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
2001-11-14
2004-06-01
Nelson, Amy J. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide confers pathogen or pest resistance
C800S278000, C800S298000, C800S320000, C800S320100, C800S320200, C800S320300, C435S419000, C435S468000, C435S320100, C536S023100, C536S023600, C536S024100
Reexamination Certificate
active
06743969
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the preparation and use of an isolated nucleic acid fragment in order to confer a resistance gene mediated defense response in plants against a fungus comprising in its genome virulent and/or avirulent AVR-Pita alleles. Chimeric genes incorporating such fragments or functionally equivalent subfragments thereof and suitable regulatory sequences can be used to create transgenic plants which can produce a resistance gene mediated defense response against a fungus comprising in its genome virulent and/or avirulent AVR-Pita alleles.
BACKGROUND OF THE INVENTION
Plants can be damaged by a wide variety of pathogenic organisms including viruses, bacteria, fungi and nematodes. The invasion of a plant by a potential pathogen can result in a range of outcomes: the pathogen can successfully proliferate in the host, causing associated disease symptoms, or its growth can be halted by the host defenses. In some plant-pathogen interactions, the visible hallmark of an active defense response is the so-called hypersensitive response (HR). The HR involves rapid necrosis of cells near the site of the infection and may include the formation of a visible brown fleck or lesion. Pathogens which elicit an HR on a given host are said to be avirulent (AVR) on that host, the host is said to be resistant, and the plant-pathogen interaction is said to be incompatible. Strains which proliferate and cause disease on a particular host are said to be virulent, in this case the host is said to be susceptible, and the plant-pathogen interaction is said to be compatible.
Genetic analysis has been used to help elucidate the genetic basis of plant-pathogen recognition for those cases in which a series of strains (races) of a particular fungal or bacterial pathogen are either virulent or avirulent on a series of cultivars of a particular host species. In many such cases, genetic analysis of both the host and the pathogen revealed that many avirulent fungal and bacterial strains differ from virulent ones by the possession of one or more avirulence (“avr” or “AVR”) genes that have corresponding “resistance” (R) genes in the host.
This avirulence gene-resistance gene model is termed the “gene-for-gene” model (Crute et al. (1985) pp 197-309 in: Mechanisms of Resistance of Plant Disease. R. S. S. Fraser, ed.; Ellingboe, (1981)
Annu. Rev. Phytopathol.
19:125-143; Flor, (1971)
Annu. Rev. Phythopathol.
9:275-296). According to a simple formulation of this model, plant resistance genes encode specific receptors for molecular signals generated by avr genes. Signal transduction pathway(s) then carry the signal to a set of target genes that initiate the host defenses. Despite this simple predictive model, the molecular basis of the avr-resistance gene interaction is still unknown.
The first R-gene cloned was the Hm1 gene from corn (
Zea mays
), which confers resistance to specific races of the fungal pathogen
Cochliobolus carbonum
(Johal et al., 1992
, Science
258:985-987). Hm1 encodes a reductase that detoxifies a toxin produced by the pathogen. Next to be cloned was the Pto gene from tomato (
Lycopersicon pimpinellifollium
) (Martin et al., 1993
, Science
262:1432-1436; U.S. Pat. No. 5,648,599). Pto encodes a serine-threonine protein kinase that confers resistance in tomato to strains of the bacterial pathogen
Pseudomonas syringae
pv. tomato that express the avrPto avirulence gene. Taking center stage now are R-genes that encode proteins containing leucine-rich-repeats (LRRs) (Jones and Jones, 1997
, Adv. Bot Res. Incorp. Adv. Plant Pathol.
24:89-167). Two classes of membrane anchored proteins with extracellular LRRs have been identified. One subclass includes R-gene products that lack a cytoplasmic serine/threonine kinase domain such as the tomato Cf-9 gene for resistance to the fungus
Cladosporium fulvum
(Jones et al., 1994
, Science
266:789-793, WO 95/18230), and the other subclass includes an R-gene product with a cytoplasmic serine/threonine kinase domain, the rice Xa-21 gene for resistance to the bacterial pathogen
Xanthomonas oryzae
(Song et al., 1995
, Science
270:1804-1806; U.S. Pat. No. 5,859,339). The largest class of R-genes includes those encoding proteins with cytoplasmic LRRs such as the Arabidopsis R-genes RPS2 (Bent et al., 1994
, Science
265:1856-1860; Mindrinos et al., 1994
, Cell
78:1089-1099) and RPM1 (Grant et al., 1995
, Science
269:843-846). These R-proteins also possess a putative nucleotide binding site (NBS), and either a leucine zipper (LZ) motif or a sequence homologous to the Toll/Interleukin-1 receptor (TIR). Table 1 has been reproduced in part from Baker et al. (1997
, Science
276:726-733) as a concise summary of classes of R-genes cloned to date and examples of cloned genes within each class.
