DE19700844A1 - Nematode resistance gene - Google Patents

Abstract

The invention concerns a nucleic acid that induces resistance to sedentary nematodes in plants of the solanaceae, chenopodiaceae and/or brassicaceae family. Said nucleic acid comprises specially a translated area, that is at least 60 % homologous to the sequence of the Hsl<pro-1>-gene of <u>beta procumbens</u>. The invention also covers the sequence of a cDNA clone and a genomic clone of said nucleic acid. Moreover, it covers a vector, which can be, for instance, a yeast artificial chromosome "YAC", containing the nucleic acid for resistance to sedentary nematodes in plants. The invention also relates to the use of the nucleic acid or the vector to induce resistance to sedentary nematodes in plants as well as a transgenic plant containing the nucleic acid or the vector. In addition, the invention concerns the protein coded by the nucleic acid, a test kit containing the nucleic acid and/or the vector and a method for a transgenic plant as well as a method for producing a nematode resistance in plants. Lastly, the invention also concerns the promoter of the nucleic acid, its use and a primer for the PCR.

Description

The present invention relates to a nucleic acid which in Plants, preferably of the Beta and / or Brassica genus, a Resistance to sedentary nematodes induced.

The present invention also relates to the DNA sequence egg nes cDNA clone and a genomic clone of this nucleic acid. The present invention further relates to a vector which e.g. B. an artificial yeast chromosome (yeast artificial chromoso me) "YAC" can be the nucleic acid for resistance against sedentary nematodes in plants. Finally be the present invention addresses the use of nucleic acid right or the vector to induce resistance to sedimentation tary nematodes in plants and a transgenic plant that contains the nucleic acid or the vector.

Furthermore, the invention relates to the ko by the nucleic acid dated protein, the nucleic acid and / or the vector ent holding test kit and a method of a transgenic plant and a method for generating a nematode resistance in Plants.

Finally, the invention relates to the resistance promoter gens.

It is known that plants from various pathogens and Pa types of rasite are attacked. It is also known that Cultivated plants are usually more susceptible to parasites than their wild relatives. You often meet plants Parasites from the family of nematodes, with a liquid filled with a skin muscle tube Pseudocyloma envelope. Nematodes are important parasites in the Harvest damage of around 150 million all over the world Cause DM per year. Nematodes are particularly harmful Genera Meloidogyne, Heterodera and Globodera, which are per manent in the roots of the infested plant, after which they have induced certain nutritional structures. The Ne  matode Heterodera schachtii has a broad host range, the includes many species of different plant families, e.g. B. the Chenopodiaceae and Brassicaceae.

The life cycle of nematodes is divided into four larval stages divided (J1-J4). The roots are from J2 juvenile stadiums infected, which migrate to the central cylinder, where they develop induction of syncytia. This extensive food structure structures result from partial cell wall degradation between the cells of the xylene parenchyma. The nematode ends its Adult life cycle after three stages. The feminine The nematodes swell and eventually destroy the sausage zelcortex while still feeding on the syncytia ren. The male stages feed upon completion of the third stage no longer and move when they awaken are towards the female stages from which they are passing Sex pheromones are attracted. The mature female stages are filled with eggs. After their death they form a cyst in which the infectious larvae (J2) in the earth for up to 10 Can survive for years.

It is believed that nematode resistance genes are an incompatible ble trigger reaction between the host and the parasite that have already been described at the cellular level. The roots of plants that carry this or these gene (s) are from J2 juvenile stages are affected, but most of the nematodes die in the late J2 stage due to the dismantling of the initiator ten syncytium. In rare cases, female stadiums s that develop a transparent appearance and stop growing. In this way, the Nemato that do not complete their life cycle.

Because the use of nematicides due to environmental policy is only possible to a limited extent, it is particularly desirable worth to confuse these resistance genes in crops chen.  

In particular, cultivated beet of the Beta genus (e.g. sugar beet, Beet, Swiss chard, beetroot) are highly susceptible to beet benzystennematodes Heterodera schachtii. It's been around since long worked on resistance to Heterodera schachtii and other phytopathogenic nematodes in plants, in particular Cultivated plants to produce, since this corresponding resistance genes are missing. The only sources of resistance are game art Beta procumbens and her close relatives B. webbiana and B. patellaris.

