NEMATODE RESISTANCE IN PLANTS

Abstract

The invention provides a gene, herein termed Hs4, which encodes a protein, also referred to as Hs4, which upon presence in plants confers resistance against plant parasitic nematodes, especially against the beet cyst nematode Heterodera schachtii Schmidt. Specifically, the invention provides the use of plants containing the Hs4 gene for cultivation in the presence of nematodes, in soil of which the presence of nematodes is unknown and in soil that is free from nematodes

Claims

1. A process for analysis of a plant in respect of resistance against nematodes, comprising analyzing nucleic acids of the plant for containing a nucleic acid sequence encoding a protein having a homology of at least 80% to SEQ ID NO: 1 and/or SEQ ID NO: 17.

2. The process according to claim 1, wherein the protein of SEQ ID NO: 1 is encoded by a nucleic acid sequence having a homology of at least 80% to SEQ ID NO: 3 or to SEQ ID NO: 2.

3. A resistance gene against plant parasitic nematodes encoding a protein having a homology of at least 80% to SEQ ID NO: 1 and/or SEQ ID NO: 17.

4. The resistance gene according to claim 3, the nucleic acid sequence encoding a protein having a homology of at least 80% to SEQ ID NO: 1 being arranged under the control of a promoter having SEQ ID NO: 4.

5. The resistance gene according to claim 3, wherein the protein is encoded by a nucleic acid sequence having a homology of at least 80% to SEQ ID NO: 3 or to SEQ ID NO: 2.

6. The resistance according to claim 3, within a plasmid vector suitable for gene transfer into plant cells.

7. The resistance gene according to claim 3 for use in protecting plants of the genera of Amaranthaceae, of Brassicaceae, of Poaceae, or of Solanaceae against nematodes.

8. A plant, genetically manipulated to contain a gene conferring resistance against plant parasitic nematodes, wherein the gene encodes a protein having a homology of at least 80% to SEQ ID NO: 1 and/or SEQ ID NO: 17.

9. The plant according to claim 8, wherein the gene encoding the protein having a homology of at least 80% to SEQ ID NO: 1 and/or SEQ ID NO: 17 is functionally arranged under the control of a promoter having SEQ ID NO: 4.

10. A plant genetically manipulated to contain a gene conferring resistance against plant parasitic nematodes, wherein the gene is under the control of a promoter having a homology of at least 80% to SEQ ID NO: 4.

11. The plant according to claim 10, wherein the gene conferring resistance against plant parasitic nematodes is comprised in a DNA portion inserted into the plant genome, which DNA portion consists of at maximum 3 kbp.

12. A process for obtaining a genetically manipulated plant, comprising crossing plants, one of which is a cultivar and the other one of which contains a resistance gene against plant parasitic nematodes encoding a protein having a homology of at least 80% to SEQ ID NO: 1, and selecting offspring plants by identifying offspring plants that contain the resistance gene against plant parasitic nematodes encoding a protein having a homology of at least 80% to SEQ ID NO: 1 and/or to SEQ ID NO: 17.

13. The process according to claim 1, wherein the offspring plant has essentially all traits of the cultivar and is genetically manipulated such that the resistance gene against plant parasitic nematodes is comprised in a DNA portion inserted into its genome, which DNA portion consists of at maximum 3 kbp.

14. A plant, genetically manipulated to comprise an expression cassette encoding a gene, characterized in that the expression cassette contains a promoter having a homology at least 80% to SEQ ID NO: 4, or the promoter comprising or consisting of SEQ ID NO: 4.

Description

[0025] The Hs4 gene of the invention has the advantage of being of plant origin, and to encode a protein that does not significantly impair yield of cultivated plants, e.g. of sugar beets.

[0026] The figures show in

[0027] FIG. 1 fluorescence microscopic images of 3-week old root clones from NEMATA expressing GFP and carrying the CRIPR-Cas9 cassette for knock-out of the Hs4 gene,

[0028] FIG. 2 a box plot showing that expression of the resistance gene of the invention correlates with reduction of infection by nematodes,

[0029] FIG. 3 shows microscopic images of hairy roots infected with nematodes, A) of susceptible sugar beet line 93161 as a positive control, B) of Nemata roots in which the Hs4 gene has been knocked out by CRISPR-Cas technology turning a resistant into a susceptible root,

[0030] FIG. 4 fluorescence microscopic images of 12-d-old hairy roots genetically manipulated to express the RFP gene as a reporter gene and the Hs4-overexpression cassette,

[0031] FIG. 5 a graphic presentation of the expression levels of the Hs4 resistance gene (left column) in relation to expression of the housekeeping gene GAPDH, and number of female nematodes, and

[0032] FIG. 6 a microscopic image of hairy roots of a clone expressing the Hs4 gene in the presence of infecting nematodes.

[0033] In the microscopic images, the bar is 1000 μm.

