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United States Department of Agriculture

Agricultural Research Service

Title: Construction of a Phage Library of Ascochyta Rabiee

Authors
item White, David
item Chen, Weidong

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: March 20, 2006
Publication Date: May 20, 2006
Citation: White, D.J., Chen, W. 2006. Construction of a phage library of ascochyta rabiee. Meeting Abstract.

Technical Abstract: BACKGROUND and OBJECTIVES Identifying and isolating pathogenic determinants of Ascochyta rabiei will provide insights into our understanding of Ascochyta blight of chickpea. To that end, we recently developed an insertional mutagenesis technique using Agrobacterium-mediated transformation to generate random mutations in A. rabiei (3). Non-pathogenic mutants were identified after screening more than 500 transformants (3) using the mini-dome pathogenicity bioassay. However, because the genome sequence of A. rabiei is not available, a genomic library is needed in order to isolate a copy of the genes disrupted in the non-pathogenic mutants. A phage library representative of the genome would be useful for isolation and characterization of DNA regions of interest. The objective of this work is to generate a phage library of A. rabiei suitable for isolation and recovery of potential pathogenicity determinants. MATERIAL and METHODS Genomic DNA from strain AR628 of A. rabiei pathotype II was digested with the restriction enzyme ApoI. DNA fragments between 7,000 and 10,000 bps were ligated to pre-digested (EcoRI) Lambda ZAPII vector arms (Stratagene), packaged using Gigapack III extracts, and amplified in E. coli strain XL1-Blue. Ten randomly selected plaques were used to determine the average size of recombinant insert DNA after plasmid rescue using the ExAssist helper phage and excising the insert DNA. RESULTS A phage library consisting of approximately 1.7 x 106 recombinants was constructed, with each recombinant containing, on average, 8,000 bp of A. rabiei DNA. A single round of amplification of the library was performed to produce a final titer of 1 x 109 pfu/ml. Recombinant DNA can be rescued from the phage in the form of a plasmid and fungal DNA can be excised from the plasmid as an ApoI fragment. CONCLUSIONS A high titer phage library has been constructed from A. rabiei DNA. Recombinants of interests can be detected by probing plaques with labeled DNA. This library will be used as a target for probes representing insertion sites in the less or non- pathogenic transformants of A. rabiei. In addition, we recently detected in A. rabiei via PCR homologs to the fungal melanin biosynthesis gene pks1 and a virulence gene cps. Now these genes can be exploited by recovering full-length copies of each for determining their functions and potential roles in pathogenicity. This represents the first genomic library of A. rabiei.

Last Modified: 7/31/2014
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