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The present invention provides a MAP kinase homologue gene, designated EhHOG, isolated fromEurotium herbariorum, a common fungal species from the extreme hypersaline environment of the Dead Sea, capable of improving tolerance of plants and other organisms to abiotic stresses such as osmotic, heat, dehydration, freezing-thawing, oxidative and salinity stress.

Inventors:Eibi Nevo, Weining Song, Yan Jin 




The present invention relates to a MAP kinase homologue gene, designated EhHOG, isolated from Eurotium herbariorum, a common fungal species from the extreme hypersaline environment of the Dead Sea, to vectors comprising it, and to transgenic plants and other organisms containing said gene. In particular, it relates to methods for conferring salt tolerance and other generalized stresses tolerance such as heat tolerance, oxidative tolerance osmotic tolerance and freezing-thawing tolerance on plants and other organisms.




The productivity of crops is greatly affected by salt stress. The progressive salinization of soil, estimated at around 20% of irrigated land, has made the genetic improvement of salt tolerance an urgent priority for the future of agriculture. Salt tolerant plants can facilitate use of marginal areas for crop production, or allow a wider range of sources of irrigation water.


The Dead Sea is one of the most saline lakes on earth (salinity about 340 g/l). The pioneering studies of Benjamin Elazari-Volcani in the 1930s on the biology of the Dead Sea revealed a variety of microorganisms, including red halophilic Archaea, unicellular green algae (Dunaliella parva Lerche), different types of bacteria, and possibly even protozoa (Volcani, 1944). Recently, filamentous fungi were isolated from surface water to 300 m depths down in Dead Sea (Buchalo et al., 1998; Nevo et al., 2003). The fungi isolated from the Dead Sea did not grow in undiluted Dead Sea samples. But these isolated fungi showed a remarkable salt tolerance and, in many cases, even had a requirement for high salt concentrations, making them halophilic. Eurotium herbariorum is the most common species isolated from Dead Sea water from the surface to 300 m in all investigated seasons (Kis-Papo et al., 2001). All these organisms needed to adapt to the extremely high salinity of the Dead Sea brines.


Exposure to high environmental osmolarity leads to dehydration in mammals; thus, consequently, cell viability decreases. To cope with this, the cells of both prokaryotic and eukaryotic microorganisms have developed mechanisms to adapt to severe osmotic changes in their environments, which is often called osmoregulation. To adapt to salt stress, microorganisms balance high external osmotic pressure by synthesizing and/or accumulating low-molecular mass compounds which are compatible with cellular function and do not inhibit the enzymes. Increased synthesis and/or accumulation of glycerol and other compatible solutes, mainly polyols, have been observed to be the major feature of fungi osmoregulation (Mager and Varella, 1993).


Eukaryotic organisms use different MAN kinase (MAPK) cascades to regulate various aspects of cellular function (Banuett, 1998; Gustin et al., 1998). MAPKs that specifically transmit environmental stress signals are also known as stress activated protein kinases. This pathway is called the high osmolarity glycerol (HOG) response pathway in Saccharomyces cerevisiae (Brewster et al., 1993). Members of this MAPK subfamily include Hog1 in S. cerevisiae, Spc1 (also called Sty1) in Schizosaccharomyces pombe, SakA in Aspergillus nidulans, and p38/JNK in the mammalian. Indeed, S. cerevisiae hog1 mutants are sensitive to high osmolarity, whereas spc1 mutations in S. pombe result in sensitivity to high osmolarity, heat shock, and oxidative stress. Activation of the HOG pathway increases the transcription of some proteins, including enzymes involved in glycerol synthesis (Albertyn et al., 1994; Norbeck et al., 1996). As a result, a high accumulation of glycerol inside the cell occurs and leads to increased internal osmolarity and restores the osmotic gradient between the cells and their environment (Akhtar et al., 1997; Norbeck & Blomberg, 1997). Therefore, HOG1 gene holds a key position in osmoadaptation of the yeast S. cerevisiae.


The presence of HOG1 homologous genes has been reported in fungal species (Degols et al., 1996), plants (Hirt, 1997), and animals (Marshall, 1994) indicating that this pathway is conserved among eukaryotes. However, no information is available in osmosensing signal transduction pathway in E. herbariorum.


In the past few years there have been large advancements in the identification of genes that are responsible for salt tolerance in halophytic plants. Today salt tolerant tomatoes have been produced using genes identified from Arabidopsis thaliana andSaccharomyces cerivisiae. Serrano and coworkers have identified two genes, HAL1 (Gaxiola et al., 1992) and HAL2 (Glaser et al., 1993) in Saccharomyces cerivisiae by selecting for genes whose overexpression leads to improved growth on saline conditions. A HAL1 homolog is present in plants, where it is induced by NaCl and abscisic acid, a plant hormone known to mediate adaptation of plants to osmotic stress (Murguia et al., 1995).


Another gene, calcineurin, or phosphoprotein phosphatase type 2B (PP2B), is a calmodulin-regulated enzyme found in many organisms, including yeast. Although its physiological functions are not well understood, it is known that yeast strains which do not contain active calcineurin proteins are more sensitive to growth inhibition by salt than are wild-type strains. Bacterial genes associated with salt tolerance have also been identified (Tarczynski et al., 1993).


The genetic manipulation of crop species with individual transgenes could lead to an improvement in tolerance level, which would be sufficient from a breeding point of view. Some transgenes, related mainly to the synthesis of osmolytes, have been introduced in tobacco (Traczynski et al., 1993) and Arabidopsis (Hayashi et al., 1997). In general, the expression of those transgenes seems to confer a low tolerance level to osmotic (water deficit) and/or salt (NaCl) stress. U.S. Pat. No. 5,859,337 describes isolation of two genes from Arabidopsis coding for STZ and STO polypeptides, capable of conferring salt tolerance to plants and other organisms. PCT Publication WO 03/097827 discloses salt-tolerant tomato plants comprising betaine aldehyde dehydrogenase (BADH) gene.


Despite the efforts toward cloning genes conferring tolerance to high saline condition, no single salt tolerance gene has been isolated from the Dead Sea microorganisms. The present invention addresses these needs.




The present invention relates to a MAP kinase homologue gene isolated from Eurotium herbariorum, herein designated EhHOG, the coding region of said EhHOG gene being represented by SEQ ID NO:1 herein, and to an isolated DNA having a nucleotide sequence differing from SEQ. ID NO: 1 in codon sequence due to the degeneracy of the genetic code. The gene EhHOG codes for the EhHOG protein of SEQ ID NO: 2.


The present invention further relates to plants and other organisms transformed with said EhHOG gene and expressing the EhHOG protein of SEQ ID NO: 2, which confers tolerance to abiotic stresses such as osmotic, high salinity, heat, freeze, dehydration, or oxidative stress