Understanding the Cellular Mechanism of the Excess Microsporocytes I (EMSI) Gene. Andrew ElBardissi, The Pennsylvania State University

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1 Understanding the Cellular Mechanism of the Excess Microsporocytes I (EMSI) Gene Andrew ElBardissi, The Pennsylvania State University Abstract: Hong Ma, The Pennsylvania State University The Excess Microsporocytes I (EMS I) gene is a leucine-rich repeat protein kinase (LRR-RPK) which has been isolated and analyzed from Arabidopsis male reproductive cells. Analysis of a mutant gene has provided sufficient evidence linking EMS I function to proper differentiation of the reproductive cells of male tissue. One of the pathways responsive to this gene is a production of excessive microsporocytes and a lack of tapetum cells. Genetic and statistical analysis has provided evidence of the responsibilities of EMS I which include tapetal cell fate and cytokenesis of microsporocytes (Ma et al., 2002). While it is undisputed that EMS I plays a critical role in the formation and differentiation of reproductive cells, the cellular mechanism responsible is still unknown. Here, investigation of the cellular mechanism is performed to provide insight as to the protein-protein interactions that may occur involving this protein. Introduction: The composition of the tissue responsible for the proper differentiation of cells for plant reproduction includes both reproductive and non-reproductive cells. The reproductive cells consist of microsporocytes which eventually differentiate into pollen grains. The non-reproductive cells consist of somatic layers of tissue including: epidermis, endomecium, middle layer and tapetum (Ma et al., 2002). These nonreproductive cells are responsible for the proper development and release of pollen grains. A newly discovered gene noted EMS I has been found to control many of the aspects of reproductive cell differentiation. A mutant of this gene has been found to contain the following differences with respect to the wild type: 1) There is an excess of microsporocytes, 2) The middle layer is maintained significantly longer (it should degenerate), 3) Male meiosis seems to occur without error, however cytokenesis does not occur which results in the failure or microsporogenesis (maturation of microsporocytes into pollen grains) (Ma et al., 2002). Because EMS I controls both the formation of pollen grains and formation of the surrounding somatic cells, EMS I is thought to control the fates of reproductive cells and their adjacent somatic cells. Gene expression has led to the conclusion that excess microsporocytes are formed at the detriment of tapetal cells indicating a direct correlation between formation of reproductive cells from nonreproductive cells. EMS I is an 1192 amino acid leucine-rich receptor protein kinase which consists of three domains: 1) Extracellular receptor domain with 30 leucine rich repeats, 2) A transmembrane domain essential for structural support, and 3) A cytoplasmic protein kinase domain.

2 Receptor protein kinases are key components of signal transduction pathways. As of now no receptor protein kinase has been found to be involved with cell differentiation and cell fate in Arabidopsis. However, preliminary results indicate the EMS I is directly involved in the differentiation of cells in male reproduction. Methodology: The purpose of this project was to analyze the properties of the EMS I gene, and determine the biochemical interactions that this gene or segments of this gene are involved in. In order to properly complete this task, research into similar Leucine-rich repeat receptor protein kinases (LRR-RPK) was performed to provide information into the type of cellular pathway that could be involved. Previous experiments yielded results that indicate that there are two main segments of LRRPK s that undergo biochemical interactions in vivo; the extracellular ligand binding segment, and the intracellular catalytic kinase domain. This experimental procedure was therefore designed around this information. Rather than analyzing a 3.6 kilobase gene (corresponding to an 1192 amino acid protein), the gene was subdivided into two segments, the respective intracellular and the extracellular portions of the protein. OMC 540 Extracellular Ligand Binding Segment OMC 542 Intracellular Kinase Segment kb.95 kb OMC 541 OMC 543 Figure 1: Shown above is a diagram of the EMS I gene. Noted are the two biochemically active segments. The divisions that were created for the gene are also noted above. The OMC 54X correspond to primers used to investigate the segments of the EMS I gene. After it was determined that the most efficient examination of EMS I would be to divide the gene into segments, phenotypically wild type plants were chosen as the donors for the gene. These plants underwent a procedure to isolate DNA via the Edwards preparation method. The DNA from these plants were used for the remainder of the experiment. In order to sufficiently study or perform any molecular technique on a fragment of DNA, a very large amount is required. Generally, there is only one way that allows one to begin with a small amount of DNA, and amplify it to a concentration that allows further analysis; this technique is the Polymerase Chain Reaction (PCR). Polymerase Chain Reaction: PCR is a procedure which uses a thermostable DNA polymerase, a primer set, and a cycling between three temperature phases to create a high concentration of DNA. Primers are very short portions (20 base pairs) of DNA at the beginning and end of the fragment of DNA that allow the polymerase to add nucleotides in a stepwise fashion. The three cycling phases of PCR are denaturation, annealing, and extension. During denaturation the double stranded DNA is heated to the point (approximately 94 degrees Celsius) that the hydrogen bonds supporting the structure of DNA are broken causing the

