A full-length enriched cDNA library and expressed sequence tag analysis of the parasitic weed, Striga hermonthica

<p>Abstract</p> <p>Background</p> <p>The obligate parasitic plant witchweed (<it>Striga hermonthica</it>) infects major cereal crops such as sorghum, maize, and millet, and is the most devastating weed pest in Africa. An understanding of the nature of its parasitism would contribute to the development of more sophisticated management methods. However, the molecular and genomic resources currently available for the study of <it>S. hermonthica </it>are limited.</p> <p>Results</p> <p>We constructed a full-length enriched cDNA library of <it>S. hermonthica</it>, sequenced 37,710 clones from the library, and obtained 67,814 expressed sequence tag (EST) sequences. The ESTs were assembled into 17,317 unigenes that included 10,319 contigs and 6,818 singletons. The <it>S. hermonthica </it>unigene dataset was subjected to a comparative analysis with other plant genomes or ESTs. Approximately 80% of the unigenes have homologs in other dicotyledonous plants including <it>Arabidopsis</it>, poplar, and grape. We found that 589 unigenes are conserved in the hemiparasitic <it>Triphysaria </it>species but not in other plant species. These are good candidates for genes specifically involved in plant parasitism. Furthermore, we found 1,445 putative simple sequence repeats (SSRs) in the <it>S. hermonthica </it>unigene dataset. We tested 64 pairs of PCR primers flanking the SSRs to develop genetic markers for the detection of polymorphisms. Most primer sets amplified polymorphicbands from individual plants collected at a single location, indicating high genetic diversity in <it>S. hermonthica</it>. We selected 10 primer pairs to analyze <it>S. hermonthica </it>harvested in the field from different host species and geographic locations. A clustering analysis suggests that genetic distances are not correlated with host specificity.</p> <p>Conclusions</p> <p>Our data provide the first extensive set of molecular resources for studying <it>S. hermonthica</it>, and include EST sequences, a comparative analysis with other plant genomes, and useful genetic markers. All the data are stored in a web-based database and freely available. These resources will be useful for genome annotation, gene discovery, functional analysis, molecular breeding, epidemiological studies, and studies of plant evolution.</p

Agrobacterium rhizogenes-Mediated Transformation of the Parasitic Plant Phtheirospermum japonicum

Background: Plants within the Orobanchaceae are an agriculturally important group of parasites that attack economically important crops to obtain water and nutrients from their hosts. Despite their agricultural importance, molecular mechanisms of the parasitism are poorly understood. Methodology/Principal Findings: We developed transient and stable transformation systems for Phtheirospermum japonicum, a facultative parasitic plant in the Orobanchaceae. The transformation protocol was established by a combination of sonication and acetosyringone treatments using the hairy-root-inducing bacterium, Agrobacterium rhizogenes and young seedlings. Transgenic hairy roots of P. japonicum were obtained from cotyledons 2 to 3 weeks after A. rhizogenes inoculation. The presence and the expression of transgenes in P. japonicum were verified by genomic PCR, Southern blot and RT-PCR methods. Transgenic roots derived from A. rhizogenes-mediated transformation were able to develop haustoria on rice and maize roots. Transgenic roots also formed apparently competent haustoria in response to 2,6dimethoxy-1,4-benzoquinone (DMBQ), a haustorium-inducing chemical. Using this system, we introduced a reporter gene with a Cyclin B1 promoter into P. japonicum, and visualized cell division during haustorium formation. Conclusions: We provide an easy and efficient method for hairy-root transformation of P. japonicum. Transgenic marker analysis revealed that cell divisions during haustorium development occur 24 h after DMBQ treatment. The protocol

Genome Sequence of Striga asiatica Provides Insight into the Evolution of Plant Parasitism

Parasitic plants in the genus Striga, commonly known as witchweeds, cause major crop losses in sub-Saharan Africa and pose a threat to agriculture worldwide. An understanding of Striga parasite biology, which could lead to agricultural solutions, has been hampered by the lack of genome information. Here, we report the draft genome sequence of Striga asiatica with 34,577 predicted protein-coding genes, which reflects gene family contractions and expansions that are consistent with a three-phase model of parasitic plant genome evolution. Striga seeds germinate in response to host-derived strigolactones (SLs) and then develop a specialized penetration structure, the haustorium, to invade the host root. A family of SL receptors has undergone a striking expansion, suggesting a molecular basis for the evolution of broad host range among Striga spp. We found that genes involved in lateral root development in non-parasitic model species are coordinately induced during haustorium development in Striga, suggesting a pathway that was partly co-opted during the evolution of the haustorium. In addition, we found evidence for horizontal transfer of host genes as well as retrotransposons, indicating gene flow to S. asiatica from hosts. Our results provide valuable insights into the evolution of parasitism and a key resource for the future development of Striga control strategies.Peer reviewe

Optimization of factors influencing stable transformation of <i>P. japonicum</i>.