Genes for resistance to various types of nematodes are found used for breeding in different types of crops (e.g. potato, tomato, wheat, oil radish). It also became a Resistance gene from the wild species Beta procumbens using species cross into the sugar beet. From this could be resistant Sugar beets were selected, which, however, had the disadvantage that they lack quality and performance were characterized. The resistant sugar beet that resulted from the crossing with Beta procumbens, have alternately large translocations from the wild beets the Procumbentes section. Their low performance and reduced quality is probably due to the fact that in addition to the resistance gene, other performance-reducing genes the wild species in these sugar beet lines. Is too the transmission of the resistance property to subsequent Ge generations incomplete. Leave in a purely breeding way these disadvantages cannot be eliminated, since breeding by Crossing can hardly select specific properties without that other - possibly disadvantageous - properties are also transmitted.

Numerous attempts have also been made to make artificial Nematode resistance in plants by a combination of "Suicide genes" with syncytia-specific promoters to in reduce. So far, however, no resistant Plants are grown.  

In addition, a natural re to identify molecular genes and genes for generation to use resistance in crops.

It was therefore an object of the present invention, a nu provide small acid that is resistant to nematodes mediated in plants. It was another task at hand gene invention, the DNA sequence underlying this gene to provide.

It was also another task according to the present Er finding the use of such a gene for induction of a To enable resistance to sedentary nematodes.

It was also an object of the present invention to specify a transgenic plant that has such resistance mediating nucleic acid contains, as well as cells, seeds or Parts of plants that contain this nucleic acid.

Finally, it was an object of the present invention Specify vectors in which the gene for resistance mediation ge gene nematodes can be effectively incorporated into plants and is included in it.

It was a further object according to the present invention the protein encoded by the nucleic acid and a test kit, which contains the nucleic acid.

After all, it was a task according to the present invention dung, a process for the production of a transgenic plant and a method of creating resistance to nematodes specify.

It was also an object of the present invention, the promo tor to indicate the expression of the above Resi stenzgens controlled.  

These tasks are performed by those specified in the claims Objects of the invention solved.

Fig. 1 shows a Northern analysis of total RNA from leaves and roots. Total RNA was isolated from leaves and roots of six week old plants that were either (1) infected or (2) uninfected. The full length 1832 cDNA was used as a probe. 20 µg of total RNA was separated in 1.3% agarose and transferred to nylon membranes. The filter was hybridized with the radioactively labeled probe at 60 ° C. overnight and then washed for 2 × 30 min at 60 ° C. in 0.2 × SSC.

According to claim 1 of the present invention, a nucleus acid in plants, preferably of the beta and / or genus Brassica, induced resistance to sedentary nematodes, provided. The provision of such a gene allowed cultivating crops that are resistant to seding tary nematodes. Such resistant crops are of course far from their non-resistant relatives because they cannot be affected by nematodes and are therefore less prone to illness. By providing lung nucleic acid that shows resistance to sedentary nema deaths in plants, it is still possible to become resistant Get plants that are of the same high quality and Performance like other, non-resistant crops exhibit. This is due to the fact that a single Nucleic acid, namely the nucleic acid for resistance to sedentary nematodes into which plants are transferred while in conventional breeding methods in addition to the desired Genes can also be transferred to other DNA sequences that are under Code circumstances for undesirable properties.

The nucleic acid which induces resistance to sedentary nematodes particularly preferably comprises a translated region which is at least 60% homologous to the sequence of the Hs1 ^Pro-1 gene from Beta procumbens. These include the homologous genes from Beta webbiana and Beta patellaris. The HS1 ^Pro-1 gene from Beta procumbens bears resistance to secondary nematodes in plants. A 60% homology with the DNA sequence listed above is already sufficient to induce the desired property of resistance to sedentary nematodes in a plant which carries this nucleic acid. Genes according to the invention can also be obtained by searching gene banks with the sequence 1832, the hybridization conditions being able to be selected as follows:

Hybridization temperature 50 ° C, preferably 60 ° C, and washing the filter in 0.5 × SSC, preferably in 0.2 × SSC for at for example 30 minutes.