[0034] The invention is now described in greater detail by way of examples. In the examples, the resistance gene product is also referred to as the Hs4 gene. Nematodes were Heterodera schachtii Schmidt that were propagated under non-sterile conditions on susceptible sugar beet plants. Fully developed brown cysts were harvested from the roots onto 50 m sieves. A 3 mM ZnCl.sub.2 solution was used to stimulate the hatching of juveniles in the dark. Nematodes were examined under a binocular microscope. Only suspensions with >90% mobile nematodes were taken as inoculum. For in vitro tests, nematodes were surface-sterilized by soaking them in 0.05% HgCl.sub.2 solution for 30 s, and were then washed four times with sterile water and resuspended in 0.2% (w/v) Gelrite (Duchefa Biochemie BV, Haarlem, the Netherlands). In all, 250 sterile nematodes were used to inoculate the hairy roots. For glasshouse resistance tests, plants were grown in 20 ml tubes filled with sterile sand (grain size 0.1-1.5 mm), sterilized at 80° C. for 3 h. Six hundred freshly hatched second-stage juveniles (J2 larvae) were added to each plant with a syringe. At 4 weeks after infection, plants were harvested and washed, and roots were examined under a binocular microscope. In the in vitro tests, root clones were inoculated with 250 nematodes and roots were examined for resistance after 4 weeks. For RNA isolation, root samples were collected 3, 6, 9 and 12 d post-inoculation (dpi), with three biological replicates per sample.

[0035] A comparison of the Hs4 protein sequence to the sequences analysed in Jager, quoted above, showed that the Hs4 gene was not among the BAC sequences analysed in Jager.

Example 1: Analysis of the Presence of the Hs4 Gene in a Plant

[0036] As an example for analytical methods for determining the presence of specific nucleic acid sequences encoding a protein having a homology of at least 80% to SEQ ID NO: 1, the DNA from leaf samples or root samples from sugar beets was purified. The DNA was analysed by PCR, using the primer of SEQ ID NO: 5 and SEQ ID NO: 6, which between them specifically hybridize to a section of the natural Hs4 gene, comprising the first exon and part of the 5′UTR of SEQ ID NO: 3. Generally, primer pairs that are specific for amplifying a portion of a known DNA sequence, can be designed using generally available computer programmes on the Hs4 gene sequence. Amplification conditions were annealing at 58° C., polymerase synthesis at 72° C. for 60 s, denaturing at 94° C. for 30 s, for 35 cycles. The amplification product had a size of 194 bp.

Example 2: Generating a Plant that Contains the Hs4 Gene by Genetic Manipulation

[0037] Transfer of the resistance gene Hs4 into plants can be made using a Ti plasmid and Agrobacterium tumefaciens or a Ri plasmid and Agrobacterium rhizogenes, wherein the plasmid comprises an expression cassette encoding the Hs4 gene product.

[0038] Alternatively, a nucleic acid sequence encoding a protein of at least 50% homology, preferably of at least 80% homology, more preferably of at least 89% homology to SEQ ID NO: 1 can be integrated into the genome of a plant using a CRISPR-Cas based method.

[0039] Another method for introducing the nematode resistance gene Hs4 into a plant which originally is devoid of the functional Hs4 gene, especially into sugar beets, more preferably into sugar beet varieties, comprises the steps of crossing the plant with a Hs4 gene containing plant and from offspring of the crossing selecting those that contain the Hs4 gene in combination with a desired combination of traits of the plant which originally is devoid of the functional Hs4 gene. The presence of the Hs4 gene can be determined by analysing plant material according to Example 1.

Example 3: Nematode Resistance is Caused by the Hs4 Gene

[0040] The sugar beet (Beta vulgaris) variety Nemata, which is known as nematode resistant, was genetically manipulated to inactivate the Hs4 gene specifically. The inactivation of the Hs4 gene directly resulted in nematode susceptibility of the plants.

[0041] In short, target sites for integration were identified manually. As target sites, SEQ ID NO: 7 and SEQ ID NO: 8 were used, which target the exon region of the sugar beet. These target sites were cloned into the pChimera vector (described by Fauser et al., 2014, kindly provided by Prof. Dr. Holger Puchta to the Plant Breeding Institute, CAU Kiel). A single guide RNA having SEQ ID NO: 9 and SEQ ID NO: 10 were cloned into the vector p201G (described by Jacobs et al. 2015, obtainable from Addgene) separately and transformed into Agrobacterium rhizogenes. Leaf stalks of the sugar beet were inoculated with the transformed A. rhizogenes, and obtained hairy roots were kept on Gamborg B5 medium for two weeks. Hairy roots were tested for mutation in or around target sites by PCR using SEQ ID NO: 11 and SEQ ID NO: 12 as primers and were confirmed by Sanger sequencing. Hairy roots were then inoculated with 250 of J2 Heterodera schachtii nematodes, and after four weeks of subsequent cultivation in the dark, female nematodes in hairy roots were counted under the binocular.

