Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
1997-05-05
2001-01-16
Bui, Phuong T. (Department: 1645)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S069100, C435S071100, C435S071200, C435S410000, C435S320100, C435S243000, C435S252330, C435S006120, C435S418000, C435S419000, C435S252300, C435S252100, C530S350000, C536S023700, C536S023100, C536S024500, C800S278000, C800S279000, C800S288000
Reexamination Certificate
active
06174717
ABSTRACT:
Partial support for the research which led to the making of the present invention was provided by funds from the United States Department of Agriculture. Accordingly, the United States government has certain statutory rights to this invention under 35 USC 200 et seq.
Plants, as well as humans and animals, suffer injury and losses due to infection by bacteria. On a worldwide basis, bacteria classified in the genera Erwinia, Pseudomonas and Xanthomonas are responsible for most losses due to bacterial plant pathogens. Many of the bacterial diseases of plants cause farmers great losses on a sporadic basis. The losses result from death, disfigurement or reduced productivity of affected plants.
Many bacterial pathogens of plants exhibit a marked degree of specificity towards the plants that they infect. For example,
Erwinia amylovora
infects apples, pears and related plants of the family Rosaceae. Other plants do not become diseased when exposed to
E. amylovora
. However, when sufficient cells of
E. amylovora
are introduced into leaf tissue of the other plants, the mesophyll tissue collapses within hours. This collapse has been called the hypersensitive response (HR), and it is considered a defense reaction of plants since, during the HR, the bacteria are delimited within the collapsed tissue, eventually die, and thus do not cause much damage to the plant as a whole.
The genes that bacterial plant pathogens require for HR-eliciting ability, are called hrp genes, for hypersensitive reaction and pathogenicity, are also required for causing disease. However, the products of hrp genes and how they function in elicitation of the HR, and in disease development, remained unknown prior to the present invention. The present invention concerns products of hrp genes (elicitors) that are responsible for the collapse seen in the HR and are required for disease development.
Interactions between bacterial pathogens and their plant hosts generally fall into two categories: (1) compatible (pathogen-host), leading to intercellular bacterial growth, symptom development and disease development in the host plant; and (2) incompatible (pathogen-nonhost), resulting in the hypersensitive response, a particular type of incompatible interaction occurring, without progressive disease symptoms. During compatible interactions on host plants, bacterial populations increase dramatically and progressive symptoms occur; during incompatible interactions bacterial populations do not increase, and progressive symptoms do not occur.
The hypersensitive response of higher plants is characterized by the rapid, localized collapse and death of tissues containing an incompatible pathogen (a microorganism that is pathogenic only on other plants) and is associated with the defense of plants against many bacteria, fungi, nematodes, and viruses [see Phytopathogenic Prokaryotes, (M. S. Mount and G. H. Lacy eds.) Academic Press, New York. pp 149-177 (1982)]. Elicitation of the hypersensitive response by bacteria was first demonstrated in 1963 when the intercellular spaces of tobacco leaves were infiltrated with 10
7
cells/ml of an incompatible pathogen. The infiltrated areas collapsed within 24-48 hours and ceased to support bacterial multiplication [see Nature 199:299 (1963)]. Thus, in the HR, the pathogen is localized and further growth is restricted.
The technique used in the laboratory to demonstrate the HR is straight-forward. The intercellular spaces of tobacco leaves are infiltrated by first puncturing a sector on a leaf with a common straight dissecting needle. Then a 1-ml capacity syringe (without a needle), containing 0.1-0.5 ml of a bacterial cell suspension (usually 10
7-10
8
viable cells/ml) of bacteria is pressed against one side of the leaf directly over the puncture. While pressing a finger on the opposite side of the leaf to stabilize it and to prevent liquid from leaking out of the punctured area, the syringe plunger is pressed gently to introduce the bacterial suspension into the leaf. Infiltration is considered successful when a water-soaked area approximately 1-4 cm
2
appears in the leaf.
A common hypothesis proposed to explain the mechanism of hypersensitive reaction induction involves the production by bacteria of a specific elicitor that reacts with a specific receptor on the plant cell. However, the molecular basis (gene and gene product) for this response to potential pathogens had been unknown prior to the present invention in spite of continued research by plant pathologists since the HR first was described in 1963.
Physiological and genetic observations suggest that the same bacterial factor that elicits the hypersensitive response in nonhosts is also required for pathogenicity in hosts.
Production of the elicitor of the hypersensitive response is controlled by a cluster of several hrp genes, which are highly conserved, and often interchangeable, among many species of plant pathogenic bacteria. Although individual and several hrp genes have been cloned by others, functional clusters of hrp genes have been cloned only from
Erwina amylovora
and
Pseudomonas syringae
. These clusters have been shown to confer on nonpathogenic bacteria the ability to elicit the hypersensitive response in tobacco and other leaves [see Mol. Plant-Microbe Interact. 4:132 (1991); J. Bacteriol 170:4748 (1988); and Beer et al., Advances in Molecular Genetics of Plant-Microbe Interactions (H. Hennecke and D. P. S. Verma eds.) Kluwer Academic Publishers, Boston, pp 53-60 (1991)].
The elicitor, according to the present invention, was initially isolated and purified from
E. coli
DH5&agr;(pCPP430), and later from a wild-type strain of
E. amylovora
, the bacterium that causes a disease of rosaceous plants, such as apple and pear, known as fire blight. According to the present invention, the name “harpin” is proposed for the hypersensitive response elicitor from
E. amylovora
; this elicitor is considered to be the archetype for a family of proteinaceous HR elicitors that are produced by many different phytopathogenic bacteria.
It is thus one aspect of this present invention to describe specific elicitor proteins isolated from bacteria, which when applied to nonhost plants, cause a toxic response that is similar to the response elicited by living cells of the bacteria that produced the proteins. A further aspect of this present invention is to isolate and describe the genes that encode the elicitor proteins, which might be used to cause plants or other organisms to produce elicitor protein, which would exert its toxic effects in a precise controlled manner.
A further aspect of this present invention is to provide sufficient characterization, and identification of these proteins to allow design and development of techniques that will inactivate, destroy, or bind with these proteins. This aspect is desirable because it is known the same proteins are required by the bacteria that produce them in order to cause disease in host plants of the bacteria. Neutralizing the toxic effects of the proteins neutralizes their roles in disease and reduces disease in plants.
A still further aspect of the present invention is to develop antibodies against these proteins, sequence the antibodies produced, construct nucleic acid sequences which when inserted properly into the genome of a plant would cause the plant to express the antibody and thus prevent bacteria from causing disease in plants.
One portion of the present invention is based on the identification of a particular hrp gene of the hrp gene cluster of
Erwinia amylovora
. That particular gene is transcribed and translated to yield the proteinaceous elicitor of the of the hypersensitive response. Another portion of the present invention deals with the identification of homologous genes from Erwinia, Xanthomonas, and Pseudomonas species that encode similar proteins to the HR elicitor from
E. amylovora
. Prior to the making of the present invention, the isolation of a proteinaceous elicitor of the hypersensitive response had not been reported. Thus, ano
Bauer David W.
Beer Steven V.
Collmer Alan
He Sheng-Yang
Laby Ron
Bui Phuong T.
Cornell Research Foundation Inc.
Nixon Pebody LLP
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