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BTH2732 Tutorial Assignment 2 Critical Review

Article 1:

Title: MSI-99, a magainin analogue, imparts enhanced disease resistance in transgenic tobacco and banana Citation: Chakrabarti, A, Ganapathi, TR, Mukherjee, PK and Bapat, VA (2003), MSI-99, a magainin analogue, imparts enhanced disease resistance in transgenic tobacco and banana, Planta 216: 587-596.

Article 2:

Title: Identification of bacterial populations in dairy wastewaters by use of 16S rRNA gene sequences and other genetic markers Citation: McGarvey, JA, Miller, WG, Sanchez, S and Stanker, L (2004), Identification of bacterial populations in dairy wastewaters by use of 16S rRNA gene sequences and other genetic markers, Applied and Environmental Microbiology 70(7): 42674275.

1. Is the study correlative or Manipulative? Justify your reasons

Paper 1: This is a manipulative or experimental study. The researchers transform the tobacco and banana plants by inserting the synthetic peptide analogue of magainin (MSI-99) to obtain the expected outcome, i.e. the expression of magainin as conferring resistance to the transgenic plants against phytopathogens. Hence the study manipulates the cause, to achieve the effect.

Paper 2: This study is correlative because, instead of interfering in a natural process to create artificial factors to get the outcome, the researchers study a natural variation in outcome caused by one or more factors. This correlative study investigates the relationship between the bacterial numbers/diversity and the different stages of hydraulic flush waste removal systems to manage animal waste in a dairy farm.

Sadaf Majid (21212163) BTH2732 Critical Review

2. a. Define "Reverse Causation" Reverse causation is a process that reverses the causality, i.e. the effect precedes the cause. For example, the effect of increased work performance would cause the income to increase. However, cometimes the principle of reverse causation is used, whereby increasing the income, which is the outcome/effect, actually increases the performance of employees (the cause). b. Define "Confounding Variables Confounding variable is also known as a third variable which can adversely affect the relationship between the independent and the dependent variable, thereby destroying the reliability of the experiment. A confounding variable is the one that is difficult to control or eliminate.

3. What is the primary research question in each paper? a. What Hypotheses do these research questions lead to? b. What possible predictions can you make for the outcome of each?

Paper 1: The primary research is whether or not the synthetic peptide MSI-99 is successfully expressed in transgenic tobacco and banana plants and therefore confers resistance against bacterial and fungal phytopathogens such as F. oxysporum f.sp. cubense and Mycosphaerella musicola. This research question leads to the hypothesis that MSI-99 inhibits the growth of pathogens infecting tobacco and banana, and therefore it can be predicted that MSI-99 may confer enhanced disease resistance in transgenic plants against a wide variety of bacterial and fungal pathogens.

Paper 2: The primary research query is to find out what is the amount and composition of the chemicals and microorganisms present in different locations (free stall floor, separator pit, and storage lagoon) of the hydraulic flush waste water treatment system in a dairy farm. This leads to the hypothesis that the bacterial population as well as bacterial diversity, including larger number of pathogenic bacteria, would be higher in the earlier stages of waste water treatment, i.e. higher on the free stall floor, lower in the separator pit and lowest in the holding lagoon water. This is

Sadaf Majid (21212163) BTH2732 Critical Review

because the manure of cattle contains many pathogenic bacteria such as Escherichia coli O157:H7, Campylobacter spp, Salmonella spp., and Mycobacterium spp. So it is likely that consumption of the crops fertilized with wastewater from the earlier stages of the wastewater treatment (example from the free stall floor) will lead to adverse health effects in the consumers.