3 strands to break apart from each other, therefore creating two single strands of DNA ready to be copied. In the annealing phase of the PCR program, the temperature is lowered to degrees Celsius (depending on the length and composition of the primers used) which allows the formation of hydrogen bonds between the primers and the single stranded template. After the double stranded DNA has been denatured and the primer has annealed to the single strands, the extension phase allows the polymerase to add nucleotides, thereby creating a new copy of the studied segment of DNA. These three phases are then repeated anywhere from 30 to 40 times to create millions of copies of target DNA. In this experiment, four different primers were used for PCR (shown in figure 1), OMC 540, OMC 541, OMC 542, and OMC 543. The portions of DNA that were studied are listed below. Primer Set Size Function OMC 540-OMC kb Ligand Binding Domain OMC 542-OMC kb Kinase Domain OMC 540-OMC kb Whole Gene Following the creation of a PCR program, the PCR products were run on a 1% agarose gel to determine the relative concentration of the DNA. The concentration of the PCR products indicate the efficiency of PCR program. Gene Cloning: After this phase of the experiment was completed successfully, the next step was to clone the gene by ligating the PCR product into a linear vector, thereby creating a circular plasmid. The vector used is shown below.

4 The PCR products were added in a 3:1 ratio with respect to the pgem-t vector (the concentration of the PCR products were determined by gel electrophoresis). The ligation of the PCR products into the vector was catalyzed by the enzyme T4 DNA ligase. After the PCR products were incorporated into the vector, the next phase of this experiment was to cause bacterial cells to uptake the newly formed circular plasmid. Bacterial Transformation: Bacterial transformation is a molecular technique which allows specially prepared bacterial cells to accept foreign DNA. In addition to allowing the uptake of this DNA, if the foreign DNA contains an origin of replication recognized by the bacteria, the bacteria will replicate its genomic DNA as well as the foreign DNA during the bacterial growth phase. In addition to the ability of replication, if the foreign DNA contains resistance to antibiotics (as does the pgem-t vector), selection among bacterial colonies can occur. After the ligation was completed, the E. coli bacterial strain XL1-Blue was made competent (able to uptake DNA) by adding Calcium Chloride (CaCl2). When CaCl2 is introduced into bacterial culture, chloride ions rush into the cell and therefore by osmosis, cause water to rush into the cell as well, causing the cell to swell, thereby making it susceptible to uptake DNA when exposed to heat shock (approximately 42 degrees Celsius). After preparation of these competent bacterial cells, the DNA ligation was