The nucleic acid having resistance is particularly preferred induced against sedentary nematodes in plants, the following DNA sequence includes:

This sequence is referred to below as No. 1832.

Also included in the present invention are nuclein acids that encode a protein that has the same nematode gives, as is the case with the above nucleic acid no. 1832 ko dated gene product, preferably all of these gene products comprise the same amino acid sequence.  

The nucleic acid is preferably a cDNA.

In a further embodiment, the nucleic acid which is the Resistance to sedentary nematodes induced in plants, a genomic DNA comprising the following DNA sequence:

Positions with (?) Can be A, G, C or T or the nucleotide is absent. The translation start is underlined. The bold letters represent the cDNA portion. In the attached sequence listing (?) the letter N.  

This sequence will be referred to as No. 1832.1 in the following. The original sequence of 5407 nucleotides, as shown in Figure 2, contained sequencing errors, but these did not affect the protein coding region.

Also included is a nucleic acid available from Ab search for a DNA bank with a DNA sequence as described above and which codes for nematode resistance as for Clone detected in 1832.

The nucleic acid preferably comes from a wild species of the sec tion Procumbentes of the genus Beta.

The nucleic acid is particularly preferably dissolved, as described above ben, resistance to sedentary nematodes of the genera Me loidogyne, heterodera and / or globodera. Most notably induction of resistance to heterodera is preferred schachtii in plants. Resistance is particularly preferred against sedentary nematodes in plants of the species Beta vulgaris in induced.

When such a gene is introduced into the plants to be changed, usually only the property of nematode resistance is influenced. No pleiotropic effects are to be expected. The performance of the breeding material remains unaffected. The transgenic plants that express the above gene show an incompatible reaction to cyst nematodes. This means that they can be used to breed resistant varieties. Since it is a natural resistance nucleic acid from wild species of the Procumbentes section of the genus Beta, no acceptance problems with regard to genetically modified plants are expected. Nematode-resistant varieties that have the gene mentioned above can lead to an increase in the proportion of host cultures in the crop rotation. In the case of sugar beet, this means theoretically that a crop with a high contribution margin can be grown to an increased extent. Furthermore, the HS1 ^Pro-1 sequence is not only active in plants of the genus Beta, but is also capable of generating nematode resistance in plants of other genera, such as, for example, in Arabidopsis thaliana.

Whether a sequence found has the potential, one Giving plant nematode resistance can be based on the usual Check tests, such as those below are explained in more detail.

According to a further preferred embodiment of the present The invention is a vector, particularly preferably an artificial ches yeast chromosome, provided (yeast artificial chromosome, YAC), which is resistant to sedentary nematodes mediated in plants and the nucleic acid as described above ben, contains.

The YAC can e.g. B. contain the following DNA sequence:

• 1. (No. 1832)
• 2. (No. 1832.1).

A 60% homology of the nucleic acid contained in the YACs Ren is sufficient to induce resistance in the plants. Preferred YACs are the following:

TABLE 1

In a preferred embodiment, resistance to Nematodes of the genera Meloidogyne, Heterodera and / or Globo dera induced in plants. It is particularly preferred resistance to Heterodera schachtii.

Resistance to sedentary nematodes is preferred in Plants of the Beta vulgaris species induced.

Furthermore, the invention is directed to the use of nu small acid or of the vector, as described above, for induction on resistance to sedentary nematodes in plants.

The invention is also directed to a transgenic plant tet, the nucleic acid or the vector as described above ben, contains.

The gene can either be transformed by standard methods under the control of a constitutive promoter or under control of the internal promoter upstream of the translated sequence is located in plants for expression be brought. This will result in an incompatible reaction with the sedentary nematodes, especially the cyst nematode hetero dera schachtii, caused.

The upstream, approximately 1500 nucleotides Pro motor region of the gene can be used for the root-specific expression of any genes in any plants. Included are promoters which are derived from the 5 'untranslated region of the HS1 ^Pro-1 gene and show the same promoter activity as the HS1 ^Pro-1 gene promoter.

In a preferred embodiment, the transgenic belongs Plant of the genus Beta or the genus Brassica. Especially the transgenic plant of the type Beta vulga is preferred ris on.  

The invention is also applicable to cells, seeds or parts of plants directs the nucleic acid or vector as above wrote, included.

