# Patent

## Title
Powdery mildew resistance providing genes in Cucumis melo

## Abstract

The present invention relates to powdery mildew resistance providing genes of the Cucumis family, and especially Cucumis melo, wherein said resistance is provided by impairment of the present genes. Further, the present invention relates plants comprising the present impaired resistance conferring genes and seeds, embryos or other propagation material thereof. Especially, the present invention relates to powdery mildew resistance conferring genes, wherein the amino acid sequence encoded by said resistance conferring gene is selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12 and SEQ ID No. 14, and amino acid sequences with more than 70% identity, preferably more than 80% identity, more preferably more than 90% identity, and most preferably more than 95% identity.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the United States national phase of International Application No. PCT/EP2012/053208 filed Feb. 24, 2012, and claims priority to International Application No. PCT/EP2011/053053, filed Mar. 1, 2011, the disclosures of which are hereby incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to powdery mildew resistance providing genes of Cucumis melo, wherein said resistance is provided by impairment of the present genes either at the expression or protein level. Further, the present invention relates to plants comprising the present resistance conferring genes and seeds, embryos or other propagation material thereof.
Powdery mildew (PM) is one of the main fungal diseases known in plants belonging to the Cucumis family such as Cucumis melo (melon), both in the field and greenhouse.
Powdery mildew diseases are generally caused by many different species of fungi of the order Erysiphales. The disease is characterized by distinctive symptoms such as white powder-like spots on the leaves and stems. Generally, the lower leaves are the most affected, but the mildew can appear on any part of the plant that is exposed above ground. As the disease progresses, the spots get larger and thicker as massive numbers of spores form, and the mildew spreads up and down the length of the plant such on the stem and even the fruits.
Severely affected leaves can become dry and brittle, or can wither and die. Because of the infection, the fruits can be smaller in size, fewer in number, less able to be successfully stored, sun scalded, incompletely ripe, and having a poor flavor. It may also predispose plants to be more vulnerable to other pathogens. Eventually, the plant can die.
Powdery mildew can, amongst others, be caused by the fungus Sphaerotheca fuliginea (recently renamed: Podosphaera xanthii also designated as Oidium erysiphoides) and/or Erysiphe cichoracearum DC (recently renamed: Golovinomyces cichoracearum also designated as Oidium chrysanthemi).
Considering the economic importance of Cucumis plant species, such as melon, there is a continued need in the art to provide powdery mildew resistance providing genes.
In view of the above need in the art, it is an object of the present invention, amongst other objects, to meet this need.
SUMMARY OF THE INVENTION
According to the present invention, this object, amongst other objects, is met by an powdery mildew resistance conferring gene as defined in the appended claim 1.
Specifically, this object of the present invention, amongst other objects, is met by a powdery mildew resistance conferring gene, wherein the amino acid sequence encoded by said resistance conferring gene is selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12 and SEQ ID No. 14, and amino acid sequences with more than 70% identity, preferably more than 80% identity, more preferably more than 90% identity, and most preferably more than 95% identity such as more than 96%, 97%, 98%, 99%; and wherein said resistance conferring gene is impaired.
The object of the present invention, amongst other objects, is additionally met by a powdery mildew resistance conferring gene, wherein the cDNA sequence transcribed from said resistance conferring gene is selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11 and SEQ ID No. 13, and cDNA sequences with more than 70% identity, preferably more than 80% identity, more preferably more than 90% identity, and most preferably more than 95% identity such as more than 96%, 97%, 98%, 99%; and wherein said resistance conferring gene is impaired.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: shows the results of the complementation assay for 35S::CmKIP1_cDNA (designated 35S::CmMlo1_cDNA) which resulted in a gain-of-function for the powdery mildew susceptibility pathway. The percentage of diseased leaf area 12 days post inoculation is shown for Arabidopsis WT, Atmlo2/6 and Atmlo2/6/12 mutants, the data represent mean±s.d. of 2 individuals. For Atmlo2+35S::CmKIP1_cDNA, Atmlo2/6+35S::CmKIP1_cDNA and Atmlo2/6/12+35S::CmKIP1_cDNA the data represent mean±s.d. of respectively 4, 4 and 9 primary transformants;
