Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide confers pathogen or pest resistance
Reexamination Certificate
1997-08-11
2002-12-17
Benzion, Gary (Department: 1649)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide confers pathogen or pest resistance
C536S023600, C536S024100, C435S069100, C800S278000
Reexamination Certificate
active
06495737
ABSTRACT:
FIELD OF THE INVENTION
The present invention pertains to the fields of plant molecular biology. More specifically, the invention relates to induction of systemic acquired disease resistance in plants involving a novel salicylic acid-independent signal transduction pathway whose activation is associated with enhancement of a plant's ability to resist microbial infection.
BACKGROUND OF THE INVENTION
Several publications are referenced in this application in parentheses in order to more fully describe the state of the art to which this invention pertains. Full citations for these references are found at the end of the specification. The disclosure of each of these publications is incorporated by reference herein.
In plants, resistance to environmental challenges, e.g. pathogens, insects and stresses, enables them to better survive in nature, Resistance to pathogen attack is often associated with the hypersensitive response (HR), in which rapid, local cell death occurs at the infection site. The formation of these necrotic lesions is associated with the restriction of pathogen multiplication and spread. In the tobacco/tobacco mosaic virus (TMV) system, a typical HR develops on TMV-inoculated leaves of the tobacco cultivar Xanthi nc, which contains the N resistance gene. By contrast, when the TMV-susceptible cultivar Xanthi is infected, the virus spreads throughout the plant and causes disease symptoms. Several days after HR formation, systemic acquired resistance (SAR) develops throughout the plant (Ross, 1961a, 1961b; Ryals et al., 1994, 1996). SAR confers enhanced resistance not only to a secondary challenge by the initial infecting pathogen, but also to a broad range of other pathogens. Therefore, plant disease resistance is associated with both local (HR) and systemic (SAR) responses.
Activation of resistance responses is associated with the induction of a large number of defense genes and the synthesis of many secondary compounds. The former include genes encoding peroxidases, glucanases, chitinases, and other pathogenesis-related (PR) proteins while the latter include phytoalexins, phenolic compounds and lignin (Bowles, 1990; Cutt and Klessig, 1992). Phytoalexins are low molecular weight antimicrobial compounds that are synthesized by the plant and accumulate at the site of infection. While most phytoalexins are synthesized via the phenylpropanoid pathway, some are synthesized by the isoprenoid biosynthetic pathway (Ebel, 1986). Phenylalanine ammonia-lyase (PAL) is the first enzyme in the phenylpropanoid pathway and it regulates the biosynthesis of flavonoids, phytoalexins and lignins (Hahlbrock and Scheel, 1989; Dixon and Lamb, 1990). Levels of PAL mRNA and enzymatic activity increase in the inoculated tissue of resistant plants after pathogen infection. In addition, when tobacco plants are treated with a fungal elicitor, PAL mRNA accumulates in stem tissues (Pellegrini et al., 1994).
Plants resisting pathogen attack also synthesize a variety of PR proteins (Linthorst, 1991; Cutt and Klessig, 1992). In tobacco, at least five families of PR genes have been identified. They are induced in both the TMV-infected leaves and the upper uninoculated leaves of resistant cultivars (Ward et al., 1991). The function of some PR proteins is still unclear; however, several have been shown to have antimicrobial activities either in vivo or in vitro (Dempsey and Klessig, 1995).