Furthermore, the invention is based on that encoded by the nucleic acid Protein directed, as well as derivatives thereof with the same resistance-imparting properties.

The invention also relates to a test kit that a Nu small acid or a vector as described above, or a Contains protein as described above. He also concerns a process for producing a plant, thereby characterized in that a nucleic acid as described above in a plant cell is introduced and a plant from the Plant cell is regenerated.

Furthermore, the present invention relates to a method to create nematode resistance in plants, thereby is characterized in that a nucleic acid as described above ben, is introduced into a nematode-sensitive plant.

The invention also relates to a promoter which expresses sion controls the nucleic acid described above and the is characterized in that it is root-specific active.

The 5′-flanking region of the gene, a 1.6 kb XbaI fragment, contains typical elements of eukaryotic promoters, such as B. the TATA box. The promoter is obviously root specific, because according to Northern analysis with leaf and root RNA only a signal with root RNA was found. This also confirm Experiments with transgenic potatoes using a fusion transformed product from the 1832 promoter and the GUS gene have been. There the roots showed a clear color craze tion, which suggested activity of the 1832 promoter.

This means that the 5′-region of the approx. 1500 nucleotides 1832 gene and derivatives thereof with corresponding promoter activi  act for the expression of any genes, especially in root tissues, different plants can be used. Application examples are the expression of genes for resistance to Nematodes and resistance to other root-borne pests lively, as well as expression of genes that are involved in the sucrose trans location and generally of genes involved in the Sucrose or inulin storage are involved. Derivatives of Promoters according to the invention are sequences that differ from the Promoter of the 1832 gene e.g. B. by deletions, insertions, ba derived exchanges, etc., the promoter properties of the 1832 gene promoter.

Finally, the invention relates to a primer for PCR, available from sequence no. 1832.1.

In the following the invention is illustrated by examples in a described, these examples reflect the scope of the inven not limit.

example 1 Cloning of the HS1 ^Pro-1 gene

For the cloning of the HS1 ^Pro-1 gene, closely linked markers were identified. A B. procumbens-specific satellite (pRK643) was cloned from one of the fragment addition lines. Southern analysis showed that all the resistant lines tested carried this satellite, suggesting that it was distributed in the region of the wild species B. procumbens genome where the gene is located. This marker was found to be helpful in identifying the translocation line with the smallest segment of the beet among a plurality of chromosomal mutants. This line was chosen for the positional cloning of the gene. The marker pRK643 co-segregated perfectly with resistance in a segregating F2 population of 241 individuals. Using this satellite marker as a probe, 3 clones were extracted from a YAC library of line A906001, which included the HS1 ^Pro-1 gene region.

Example 2 Identification of the transcribed sequences of the YACs

To identify the transcribed sequences of the YACs, was a cDNA library from the roots of with nematodes infected A906001 plants and created with the three YACs screened, resulting in the isolation of three cDNA clones, namely numbers 1832, 1845 and 1859. The clone in 1849 showed egg ne hybridization with sugar beet DNA during the clone 1859 multiple band patterns with the DNA of both susceptible as well as resistant beets. The continuing work focused on the cDNA 1832 because:

• 1. This cDNA gave a single copy signal with DNA of the resistant lines, while no signal with DNA from the susceptible sugar beet was visible, which allows the assumption that this gene is not present in cultivated beets. All monosomal addition lines carrying the HS1 ^Pro-1 gene gave a signal with this probe.
• 2. It showed complete co-segregation with resistance property in the segregating F2 populations.
• 3. A 1.6 kb transcript was resistant only in roots Plants present as shown by Northern analysis. A significantly stronger hybridization signal was compared found to uninfected roots with RNA from roots that were infected with Heterodera schachtii.
• 4. Sequence analysis of the predicted polypeptide showed Mo tives that are typical of recently cloned resistance gene products.

Taking these results together, the clone represented 1832 a wild beet-specific gene that only expri in roots is lubricated and stimulated after nematode infection.