4. Briefly outline the practical steps taken to carry out the investigation

Paper 1: The anti-fungal activity of MSI-99 was tested in vitro, whereby cultures of Fusarium oxysporum f.sp cubense race 2 were grown on potato dextrose agar (PDA) at 30C and incubated for 5 days, after which the spores were collected and spore count determined via a haemocytometer. Inhibition of spore germination was studied by diluting the spores to 400 spores/l. MSI-99 was serially diluted in water to 0, 4, 8, 16, 32, 64, 128 and 256 g/ml. 1 l of the diluted spore suspension was incubated in 100 l of the mentioned concentrations of peptide solution in cavity slides, which were then placed on moist sterile blotting paper and incubated overnight at 30C. After that the germinating spores were counted and the percentage germination was calculated. In order to observe growth inhibition, MSI-99 was diluted to the same concentrations mentioned above using PDB. 10 l aliquots were observed microscopically, followed by continued incubation, with regular observation of fungal growth every 24 hours. Plant transformation vectors were created using standard procedure, whereby the respective E. coli and Agrobacterium tumefaciens strains were grown, and then transformed by electroporation. An additional Methionine (ATG), which does not occur in naturally occurring magainin, was added to the synthetic gene for MSI-99 to promote initiation of translation. The synthetic gene was inserted into the plasmids pSAN164 and pSAN168. In the latter plasmid, a secretary signal was combined with the peptide to enable it to reach extracellular spaces. The synthetic gene along with the promoter was released from the plasmids via double digestion by HindIII-SacI. The promoter and the green fluorescent protein were also removed from the transformation vector by HindIII-SacI digestion. MSI-99 and the ubq3promoter were cloned to the appropriate sites in order to obtain plant transformation vectors pMSI164 and pMSI168.

Sadaf Majid (21212163) BTH2732 Critical Review

Tobacco and Banana transformation: Tobacco was used as a model system to confirm that the expression cassettes were functioning properly. Leaves from the greenhouse plants, grown from Nicotiana tabacum seeds obtained from the Central Tobacco Research Institute, India, were transformed via A. tumefaciens strain EHA105:pSAN168. Banana transformation was also achieved by A. tumefaciens using embryogenic cells of the banana cultivar Rasthali. This was basically done by co-cultivation of A. tumefaciens strain EHA105 having a pMSI164 or pMSI168. Growth of transformed cells was promoted by Cefotaxime in M2 medium for 3 days, followed by development of embryo, enhanced by cefotaxime and geneticin in RR medium. The chemicals cefotaxime, geneticin and naphthalene-1-acetic acid in MS medium favored the development of plantlets from the germinating embryos. After two months of growth in the greenhouse, the plantlets were utilized for molecular analysis and bioassay studies. Tobacco and banana transgenics were analysed by the PCR. Nucleic acids from leaves of hardened transformed and untransformed control plants were isolated using a modified CTAB method. The products of PCR were analysed on 2% agarose gel using TAE buffer. PCR products were analysed by southern blotting. Reverse transcription PCR was carried out on transgenic tobacco and banana, and they were re-analysed by southern blotting. After that a bioassay of tobacco transgenics for disease resistance was carried out and the data were analysed. Transgenic banana plants were screened for resistance to fusarium wilt, followed by a bio-assay against Mycosphaerella musicola.

Paper 2: This study required sampling of manure and two different wastewater samples. These were collected over a period of eight months (November 2002 July 2003) from a large Californian dairy farm. 1 gram samples of manure were collected from 25 different locations on the free stall floor, whereas the water was sampled at a volume of 1 liter from the separator pit and circulated holding lagoon separately. All the samples were stored overnight at 4C to prevent microbial degradation until further analysis. Bacterial pellets were obtained by high-speed centrifugation of wastewater. Then 0.25 g samples of the pellet and solid manure were frozen at 80C. There were 10 replicates for each 0.25 g sample.

Sadaf Majid (21212163) BTH2732 Critical Review

Viable bacterial count was performed by plating and incubation of diluted samples on brain-heart infusion agar, while coliform bacteria were enumerated by plating and incubation on MacConkey agar. Water samples were chemically analyzed by the A&L West Agricultural Labs. In the next step, DNA from the samples was extracted, pooled, precipitated and finally suspended in sterile water at 50 ng/l. The non-culture based method included PCR amplification of 16S rDNA sequences, which was done by the use of eubacterium-specific primers 27f and 1392r. The products of PCR were purified and cloned, and then transformed by heat shock at 42C for 30 seconds. PCR bias was minimized by performing 7 PCRs for each sample, and collecting 96 clones. The templates were then prepared and sequenced. Editing of DNA sequences was performed manually by correcting wrongly identified bases and trimming the sequence using Chromas software. Sequences longer than 500 bases were used, with an average size of 600 base pairs. Using the FORMATDB program, a BLASTable 16s rDNA nucleotide database was constructed, so that comparison could be made with the predicted 16s rDNA sequences from this study, using FASTA-formatted file. A dendrogram was constructed based on phylogenetic and molecular evolutionary analyses. The diversity within the libraries was analyzed via rarefaction using the analytical approximation algorithm with 95% confidence intervals. Sequence-specific PCR was used to analyze the DNA from manure or wastewater pellets for presence of pathogens such as Salmonella, E. coli O157, Campylobacter spp., Yersinia spp., Listeria spp., pathogenic E. coli, toxigenic Staphylococci, and Clostridium perfringens.

