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
2000-11-28
2004-09-14
McElwain, Elizabeth F. (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, C800S295000, C435S320100, C435S419000, C435S468000, C526S085000, C526S085000
Reexamination Certificate
active
06791007
ABSTRACT:
The present invention relates to stimulating a defence response in plants, with a view to providing the plants with enhanced pathogen resistance. More specifically, it has resulted from cloning of the barley Mlo gene, various mutant mlo alleles, and a number of homologues from various species. The Mlo gene has been isolated using a positional cloning approach which has never previously been successful in Barley. Details and discussion are provided below. Wild-type Mlo exerts a negative regulatory function on a pathogen defence response, such that mutants exhibit a defence response in the absence of pathogen. In accordance with the present invention, down-regulation or out-competition of Mlo function may be used to stimulate a defence response in transgenic plants, conferring increased pathogen resistance.
Mutations have been described in several plants in which defence responses to pathogens appear to be constitutively expressed. Mutation-induced recessive alleles (mlo) of the barley Mlo locus exhibit a leaf lesion phenotype and confer an apparently durable, broad spectrum resistance to the powdery mildew pathogen,
Erysiphe graminis
f sp hordei.
Resistance responses to the powdery mildew pathogen have been genetically well characterized (Wiberg, 1974; Søgaard and Jørgensen, 1988; Jørgensen, 1994). In most analyzed cases resistance is specified by race-specific resistance genes following the rules of Flor's gene-for-gene hypothesis (Flor, 1971). In this type of plant/pathogen interaction, resistance is specified by and dependent on the presence of two complementary genes, one from the host and one from the fungal pathogen. The complementary genes have been termed operationally (pathogen) resistance (“RA”) gene and avirulence gene, respectively. Most of the powdery mildew resistance genes (Mlx) act as dominant or semidominant traits (Jøgensen, 1994).
Monogenic resistance mediated by recessive (mlo) alleles of the Mlo locus is different. Apart From being recessive, it differs from race-specifc resistance to single pathogen strains in that (i) it confers broad spectrum resistance to almost all known isolates of the pathogen (ii) nlo resistance alleles have been obtained by mutagen treatment of any tested susceptible wild type (Mlo) variety, and (iii) mao resistance alleles exhibit a defence mimic phenotype in the absence of the pathogen (Wolter et al., 1993). Thus, the genetic data indicate the Mlo wild type allele exerts a negative rmedatory function on defence responses to pathogen attack.
Resistance mediated by alo alleles is currently widely used in barley breeding and an estimated 10 million hectares are annually planted in Europe with seeds of this genotype. A ‘mlo’ like, inherited resistance to powdery mildew in other cereal plants has not been reported so far although the fungus is a relevant pathogen in wheat (attacked by
rzysiphe graminiu
f sp
triticl
), oat (attacked by
E. g
. f sp
avenae
), and rye (attacked by
.E g
. f sp
secalis
). Because cereals are morphologically, genetically and biochemically highly related to each other (Moore et al., 1995), one would predict the existence of homologous genes in these species. The failure to have found a ‘mlo’ like, inherited resistance in wheat and oat is probably due to their hexaploid genomes, making it difficult to obtain by mutagenesis defective alleles in all six gene copies, and the chance of all such mutations occurring in Nature is remote. The failure to have found a mlo equivalent in other cereals is probably due to insignificant amount of mutational analysis in these species and complications as a result of their outbreeding nature (e.g. rye).
RFLP markers closely linked to Mlo on barley chromosome 4 were previously identified on the basis of a mlo backcross line collection containing mlo alleles from six genetic backgrounds (Hinze et al., 1991). The map position of Mlo on the basis of RFLP markers was consistent with its chromosomal localization as determined by a previous mapping with morphological markers (Jørgensen, 1977).
Having identified an ~3 cM genetic interval containing Mlo bordered by genetic markers, we decided to attempt to isolate the gene via positional cloning.
