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[sgn.git] / mason / secretom / detail / functional_screens.mas

<& /page/page_title.mas, title => 'Functional Screens to Identify Secreted Proteins' &>

<div class="page_introduction">
  <p>
  We have been developing and applying functional screens to identify
  genes encoding secreted proteins. The genes are identified based on
  the ultimate localization of the encoded protein, utilizing protein
  markers whose activities confirm targeting to the cell surface.
  </p>
</div>

<&| /page/info_section.mas, title => 'Yeast Secretion Trap (YST) Screen' &>

  <div style="width: 330px; margin: 0; margin-right: 2em; float: left" class="captioned_image caption_right">

    <img src="/documents/img/secretom/YST_control_170.jpg" />

    <p>
    Sucrose plate with no yeast colonies growing (negative control).
    </p>

  </div>

  <p>
  The YST screen can be used to test a specific cDNA of interest, or to
  screen a cDNA library.  The screen uses invertase, an enzyme that
  hydrolyzes sucrose to generate glucose and fructose, as a reporter
  gene/protein and yeast (<span class="species_binomial">S. cerevisiae</span>)
  as a host. If yeast cells are streaked onto plates containing sucrose
  as the sole carbon source, they must secrete invertase to break down
  the sucrose in order to grow, and so an invertase-deficient yeast
  mutant will be unable to grow.
  </p>


  <div style="width: 330px; margin: 0; margin-right: 2em; float: left" class="captioned_image caption_right">

    <img src="/documents/img/secretom/YST_transformed_170.jpg" />

    <p>
    Sucrose plate showing yeast colonies transformed with the YST vector library.
    </p>

  </div>


  <p>

  This system can also identify plasma membrane (PM)-spanning or
  PM-anchored proteins if the orientation of the fusion protein is such
  that the invertase component is external to the cell. The resulting
  yeast transformants can then be isolated and the plasmids containing
  the heterologous transgenes sequenced, allowing identification of the
  secreted protein. We typically also confirm the localization of
  candidates of interest by transient expression as GFP fusion proteins
  in onion epidermal cells, coupled with staining using the FM-64 dye to
  co-stain for the plasma membrane,

  </p>

</&>

<&| /page/info_section.mas, title => 'Necrosis Inducing Protein (NIP) Secretion Trap Screen' &>

  <div style="width: 350px; float: right; margin: 0 0 10px 20px;" class="captioned_image caption_left">

    <img src="/documents/img/secretom/N_benth_NIP_x210.jpg" />

    <p style="text-align: left">
    A <span class="species_binomial">Nicotiana benthamiana</span> leaf
    showing regions of HR indicating the presence of a secreted
    protein fused to NIP.
    </p>

  </div>

  <p>
  We have also developed a new technique termed the NIP secretion trap
  screen. Briefly, this involves ligating cDNAs in frame at the 5&rsquo;
  end of the DNA sequence encoding a gene termed necrosis-inducing
  protein (NIP) that has been truncated to remove the native secretory
  signal peptide. NIP was identified from the oomycete Phytophthora
  sojae, as a protein that induces a hypersensitive response (HR),
  resulting in cell death, in leaves of some plants when present in the
  apoplast. It does not cause HR when expressed in the plant
  cytosol. Constructs containing individual cDNA::Nip fusions are
  transformed into <span class="species_binomial">Agrobacterium
  tumefaciens</span>, which is then infiltrated into the apoplastic
  compartment of Nicotiana benthamiana leaves. Any proteins encoding SPs
  will be secreted from infected plant cells as NIP fusion proteins,
  causing a readily detectable HR.
  </p>

  <p>
  The Nip fusion assay is a convenient and rapid means to confirm
  secretion of proteins in planta and complements the YST screen.
  </p>

</&>

<&| /page/info_section.mas, title => 'Objectives', is_subsection => 0 &>

  <div style="width: 230px; margin: 0 0 10px 20px; float: right" class="captioned_image">

