Transcripts
expressed during early haustorium development in Triphysaria.
In order to isolate transcripts expressed in parasite roots early in haustorium development, aseptic Triphysaria seedlings were exposed to Arabidopsis roots, host root exudates, or the purified haustorial inducing factors 2,6-dimethoxybenzoquinone or peonidin. Triphysaria roots were in contact with host factors from zero to five hours at which time root tips were harvested and mRNA isolated. As seen in the photo below, these times encompass the three earliest morphological features of haustorium development in Triphysaria: 1) the rapid (within 30 minutes) cessation of root tip cell division and elongation; 2) the initiation of epidermal cell expansion and development into haustorial hairs, and; 3) the isotropic expansion of cortical cells underlying the proliferating hairs. The haustorium penetrates the host root 12-24 hours after contact so the transcripts represented in these libraries are expressed prior to host invasion. cDNA libraries were prepared from two species of Triphysaria; the self pollinating T. pusilla and the out-crossing, self incompatible T. versicolor.
Early haustorium development in Triphysaria root tip exposed to host
root exudates
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Two types of cDNA libraries have been analyzed. “Full length” libraries were enriched for full length transcripts using Clontech SMART cDNA technology. The cDNA inserts were normalized for transcript abundance using the duplex-specific kamchatka crab nuclease. The cDNAs were cloned into the vector pDNR-LIB which allows transfer of cloned inserts into Cre-Lox acceptor vectors.
A second type of cDNA library used Suppressive Subtraction Hybridization (SSH) to enrich for transcripts either up or down regulated during early haustorium development. Nine SSH libraries were made from T. versicolor root tips exposed to Arabidopsis roots, Arabidopsis root extracts, or the purified haustorial inducers. Libraries enriched for root tip specific transcripts were similarly made. The SSH products were cloned into the Gateway entry vector pCR8/GW/topo.
The cDNA libraries were purified and bi-directionally sequenced by the DOE Joint Genome Institute as part of their Community Sequence Program. Trace files were base-called using Phred (version 0.990722.g) and low quality sequences were removed based on a Phred p value ≤ 0.05. The sequences were masked for vector, linker sequences, and repetitive sequences (excluding poly A and poly T) based on alignments generated by BLASTN program accessed through the PyMood Sequence Processor (Allometra, Davis, CA) (Alexander Kozik, pers. comm.). Sequences less than 100 nts were discarded from further analyses. Approximately five percent of the clones had linker sequences internal to an ORF sequence that were determined to be chimeras generated by the ligation of multiple SSH fragments into a single plasmid. The chimeric sequences were computationally digested into independent ESTs. The finished ESTs were submitted to NCBI’s dbEST repository.
ESTs were assembled into contigs using the cap3 program at default parameters. The contigs can be access below.
T. pusilla EST sequences from normalized, SMART library (59,549 reads)
T. pusilla contigs from normalized, SMART library (11,681 contigs)
T. versicolor EST sequences from SSH libraries (49,006 reads)
T. versicolor contigs from SSH libraries (6,364 contigs)