Example 3 Genetic complement analysis

Hair roots were used for the genetic complementation analysis cultures obtained by induction with Agrobacterium rhizogenes and used. The hair root cultures of the sugar beet proved itself as a suitable substrate for root pathogens. The compati ble reaction of the susceptible as well as the incompatible reaction The resistant roots on cyst nematodes become in hair roots Beet crops are maintained. A vulnerable beet line (No. 93161p) was with the 1450 base pair (bp) cDNA 1832 using an A. rhizogenes-mediated Gene transfers transformed. The genetic modification of the Transformants were determined by GUS assay and DNA blot analysis confirms. After inoculation with J2 juveniles, six were independent roots transformed from one another that 1832 gene expressed and the same incompatible response as the resistant line A906001 showed up while nematodes regularly developed and based on the vulnerable controls the hair roots that did not contain the gene. susceptibility was able to transform from a resistant root culture with an antisense construct of cDNA 1832 restored will.

These experimental data demonstrate that resistance in hair roots from the 93161p line depends on the expression of the 1832 gene. The isolated gene is referred to as the HS1 ^Pro-1 gene because it transfers the nematode resistance to the susceptible sugar beet line in such a way that it completely matches the resistance in the A906001 line.

Example 4 Sequencing of the cDNA

Sequencing of the entire cDNA and the corresponding one genomic clones showed an open reading frame without introns of 846 bp, which is a predicted gene product of 282 amino acids encoded what with the data obtained from the RNA blots matches.

Example 5 Structural analysis of the amino acid sequence

The amino acid sequence of the predicted polypeptide can be divided into four different subdomains. A suspected signal peptide (domain A) can be defined at the N-terminus, who is believed to have the task of directing the protein to the cytoplasmic membrane. A leucine-rich region (domain C), which is arranged in imperfect, leucine-rich, repeating units, is clearly shown at the N-terminus of the HS1 ^Pro-1 polypeptide. The repeating leucine-rich units (LRR) are part of the protein-protein interaction and were found in previously cloned resistance genes from plants, e.g. B. the RPS2 gene from A. thaliana. Their function varies from mutative recognition sites in receptor-like molecules that are located extracellularly to catalytic domains of enzymes that are active in the cytoplasm. Similar to other LRRs identified in plant resistance genes, the LRRs of the HS1 ^Pro-1 polypeptide are poorly conserved compared to the consensus sequence of the LRR consensus superfamily. The LRRs of the HS1 ^Pro-1 polypeptide are characterized by a 20aa consensus motif (xLxxaxxaxLxxLxxaxxxL; L = leucine or iso leucine, a = aliphatic or aromatic aa, x = each aa). The leucine and aliphatic residues at positions 2, 5 and 16 are located in the same position as in the LRR superfamily consensus. The highly conserved asparagine at position C is substituted with a leucine / isoleucine. This asparagine also falls within the consensus LRRs of the RPS2 polypeptide. The hydrophobic domain of 17aa of the HS1 ^Pro-1 gene (domain F) indicates a transmembrane segment. The C-terminal domain contains aa with positively charged residues and a putative N-glycosylation site.

The Elicitor receptor model of the plant pathogenic interaction indicates that the products of the resistance genes act as specific receptors for pathogenic triggers according to the gene-for-gene hypothesis. Sequence analysis of HS1 ^Pro-1 indicates that it is involved in gene-for-gene resistance as part of a cascade of defense responses. The predicted polypeptide consists of imperfect LRRs located at the N-terminus with an additional signal peptide, a putative transmembrane-spanning domain and a positively charged C-terminus, and thus fits into the second group of plant resistance genes.

Similar protein structures between HS1 ^Pro-1 and the resistance gene Cf-9 from the tomato could be predicted, although no significant sequence homology was found. As a possible type of resistance reaction, the extracytoplasmic LRRs can act as receptor-recognizing putative triggers. Nematodes are known to produce secretions that can interact with membrane-bound plant receptors. The positively charged C-terminus may interact with the cytoplasmic components for signal transmission. Alternatively, as a protein located in the cytoplasm, it can act as a receptor for triggers injected into the cell via the nematode's mouth prick.

By cloning the first plant gene that is involved in nematode resistance, it should be better possible to understand the process of host-specific defense against nematodes. In addition, the isolation of the HS1 ^Pro-1 gene offers the possibility of transferring resistance to host species of agricultural importance in which no allelic form of the gene is present.