[0042] FIG. 1 shows fluorescence microscopic images of 3-week old root clones from Nemata expressing the reporter GFP and the CRISPR-Cas9 cassette for knock-out of the Hs4 gene.

[0043] These images show expression of the GFP reporter gene, indicating presence of the knock-out cassette for Hs4.

[0044] FIG. 2 shows results from nematode resistance tests with four of the Nemata clones that were manipulated with the CRISPR-Cas9 knock-out cassette for the Hs4 gene. The number of females/cysts indicates susceptibility, and a low number of females/cysts indicates nematode resistance. The originally resistant control Nemata (NEMATA) and the susceptible beet (093161) were used as controls. The data show that knock-out of the Hs4 gene yielded significantly higher susceptibility, resp. abolished the resistance of the original NEMATA. FIG. 3 shows microscopic images of hairy roots infected with nematodes, A) of susceptible sugar beet line 93161 as a positive control, B) of Nemata roots in which the Hs4 gene has been knocked by CRISPR-Cas technology turning a resistant into a susceptible root. In FIG. 3, whitish beet cyst nematodes are indicated by arrows.

[0045] The susceptibility of the plants after specific inactivation of the Hs4 gene demonstrates that this gene confers resistance against nematodes.

[0046] Analysis of the originally nematode resistant sugar beet was by PCR, showing the presence of the Hs4 gene.

Example 4: The Hs4 Gene Confers Nematode Resistance to Sugar Beets

[0047] As examples for species that is not related to Patellifolia procumbens, a non-resistant, i.e. nematode susceptible sugar beet was provided with a nucleic acid construct expressing Hs4.

[0048] The Hs4 encoding nucleic acid sequence was cloned under the control of the constitutive 35S promoter and transformed into roots of the susceptible sugar beet line. The plasmid containing the expression cassette for Hs4 has a nucleic acid sequence of SEQ ID NO: 13 (pBin35SRed).

[0049] As a reporter, the DsRed gene under the control of the CsVMV promoter was co-transformed.

[0050] For transformation the protocol as outlined in Example 3 was used. The primer-binding sites indicated can be used for designing complementary primers for PCR amplification of the intermediate nucleic acid section.

[0051] FIG. 4 shows fluorescence microscopic images of 12-d-old hairy roots from the susceptible beet line 093161, which was genetically manipulated to express the DsRed gene and carrying the Hs4-overexpression cassette from the pBin35SRed vector.

[0052] In total, 11 DsRed expressing roots were observed, and after infection with H. schachtii, different infection rates, corresponding to different expression rates of Hs4, were observed. It was found that resistance against nematodes correlated to expression of Hs4, with high expression of Hs4 conferring complete resistance, low expression of Hs4 yielding moderate resistance. The one clone that did not express Hs4 was highly susceptible.

[0053] FIG. 5 shows a graphic representation of these analytical results, giving expression levels of the Hs4 resistance gene product (left column) in relation to expression of the housekeeping gene GAPDH and number of female nematodes (right column) present on the hairy root clones (OEX1, OEX2, OEX3, OEX4, OEX5, OEX6, OEX7, OEX8, OEX9, OEX10), and the original susceptible control (093161).

[0054] As an example for a plant that is nematode resistant due to genetic manipulation to express the Hs4 gene as the only resistance gene, FIG. 6 shows a microscopic image of hairy roots of clone OEX7 in the presence of infecting nematodes. FIG. 6 confirms that the resistance conferred by expression of the Hs4 gene results in the absence of infection with the beet cyst nematode.

[0055] These data show that the Hs4 gene product confers resistance against nematodes to sugar beets.

Example 5: The Hs4 Gene Confers Nematode Resistance Works into Plants of Different Genera

[0056] As an example for another plant genus, Brassicaceae, Arabidopsis thaliana, was transformed with an expression cassette encoding the Hs4 protein. The expression cassette contained the nucleic acid sequence encoding Hs4 under the control of the nematode inducible Hs1 promoter (nucleotides 9 . . . 1477 of SEQ ID NO: 15).

[0057] In short, the expression cassette was transformed into A. thaliana by the floral dip method. Seeds expressing the dsRed gene were selected. Plants were grown in the climate chamber and the T2 seeds were harvested. Two T2 populations were grown under sterile conditions and plants exhibiting the dsRed gene were inoculated with H. schachtii J2 larvae. The average number of females which had developed after 4 weeks was 1.4 and 14.7, respectively, while the control line Col0 exhibited 23.7 females on the average. This demonstrates that both populations are resistant to H. schachtii, but with varying degrees. One population exhibited almost complete resistance while resistance in the other population was moderate. This results typically reflects different integration sites and thus different expression intensities of the Hs4 gene. These results demonstrate that expression of Hs4 conferred complete resistance to this member of the Brassicaceae against nematode infection. The plasmid containing the expression cassette for Hs4 has a nucleic acid sequence of SEQ ID NO: 15 (hs1_hs4_pbin35sred).