1. ABSTRACT MATERIALS AND METHODS DISCUSSION BACKGROUND REFERENCES
STUDY OF ORYZA SATIVA GENES IN ARABIDOPSIS TO ADVANCE UNDERSTANDING OF PATHOGENIC DEFENSES IN BARLEY Garrett Hall1, Jesse Wilcox2, Jackson Moeller3, Jaime Dittman3, and Steve Whitham3 1Southeast Polk High School, Pleasant Hill, Iowa ,2Plant Valley Southwoods High School, West Des Moines, Iowa, and 3 Department of Plant Pathology and Center for Plant Responses to Environmental Stress, Iowa State University, Ames IA 50011
ABSTRACT
To find important differences between plants that are resistant to pathogen attack and those that are susceptible, microarrays were previously conducted to describe the response of the monocot plant Hordeum vulgare, barley, to Blumeria graminis f. sp hordei (Bgh),  powdery mildew fungus. From these microarrays, over 200 genes were identified that may be associated with plant defenses due to the mRNA accumulation in cases of resistance to the pathogen. To determine the function of these genes, we are studying genes of highly similar genetic sequence in Oryza sativa and the model plant Arabidopsis. Although much is known about barley, the resources of Arabidopsis allow for easier investigation of the genes of interest. Through the over-expression of these genes we hope to gain a better understanding of their function. With this approach using monocot and dicot plants and their pathogens, we aim to find and characterize plant genes that are of widespread importance to a plant's resistance to plant pathogens. To get to this point a gene is inserted into an Agrobacterium vector and is used to transform the Arabidopsis plant. These mutants will then be challenged with strains of the bacterial pathogen Pseudomonas syringae pv. tomato to assess whether or not these genes have importance in limiting the growth and spread of this pathogen. If these genes have importance in the Arabidopsis-Pseudomonas interaction, it is our expectation that the corresponding barley genes will have similar functions in resistance to powdery mildew as well.  
MATERIALS AND METHODS
The processes used in our research focused on using different vectors to move our target gene into Agrobacteria which would later be used to transform Arabidopsis plants.
Oryza sativa and Arabidopsis homologues retrieved; cDNA clone selected.
PCR gene amplification BP Clonase Reaction & E. coli transformation LR Clonase Reaction & E. coli transformation
transformation of Agrobacteria Growth of Agrobacteria Transformation of Arabidopsis
Harvest Seed Growth of seed to Check for Over-expression
A PCR product was obtained and sent to a BP Clonase reaction using the pDONR207 vector. Once the BP had been quantified using a Nano Drop it was then carried into the LR Clonase reaction using the pCB2004 vector.
When the LR reaction had been validated by PCR it was then sent to the Iowa State University DNA Facility for sequencing. Upon retrieving the sequence data, the AlignX program was used to verify base pairs. If the sequences matched, the LR reactions were transformed to Agrobacteria cultures. These cultures were then used to dip Arabidopsis flowers with the hope of transforming the future generation.
DISCUSSION
Genes that have potential pathogenic resistance functions were sequenced following cloning into our plant over-expression vector. We analyzed the nucleotide sequence and made a comparison to the gene’s open reading frame to determine if the sequence generated was correct. One problem that we encountered in this process was a number of samples did not match the open reading frame, but the forward strand matched the reverse strand. From this information, we concluded that the forward and reverse strands were from the same clone, but the clone that we had sequenced was not the clone we expected. With a BLAST search of the Arabidopsis genome, some of the samples were in the same gene family as the gene we were interested in. With further investigation and testing, a likely cause of the wrong clones was a contamination or labeling error from the center they were purchased from.
The genes that were sequenced and matched the clones we expected were electroporated into Agrobacteria. Arabidopsis florets were then dipped with the Agrobacteria cultures. Unfortunately, with a six week investigation, we were unable to ascertain if the clones were incorporated and expressed in the Arabidopsis plants. Our prediction is that a portion of the seeds from the dipped Arabidopsis plants would express the herbicide resistance gene that is associated with the gene of interest. RNA could then be extracted and analyzed to determine if the gene of interest was incorporated into the plant and if that gene is over-expressed compared to a control.
BACKGROUND
Leaf spots, wilting, tumors, cankers, rotting, and rust are terms that cause alarm to those involved in crop production. Large amounts of resources are used to combat the pathogens that cause these symptoms, but chemicals only help so much.  Pathogens may adapt to these chemicals over time. If new means of plant protection are established, the agriculture industry may not lose as much yield each year to pathogens. Recombinant DNA technology allows the enhancement of plant responses to a pathogen.  Genes of interest from one plant may be transferred to another to increase the recipient plants’ ability to defend itself. The Whitham lab at Iowa State University is currently working on a model pathogen system in Arabidopsis to discover plant genes responsible for pathogen defense. These results can later be used to help protect barley and other crops.  Emphasis is placed on genes that are more likely to have large effects on pathogen resistance such as signal transduction components. Around two hundred genes of interest have been selected for over-expression to determine gene function.
REFERENCES
Caldo, R.A., Nettleton, D. and Wise, R.P. (2004) Interaction-dependent gene expression in Mla-specified response to barley powdery mildew. Plant Cell, 16, 2514–2528.
Clough S and Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:
RESULTS
During our six week summer experience, research that had been occurring for 3 years was continued.  The results from our investigation included 35 total genes from Oryza sativa and Arabidopsis.  The genes were inserted into a BP vector after PCR amplification.  From this BP plasmid, pDONR207, 30 of those samples were inserted in the LR plasmid, pCB2004.  Once the samples were verified, they were sequenced and analyzed. In total, 30 DNA clones were taken from clones at the beginning of the process to DNA sequencing during the six weeks.  Arabidopsis plants were dipped with 15 separate Agrobacteria cultures that were created from the LR plasmid samples.
BP Clonase Reactions (pDONR207)
A total of 35 clonase reactions
were able to be sent to LR Reaction
LR Clonase Reactions (pCB2004)
A total of 30 clonase reactions
Were able to be sent to sequencing
Agrobacteria Solutions
15 solutions each with a separate gene of interest were used to transform Arabidopsis.
ACKNOWLEDGEMENT
We would like to thank the following people from the Whitham lab for their graciousness and support during our experience: Jackson Moeller, Jaime Dittman, Nathan Bestor, Chunling Yang, and Steve Whitham.
RESEARCH GOALS
The main goal of this research is to identify the role of specific genes in pathogenic resistance. The results of this investigation could then be applied to pathogen resistance in barley and other crops.
Our specific goal for this project was to clone genes of interest into a plant over-expression vector for stable over-expression in Arabidopsis.