Example 6 Identification and characterization of the HS1 ^Pro-1 promoter

A 1.6 kb XbaI fragment was isolated from a lamda DASHII bank with a PCR fragment from the 5′-region of the HS1 ^Pro-1 gene, which is the 5′-flanking region of the gene and the sequence with the number 1832. The isolated promoter sequence contains the typical elements of eukaryotic promoters, such as the TATA box with the sequence TACATAAA in position -33 before the start of transcription or the 5'-end of the cDNA.

The promoter identified is root-specific, since in Nort hern blots a signal with the sample from the 5′-range of 1832 gene is found only in root tissue.

Furthermore, constructs containing the invention Promoter and a GUS reporter gene exclusively a color freak tion of roots of transformed potatoes or tobacco.

In addition, the promoter can be induced by nematodes. This shows a comparison of the transcriptional activity of sugar beet infected and uninfected with H. schachtii root. This suggests that the invention Promoter binds transcription factors derived from nematodes men or which are formed as a result of the infection.

The experiments mentioned demonstrate the root specificity of the HS1 ^Pro-1 promoter.

Sequence listing

Claims (25)

1. Nucleic acid found in plants, preferably the genus Beta and / or Brassica, resistance to sedentary Ne can induce matodes. 2. Nucleic acid according to claim 1, characterized in that it comprises a translated sequence which is at least 60% homologous to the protein coding sequence of the Hs1 ^Pro-1 gene from B. procumbens. 3. Nucleic acid according to claim 1 or 2, characterized in that the HS1 ^Pro-1 gene comprises the following sequence: 4. Nucleic acid according to one of claims 1 to 3, characterized characterized in that it is a DNA, preferably a cDNA, is. 5. Nucleic acid according to claim 1 or 2, characterized in that it is a genomic DNA which comprises the following sequence: 6. Nucleic acid according to one of claims 1 to 5, characterized ge indicates that it is available by searching one DNA bank with a DNA sequence as in claim 3 and / or 5 specified. 7. Nucleic acid according to one or more of the preceding Claims, characterized in that they are a wild species comes from the Procumbentes section of the Beta genus. 8. Nucleic acid according to one or more of the preceding An sayings, characterized in that they have a resistance against sedentary nematodes of the genera Meloidogyne, Hete rodera and / or globodera induced. 9. Nucleic acid according to one or more of the preceding An sayings, characterized in that they have a resistance induced against Heterodera schachtii.   10. Nucleic acid according to one or more of the preceding An sayings, characterized in that they have a resistance against sedentary nematodes in plants of the Beta vulga species ris induced. 11. Vector containing a nucleic acid according to one of the An sayings 1 to 10. 12. Vector according to claim 11, characterized in that it is a YAC vector. 13. Use of a nucleic acid and / or a vector according to one or more of the preceding claims for inductance on resistance to sedentary nematodes in plants. 14. Transgenic plant containing a nucleic acid and / or a vector according to one or more of the preceding an claims. 15. Transgenic plant according to claim 14, characterized net that they are of the genus Beta or the genus Brassica listened to. 16. Transgenic plant according to claim 14, characterized net that she belongs to Beta vulgaris. 17. Cell, seeds or parts of plants containing a nucleus acid according to one of claims 1 to 10 and / or one Vector according to one of claims 11 or 12. 18. Protein encoded by a nucleic acid according to one of the Claims 1 to 10. 19. Test kit containing a nucleic acid according to one of the An say 1 to 10 and / or a vector according to one of the An say 11 or 12 and / or a protein according to claim 18.   20. Process for the production of a plant, characterized thereby records that a nucleic acid according to any one of claims 1 up to 10 is introduced into a plant cell and one Plant is regenerated from the plant cell. 21. Method for generating nematode resistance in Pflan zen, characterized in that a nucleic acid after one of claims 1 to 10 in a nematode sensitive Plant is introduced. 22. Root-specific promoter, obtainable by searching egg ner gene bank with the sequence as stated in claim 5, and derivatives thereof with the same promoter activity. 23. Promoter according to claim 22, characterized in that it controls the expression of the HS1 ^Pro-1 gene. 24. Use of the promoter according to claim 22 or 23 for the Expression of genes. 25. Primer for PCR, available from sequence no. 1832.1.