Isolation of Candidate Genes in Host- Phelipanche aegyptiaca Interaction by Genome Walking

Introduction Broomrape (Phelipanche aegyptiaca) is one of the most important plant parasitic species which causes significant yield loss of different crops by colonizing roots and uptaking nutrients from the host plants. Haustoria attachment stage is the most important stage to study molecular mechanism of plant-parasite interaction. Identifying key genes in haustoria attachment stage may reveal novel strategies to control Broomrape. Transcriptome studies by Next-generation (high throughput, deep) sequencing have become an important tool in the molecular biology of plants in recent years. All stage-specific RNA-seq data are available on the plant parasite genome project database (http://ppgp.huck.psu.edu). Differential gene expression in haustoria attachment stage can detect candidate parasitism genes and contribute to understanding molecular basis of plant-parasite interaction. This information may reveal novel genetic strategies such as HIGS to control Phelipanche aegyptiaca efficiently.Materials and MethodsAnalysis of Transcriptome data from the plant parasite genome project database (http://ppgp.huck.psu.edu) at Haustoria attachment stage and imbibed seed stage revealed 391 gene transcripts with differential expression in these stages (unpublished data). Among these transcripts, four transcripts with unknown functions were detected with a high fold change in expression in the haustoria attachment stage. In order to predict possible roles of these transcripts in broomrape-host interactions, we used genome walking method to extend these transcripts. DNA was extracted from Phelipanche aegyptiaca stem using CTAB method. The quantity and quality of DNA samples were determined using the NanoDrop and agarose gel electrophoresis. DNA was digested by four restriction enzymes, DraI, EcoRV, StuI, PvuII. Four DNA libraries were purified using SDS protocol and ligated to GenomeWalker Adaptor (GenomeWalker Adaptor 1 and GenomeWalker Adaptor 2). Gene specific primers (GSPs) were designed using Primer3plus for Oa548, Oa3391, Oa1635, Oa424 transcripts. Primary PCR was done using gene-specific primer 1 (GSP1) and adaptor primer 1 (AP1). 1 µl of each primary PCR were diluted into 49 µl of deionized water. Diluted primary PCR products were used as template for Secondary PCR. Primary PCR was done to amplify the unknown sequence using gene-specific primer 2 (GSP2) and adaptor primer 2 (AP2). Secondary PCRs desired bands were extracted form agarose gel using Genet Bio k-8000 kit. Extracted products were ligated to pTG19-T vector. Recombinant vectors were cloned to Escherichia coli competent cells using heat shock procedure and then cultured on LB plates. Colonies that contain recombinant vectors were detected using blue-white screening. Colony PCR was done to confirm the presence of inserted sequences. Selected colonies were incubated in 37C in LB media containing 100 microgram per ml Ampicillin. Plasmid extraction was done by Silica procedure. After sequencing by M13F and M13R, complete sequences were assembled using CAP contig assembly software. Fgenesh online software was used to predict gene structure by selecting Arabidopsis thaliana as organism. Gene prediction was done by AUGUSTUS. Complete sequences were more analyzed using Blast, CDD, Phobius prediction, HMMER, InterProScan.Results and DiscussionIn this study we successfully extended the genomic sequences for two candidate transcript that showed increase expression in attachment stage. The sequence of Oa1635 and Oa424 transcripts was extended from the end of the 5`using the genome-walking method to determining the DNA sequence of unknown flank genomic regions and study the role of these sequences in plant-parasite interaction. Using this technique, 587bp and 165bp of new DNA sequences were obtained for Oa424 and Oa163, respectively. Analysis of homology using BLASTX algorithm for Oa424 showed 71.57% similarity (e-value: 2e-41) with unknown protein (XP_011081407.1) containing the transposase domain. Also, the results of CDD tool predicted the DDE_Tnp_ISL3 domain in position between 257 and 466 bp (e-value: 7.31e-14). For Oa1635 transcript, the results of homology analysis using BLASTX algorithm showed 72.73% (2e-9) similarity with retrovirus-related polyprotein sequence from transposon tnt 1-94 (GFP84907.1). As the regulatory function of proteins with mutant-like transposase domains, the two transcripts Oa424 and Oa1635 may play a key role in haustoria development and plant-parasitic interaction. Signal peptides have observed in these sequences suggesting that these transcripts encodes secretory proteins from haustoria to plant-parasite interaction.ConclusionBioinformatics analysis on extended sequence, identified transposase domains which may have regularity role in parasitic process such as haustoria development or penetration. These genes may play important roles in plant-parasite interaction and developing molecular strategies to control this parasitic plant.