Identification of Pathogen Defense Genes in Cereal Plants

1. Identification of Pathogen Defense Genes in Cereal Plants Abstract This project was designed to identify the knockout genes responsible for causing two mutant strains of barley to be susceptible to powdery mildew. Based on bioinformatic data, primers were designed to target genes thought to be involved in defense. PCR was performed to locate the deleted gene in both mutants. Simultaneously, an F2 (wild type x mutant) population was grown and phenotypes were recorded for several days after inoculation with the Blumeria graminis f. sp. hordei (Bgh) isolate cc148 to identify individuals displaying the mutant and wild type phenotypes. No deletions were identified, the F2 generation was screened for co-segregation. This screen supports the deletion as responsible for causing susceptibility. Methods Background Plant diseases are one of the greatest problems to crop production worldwide. Genomic research such as gene deletion studies provide the information necessary to control these diseases (Zhang, 2006). Previously, fast neutron mutagenesis was conducted on wild type (C.I. 16151) seeds to randomly knock out chunks of genomic DNA. The progeny of these seeds were then planted and inoculated with the powdery mildew isolate 5874 (Blumeria graminis f. sp. hordei). Plants displaying cell death symptoms or sporulating colonies were selected. Seeds from these plants were then planted and inoculated with the same fungal isolate. RNA samples were collected at six time points after inoculation and hybridized to a Barley1 GeneChip (Close et. al. 2003). Bioinformatic analysis was conducted to determine the genes, based on expression patterns, most likely to have been knocked out in m9467 and m9468. Primers were designed for those 48 genes and PCR (polymerase chain reaction) was used to identify the deletions. Results Ehren Whigham1, John Upah1, Greg Fuerst2,4, Matt Moscou3, Karin Werner2,4, Liu Xi3, Amy Schwartz3, and Roger Wise3,41 - Secondary Biology Teacher Intern, Iowa State University Plant Pathology, Iowa State University, Ames, IA 500112 - Technician-USDA, Department of Plant Pathology, Iowa State University, Ames, IA 500113 - Department of Plant Pathology and Center for Plant Responses of Environmental Stresses, Iowa State University, Ames, IA 500114 - Corn Insects and Crop Genetics Research, USDA-ARS, Iowa State University, Ames, IA 50011 Discussion Research Hypothesis To locate the deleted gene in mutants of C.I. 16151, and to determine if the deleted gene co-segregates with the mutant phenotype. Analysis of the PCR results for m9467 and m9468 using the designed primers yielded no deletions in either mutant. Possible explanations include no deletion present at the target location, multiple copies of the gene in the genome, and ineffective primer design. Phenotype data compiled for both mutants indicates which plants express the mutant phenotype and which express the wild type phenotype. This data will be useful when screening for co-segregation after the gene deletions for both mutants are identified. Future work includes the design of new primer sets and redesign of existing primer sets. When the deletion is found, screening of the F2 population for co-segregation will be possible. No deletions were found in either mutant (m9467 or m9468) after PCR screening 48 candidate genes under three annealing temperatures: 52, 55, and 58ºC. m9467, 52oC annealing temperature m9468, 52oC annealing temperature References Close, TJ, S Wanamaker, R Caldo, SM Turner, DA Ashlock, JA Dickerson, RA Wing, GJ Muehlbauer, A Kleinhofs and RP Wise. 2004. A new resource for cereal genomics: 22K barley GeneChip comes of age. Plant Phys. 134:960-968. Zhang, L, T Fetch, J Nirmala, D Schmierer, R Brueggeman, B Steffenson, and A Kleinhofs. 2006. Rpr1, a gene required for Rpg1-dependent resistance to stem rust in barley. Theor. Appl. Genet. 113:847-855. Nine individuals from the m9467 x C.I. 16151 F2 population exhibited the mutant phenotype of necrosis (cell death) and tip wilt. Similarly, six individuals from the m9468 x C.I. 16151 F2 population exhibited the mutant phenotype. DNA was extracted from two fast-neutron derived barley mutants (m9467 and m9468) and the wild-type (C.I. 16151) using 2X CTAB. Sample concentrations were determined using a spectrophotometer and diluted for use as template in subsequent PCR reactions. To find possible gene deletions within the mutants, PCR was conducted at multiple annealing temperatures, and possible deletions were repeated using a gradient thermocycler. To determine if any deletions co-segregated with the inoculated mutant phenotypes, F2 generation plants were subjected to powdery mildew and phenotyped for necrosis and tip wilt. DNA was extracted for all individuals of these populations using the methods described above. Acknowledgement We would like to thank the Plant Genomics Outreach Program at Iowa State University especially Adah Leshem-Ackerman and Jay Staker for their support. In addition, we would like to thank the Biotechnology Outreach Education Center and the Office of Biotechnology for their generous assistance with equipment, teaching and support. Mostly, we would like thank the members of the Wise Lab including our PI Roger Wise, Matt Moscou, Karin Werner, and especially Greg Fuerst for all of their support, guidance, and patience. C.I. 16151 m9467 m9468

