Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Plant proteins – e.g. – derived from legumes – algae or...
Reexamination Certificate
1999-10-12
2001-06-26
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Plant proteins, e.g., derived from legumes, algae or...
C435S069100, C435S252300, C435S320100, C536S023100, C536S023600, C530S350000
Reexamination Certificate
active
06252049
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a cDNA clone, designated to icc13 (incompatible
Capsicurn annuuin
-
Colletotrichum gloeosporioides
interactions) gene and individual component; thereof including its coding region and its gene product; modification thereto; application of said gene, coding region and modifications thereto; DNA construct, vectors and transformed plants each comprising the gene or part thereof.
Plants are continuously threatened by fungi and other microbial pathogens and deploy a number of strategies to defend them against pathogen infection. The defense responses are complex and include the formation of antimicrobial phytoalexins (Van Etten et al., 1989) and pathogenesis-related (PR) proteins (Linthorst, 1991; Ponstein et al., 1994), and the deposition of lignin to cell wall (Dean and Kúc, 1988). One of the most efficient ways of disease resistant reactions against potential pathogens is the hypersensitive reaction (HR). HR prevents further invasion and colonization of pathogens, which is known to be incompatible interactions between plant and pathogen (Goodman and Novacky, 1994).
Fruit ripening represents a genetically synchronized system that involves development unique to plant species. The phenomenon of ripening includes changes in color, texture, respiration rates, flavor, and aroma (Giovannoni, 1993). Generally, ripe fruit increases susceptibility to pathogen infection (Prusky et al., 1991; Swinburn, 1983). Therefore, fruits as one of the reproductive organs of plants need protection against pathogens to maintain fruit integrity and seed maturation. The expression of PR proteins is developmentally regulated during the formation of flower and fruit (Neal et al., 1990; Tattersall et al., 1997). Recently, several antifungal proteins that are responsible for protection against pathogens during fruit ripening were identified (Fils-Lycaon et al., 1996; Meyer et al., 1996; Salzman et al., 1998).
Colletotrichum gloeosporioides
(Penz) is the casual agent of anthracnose diseases on fruit crops (Daykin, 1984; Dodd et al., 1991; Prusky et al., 1991) such as pepper (
Capsicum annuum
L.) (Kim et al., 1986; Manandhar et al, 1995). In order to successfully infect the host plants, the fungus produces the appressorium from the germinating conidiunm Subsequently, an infection hypha from the appressorium penetrates the cuticle and epidermal cell of the host surfaces. In Colletotrichum fungi, the topography of the plant surface was thought to be a physical signal for inducing the appressorial formation (Staples and Macko, 1980). The surface wax of avocado fruit was reported to act as a chemical signal for the appressorial formation in
C. gloeosporioides
(Podila et al., 1993). The genes expressed during the appressorium formation of
C. gloeosporioides
induced by the host surface wax were cloned (Hwang and Kollatukudy, 1995). However, plant responses to the formation of appressorium and infection hypha of the fungus has not been studied.
In our previous study, we found that an isolate of
C. gloeosporioides
showed the incompatible interactions with ripe-red pepper fruit and the compatible interactions with unripe-mature-green fruit (Oh et al., 1998). To investigate the molecular mechanism involved in the incompatible interactions between pepper and
C. gloeosporioides
, we isolated the icc13 cDNA that is specifically expressed in the ripe fruit of pepper, but not in the unripe fruit, using mRNA differential display method. The expression of icc13 gene was studied based on fungal morphogenesis in the initial infection process during the incompatible interactions. We also found that the icc13 mRNA is induced by wounding and exogenous jasmonic acid during ripening.
SUMMARY OF THE INVENTION
The object of the present invention is to provide an isolated cDNA clone encoding for ICC13 protein from
Capsicum annuum
having nudeotide sequence as shown in SEQ ID No. 1, and an ICC13 protein from the incompatible interactions between plant and microorganism encoded by said cDNA sequence number 22 to 1005 of said cDNA clone having amino acid sequence as shown in SEQ ID No. 2.
Another object of the present invention is to provide a protein from the incompatible interactions between plant and microorganism, wherein the amino acid sequence has at least 80% homology to said ICC13 protein, and a protein from the incompatible interactions between plant and microorganism comprising pentapeptide motifs -His-Gly-Gly-ly-Phe- and -Gly-X-Ser-X-Gly- same as said protein, wherein X means any anuno acid.
The further object of the present invention is to provide a protein comprising the an-ino acid sequence obtained by the expression of a modified recombinant DNA sequence.
The present invention relates to a cDNA clone, designated to icc13, the sequence of which is depicted in SEQ ID No. 1. The anthracnose fuingus,
Colletotrichum gloeosporioides
, was previously shown to have incompatible interactions with the ripe fruit of pepper (
Capsicum annuum
). We isolated a novel gene icc13 from this incompatible interaction using mRNA differential display method. The amino acid sequence of the encoded protein (SEQ ID No. 2) showed limited but significant sequence homology to both lipases and esterases of prokaryotic organisms, and contains the HGGGF and GXSXG motifs as well as the catalytic triads of amino acid residues. The icc13 gene was highly expressed in the ripe fruit at 48 h after infection with the fungus, but was not expressed in the unripe fruit that showed compatible interactions. The examination of the fungal morphogenesis showed that infection hypha from appressorium invaded and colonized the epidermal cells of the unripe fruit, but not on those of the ripe fruit. These results suggest that the icc13 gene product may be involved in a defense mechanism against the fungal invasion and colonization after infection hypha penetrated into the outer-epidermal cells of the fruit. In addition, the icc13 mRNA was induced only in the ripe fruit by wounding and jasmonic acid treatment. The icc13 gene can be cloned into an expression vector to produce a recombinant DNA expression system suitable for insertion into cells to form a transgenic plant transformed with this gene. The icc13 gene of this invention can be also used to produce transgenic plants which exhibit enhanced resistance against phytopathogens, including fungi, bacteria, viruses, nematodes, mycoplasmalike organisms, parasitic higher plants, flagellate protozoa, and insects.
REFERENCES:
Salzman et al. Plant Physiol. 117 : 465-472 (1998).*
Meyer et al. Plant Physiol.. 112 : 615-622 (1996).
Ko Moon Kyung
Kostenyuk Igor
Oh Boung-Jun
Achutamurthy Ponnathapu
Kenyon & Kenyon
Korea Kumho Petrochemical Co. Ltd.
Saidha Tekchand
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