Increases in salicylic acid (SA) levels also correlate with resistance to pathogen attack (Malamy et al., 1990; Metraux et al., 1990; Rasmussen et al., 1991). In tobacco resisting TMV infection, endogenous SA levels increase in both inoculated and uninoculated leaves. These increases correlate with the HR and SAR and precede the induction of PR genes (Malamy et al., 1990). A correlation between SA accumulation and resistance to pathogen attack has also been shown in other plants, e.g. cucumber and Arabidopsis (Metraux et al., 1990; Rasmussen et al., 1991; Uknes et al., 1993; Lawton et al., 1994; Summermatter et al., 1995; Dempsey et al., 1997). In addition, exogenously applied SA enhances resistance and induces nine gene families whose expression is associated with SAR in TMV-inoculated tobacco (Ward et al., 1991). Elevated SA levels also correlate with the activation of plant defenses in temperature shifted tobacco. At temperatures above 28° C., development of resistance to TMV is blocked and tobacco plants become systemically infected (Kassanis, 1952). At this elevated temperature, there is no detectable increase in SA levels, no PR protein synthesis and no HER after TMV infection (Gianinazzi, 1970; Yalpani et al., 1991; Malamy et al., 1992). However, when these infected tobacco plants are shifted to lower temperatures, resistance to TMV is restored. Moreover, a rapid and dramatic increase in SA levels precedes both PR gene expression and the appearance of a HR (Malamy et al., 1992).
Mutant analysis in Arabidopsis also provides support for SA's importance in disease resistance. Several mutants (e.g. npr1, nim1, and sai1), have been isolated which are unable to activate PR gene expression after treatment with SA or INA (2,6-dichloroisonicotinic acid, a synthetic functional analog of SA; Conrath et al., 1995; Durner and Klessig, 1995; Vernooij et al., 1995; Malamy et al., 1996). These mutants show greater susceptibility to bacterial and fungal pathogens (Cao et al., 1994; Delaney et al., 1995; Shah et al., 1997). conversely, mutants with elevated levels of SA, including the lesion mimic mutants acd2 and the 1sd series (Dietrich et al., 1994; Greenberg et al., 1994), as well as cpr1 (Bowling et al., 1994) and cep1 (Klessig et al., 1996), constitutively express these PR genes and exhibit enhanced resistance to pathogens.
Some of the strongest evidence supporting a signaling role for SA comes from the analysis of transgenic tobacco and Arabidopsis plants that contain the bacterial nahG gene encoding salicylate hydroxylase. Since salicylate hydroxylase degrades SA, these transgenic NahG plants are unable to accumulate SA. Following inoculation with TMV, NahG tobacco plants exhibit substantially reduced induction of PR gene expression and fail to develop SAR. Furthermore, they show enhanced susceptibility to infection with both virulent and avirulent pathogens (Gaffney et al., 1993; Delaney et al., 1994; Vernooij et al., 1994). Thus, SA appears to be required for an effective resistance response.
While SA mediates resistance to certain pathogens, other signal transduction pathways also appear to be involved in plant defense response. Both ethylene and jasmonates have been implicated as signal molecules that induce PR proteins during plant defense responses (Boller et al., 1983; Boller, 1991; Xu et al., 1994). Furthermore, several recent reports have shown that induction of a number of defense genes by pathogens, elicitors or abiotic agents (soluble sugars) is mediated by a SA-independent pathway(s) (Herbers et al., 1996; Penninckx et al., 1996; Pieterse et al., 1996; Vidal et al., 1997).
Compositions and methods that enhance disease resistance in plants are agronomically important and highly desirable. While SA is a key component in a plant's capacity to withstand environmental stresses, mounting evidence suggests that a second pathway leading to systemic acquired disease resistance exists. Thus, a need exists to identify and characterize the genes and encoded proteins involved in this novel, second pathway.
SUMMARY OF THE INVENTION
The present invention relates to a new signal transduction pathway in plants which is associated with and leads to the development of systemic acquired resistance (SAR). Activation of this pathway results in the induction of a diverse group of genes which are expressed in both uninfected and infected tissues in a salicylic acid (SA)-independent manner. These genes are termed SIS for SA-independent systemically induced.
According to one aspect of the invention, a method for identifying SIS genes is provided. The method uses the following steps: (a) providing pairs of plants, eac
Guo Ailan
Klessig Daniel F.
Benzion Gary
Dann Dorfman Herrell and Skillman
Rutgers The State University of New Jersey
Zaghmout Ousama M-Faiz
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