    <img src="/documents/img/secretom/onion_cyto_secreted_200.jpg" />

    <p style="text-align: left">
    A cytoplasmic protein (green) and a secreted protein (red) in
    plasmolyzed onion cells. Scale bar represents 50 microns.
    </p>

  </div>

  <ul>
    <li>
    Screen a range of tomato tissues for genes encoding secreted
    proteins, focusing particularly on fruit development and leaves at
    various stages of infections with <span class="species_binomial">
    Phytopthora infestans</span>.
    </li>
    <li>
    Annotate the corresponding genes and confirm their localization in
    the cell wall by transient expression as fluorescent fusion proteins
    in onion epidermal cells.
    </li>
    <li>
    Further examine the localization and trafficking pathways of
    candidates that show unexpected localization in the apoplast.
    </li>
  </ul>



  <p>
  To date we have identified both known cell wall localized proteins as
  well as a number of proteins whose presence in the apoplast is
  unexpected, based on functional annotation, or the absence of a
  predicted secretory signal peptide, suggesting alternative
  non-canonical secretion pathways, as have describe in yeast and
  mammalian cells. We have also identified several proteins that show
  dual localization in the wall an intracellular compartments, and we
  are now characterizing some of the complexity in the secretory
  pathway.
  </p>

</&>

<&| /secretom/section_templates/data_items.mas &>
- text: "Tomato (cv. Ailsa Craig) fruit (multiple growth and ripening stages): YST clones"
  ref: /download/data/secretom/Functional_screens/Tomato_fruit_YST_clones.xlsx
- text: "Tomato (cv. Ailsa Craig) leaves infected with Phytophthora infestans: YST clones"
  ref: /download/data/secretom/Functional_screens/Tomato_and_Pinfestans_YST_clones.xls
</&>

<&| /secretom/section_templates/publications.mas &>

Lee, S.J. and Rose, J.K.C. (2012) A yeast secretion trap assay for identification of secreted proteins from eukaryotic phytopathogens and their plant hosts. Methods in Molecular Biology 835:519-30.

Lee, S.-J. and Rose, J.K.C. (2011) Characterization of the plant cell wall proteome using high throughput screens. In The Plant Cell Wall. Methods and Protocols. Methods in Molecular Biology. 715: 255-272.

Yeom, S.-I., Baek, H.-K., Oh, S.-K., Kang, W.-H., Lee, S.-J., Lee, J.M., Seo, E., Rose, J.K.C., Kim, B.-D. and Choi, D. (2011) Title: Use of a secretion trap screen in pepper following Phytophthora capsici infection reveals novel functions of secreted plant proteins in modulating cell death. Molecular Plant Microbe Interactions 24: 671-684.

McCann, M.C. and Rose, J.K.C. (2010) Blueprints for building plant cell walls. Plant Physiology 153: 365.

Rose, J.K.C. and Lee, S.-J. (2010) Straying off the highway: trafficking of secreted plant proteins and complexity in the plant cell wall proteome. Plant Physiology 153: 433-436.

Mueller, L.A. et al. (2009) A snapshot of the emerging tomato genome sequence. The Plant Genome 2:  78-92.

Tyler, B.M. et al. (2006) Phytophthora genome sequences uncover evolutionary origins and mechanisms of pathogenesis. Science 313: 1261-1266.

Isaacson, T., Saravanan, R.S., He, Y., Damasceno, C.M.B., Catal&agrave;, C., Saladi&eacute;, M. and Rose, J.K.C. (2006) Sample extraction techniques for enhanced proteomic analysis of plant tissues.  Nature Protocols 1: 769-774.

Lee, S.-J. Kelley, B., Damasceno, C.M.B., St. John, B., Kim, B.-S. Kim, B.-D. and Rose, J.K.C. (2006) A functional screen to characterize the secretomes of eukaryotic phytopathogens and their hosts in planta.  Molecular Plant Microbe Interactions 12: 1368-1377.

Lee, S.J., Kim, B.-D. and Rose, J.K.C. (2006) Identification of eukaryotic secreted and cell surface proteins using the yeast secretion trap screen. Nature Protocols 1: 2439-2447.
</&>