FIG. 2: shows the results of the complementation assay for 35S::CmKIP1_cDNA (designated 35S::CmMlo1_cDNA) which resulted in a gain-of-function for the powdery mildew susceptibility pathway. The level of resistance observed for Atmlo2/6 and Atmlo2/6/12 mutants and for primary transformants Atmlo2+35S:: 35S::CmKIP1_cDNA, Atmlo2/6+35S:: 35S::CmKIP1_cDNA and Atmlo2/6/12+35S:: 35S::CmKIP1_cDNA is presented in comparison to the Arabidopsis WT phenotype;
FIG. 3: shows complementation of Atmlo2/6/12 with the 35S::CmKIP2_cDNA which results in gain-of-function for the powdery mildew susceptibility pathway. (A) Susceptible interaction phenotype of G. orontii on leaves of WT Columbia; clear hyphae and conidiophore development. (B) Resistant interaction phenotype of G. orontii on leaves of Atmlo2/6/12; spores on the leaf surface have germinated but no further development is observed. (C, D, E) Intermediate interaction phenotypes observed on Atmlo2/6/12 after complementation with 35S:: CmKIP2_cDNA; on some primary transformants the germinated spores do not further develop (C), on other primary transformants germination of spores is observed, hyphae do develop and conidiophores are formed (D,E).
DESCRIPTION OF THE INVENTION
Impaired resistance conferring gene according to the present invention is meant to indicate a gene providing a reduced, or even absent, susceptibility to powdery mildew caused by fungi indicated by powder-like spots on the leaves and stems, such as fungi belonging to the order Erysiphales such as Sphaerotheca fuliginea (recently renamed: Podosphaera xanthii also designated as Oidium erysiphoides) and/or Erysiphe cichoracearum DC.
Impaired resistance conferring gene according to the present invention are mutated genes. The mutation of the present genes can through different mechanisms results in impairment. For example, mutations in protein encoding DNA sequences may lead to mutated, truncated or non-functional proteins. Mutations in non-coding DNA sequences may cause alterantive splicing, translation or protein trafficking. Alternatively, a mutation resulting in an altered transcriptional activity of a gene, which determines the amount of mRNA available for translation to protein, may results in low levels, or absence, of proteins. Additionally, the impairment of gene function may be caused after translation, i.e. at protein level.
Impairment according to the present invention is also indicated by observing a powdery mildew resistance in a Cucumis melo plant comprising a gene which as mutated at the protein level as compared to the SEQ ID Nos. provided herein or no expression of the SEQ ID Nos. provided herein is observed.
Impaired is also indicated herein as a non-functional gene or protein. Although the function of the present genes is not yet identified, a non-functional gene or protein can be readily determined by establishing powdery mildew resistance (non-functional) or powdery mildew susceptibility (functional) in a plant. A powdery mildew resistance (non-functional) plant is indicated by comprising a gene which as mutated at the protein level as compared to the SEQ ID Nos. provided herein or no expression of the SEQ ID Nos. provided herein is observed.
Functional and non-functional genes or proteins can also be determined using complementation experiments. For example, transforming a resistant powdery mildew Cucumis melo plant with any of the present genes or proteins will result in a powdery mildew susceptible Cucumis melo plant when the gene or protein is functional while the Cucumis melo plant will remain resistant when the gene or protein is non-functional.
According to the present invention, the present powdery mildew resistance conferring genes provide powdery mildew resistance when the present genes are impaired. Impaired according to the present invention can be indicated by the absence, or decrease of a functional, or non-muted, protein identified herein as SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12 or SEQ ID No. 14. In the art, many mechanisms are known resulting in the impairment of a gene either at the transcription, translation or protein level.
For example, impairment at the transcription level can be the result of one or more mutations in transcription regulation sequences, such as promoters, enhancers, and initiation, termination or intron splicing sequences. These sequences are generally located 5′ of, 3′ of, or within the coding sequence represented by SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11 or SEQ ID No. 13. Impairment can also be provided by a deletion, rearrangement or insertion in the present genes.
Impairment at the translation level can be provided by a premature stop-codons or other RNA→protein controlling mechanisms (such as splicing) or posttranslational modifications influencing, for example, protein folding or cellular trafficking.
Impairment at the protein level can be provided by truncated, misfolded or disturbed protein-protein interactions.
Independent of the underlying mechanism, impairment according to the present invention is indicated by an decrease or absence a functional protein according to SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12 or SEQ ID No. 14.
According to a preferred embodiment, impairment according to the present invention is provided by one or more mutations in the present genes resulting in the absence of a protein expression product. As indicated, these mutations can cause a defective expression at the transcription or translation level.
According to another preferred embodiment, impairment according to the present invention is caused by one or more mutations in the present genes resulting in a non-functional protein expression product. A non-functional protein expression product can, for example, be caused by premature stop-codons, incorrect translation or posttranslational processing or by insertions, deletions or amino acid changes.
Using molecular biology methods, impairment of the present genes can also be accomplished by gene silencing, for example using siRNA or knocking out of the present genes. Methods based on EMS or other mutagenic chemical compounds capable of randomly change nucleic acids into other nucleotides are also contemplated within the context of the present invention. Detection of such mutations typically involves high sensitivity melting curve analyses or nucleotide sequencing-based TILLING procedures.
The present invention relates to nucleotide and amino acid sequences with more than 70%, preferably more than 80%, more preferably more than 90% and most preferably more than 95% sequence identity either at the nucleotide level or the amino acid level.
Sequence identity as used herein is defined as the number of identical consecutive aligned nucleotides, or amino acids, over the full length of the present sequences divided by the number of nucleotides, or amino acids, of the full length of the present sequences and multiplied by 100%.
For example, a sequence with 80% identity to SEQ ID No. 1 comprises over the total length of 1713 nucleotides of SEQ ID No. 1 1370 or 1371 identical aligned consecutive nucleotides, i.e., 1370 or 1371/1713*100%=80%.
According to the invention, the present genes are derived from Cucumis melo. 
According to another aspect, the present invention relates to Cucumis melo plants comprising in their genome the present impaired powdery mildew resistance conferring genes, i.e., plants not expressing a functional protein selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12 and SEQ ID No. 14, and amino acid sequences with more than 70% identity, preferably more than 80% identity, more preferably more than 90% identity, and most preferably more than 95% identity.
In general, and preferably, the present plants will be homozygous for the present impaired genes, i.e., comprising two impaired powdery mildew resistance conferring genes, wherein the cDNA sequence transcribed from said resistance conferring gene is selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11 and SEQ ID No. 13, and cDNA sequences with more than 70% identity, preferably more than 80% identity, more preferably more than 90% identity, and most preferably more than 95% identity.
Considering the benefits of the present plants, i.e., providing powdery mildew resistance in melon plants, the invention also relates to seeds, plant parts or propagation material capable of providing the present powdery mildew resistant melon plants which seeds, plant parts or propagation material comprise one or more of the present powdery mildew resistance conferring genes, i.e., impaired powdery mildew resistance conferring genes, wherein the cDNA sequence transcribed from said resistance conferring gene is selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11 and SEQ ID No. 13, and cDNA sequences with more than 70% identity, preferably more than 80% identity, more preferably more than 90% identity, and most preferably more than 95% identity.
According to yet another aspect, the present invention relates to isolated nucleotide sequences selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11 and SEQ ID No. 13, and nucleotide sequences with more than 70% identity, preferably more than 80% identity, more preferably more than 90% identity, and most preferably more than 95% identity.
According to still another aspect, the present invention relates to isolated amino acid sequences selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12 and SEQ ID No. 14, and amino acid sequences with more than 70% identity, preferably more than 80% identity, more preferably more than 90% identity, and most preferably more than 95% identity.
The present invention also relates to the use of one or more of the present powdery mildew resistance conferring genes, one or more of the present isolated nucleotide sequences, or one or more of the present isolated amino acid sequences for providing a powdery mildew resistant melon plants (Cucumis melo). As indicated, the present use is based on impairment, either at the expression or protein level, of the genes described herein and can be readily determined by the presently provided cDNA and amino acid sequences optionally in combination with determination of the presence or absence of powdery mildew resistance and/or in combination with complementation assays.



The present invention will be further described in the examples below of preferred embodiments of the present invention. In the example, reference is made to figures wherein:



EXAMPLES
Example 1
Complementation of Arabidopsis thaliana Atmlo Mutant Plants
Material and Methods
Full length cDNA of melon CmKIP genes were cloned either in pBINPLUS or Gateway vectors under control of a 35S promoter using standard cloning techniques.
In order to analyze the in planta function of identified melon powdery mildew resistance genes, via the Nottingham Arabidopsis Stock Centre (University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, United Kingdom, seeds were obtained of Arabidopsis thaliana ecotype Columbia (Col) mutant line NACS ID N9707 (mlo2-5 single mutant), N9710 (mlo2-5, mlo6-2 double mutant) and N9715 (mlo2-5, mlo6-2, mlo12-1 triple mutant).
The seeds were cultivated in soil, DNA was extracted and PCR assays with primers flanking the mutations (Table 1) were used to screen 4-week-old plants for the presence of the mutations as described at The Arabidopsis Information Resource (TAIR).







TABLE 1







Primer pairs used to identify WT gene versus


mutated Mlo homologue in Arabidopsis mutant lines


(5′ to 3′).










Primer pair
Primer pair



WT gene
mutated gene














Mlo2-
acgtggaagtc
attcgttaccg
ttcataaccaa
acgtggaag


5
gtgggaggaag
ggagcaaaatg
tctcgatacac
tcgtgggag



a
c
(SEQ ID NO:
gaaga



(SEQ ID NO:
(SEQ ID NO:
17)
(SEQ ID



15)
16)

NO: 18)


 


Mlo6-


ttcataaccaa
acaagaact


2


tctcgatacac
ggtttcatt





(SEQ ID NO:
tagca



 
 
19)
(SEQ ID



 
 

NO: 20)


 


Mlo12-
tggagcaagtc
tcagtgggctg
ggtgcagcaaa
tggagcaag


1
tacctttaccc
cattcacacaa
acccacacttt
tctaccttt



tctgg
a
tacttc
accctctgg



(SEQ ID NO:
(SEQ ID NO:
(SEQ ID NO:
(SEQ ID



21)
22)
23)
NO: 24)









The floral dip method was used to transform Arabidopsis thaliana ecotype Columbia and the single, double and triple mutants with constructs harboring the putative melon Mlo orthologs. After selection of the transformant plants on kanamycin, analyses were performed to identify the resistant versus susceptible phenotypes upon powdery mildew (Golovinomyces orontii) infection.
Dr. R. Panstruga (MPI, Cologne, Germany) kindly provided Arabidopsis thaliana ecotype Columbia leaves infected with Golovinomyces orontii. To maintain a fresh growing Golovinomyces orontii culture, leaves with sporulating Golovinomyces orontii were used every 10-14 days to rub-infect fresh Arabidopsis thaliana ecotype Columbia leaves. In addition, the mlo single, double and triple mutants and their progeny, complemented with the melon Mlo candidate genes, were inoculated with sporulating Golovinomyces orontii in order to confirm the powdery mildew susceptibility levels.
Results
To confirm the predicted involvement of the identified CmKIP genes in susceptibility to powdery mildew, full length cDNA sequences of CmKIP1, CmKIP2, CmKIP3 and CmKIP4 were expressed in Arabidopsis using the ubiquitous 35S promoter. In addition, also a full length genomic fragment was expressed for CmKIP2 using the 35S promoter.
The 35S::CmKIP1_cDNA, 35S::CmKIP2_cDNA, 35S::CmKIP2_genomic, 35S::CmKIP3_cDNA and 35S::CmKIP4_cDNA constructs were transformed to Arabidopsis mutant line NACS ID N9707 (mlo2-5 single mutant), N9710 (mlo2-5, mlo6-2 double mutant) and N9715 (mlo2-5, mlo6-2, mlo12-1 triple mutant). In addition, to examine epistatic effects of CmKIP overexpression in Arabidopsis, also WT Columbia plants were transformed with the same series of constructs.
FIG. 1 and FIG. 2 show the results obtained for the complementation assay for the 35S::KIP1_cDNA construct. The Arabidopsis mutant Atmlo2 shows reduced susceptibility compared to WT Columbia (Col-0) (data not shown). The double mutant Atmlo2/6 is even more reduced in susceptibility to Golovinomyces orontii. The triple mutant Atmlo2/6/12 is fully resistant to Golovinomyces orontii infection.
Primary transformants of Atmlo2/6/12 with the 35S::CmKIP1_cDNA constructs retained susceptibility to Golovinomyces orontii infection to the levels of susceptibility compared to the Atmlo2/6 double mutant. Complementation of the Atmlo2/6 double mutant of Arabidopsis with the 35S::CmKIP1 construct resulted in plants with near WT levels of infection with Golovinomyces orontii. 
A quick and strong development of the fungus resulting in sporulating hyphae was detected. Complementation of the Atmlo2/6/12 triple mutant of Arabidopsis with the 35S::CmKIP1_cDNA construct resulted in clear visible powdery mildew infections on inoculated leaves for most of the primary transformants analyzed. The retaining of susceptibility to Golovinomyces orontii of the Arabidopsis triple mutant previously shown to be full resistant to Golovinomyces orontii indicates that the identified CmKIP1 gene is able to reestablish the powdery mildew susceptibility pathway in Arabidopsis. With this result we showed we were able to identify CmKIP1, a gene functional in the powdery mildew susceptibility pathway.
FIG. 3 shows the results obtained for the complementation assay for the 35S::CmKIP2_cDNA construct. Primary transformants of Atmlo2/6/12 with the 35S::CmKIP2 cDNA constructs do in a few cases retain minor susceptibility symptoms to Golovinomyces orontii infection. However, this susceptibility is difficult to detect by eye and not all primary transformants show this weak susceptibility phenotype. To study in more detail the development of Golovinomyces orontii, microscopic studies were performed.
Leaves were washed for 48 hours in 70% EtOH and stained with CBB. With this microscopic screening a clear difference was observed between the powdery mildew resistant AtMlo2/6/12 mutant line and this line complemented with 35S::CmKIP2_cDNA.
Of the analyzed complemented lines, several showed clear development of hyphea in the leaves while all germinating spores on the triple mutant are stopped immediately after penetration in the leaves. On the 35S::CmKIP2_cDNA complemented lines the clear development of hyphae was detected which indicate that the CmKIP2 gene is able to reestablish the powdery mildew susceptibility pathway in Arabidopsis. 
Comparable results, i.e. complementation in Arabidopsis, was observed, for the CmKIP3 overexpression construct.







TABLE 2







cDNA and amino acid sequences










powdery mildew

Sequence



gene identity
Plant
type
SEQ ID No.













CmKIP1
Cucumis melo
cDNA
1




aa
2


CmKIP2
Cucumis melo
cDNA
3




aa
4


CmKIP3
Cucumis melo
cDNA
5




aa
6


CmKIP4
Cucumis melo
cDNA
7




aa
8


CmKIP6
Cucumis melo
cDNA
9




aa
10


CmKIP7
Cucumis melo
cDNA
11




aa
12


CmKIP9
Cucumis melo
cDNA
13




aa
14

## Claims

1. A full-length cDNA transcribed from a nucleotide sequence having the sequence of SEQ ID NO: 1.

2. A method for obtaining a melon plant that is resistant to powdery mildew, comprising introducing one or more mutations to a nucleotide sequence having the sequence of SEQ ID NO: 1 in a melon plant that is susceptible to powdery mildew, wherein the mutation results in a decrease, absence, or loss of function of a protein having the amino acid sequence of SEQ ID NO: 2 and resistance to powdery mildew.

3. The method according to claim 2, wherein the one or more mutations cause the absence of a protein.

4. The method according to claim 2, wherein the one or more mutations cause a non-functioning protein.

5. The method according to claim 2, wherein the one or more mutations cause gene silencing.

6. The method according to claim 2, wherein the one or more mutations are at least three mutations.

7. The method according to claim 2, wherein the resistance is to powdery mildew caused by Golovinomyces orontii.