5 added to the bacterial culture. This mixture was then heat shocked, and then incubated at 37 degrees Celsius (optimum bacterial growth temperature) to allow the cells to recover. After the cells were allowed to recover, they were plated onto Ampicillin positive growth medium agar plates. Therefore, only those bacterial cells that contain the gene for Ampicillin resistance would have the ability to grow on the plates. In order to identify which colonies on the plates were positive, PCR was performed with the respective primers. When positive colonies were found for each construct by PCR, verification of the insertion was done by restriction enzyme digestion. DNA Sequencing: After positive colonies were identified, they were inoculated into a growth medium and allowed to grow until the proper cell density was achieved. Once a sufficient amount of bacterial cells were obtained, the plasmid DNA was isolated by the lysis by alkali method. This plasmid DNA was then sequenced using the dideoxy method. After the sequencing was completed, analysis of the sequence was performed to assure that no point mutations, deletions or insertions had occurred. Excision of Insertion via Restriction Enzymes: When mutation free insertions were found, they were excised from the vector using the restriction enzymes Sal I and Srf I (figure 2). This digestion reaction caused the formation of two products: a 3kb pgem-t vector and a.95kb, 2.2kb or 3.6kb insertion. For the.95 kb and 2.2 kb insertion a 1% Agarose gel caused sufficient separation of the DNA to isolate the two products. The 3.6 kb insertion (whole gene) however, was close in size to the pgem-t vector and therefore a 1.5% Agarose gel was used to separate the two products. The insertions were then cut out of the Agarose gel and purified. At the end of this portion of the experiment, three constructs that were mutation free were present. Each construct was a linear segment of DNA with Srf I and Sal I restriction sites present at the 5 and 3 ends respectively. Yeast Two-Hybrid The final phase of this experiment was a procedure that identifies interactions between proteins. Yeast two-hybrid is a system that contains a bait protein and a library of proteins. Interactions that occur between proteins are identified by a reporter gene. In order to perform this experiment, the respective segment of DNA was required to be inserted into the pbd-gal 4 Vector shown below.

6 Figure 3: pbd-gal4 Yeast Two hybrid Vector. Taken from Stratagene. The pbd-gal4 is a circular plasmid with chloramphenicol resistance. In order to insert the DNA segment required, DNA restriction enzymes Sal I and Srf I were used to digest both the vector and the segment thereby creating compatible ends so the DNA fragment could be inserted into the vector via T4 DNA ligase. Following ligation into the pbd-gal4 vector, transformation into the E. coli XL1-Blue was once again performed. Because the pbd-gal4 vector contains chloramphenicol resistance rather than ampicillin, agar plates with chloramphenicol were used to grow the transformed bacterial cultures and screen for transformants. Once again, PCR was performed on the plated colonies until a positive one was identified. These p ositive colonies were then sequenced one last time to determine that no mutations were present and that the insertion was properly ligated into the vector. After all positive colonies were found with no sequence mutations, preparation of the yeast began. In this phase, the YRG-2 host strain was plated onto a YPAD agar plate for growth. The yeast cells that were grown on the YPAD plate were then plated on SD agar plated with amino acid nutritional deficiencies to determine the phenotype. Only those yeast colonies that display the correct phenotype were used for the yeast two hybrid. Once the yeast was identified and the pbd-gal4 plasmid was ready for insertion, the yeast cells were transformed with the plasmid DNA. Following successful transformation, the bait construct (pbd-gal4 with insertion) was isolated along with the library plasmid containing genes that were suspected to interact with the bait construct. Once interactions between the proteins occured, identification of the genes responsible for the interaction was performed.

7 Results: Figure 4: 1% Agarose gel of PCR products. Lane 1 contains OMC 540-OMC541 Primer, Lane 2 contains OMC 542-OMC543 Primer, and lane 3 contains OMC 540- OMC 543 Primers. Figure 5: 1% Agarose gel of bacterial colony screening. The first three lanes contain DNA from the primers OMC 540 OMC 541, lanes 4 through 6 contain the primers OMC 542 OMC 543, and the final three lanes contain the primers OMC 540-OMC 543

8 Figure 6: DNA sequencing results via the dideoxy method for the construct , this construct did have an amino acid changing mutation. Figure 7: Restriction enzyme digestion on the OMC 540-OMC 541 construct. Conclusion: While it has been determined that the EMS I gene is responsible for cell differentiation, the exact pathway by which it occurs has yet to be determined. PCR was used to amplify two main segments of the gene (and also the whole gene), for further analysis. In addition to PCR, transformation and DNA sequencing has yielded three