<p>Frequency of stable transformation in <i>P. japonicum s</i>eedlings submitted to different treatments. <b>A</b> Transformation efficiency in 3-day-old seedlings infected with strains ATCC15834, LBA1334 and AR1193 and co-cultivated for 2 or 7 days. The data show representative results from one of two independent experiments using 20 to 60 plants each. <b>B</b> Effect of acetosyringone (AS) on transformation efficiency. <i>S</i>eedlings were co-cultivated with <i>A. rhizogenes</i> strains ATCC15834 and LBA1334 in media with or without 100 µM AS for 2 days for LBA1334, and 7 days for ATCC15834. 4–5 weeks after the inoculation the transformation frequency was scored. The data show representative results from one of at least two independent experiments using 20 to 60 plants each.</p

Efficiency of stable transformation after the addition of Silwet L-77 and/or NAA into bacterial suspension.

<p>Efficiency of stable transformation after the addition of Silwet L-77 and/or NAA into bacterial suspension.</p

<i>Agrobacterium rhizogenes</i> strains.

<p><i>Agrobacterium rhizogenes</i> strains.</p

Detection of transgenes by PCR and RT-PCR.

<p><b>A-B</b> PCR analysis of genomic DNA isolated from <i>P. japonicum</i> fluorescent hairy roots (F) and non-transformed tissues (NT). <b>A</b>. Amplification of <i>GFP</i> and <i>rolB</i> fragments with expected sizes (380 bp and 780 bp, respectively). <b>B</b>. No amplification of <i>virD1</i> fragment (450 bp) in fluorescent tissues (F), as positive control a diluted ATCC15834 bacterial suspension (Ar) was used. <b>C</b>. Southern blot of genomic DNA extracted from fluorescent (F) and non-transformed (NT) <i>P. japonicum</i> roots. DNA was digested with <i>Eco</i>RI. The positive control (Pl) corresponds to linearised pBCR101 plasmid (30 ng). <b>D</b>. RT-PCR analysis of the <i>rolB</i> gene using total RNAs extracted from <i>P. japonicum</i> fluorescent roots (F) and non-transformed tissues (NT).</p

Transgenic root retains parasitic competence.

<p><b>A</b> and <b>B</b>. Haustorium development in transgenic hairy roots following 2-day exposure to 10 µM DMBQ observed under bright field (A) and fluorescent (B) microscopy. The white arrows point to haustoria developing on a transformed root and the black arrow points to a haustorium in a non-transformed root. <b>C</b> and <b>D</b>. Haustorial connection with host rice observed under bright field (C) and fluorescence (D) microscopy. <b>E</b> and <b>F</b>. Haustorial connection with host maize observed under bright field (E) and fluorescence (F) microscopy. White arrows indicate haustorial connection of transgenic roots to hosts. H: host, P: parasite. Bars correspond to 0.5 mm.</p

A flowchart for hairy root transformation in <i>P. japonicum</i>.

<p>A flowchart for hairy root transformation in <i>P. japonicum</i>.</p

Stable and transient transformation of <i>P. japonicum</i> mediated by <i>A. rhizogenes</i>.

<p>Transformation induced by <i>A. rhizogenes</i> ATCC15834. <b>A</b>. Five-week-old plants showing accumulation of black substance(s) at the wound site after hypocotyl-cutting infection method. <b>B</b>. Transformed roots emerged from cotyledons 5 weeks after <i>A. rhizogenes</i> inoculation by the SAAT method. The black arrow indicates transformed roots. <b>C</b> and <b>D</b> GFP-fluorescing transformed roots observed under bright field (C) or fluorescent (D) microscopy. The black arrows point to fluorescent roots and the white arrow to non-fluorescent root. <b>E</b> and <b>F</b> Transiently-transformed cotyledons observed under bright field (E) or fluorescent (F) microscopy. <b>G</b> and <b>H</b> Confocal micrograph of cotyledon leaves. Non-transformed (G) and transiently-transformed (H). Red color corresponds to autofluorescence from chlorophyll. White bars correspond to 2 mm and yellow bars to 20 µm.</p