However, there is no documented example of a successful positional cloning attempt of a barley gene. We were faced with a number of difficulties.
Firstly, the genome of barley (5.3×10
9
bp/haploid genome equivalent; Bennett and Smith, 1991) has almost double the size of the human genome and because the total genetic map covers ~1.800 cM (Becker et al., 1995) we were confronted with a very unfavourable ratio of genetic and physical distances (1 cM corresponds to ~3 Mb).
Seconcly, a high resolution genetic map had to be constructed around Mlo enabling the positioning of linked markers with a precision of better than 0.1 cM.
Thirdly. we aimed to physically delimit the target gene and both flanking DNA markers on individual large insert genomic clones, a procedure later termed “chromosome landing”. (Tanksley er al., 1995). For this pupose, a complete barley YAC library from barley Megabase DNA had to be constructed with an average insert size of 500-600 kb, which was unprecedented.
Fourthly, we had to prepare unusual genetic tools that enabled us to identify the Mlo gene within a physcally delimited region without tne need for a time consuming. generation of barley transgenLc plants and testing of different candidate genes. We used for our studies ten characterized radiation- or chemically-induced mlo mutants (Jørgensen, 1992). For a conclusive chain of evidence of the gene isolation we decided to depend upon a functional restosation of the wild type Mlo allele starting out from characterized mlo defective alleles. For this purpose, we performed mlo heteroallelic crosses and isolated susceptible intragenic Mlo recominants. The sequence analysis of these proves the function of the described gene.
The cloning of the barley Mlo gene and bomlogues, including homologues from other plant species, gives rise to a number of practical applications, reflected in the varsous aspects of the present invention.
According to a first aspect of the present invention there is provided a nucleic acid molecule comprising a nucleotide sequence encoding a peptide with Mlo function. Those skilled in the art will appreciate that “Mlo function” refers to the ability to suppress a defence response, said defence response being race and/or pathogen independent and autonomous of the presence of a pathogen, such as, for example, the Mlo gene of barley, the Acd gene and the Lsd gene of Arabidopsis.
mlo mutations that down-regulate or disrupt functional expression of the wild-type Mlo sequence are recessive, such that they are complemented by expression of a wild-type sequence. Thus “Mlo function” can be determined by assessing the level of constitutive defence response and/or susceptibility of the plant to a pathogen such as, for example, powdery mildew or rust (e.g. yellow rust). Accordingly, a putative nucleotide sequence with Mlo function can be tested upon complementation of a. suitable mlo mutant. The term “mlo function” is used to refer to sequences which confer a mlo mutant phenotype on a plant.
The capitalisation of “Mlo” and non-capitalisation of “mlo” is thus used to differentiate between “wild-type” and “mutant” function.
A mlo mutant phenotype is characterised by the exhibition of an increased resistance against one or more pathogens, which is race and/or pathogen independent and autonomous of the presence of a pathogen.
The test plant may be monocotyledonous or dicotyledonous. Sutable monocots include any of barley, rice, wheat, maize or oat, particularly barley. Suitable dicots include Arabidopsis.
Nucleic acid according to the invention may encode a polypeptide comprising the amino acid sequence shown in
FIG. 2
, or an allele, variant, derivarive or mutant, or homologue, thereof.
Nucleic acid according to the present invention may have the sequence of a Mlo geae of barley, or be a mutant, variant (or derivative) or allele cf the sequence pr
Büschges Rainer
Groenendijk Johannes S.
Panstruga Ralph
Schulze-Lefert Paul M. J.
Simons Augustinus F. M.
Ibrahim Medina A.
McElwain Elizabeth F.
Nixon & Vanderhye PC
Plant Bioscience Limited
LandOfFree
Polynucleotide and its use for modulating a defence response... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Polynucleotide and its use for modulating a defence response..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Polynucleotide and its use for modulating a defence response... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3195205