Preliminary Results

1. Figure 3.A) PCR amplification of the pGEM vector using the T7 and T3 universal primers. B) Southern blot hybridization of the pGEM vector to tagged isolates (T1,2,6, and 7) of PRTUS3 (wild type P3). Cloning and Characterization of Avr1 from Puccinia triticina Pacheco and D.B. Hays  Texas A&M University, college station, TX, USA. Abstract Leaf rust is the most common and one of the most important cereal diseases of the world. Current leaf rust control has consisted of breeding for resistant cultivars by identifying Lr genes in the host. These cultivars quickly become susceptible to infection due to the tremendous extant genetic diversity of the pathogen that allows it to overcome resistant cultivars in 2-4 years. Developments of alternate methods of control are limited since little is known about Puccinia’s genome and plant-pathogen interaction. Construction of a genome-wide physical map is important in order to fully understand the infection mechanism of the pathogen and its interaction with the host. In an effort to discover more about the genetic potential of leaf rust in terms of AVR and VIR gene regulation and create novel plant resistance breeding strategies in the future, we have proposed a study of the pathogen’s genome by constructing a BIBAC library and a physical map of the pathogen. The BIBAC library is being constructed from pathotype PRTUS 3 which has AVR-1 disrupted using T-DNA mutagenesis via particle bombardment. The characterization of the AVR-1 in the BIBAC library will serve as a point of reference for cloning heterologous AVR and VIR genes, and defining their regulation and mode of inheritance and recombination. • Pucciniaurediniospores were bombarded with construct. • Trnsformantsconfirmend by phyenotyping, PCR and southern blott. • Growth and collection of fungal tissue • Wheat Plants at the 2-3 leaf stage were inoculated with PRTUS3-Tag6. • Spore count was increased, collected and cryopreserved . • Germination of the spores was induced in a nonanol-containing solution. • The resulting mycelial mat was collected and used for nucleic acid isolation. • High molecular weight DNA was extracted • Construct a BIBAC library • HMW DNA will be fragmented by using restriction enzymes BamHI and HindIII . • PFGE Size selected fragments will be cloned using the vector pCLD04541. • The clones will be arrayed in 384-well microtitre dishes as glycerol stocks and stored at the TAMU GENEfindergenomic resources center. • Construct a BIBAC physical map • Clones will be selected from the library previously collected. • DNA will be isolated, purified and digested and end labeled with fluorescent dye and fractionated on a capillary electrophoresis based automatic sequencer. • The fingerprints will be visualized on an automatic DNA sequencer and digitized into a computer. • Clones will be assembled. • ESTs will be mapped to the physical map • Using the overgo hybridization technology combined with the probe pooling strategy. • To Characterize Avr 1 and identify homologous Avr genes • Contigs will be blotted onto a nylon membrane • Avr genes will be located via hybridization with the vector tag and will be sequenced. • Genes that flank the vector and that have sequence homology to known Avr/Vir genes will be used to design primers. • Transient expression of Avr • Genes will be cloned to expression vectors that will be used to transform Thatcher lines containing lr1 to determine recognition of Avr1 by resistance genes. • Transient expression assays will be performed for the cloned Avr gene/ GFP constructs in wheat leaves and Thatcher lines. • Thatcher and lines that do not contain the lr1 gene are expected to fluoresce where as those containing Lr 1 gene are not since the corresponding R gene kills the cell. Introduction Fungal pathogens of the genus Puccinia cause yield losses of millions of dollars annually, and are among the most economically important pathogens of wheat and other cereals. A major effort has been made to identify and characterize host genes that confer pathogen resistance. However, these efforts have proven to be futile since the tremendous genetic diversity of the pathogen allows it to overcome resistant cultivars in two to four years. Due to this diversity, different approaches are needed to create resistant cultivars. Although extensive studies in wheat have identified host resistance genes that may be used to develop resistant cultivars, the gap in knowledge of the genetic information and mechanism of infection of this pathogen hinders development of novel methods of host resistance since we do not understand the pathogen’s molecular interaction with the plant’s cell in which it modifies its metabolism to extract nutrients. Methodology Preliminary Results An Avr1 gene has been tagged and disrupted in the pathotype PRTUS3 via particle bombardment. Disruption using particle bombardment of pathotype PRTUS3 urediniospores using a modified pGEM vector as a tag construct was used. PRTUS3 contains avirulencegenes 1, 2a, 2b, 2c, 2d, 3b, 3c, 9, 11, 16, 17, 19, and 24. The wheat variety “Karl 92” contains Lr 1, 10, and 13. Disruption was phenotypicaly observed by infecting “Karl 92” with the pathogen. When this occurred virulent pustules were observed on the previously resistant plant indicating a change in the pathotype corresponding to the Avr 1 gene. Putative transformants were confirmed for the presence of plasmid by PCR and southern blot. These were collected and cryopreserved and are being used for inoculum. Discussion New strategies for development of effective methods of pathogen resistance are necessary to compete with the rapidly increasing resistant strains of rust. This can only be achieved by thoroughly understanding the molecular mechanism by which a pathogen grows and develops. Many genetic studies to completely understand the pathogen are necessary. By studying avr genes in this pathotype we can begin to understand the mechanisms of interaction and genetic manipulation in the cascade of events and the pool of expressed genes in a host/pathogen interaction. We will use this information to engineer durable host resistance by engineering the Avr1 gene itself into the appropriate elite wheat background or by manipulating basic metabolic processes and by developing non specific anti-fungal compounds. Furthermore, understanding leaf rust genome will not only be useful for eradication of wheat leaf rust but will also prove useful for the eradication cereal rusts and also other rust that plague economically important crops such as coffee and soybean that are seriously threatened by new emerging, aggressive strains of rust. Literature Cited Figure 1.A) Scheme for tagging leaf rust avirulence gene 1. B) Virulent pustule isolate TAG2 isolated from Karl92 inoculated with PRTUS3 urediniospores that were bombarded with a modified pGEM vector, and the reaction of PRTUS3 and TAG2 on Karl 92. PRTUS 3 WILDTYPE Infection rating P3 TAG2 • Cloutier, S., B. D. McCallum, C. Loutre, T. W. Banks, T. Wicker, C. Feuillet, B. Keller, and M. C. Jordan. 2007. Leaf rust resistance gene Lr1, isolated from bread wheat (Triticumaestivum L.) is a member of the large psr567 gene family. Plant Molecular Biology 65 (1-2):93-106. • Meksem, Khalid, and Günter Kahl. 2005. The handbook of plant genome mapping : genetic and physical mapping. Weinheim ; [Great Britain]: Wiley-VCH. • Webb, C. A., and J. P. Fellers. 2006. Cereal rust fungi genomics and the pursuit of virulence and avirulence factors. Fems Microbiology Letters 264 (1):1-7. • Wu, C., S. Sun, P. Nimmakayala, F. A. Santos, K. Meksem, R. Springman, K. Ding, D. A. Lightfoot, and H. B. Zhang. 2004. A BAC- and BIBAC-based physical map of the soybean genome. Genome Res 14 (2):319-26.