TABLE 1
Isolated plant resistance genes
1
Class
R gene
Plant
Pathogen
Structure
1
RPS2
Arabidopsis
Pseudomonas syringae
LZ-NBS-LRR
pv. Tomato
RPM1
Arabidopsis
P. syringae
pv.
LZ-NBS-LRR
Maculicola
Prf
Tomato
P. syringae
pv. Tomato
LZ-NBS-LRR
N
Tobacco
Tobacco mosaic virus
TIR-NBS-LRR
L
6
Flax
Melampsora lini
TIR-NBS-LRR
M
Flax
M. lini
TIR-NBS-LRR
RPP5
Arabidopsis
Peronospora parasitica
TIR-NBS-LRR
I
2
Tomato
Fusarium oxysporum
NBS-LRR
2
Pto
Tomato
P. syringae
pv. Tomato
Protein kinase
3
Cf-9
Tomato
Cladosporium fulvum
LRR-TM
Cf-2
Tomato
C. fulvum
LRR-TM
HS1
pro-1
Sugar beet
Heterodera schachtii
LRR-TM
4
Xa21
Rice
Xanthomonas oryzae
LRR, protein
kinase
pv.
Oryzae
5
Hm1
Maize
Cochilobolus carbonum
,
Toxin
race 1
reductase
1
This Table has been reproduced in part from Baker et al. (1997, Science 276:726-733). References for each of the listed R-genes can be found in this review article.
Nucleotide binding sites (NBS) are found in many families of proteins that are critical for fundamental eukaryotic cellular functions such as cell growth, differentiation, cytoskeletal organization, vesicle transport, and defense. Key examples include the RAS group, adenosine triphosphatases, elongation factors, and heterotrimeric GTP binding proteins called G-proteins (Saraste et al., 1990,
Trends in Biochem. Science
15:430). These proteins have in common the ability to bind ATP or GTP (Traut, 1994
, Eur. J. Biochem.
229:9-19).
It has long been hypothesized that the rice blast system represented a classical gene-for-gene system as defined by H. H. Flor (Flor, 1971
, Annu. Rev. Phytopathol.
19:125-143). Genetic analyses needed to identify AVR-genes in the rice blast pathogen,
Magnaporthe grisea
, has been hampered by the low fertility that typifies
M. grisea
field isolates that infect rice. Genetic crosses between poorly fertile
M. grisea
rice pathogens and highly fertile
M. grisea
pathogens of other grasses (such as weeping lovegrass,
Eragrostis curvula
, and finger millet,
Eleusine coracana
) have provided laboratory strains of the fungus with the level of sexual fertility required for identifying AVR-genes (Valent et al., 1991
, Genetics
87-101). Rare fertile rice pathogens have since allowed demonstration of a one-to-one genetic or functional correspondence between blast fungus AVR-genes and particular rice R-genes (Silue et al., 1992
, Phytopathology
82:577-580).
Interest in the rice blast pathosystem is keen because rice blast disease, caused world-wide by the fungal pathogen
Magnaporthe grisea
(Hebert) Barr (anamorph
Pyricularia grisea
Sacc.), continues as the most explosive and potentially damaging disease of the rice crop despite decades of research towards its control. Manipulation of blast resistance genes remains one of the primary targets in all rice breeding programs, as fungal populations evolve to defeat deployed resistance strategies (See The Rice Blast Disease, 1994, ed. Zeigler, Leong and Teng, CAB International, Wallingford).
Commercial fungicide usage to supplement genetic control strategies began around 1915 when rice farmers used inorganic copper-based fungicides (Chapter 29 in The Rice Blast Disease, 1994, ed. Zeigler, Leong and Teng, CAB International, Wallingford). The fungicides used to control blast diseas
Bryan Gregory
Valent Barbara
E. I. du Pont de Nemours and Company
Ibrahim Medina A.
Nelson Amy J.
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