Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
2002-03-18
2004-07-27
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C800S287000, C800S298000, C800S290000, C800S319000, C536S023600, C536S023100, C435S468000, C435S419000, C435S320100
Reexamination Certificate
active
06768041
ABSTRACT:
REFERENCE TO SEQUENCE LISTING SUBMITTED ON COMPACT DISC
This application incorporates by reference in its entirety the Sequence Listing that is provided in duplicate on compact discs that accompany the application. Each CD contains the following file: 1020C2, having a date of creation of Mar. 18, 2002 and a file size of 2.90 MB.
FIELD OF THE INVENTION
This invention relates to the field of modifying the responses of plant cells to external signals, such as environmental changes, and developmental cues. More specifically, this invention provides isolated polynucleotides encoding polypeptides that are integrally located in plant cell membranes and that mediate cellular signaling processes.
BACKGROUND OF THE INVENTION
Plants progress through set developmental programs throughout the course of their lifetimes. This is particularly evident in embryogenesis and floral development. There are a variety of signal molecules produced by certain cells in the plant to which other cells, particularly in the meristematic regions, arc poised to respond. These signal molecules trigger distinct sets of developmental programs at specific times that lead to the formation of, for example, flowers or cotyledons. In addition to the programmed developmental pathways, plants are exposed to a variety of environmental stimuli such as changes in temperature and amount of sunlight, availability of water, wounding from mechanical injury and attack by pathogens. Environmental factors, such as exposure to light, heat, cold, drought, etc., activate the expression of genes and synthesis of proteins and other compounds essential for an appropriate response to the environmental signal and thereby, the healthy development of the plant. These responses, like the developmental pathways, are mediated by signal molecules (hereinafter referred to as ligands).
To respond to these ligands, plant cells produce surface receptor proteins that serve as sensors, regulators; and/or transducers of cell signals. The intracellular transduction of a signal is often transmitted via a phosphorylation cascade of molecules that culminates in the transcription of genes to elicit the appropriate cellular response either for normal development or against environmental challenge.
One major class of receptor proteins is the single-transmembrane family, of which there are several subclasses. These proteins are characterized by three domains: an extraccllular signal molecule recognition/binding domain, a single cell membrane-spanning domain and an intracellular signal transduction domain which is usually a protein kinase. Many, but not all, plant single transmembrane proteins belong to the subclass known as receptor-like kinases (RLKs). The intracellular kinase domains of plant RLKs are all serine/threonine protein kinases, while the extracellular domains of RLKs are of different types. One type of RLK is characterized by the presence of the extracellular S-domain, originally described in self-incompatibility-locus glycoproteins that inhibit self-pollination. The S-domain is recognized by an array of ten cysteine residues in combination with other conserved residues. Another class of RLKs has an extracellular domain distinguished by leucine rich repeats (LRR) that are involved in protein-protein interactions. Binding of ligands to the extracellular domain is followed by receptor dimerization, autophosphorylation and the activation of a series of intracellular proteins which serve to transduce the signal to the nucleus. The structure of plant RLKs is very similar to receptors found in cell signaling pathways in animal systems.
One example of a plant RLK is the Xa21 gene, which confers resistance to the plant pathogen
Xanthomonas oryzae
pv.
oryzae
race 6. This gene was cloned using genetic means comparing Xanthomonas-sensitive and resistant strains of rice (Song et al.,
Science
270:1804-1806, 1995), and has been subsequently shown to confer resistance to Xanthomonas in Arabidopsis. The 1025 amino acid protein shows a number of features with similarity to known protein domains including a NH
2
-terminal 23 amino acid residue signal peptide, indicating that the protein is directed to the plasma membrane. Amino acids 81 to 634 contain 23 imperfect copies of a 24-amino acid LRR. Amino acids 651 to 676 encode a 26-amino acid hydrophobic segment that is likely to form a membrane-spanning domain. The C-terminal amino acids contain a putative intracellular serine threonine kinase domain carrying 11 subdomains with all 15 invariant amino acids that are typical of protein kinases. Subdomains VI and VIII are indicative of serine-threonine phosphorylation specificity. Xa21 has strong similarities to other RLKs, such as the Arabidopsis receptor-like kinase proteins RLK5 (HAESA) and TMK1, showing conservation of both the LRR and protein kinase domains. It is not yet known to what protein Xa21 transduces its pathogen recognition signal.
Another family of membrane receptor molecules expressed by plant cells is histidine kinases (HKs). HKs have been known for some time in bacterial signal transduction systems, where they form one half of a two-component signaling system. The bacterial HK serves as a sensor molecule for extracellular signals, such as changes in osmoticum, nutrients and toxins. The HK autophosphorylates on a histidine residue in response to ligand binding. This phosphohistidine donates its phosphate group to an aspartate residue of the second member of the two component system, known as the response regulator (RR). The phosphorylated RR then goes on to further transduce the signal, by binding other proteins as regulatory subunits, thereby either activating or inactivating them, depending on the specific circumstance. Alternatively, the phosphorylated RR binds DNA in a sequence-specific manner, serving to directly activate specific genes which code for proteins that mediate the response to the extracellular stimulus. In certain cases, HKs have a composite structure. Specifically, these proteins contain RR domains at their carboxy termini. The phosphohistidine of the HK transfers its phosphoryl group to the active site aspartate residue of this RR domain. In these cases, since the RR domain is membrane-bound, the signal cannot be transduced directly by RR binding to DNA. Instead, histidine phosphotransfer (HPt) proteins serve to further transduce the signal. The phosphoaspartate of the composite HK/RR protein donates the phosphate group to an active site histidine in the HPt protein. The HPt phosphohistidine in turn donates the phosphate group to a true RR, which then modulates activities of other proteins or activates gene expression in response to the external signal.
Like bacteria, plant cells have several two-component signaling systems which consist of a sensor element HK and a RR. In addition, composite HK proteins with RR domains at their carboxy termini (hereinafter referred to as hybrid HK/RR proteins) are found in both bacteria and plants. The HK proteins are distinguished by well-conserved amino acid motifs that occur in a specific order. From the amino terminus, the conserved regions are identified as the H, N, G1, F and G2 boxes. These motifs are usually found within a 200-250 amino acid span of the protein. The G1, F and G2 boxes are thought to be involved in nucleotide binding. As in bacteria, upon receiving the extracellular ligand, the HK is autophosphorylated on the histidine residue contained in the H box. The phosphate group is subsequently transferred to the RR. Alternatively, some HKs constitutively autophosphorylate their histidine residues and this activity is suppressed by binding of the extracellular ligand. All HKs are believed to phosphorylate a RR, as an obligate part of signal transduction.
RRs are characterized by the absolute conservation of an aspartate which is phosphorylated by the phosphohistidine of the HK, and a conserved lysine residue. Unlike bacteria, RRs in plants have not been shown to bind DNA directly. Rather, all the plant RR's characterized to date appear to transduce the signal into prot
Nieuwenhuizen Nicolaas
Strabala Timothy
Baum Stuart F.
Fox David T.
Genesis Research and Development Corporation Limited
Sleath Janet
Speckman Ann W.
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