skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


Title: An artificial host system enables the obligate parasite Cuscuta campestris to grow and reproduce in vitro
Abstract Cuscuta campestris is an obligate parasitic plant that requires a host to complete its life cycle. Parasite–host connections occur via a haustorium, a unique organ that acts as a bridge for the uptake of water, nutrients, and macromolecules. Research on Cuscuta is often complicated by host influences, but comparable systems for growing the parasite in the absence of a host do not exist. We developed an axenic method to grow C. campestris on an artificial host system (AHS). We evaluated the effects of nutrients and phytohormones on parasite haustoria development and growth. Haustorium morphology and gene expression were also characterized. The AHS consists of an inert, fibrous stick that mimics a host stem, wicking water and nutrients to the parasite. It enables C. campestris to exhibit a parasitic habit and develop through all stages of its life cycle, including production of new shoots and viable seeds. The phytohormones 1-naphthaleneacetic acid and 6-benzylaminopurine affect haustoria morphology and increase parasite fresh weight and biomass. Unigene expression in AHS haustoria reflects processes similar to those in haustoria on living host plants. The AHS is a methodological improvement for studying Cuscuta biology by avoiding specific host effects on the parasite and giving researchers full control of the parasite environment.  more » « less
Award ID(s):
1645027
PAR ID:
10321486
Author(s) / Creator(s):
; ;
Date Published:
Journal Name:
Plant Physiology
ISSN:
1532-2548
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; ; ; ; ; ; ; et al (, G3: Genes, Genomes, Genetics)
    Wong, A (Ed.)
    Abstract We present the first chromosome-level genome assembly and annotation for the genus Cuscuta, a twining and leafless parasitic plant of the morning glory family (Convolvulaceae). C. campestris, the study species, is a widely studied model parasite, due in part to its worldwide occurrence as a weed of agricultural and natural plant communities. The species has served as a model parasite for studies of parasite biology, haustorium development, growth responses to chemical and light stimuli, gene content and expression, horizontal gene transfer, and interspecies RNA movement and has a recently developed transformation system. The 505 Mb (1C) genome is assembled into 31 chromosomes and supports annotation of 47,199 protein-coding genes, 214 small RNA loci (including 146 haustoria-specific miRNAs), and 3,238 interspecies mobile mRNA loci. C. campestris is a recent tetraploid with a high retention of duplicated genes and chromosomes, with less than 8% nucleotide divergence between homoeologous chromosomes. We also show that transformation of C. campestris with the RUBY marker system allows visualization of transformed Cuscuta-derived fluorescent mobile molecules that have entered the host stem. This genome, with an associated genome browser and BLAST server, will be of value for scientists performing fundamental research in a wide range of molecular, developmental, population, and evolutionary biology, as well as serve as a research tool for studying interspecies mobile molecules, generating genetic markers for species and genotype identification, and developing highly specific herbicides. 
    more » « less
  2. ; ; ; ; ; ; ; (, New Phytologist)
    Summary Cuscuta campestris, a stem parasitic plant, has served as a valuable model plant for the exploration of plant–plant interactions and molecular trafficking. However, a major barrier toC. campestrisresearch is that a method to generate stable transgenic plants has not yet been developed.Here, we describe the development of aCuscutatransformation protocol using various reporter genes (GFP, GUS, or RUBY) and morphogenic genes (CcWUS2andCcGRF/GIF), leading to a robust protocol forAgrobacterium‐mediatedC. campestristransformation.The stably transformed and regenerated RUBYC. campestrisplants produced haustoria, the signature organ of parasitic plants, and these were functional in forming host attachments. The locations of T‐DNA integration in the parasite genome were confirmed through TAIL‐PCR. TransformedC. campestrisalso produced flowers and viable transgenic seeds exhibiting betalain pigment, providing proof of germline transmission of the RUBY transgene. Furthermore, RUBY is not only a useful selectable marker for theAgrobacterium‐mediated transformation, but may also provide insight into the movement of molecules fromC. campestristo the host during parasitism.Thus, the protocol for transformation ofC. campestrisreported here overcomes a major obstacle toCuscutaresearch and opens new possibilities for studying parasitic plants and their interactions with hosts. 