5. Does each set of experiments have controls? Briefly define the controls used and how they give confidence to the results.

Paper 1: For the in vitro antifungal activity of MSI-99, the controls were the growth cultures on PDB in the absence of MSI-99. Mycelial growth at 16 g/ml was found to be less than that of the control, and none at 128 and 256 g/ml. Inclusion of a control ensured a valid comparison between the fungal spore germination and growth of mycelia grown in the presence and absence

Sadaf Majid (21212163) BTH2732 Critical Review

of MSI-99. It gave confidence to the results by verifying that it was indeed the presence of MSI99 that inhibits fungal growth. To confirm foreign gene integration, the negative controls used for this experiment were the untransformed tobacco and banana plants. Comparison of the results for the transformed and untransformed plants helped in confirming the effect of MSI-99. The positive controls used were the plasmids, i.e. the plant transformation vector PSI164 or PSI168. For the positive control, a predicted MSI-specific band of 147 bp for PSI164 transformants, and 180 bp for PSI168 transformants was expected. The presence of these transgene-specific bands in the transgenic tobacco and banana plants, and their absence in the untransformed plants, confirmed that MSI-99 was functional in the transgenic plants. The researchers also verified the identity of the MSIspecific bands in the control plasmids and the transgenic plants by blotting the PCR products on a nylon membrane and probing them with a radioactively labelled MSI99 gene and nos terminator. This resulted in amplified products of the expected size hybridized with the probe, thereby establishing the transgenic nature of the transformed plants. The result for the expression of transgene by RT-PCR was confirmed by separately subjecting the total RNA samples to PCR without reverse transcription, using the same primers. There were no products, which verified that RNA was free of DNA contamination, and that the PCR products by RT-PCR were due to the foreign gene transcript only. The resistance of tobacco against the fungal pathogens Botrytis cinerea, Alternaria alternate and Sclerotinia sclerotiorum, was significantly higher than in controls. In the screening of transgenic plants for resistance to fusarium wilt, untransformed plants derived from somatic embryos, of cultivar Rasthali, were used as the susceptible control. The tolerant control was the Basrai plants of similar age. The result for inoculation of transgenic banana plants and the control plants with F. oxysporum f.sp. cubense indicated that the fungus infected the transgenics and the susceptible control plants. The resistant control plants were able to grow normally, with no symptoms of fusarium wilt. The tolerant transgenics also indicated no discoloration of vascular tissues, as opposed to susceptible controls. On the whole, the results of this study provide important findings for agricultural and crop science. Sufficient controls were used to establish the successful activity of the synthetic peptide, which may be used to impart disease resistance against a variety of phytopathogens to important commercial crops.

Sadaf Majid (21212163) BTH2732 Critical Review

Paper 2: For the PCR detection of pathogens, a positive control for the PCR assays was used in the form of clinical isolates from the veterinary diagnostic laboratory. This verified the identity of the pathogens detected by the sequence-specific PCR. Basically no other controls were used for the bacterial counts in the three different variables used. But comparison of the bacterial counts and composition, as well as chemical composition, was made between the manure, and the wastewater from the storage pit and the lagoon. The lack of negative controls may be justified by the fact that the sampling was time-consuming, and that a lot of complex procedures were involved due to sequencing and PCR analysis; therefore inclusion of controls would not have been economical. Without the controls, the study is more comparative. A negative control which could have been included for the wastewater samples is distilled water, but setting a negative control for manure may prove to be a difficult task. Overall the results of the study were fairly confident and were a significant contribution in the field of medical science and pathology.