that did not synthesize fumonisin but contained the gene in their genome. The PCR method allowed the direct detection of fungal contamination in ground corn and could be employed to screen for the presence of potential mycotoxigenic fusaria.

Suitability of real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay for cry9C detection in Mexican corn tortillas: fate of DNA and protein after alkaline cooking.

Alkaline-cooked corn, called nixtamal, is the basis for many traditional corn products such as tortillas, chips, and taco shells that are used widely in Mexico and Central America and in the preparation of snack foods that are consumed globally. To assess the effects of alkaline and thermal treatments on the detectability of DNA and protein for the presence of genetically modified sequences, various nixtamalized products were prepared from blends of conventional white corn containing 0.1, 1.0, and 10% transgenic corn (event CBH 351, StarLink). Real-time quantitative polymerase chain reactions (RTQ-PCR) and immunoassays were used to determine the cry9C gene and protein, respectively, in unprocessed corn kernels, freshly prepared alkaline-cooked and ground corn (masa), masa flour, tortillas prepared from masa by heat treatment, chips prepared from damp masa dough by deep frying, and from tortillas processed at high (200 degrees C) and low temperatures (70 degrees C). In spite of progressive degradation of genomic DNA during processing, RTQ-PCR genetic analysis allowed detection and quantification of the cry9C gene in all products prepared from 10, 1, and 0.1% StarLink corn, except deep-fried chips containing 0.1% StarLink. Enzyme-linked immunosorbent assays readily detected <1 ppm cry9C protein in all blends of unprocessed corn (10, 1, and 0.1% StarLink) as well as in nonfried tortilla and masa products. This technique was not suitable for thermally treated nixtamalized products containing <1% transgenic corn.