Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1998-03-09
2002-05-14
Fox, David T. (Department: 1649)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C435S069100, C435S320100, C435S418000, C435S419000, C435S470000, C536S023600, C536S023700, C800S279000, C800S288000, C800S293000, C800S298000, C800S320000
Reexamination Certificate
active
06388175
ABSTRACT:
FIELD OF THE INVENTION
THIS INVENTION relates to the control of leaf scald disease and the inactivation of the phytotoxin albicidin in plants and particularly in sugarcane.
BACKGROUND ART
Leaf scald is a major disease of sugarcane which occurs in more than 50 countries (Chen et al., 1991, Report of the Taiwan Sugar Research Institute 0 (132), 19-27; Comstock and Shine, 1992, Plant Disease 76 (4), 426; Grisham et al., 1993, Plant Disease, 77 (5), 537; Irvine et al., 1993, Plant Disease, 77 (8), 846). The causal agent has been identified as
Xanthomonas albilineans. X. albilineans
produces a family of antibiotics and phytotoxins called albicidins. Albicidins selectively block DNA replication in bacteria and chloroplasts. Albicidin is the subject of U.S. Pat. No. 4,525,354. Mutants of
X. albilineans
which do not produce albicidins do not produce chlorotic or any systemic disease symptoms in inoculated sugarcane (Birch and Patil, 1987, Physiol. Molec. Plant Pathol., 30, 199-206). This indicates that albicidins are responsible for the chlorotic symptoms on
X. albilineans
infected sugarcanes, and play an important role in sugarcane leaf scald disease.
Two different mechanisms of albicidin resistance have been identified in bacteria. One mechanism involves the loss of cell permeability in some
Escherichia coli
mutants to albicidin (Birch et al., 1990 J. Gen. Microbiol., 136, 51-58). The other involves the inactivation of albicidin by the formation of a reversible protein-albicidin binding complex. This formation of a reversible binding complex has been shown in
Klebsiella oxytoca
to involve the albicidin resistance protein AlbA (Walker et al., 1988, Molec. Microbiol., 2 (4), 443-454) and in
Alcaligenes denitrificans
(Basnayake and Birch, 1995, Microbiology, 141) to involve the albicidin resistance protein AlbB. Unfortunately, however, these proteins do not irreversibly inactivate albicidin and consequently are not considered to be efficacious candidates for controlling leaf scald disease.
Leaf scald disease is an economically important disease and causes a large commercial loss in the sugarcane industry where susceptible cultivars are grown. As a result, ways of effectively combatting the disease are of great economic significance. For example, leaf scald resistance in plants is an essential requirement for every commercial Australian sugarcane variety. Selection for this resistance has unavoidably had a significant impact on the breeding program by reducing the value of some desired crosses and leading to the rejection of what would be otherwise outstanding new varieties. It takes about 10 years for breeding a new sugarcane variety and rejection of one variety in the final stage of the breeding program would cost the industry around $1 million. The recent development of a sugarcane genetic transformation system (Franks and Birch, 1991, Aust. J. Pit. Physiol., 18, 471-480); Bower and Birch, 1992, Plant J., 2, 409-416) has enabled the molecular improvement of sugarcane varieties and provided a supplementary mechanism to the conventional breeding programs.
SUMMARY OF THE INVENTION
The current invention arises from the unexpected discovery of an albicidin detoxification enzyme produced from a bacterium. It was further found that the albicidin detoxification enzyme was secreted extracellularly. Unlike the previously described albicidin binding protein AlbA and AlbB, inactivation of albicidin by the enzyme was irreversible in the sense that albicidin toxin activity was not restored upon protein denaturing treatment such as boiling. The bacterium that produced the albicidin detoxifying enzyme was identified as a strain of
Erwinia herbicola
also known as
Pantoea dispersa.
It is therefore an object of the invention to provide an albicidin detoxification enzyme for use in treating plants infected with leaf scald disease or reducing the probability of plants becoming infected with leaf scald disease.
It is a further object of the invention to provide a DNA sequence encoding an albicidin detoxification enzyme for the generation of transgenic plants and plant cells which are substantially resistant to albicidin such that resistance to leaf scald disease is substantially effected. Thus, it is yet another object to provide a transgenic plant substantially resistant to leaf scald disease.
Accordingly, in one aspect of the invention, there is provided an albicidin detoxification enzyme.
The term “albicidin detoxification enzyme” as used herein refers to a protein which catalyses the conversion of an albicidin to one or more non-toxic products wherein subsequent removal or destruction of the protein does not result in restoration of the albicidin from the non-toxic product(s). Accordingly, a protein being an albicidin detoxification enzyme may be distinguished from a protein which inactivates albicidin merely by binding reversibly thereto (eg. AlbA and AlbB) by subjecting a mixture of the protein and an albicidin to a protein denaturation step such as boiling. If the protein is an albicidin detoxification enzyme, then albicidin activity lost or reduced upon treatment with the protein is not restored by protein denaturation. Such “enzymatic detoxification” is highly advantageous because it provides a more effective and substantially permanent protection against albicidin toxicity than other mechanisms mentioned heretofore which are reversed upon denaturation of a molecule which merely binds reversibly to albicidin. It will also be appreciated that enzymatic detoxification may be highly beneficial in that an albicidin detoxification enzyme can progressively detoxify multiple albicidin molecules in contrast to albicidin binding molecules which merely bind albicidin without catalytic breakdown or modification thereof.
The albicidin detoxification enzyme is preferably a hydrolase. A suitable albicidin detoxification enzyme includes, but is not limited to, an AlbD polypeptide comprising the sequence of amino acids as shown in
FIG. 3A
(SEQ ID NO:1).
Alternatively, the AlbD polypeptide is an “AlbD polypeptide homolog”. Thus, the invention contemplates polypeptides which are functionally similar to the AlbD polypeptide. Such polypeptides may contain conservative amino acid substitutions compared to the AlbD polypeptide of
FIG. 3A
(SEQ ID NO:1).
The AlbD polypeptide homolog may be obtained from any suitable organism such as a eukaryotic cell including a yeast cell. Preferably, the AlbD polypeptide homolog is obtained from a bacterium such as, for example, an Erwinia or Pantoea strain. Alternatively, the AlbD polypeptide or polypeptide homolog thereof may be obtained by first isolating a DNA sequence encoding a polypeptide of the AlbD type as for example described hereinafter.
An albicidin detoxification enzyme of the invention may be prepared by a procedure including the steps of:
(a) ligating a DNA sequence encoding an albicidin detoxification enzyme or biological fragment thereof into a suitable expression vector to form an expression construct;
(b) transfecting the expression construct into a suitable host cell;
(c) expressing the recombinant protein; and
(d) isolating the recombinant protein.
As used in this specification, an expression construct is a nucleotide sequence comprising a first nucleotide sequence encoding a polypeptide, wherein said first sequence is operably linked to regulatory nucleotide sequences (such as a promoter and a termination sequence) that will induce expression of said first sequence. Both constitutive and inducible promoters may be useful adjuncts for expression of an albicidin detoxification enzyme according to the invention. An expression construct according to the invention may be a vector, such as a plasmid cloning vector. A vector according the invention may be a prokaryotic or a eukaryotic expression vector, which are well known to those of skill in the art.
Suitable host cells for expression may be prokaryotic or eukaryotic. One preferred host cell for expression of a polypeptide according to the invention is a bacterium. The bacterium used may be
Esc
Birch Robert
Zhang Lianhui
Akin Gump Strauss Hauer & Feld L.L.P.
Fox David T.
Mehta Ashwin D.
The University of Queensland
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