2. Centromere distance (cM) E3546-13 Rom1, E4044-5 map position MWG503 281 (190) 2.1 Ant2 0.2 MWG892 0.7 Rar1, MWG87 0.015 MWG876 2.2 cMWG694 0.8 MWG2123 Telomere NaN3 NaN3 rom1 (rar1 rom1) M100 (rar1 Rom1) Mla12 (Rar1 Rom1) Transcript Based Cloning of Rom1 Results After amplifying Rom1 candidate gene using the designed primers eight samples were selected for cloning as illustrated in Figure 1. Eventually, three were picked from plating to be sequence. Background The most common plant defense against pathogen attack involves the entire plant being resistant to a specific pathogen. In other cases the plant resistance is gene specific. The resistance to powdery mildew in barley has been found to be gene specific in some cases. For example, the Rar1 gene was identified to cause resistance in Sultan V (Freialdenhoven et al 2005). However, a mutation of the rar1 gene in Sultan V caused susceptibility to pathogens in M100. It is believed that a further mutation between M100 and rom1 caused resistance again. Using Transcript Based Cloning we can determine if a mutation occured Sultan V to M100. Methods After harvesting several varieties of Barley, DNA was extracted and then prepared for amplification using the CTAB Fresh Frozen Prep. Next the selected genes were amplified using PCR with Designed Rom1 and M100 The amplified genes were inserted in a vector and cloned using ligation and transformation. After cloning the cultures were plated on ampicillin agar. Success colonies were then picked and a mini Prep was used to prepare for sequencing. ABSTRACT This project was designed to locate the knockout gene responsible for causing two mutant strains of barley to be susceptible to powdery mildew. After bioinformatics analysis, primers were designed to target genes thought to be involved in defense. PCR was performed to locate the deleted gene in both mutants. Simultaneously, an F2 (wild type x mutant) population was grown and phenotypes were recorded for several days after inoculation with fungus to identify mutant and wild type individuals. After a deletion was identified, the F2 generation was screened for co-segregation. This screen supports the deletion as responsible for causing susceptibility. Dennis Vaughn1,John Jackson1, Matt Moscou2,4,Karin Werner2,4,Yan Meng3, Pingsha Hu3, Rico Caldo3 and Roger Wise3,41 - Secondary Biology Teacher Intern, Iowa State University Plant Pathology, Iowa State University, Ames, IA 500112 - Technician-USDA, Department of Plant Pathology, Iowa State University, Ames, IA 500113 - Department of Plant Pathology and Center for Plant Responses of Environmental Stresses, Iowa State University, Ames, IA 500114 - Corn Insects and Crop Genetics Research, USDA-ARS, Iowa State University, Ames, IA 50011 Discussion We identified differentially expressed genes between M100 and rom1. Fourteen genes were physically mapped to chromosome 2(2H). We utilized the ELP algorithm MarkerMaker to place 6,425 genes on the genetic map. Two genes were identified that mapped proximal to the centromere from marker MWG503 and were differentially expressed between m100 and rom1. We are currently sequencing PCR products and will continue our investigation. Extract DNA from tissue Design PCR primers PCR QIAquick PCR Purification / Gel Extraction Ligation References Freialdenhoven, Andreas. (2005) Barley Rom1 Reveals a Potential Link Between Race-Specific and Nonhost Resistance to Powdery Mildew Fungi. Cho, Seungho. (2006) Transcriptome Analysis and Physical Mapping of Barley Genes in Wheat-Barley Chromosome Addition Lines Transformation Plating Picking Colonies PCR Validation Overnight Cultures Mini-Prep Sequence Acknowledgement I would like to thank the Plant Genomics Outreach Program at Iowa State University especially to Adah Leshem-Ackerman and Jonathan Wendel for their support. In addition, I would like to thank the Biotechnology Outreach Education Center and the Office of Biotechnology for their generous assistance with equipment, teaching and support. Mostly, I would like thank the members of the Wise Lab including my mentor Roger Wise, Rico Caldo, Yan Meng, Pingsha Hu, Karin Werner, and especially Matt Moscou for their support, guidance, and patience. Research Question/Hypothesis To determine if the gene identified as the deletion is responsible for the necrosis phenotype associated with susceptibility to white powdery mildew.