    more » « less
  3. ; ; ; ; (, Plants)
    Cuscuta spp. are obligate parasites that connect to host vascular tissue using a haustorium. In addition to water, nutrients, and metabolites, a large number of mRNAs are bidirectionally exchanged between Cuscuta spp. and their hosts. This trans-specific movement of mRNAs raises questions about whether these molecules function in the recipient species. To address the possibility that mobile mRNAs are ultimately translated, we built upon recent studies that demonstrate a role for transfer RNA (tRNA)-like structures (TLSs) in enhancing mRNA systemic movement. C. campestris was grown on Arabidopsis that expressed a β-glucuronidase (GUS) reporter transgene either alone or in GUS-tRNA fusions. Histochemical staining revealed localization in tissue of C. campestris grown on Arabidopsis with GUS-tRNA fusions, but not in C. campestris grown on Arabidopsis with GUS alone. This corresponded with detection of GUS transcripts in Cuscuta on Arabidopsis with GUS-tRNA, but not in C. campestris on Arabidopsis with GUS alone. Similar results were obtained with Arabidopsis host plants expressing the same constructs containing an endoplasmic reticulum localization signal. In C. campestris, GUS activity was localized in the companion cells or phloem parenchyma cells adjacent to sieve tubes. We conclude that host-derived GUS mRNAs are translated in C. campestris and that the TLS fusion enhances RNA mobility in the host-parasite interactions. 
    more » « less
  4. ; ; ; ; (, The Plant Cell)
    Abstract Small regulatory RNAs can move between organisms and regulate gene expression in the recipient. Whether the trans-species small RNAs being exported are distinguished from the normal endogenous small RNAs of the source organism is not known. The parasitic plant Cuscuta campestris (dodder) produces many microRNAs that specifically accumulate at the host–parasite interface, several of which have trans-species activity. We found that induction of C. campestris interface-induced microRNAs is similar regardless of host species and occurs in C. campestris haustoria produced in the absence of any host. The loci-encoding C. campestris interface-induced microRNAs are distinguished by a common cis-regulatory element. This element is identical to a conserved upstream sequence element (USE) used by plant small nuclear RNA loci. The properties of the interface-induced microRNA primary transcripts strongly suggest that they are produced via U6-like transcription by RNA polymerase III. The USE promotes accumulation of interface-induced miRNAs (IIMs) in a heterologous system. This promoter element distinguishes C. campestris IIM loci from other plant small RNAs. Our data suggest that C. campestris IIMs are produced in a manner distinct from canonical miRNAs. All confirmed C. campestris microRNAs with documented trans-species activity are interface-induced and possess these features. We speculate that RNA polymerase III transcription of IIMs may allow these miRNAs to be exported to hosts. 
    more » « less
  5. ; ; ; ; ; ; ; ; (, mSphere)
    Moreno, Silvia N. (Ed.)
    ABSTRACT During their parasitic life cycle, through sandflies and vertebrate hosts, Leishmania parasites confront strikingly different environments, including abrupt changes in pH and temperature, to which they must rapidly adapt. These adaptations include alterations in Leishmania gene expression, metabolism, and morphology, allowing them to thrive as promastigotes in the sandfly and as intracellular amastigotes in the vertebrate host. A critical aspect of Leishmania metabolic adaptation to these changes is maintenance of efficient mitochondrial function in the hostile vertebrate environment. Such functions, including generation of ATP, depend upon the expression of many mitochondrial proteins, including subunits of cytochrome c oxidase (COX). Significantly, under mammalian temperature conditions, expression of Leishmania major COX subunit IV (LmCOX4) and virulence are dependent upon two copies of LACK , a gene that encodes the ribosome-associated scaffold protein, LACK ( Leishmania ortholog of RACK1 [receptor for activated C kinase]). Targeted replacement of an endogenous LACK copy with a putative ribosome-binding motif-disrupted variant (LACK R34D35G36 →LACK D34D35E36 ) resulted in thermosensitive parasites that showed diminished LmCOX4 expression, mitochondrial fitness, and replication in macrophages. Surprisingly, despite these phenotypes, LACK D34D35E36 associated with monosomes and polysomes and showed no major impairment of global protein synthesis. Collectively, these data suggest that wild-type (WT) LACK orchestrates robust LmCOX4 expression and mitochondrial fitness to ensure parasite virulence, via optimized functional interactions with the ribosome. IMPORTANCE Leishmania parasites are trypanosomatid protozoans that persist in infected human hosts to cause a spectrum of pathologies, from cutaneous and mucocutaneous manifestations to visceral leishmaniasis caused by Leishmania donovani . The latter is usually fatal if not treated. Persistence of L. major in the mammalian host depends upon maintaining gene-regulatory programs to support essential parasite metabolic functions. These include expression and assembly of mitochondrial L. major cytochrome c oxidase (LmCOX) subunits, important for Leishmania ATP production. Significantly, under mammalian conditions, WT levels of LmCOX subunits require threshold levels of the Leishmania ribosome-associated scaffold protein, LACK. Unexpectedly, we find that although disruption of LACK’s putative ribosome-binding motif does not grossly perturb ribosome association or global protein synthesis, it nonetheless impairs COX subunit expression, mitochondrial function, and virulence. Our data indicate that the quality of LACK’s interaction with Leishmania ribosomes is critical for LmCOX subunit expression and parasite mitochondrial function in the mammalian host. Collectively, these findings validate LACK’s ribosomal interactions as a potential therapeutic target. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email