9 constructs which are mutation free. These three constructs were then inserted into the pgem-t vector and finally into the pbd-gal4 vector to determine the protein-protein interactions that occur, which would then give insight into the cellular pathway involved in this cell differentiation. The yeast two hybrid portion of this experiment has yet to be completed. Understanding the mechanism of this interaction will have many effects on the understanding of biochemistry and more specifically receptor protein kinases. The fact that a non-reproductive cell has the ability to differentiate into a reproductive cell through a pathway central to this leucine-rich repeat protein kinase will provide further information into how this mechanism may occur in wide variety of organisms. Discussion: The purpose of this investigation is to identify the cellular mechanism central to the EMS I gene. In order to achieve this task many experiments were performed, with both successful and unsuccessful results. In the first segment of this experiment, PCR optimization played a crucial role in achieving success cloning the gene (or gene segment). It was found that two different PCR programs were required due to the variation in lengths of the studied portions of DNA. The.95 kb and 2.2kB fragments of DNA produced optimal results with an extension time of 1:30, whereas the whole gene (3.6kB) produced optimal results with an extension time of 2:00. The denaturation and annealing phases of the PCR program needed no modification from the standard and were therefore identical for all three fragments of DNA (figure 4). Following a successful PCR program, it was determined that the maximum number of bacterial transformants was seen the quicker the ligation reaction was performed. In this particular experiment, the ligation reaction was set up immediately after PCR was completed. The vector used for the ligation was the pgem-t vector created by Promega. This vector contains certain properties which made it a good candidate for this experiment. For example, this vector contains a gene for antibiotic resistance (ampicillin). Ampicillin resistance allowed for screening colonies by only allowing those bacterial cells which contained the gene for ampicillin resistance to grow on the agar growth medium plates. Numerous colonies from each construct were cloned into the pgem-t vector. The insertions in the vector were initially verified by PCR and finally by restriction enzyme digestion (figure 5). After the positive colonies were identified, DNA sequencing was performed on the positive constructs to verify that no mutations were present. Due to the fact that PCR has a high error rate a large number of colonies were sequenced until a mutation free insertion was found (figure 6). Those constructs which were found to be mutation free were then used for the next segment of the experiment. While the pgem-t vector does not have a restriction enzyme site for Srf I (figure 2), preliminary experimental analysis provided the conclusion that the only possible way of using restriction enzymes that would work with both the pgem-t vector and the pbd- GAL4 vector would be to introduce a restriction enzyme site into the DNA primer. Therefore, an Srf I site was created within the primers, thereby providing a means of excising the insertion from the pgem-t vector, and creating compatible ends for use with the pbd-gal4 vector to allow for ligation.

10 After the insertions were excised and ligated into the pbd-gal4 vector, transformation of the vector into the bacterial strain E. coli XL1-Blue was completed. This transformation procedure was very similar to the initial bacterial transformation with the exception that the pbd-gal4 vector expressed chloramphenicol resistance rather than ampicillin resistance. When positive colonies were identified by PCR and restriction enzyme digestion, they were sequenced once again to assure that no frame shift mutations had occurred and also to verify that the insertions were placed in to the correct orientation in the plasmid. This aspect of the experiment was very important due to the fact that this is the plasmid that will allow for translation of the gene and therefore cause interaction of the protein with other proteins. If a frame shift mutation was present a completely different protein would be expressed and therefore produce false results. This is the current status of this experiment. The remaining portion of this experiment is dependant on transforming the YRG-2 yeast strain with the pbd-gal4 plasmid and also the PAD-GAL4 plasmid (containing the library of proteins) to isolate and analyze the proteins that interact with the EMS I protein.

11 Works Cited: Ma, H. et al The EXCESS MICROSPOROCYTES 1 gene encodes a putative leucine-rich repeat receptor protein kinase that controls somatic and reproductive cell fates in Arabidopsis anther. Genes and Development Not Yet Released. Promega pgem-t Vector Cloning System. Stone, J.M. and Walker, J.C Plant protein kinase families and signal transduction. Plant Physiology 117: Stratagene HybriZAP-2.1 Two-Hybrid Predigested Vector Kit. Zhang, Xiarorong Leucine-rich Repeat Receptor-like Kinases in Plants. Plant Molecular Biology Reporter 16: