Powdery Mildew Resistant Grapevine Plant

Abstract

Provided herein is a powdery mildew resistant grapevine plants and methods for providing powdery mildew resistance to susceptible grapevine plants. Specifically, provided herein are grapevine plants including in their genome an impaired Erysiphe necator resistance conferring gene, wherein the corresponding not impaired Erysiphe necator resistance conferring gene designated VvMLO13 encodes a protein including the amino acid sequence of SEQ ID No. 1, or proteins having 95% sequence identity therewith. The impairment results in an absence of a protein comprising the amino acid sequence of SEQ ID No. 1, or proteins having 95% sequence identity therewith, in the grapevine plant and wherein the grapevine plant is resistant to powdery mildew.

Claims

1. A powdery mildew resistant grapevine plant comprising in its genome an impaired Erysiphe necator resistance conferring gene, wherein a corresponding unimpaired Erysiphe necator resistance conferring gene designated VvMLO13 encodes a protein comprising the amino acid sequence of SEQ ID No. 1 or a protein having 95% sequence identity therewith, wherein the impairment results in an absence of a protein comprising the amino acid sequence of SEQ ID No. 1, or a protein having 95% sequence identity therewith, in said grapevine plant and wherein the grapevine plant is resistant to powdery mildew.

2. The grapevine plant according to claim 1, wherein the absence of a protein comprising the amino acid sequence of SEQ ID No. 1 comprises one or more mutations in the nucleotide sequence of a cDNA comprising SEQ ID No. 2.

3. The grapevine plant according to claim 1, wherein the absence of a protein comprising the amino acid sequence of SEQ ID No. 1 comprises one or more mutations in the unimpaired Erysiphe necator resistance conferring gene designated VvMLO13 resulting in an absence of expression thereof.

4. The grapevine plant according to claim 2, wherein the one or more mutations comprise one or more deletions, insertions or substitutions in the nucleotide sequence of a cDNA comprising SEQ ID No. 2.

5. The grapevine plant according to claim 4, wherein the absence of a protein comprising the amino acid sequence of SEQ ID No. 1 comprises the impaired Erysiphe necator resistance conferring gene having a nucleotide sequence comprising SEQ ID No. 3, SEQ ID No. 4, or SEQ ID No. 5, or combinations thereof, or a protein comprising an amino acid sequence comprising SEQ ID No. 6, SEQ ID No. 7, or SEQ ID No. 8, or combinations thereof.

6. The grapevine plant according to claim 5 wherein the absence a protein comprising the amino acid sequence of SEQ ID No. 1 comprises the impaired Erysiphe necator resistance conferring gene having a nucleotide sequence comprising SEQ ID No. 3 and SEQ ID No. 4, SEQ ID No. 3 and SEQ ID No. 5, SEQ ID No. 4 and SEQ ID No. 5, SEQ ID No. 3 and SEQ ID No. 3, SEQ ID No. 4 and SEQ ID. No. 4, or SEQ ID No. 5 and SEQ ID No. 5.

7. The grapevine plant according to claim 1, wherein the grapevine plant further comprises in its genome one or more additional Erysiphe necator resistance conferring genes, preferably one or more Erysiphe necator resistance conferring genes selected from the group consisting of VvMLO6, VvMLO7 and VvMLO11.

8. A method for providing a powdery mildew resistant grapevine plant, comprising mutating a gene designated VvMLO13 encoding a protein comprising the amino acid sequence of SEQ ID No. 1 in a powdery mildew susceptible grapevine plant, thereby providing a grapevine plant that is resistant to powdery mildew.

9. The method for providing a powdery mildew resistant grapevine plant according to claim 8, wherein the step of mutating the gene designated VvMLO13 encoding a protein comprising the amino acid sequence of SEQ ID No. 1 comprises introducing a deletion and/or insertion of substitution in a cDNA sequence comprising the nucleotide sequence of SEQ ID No. 2.

10. A seed, fruit, or plant part of a grapevine plant according to claim 1.

11-13. (canceled)

14. A method for identifying a powdery mildew resistant grapevine plant comprising detecting any of SEQ ID Nos. 3 to 5 in the genome of the grapevine plant.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention will be further detailed in the example below. In the example, reference is made to figures wherein:

[0030] FIG. 1: shows the results of a PM assay on detached leaves of wild type and mlo13 plants. Wild type leaves show complete sporulation, mlo13 leaves are clean of PM sporulation at 14 dpi;

[0031] FIG. 2: shows hyphal growth of PM visualized using specific staining on a microscopic image. PM infection on leaves at 14 dpi in the wild type (on the right) and the mlo13 mutant (on the left). There is clearly less hyphal growth in the mutant compared to its wild type;

[0032] FIG. 3: shows a PM assay on detached leaves of wild type (on the left) and mlo13 leaves (on the right). Wild type leaves show complete sporulation, mlo13 leaves are clean of PM sporulation at 13 dpi;

[0033] FIG. 4: shows quantification of PM sporulation in wild type and mlo13 mutant leaves. Percentage of leaf area covered by PM sporulation was determined for 3 wild type leaves and 3 mlo13 leaves 10 dpi.

DESCRIPTION OF THE INVENTION

Example

[0034] Material and Methods

[0035] Leaves to be tested in a detached-leaf-assay were taken from grapevine plants grown in pots till they reached a stage of at least 8-10 leaves per stem. From the top of each stem, the second, third and fourth leaf were used as test leaves. They were surface-sterilized in a bath of 1% bleach for 2 minutes, and then rinsed three times in sterile water, by soaking them for two minutes, and then let to dry in sterile conditions (laminar flow hood).

[0036] A 1% agar layer of about 1 cm was poured in a sterile plastic box, or sterile plates, and then covered by sterile filter paper. Test leaves were laid on the paper ensuring that the petiole is sticking in the underneath agar layer. Using a PM infected leaf with visible sporulation on its surface as inoculum, E. necator spores were distributed on the test leaves by the aid of an air pump.

Box/plates were covered by a lid and stored in a growth chamber conditions with the following settings: 16 h light period, 21° C. and 21% RH. Scoring was performed at several timepoints indicated in the figures by calculating the surface area of leaves covered by powdery mildew or making pictures of the leaves.

[0037] PM hyphae were visualized by aniline blue coloration on infected leaves previously treated with ethanol:(glacial)acetic acid 3:1 as described in detail in “Pessina et al., 2016)”. Leaf sections were mounted on glass slides and observed with a microscope Leica MZ16F.

Results

[0038] Mutant plants were generated by Agrobacterium-mediated transformation of young embryogenic calli of cv. Crimson seedless. Binary vectors constitutively expressing the CRISPR/Cas9 machinery were used to specifically target VvMLO13. Plants regenerated from such calli were then selected for kanamycin resistance and their DNA analyzed by next-generation sequencing (Illumina®).

[0039] Plants were obtained edited in ML013 after NextGen sequencing confirmation (minimal sequencing depth 1000× coverage). Several mlo13 alleles were obtained after sequencing and used for these experiments. The mlo13 mutant used in experiment 1 is biallelic with 1 bp insertion for one allele and 1 bp deletion for the other allele both causing frame shift mutations. For experiment 2, another mlo13 was used, this mutant contains a 3 bp deletion for one allele and an 1 bp deletion for the other allele.

[0040] Detached leaves from Crimson seedless were used in a PM assay as described in the M & M section.

Experiment 1:

[0041] FIG. 1 shows 2 detached leaves of wild type and mlo13 mutant plants. Whereas the wild type shows PM sporulation, the mlo13 mutant does not show any sporulation. Picture was taken at 14 dpi. To further analyze the phenotype of the wild type and mlo13 mutants, histological analysis was performed on the same leaf material by visualizing PM hyphae using aniline blue. As seen in FIG. 2, in the wild type hyphae are present all over the leaf surface where they form dense structures, while on the mlo13 mutant they are visible in a limited number. This clearly illustrates that the mlo13 hardly supports pathogen growth.

Experiment 2:

[0042] FIG. 3 shows an example of wild type leaf and mlo13 leaf that were used in a PM assay. Picture of the leaves were taken 10 dpi and show severe sporulation on the wild type leaf. The mlo13 mutant is resistant to PM as seen by the strongly reduced or absence of sporulation. To further analyze this, 3 wild type leaves and 3 mlo13 leaves were used for quantitative analysis. FIG. 4 shows percentage of the leaf area covered with PM sporulation for 3 wild type leaves and 3 mlo13 mutant leaves. Wild type leaves have at least 80% of the surface area covered in PM sporulation while in the mlo13 this was absent or greatly reduced to maximum 5% area.