Transcriptional responses in the hemiparasitic plant Triphysaria versicolor to host plant signals

Abstract

Parasitic plants in the Scrophulariaceae use chemicals released by host plant roots to signal developmental processes critical for heterotrophy. Haustoria, parasitic plant structures that attach to and invade host roots, develop on roots of the hemiparasitic plant Triphysaria versicolor within a few hours of exposure to either maize (Zea mays) root exudate or purified haustoria-inducing factors. We prepared a normalized, subtractive cDNA library enriched for transcripts differentially abundant in T. versicolor root tips treated with the allelopathic quinone 2,6-dimethoxybenzoquinone (DMBQ). Northern analyses estimated that about 10% of the cDNAs represent transcripts strongly up-regulated in roots exposed to DMBQ. Northern and reverse northern analyses demonstrated that most DMBQ-responsive messages were similarly up-regulated in T. versicolor roots exposed to maize root exudates. From the cDNA sequences we assembled a unigene set of 137 distinct transcripts and assigned functions by homology comparisons. Many of the proteins encoded by the transcripts are predicted to function in quinone detoxification, whereas others are more likely associated with haustorium development. The identification of genes transcriptionally regulated by haustorium-inducing factors provides a framework for dissecting genetic pathways recruited by parasitic plants during the transition to heterotrophic growth.

Figure 1

Time course of haustorium development. T. versicolor were grown on agar media in vertically oriented petri dishes so that their roots grew along the surface of the agar. The roots were then exposed to 10 μm DMBQ and their tips photographed at different times under 2× magnification with a dissecting microscope. The pictures in the figure are a time lapse of the same root. The arrowhead to the right in each picture represents the position of the root tip when the treatment was applied.

Figure 2

Northern analysis using early DMBQ-induced transcript (EDIT) probes. Northern filters were prepared from mRNA extracted from T. versicolor root tips exposed to either water (left lane [−]) or DMBQ (right lane [+]) for 5 h. Inserts of 30 cDNAs were PCR amplified, labeled with ³²P, and used as hybridization probes. The putative functions of these clones as predicted from virtual translations are given in Table I.

Figure 3

Transcription regulation by maize root exudate. A northern blot was prepared containing RNA isolated from T. versicolor roots mock treated with water (−) or with maize root exudate (+) for 4 h. The blot was sequentially probed with clones 3K19, 1H09, 1L17, and 3F08.

Figure 4

Probe interrogation of EDIT arrays. Inserts from 141 cDNA clones were spotted onto nylon membranes in duplicate. The same filter was sequentially hybridized with forward- and reverse-subtracted probes (A and B), and with non-subtracted probes made from complex root mRNA (C). In A and C, the probes were made from T. versicolor root tips exposed to DMBQ for 2 to 5 h. In B, the probes were made from T. versicolor root tips exposed to maize root exudate. The cDNAs spotted at each position is found in supplemental data online at www.plantphysiol.org.

Figure 5

Regulation of TvPirin by DMBQ. A northern blot containing RNA isolated from T. versicolor root tips 0, 0.5, 1, 5, and 24 h after exposure to DMBQ was probed sequentially probed with TvPirin (1L17) and an unsequenced, constitutively expressed cDNA.