DOWNY MILDEW RESISTANT LETTUCE MUTANT
20210348187 · 2021-11-11
Inventors
Cpc classification
C12N9/1205
CHEMISTRY; METALLURGY
C12N15/8279
CHEMISTRY; METALLURGY
International classification
Abstract
The invention relates to lettuce plants comprising in its genome a mutant homoserine kinase allele in homozygous form, whereby the plants are resistant against Bremia lactucae.
Claims
1. A plant of the species Lactuca sativa which is homozygous for a mutant allele of the homoserine kinase gene, which mutant allele a) encodes a mutant homoserine kinase protein comprising one or more amino acids inserted, deleted or replaced compared to the wild type protein of SEQ ID NO: 1, or b) has reduced gene expression compared to the wild type allele, wherein said mutant allele results in the leaf tissue of said lettuce plant comprising an amount of L-homoserine of at least 10.0 nmol per mg fresh weight, while a plant heterozygous for the mutant allele or lacking the mutant allele does not comprise L-homoserine in the leaf tissue.
2. The plant according to claim 1, wherein the plant is homozygous for the wild type allele of the homoserine kinase gene encoding the homoserine kinase protein of SEQ ID NO: 3.
3. The plant according to claim 1, wherein the mutant allele encodes a mutant homoserine kinase protein which comprises an amino acid selected from the group N64 (Asn at position 64 of SEQ ID NO: 1), D70 (Asp at position 70 of SEQ ID NO: 1), N118 (Asn at position 118 of SEQ ID NO: 1), C119 (Cys at position 119 of SEQ ID NO: 1), K144 (Lys at position 144 of SEQ ID NO: 1), G149 (Gly at position 149 of SEQ ID NO: 1), G151 (Gly at position 151 of SEQ ID NO: 1), L152 (Leu at position 152 of SEQ ID NO: 1), G153 (Gly at position 153 of SEQ ID NO: 1), S154 (Ser at position 154 of SEQ ID NO: 1), S155 (Ser at position 155 of SEQ ID NO: 1), S158 (Ser at position 158 of SEQ ID NO: 1), D196 (Asp at position 196 of SEQ ID NO: 1), N197 (Asn at position 197 of SEQ ID NO: 1), T241 (Thr at position 241 of SEQ ID NO: 1), R245 (Arg at position 245 of SEQ ID NO: 1), R293 (Arg at position 293 of SEQ ID NO: 1) and A320 (Ala at position 320 of SEQ ID NO: 1) being replaced by a different amino acid or being deleted.
4. The plant according to claim 1, wherein the mutant allele encodes a mutant homoserine kinase protein which comprises the amino acid Arginine (Arg, R) at amino acid number 245 of SEQ ID NO: 1 being replaced by a different amino acid.
5. The plant according to claim 1, wherein the leaf tissue comprises an amount of L-homoserine of at least 15.0 nmol per mg fresh weight.
6. The plant according to claim 1, wherein said L-homoserine in the leaf tissue confers resistance against Bremia lactucae.
7. The plant according to claim 1, wherein the amount of said L-homoserine in the leaf tissue is high enough to prevent sporulation of Bremia lactucae under conditions where the susceptible control plant shows 100% sporulation.
8. The plant according to claim 1, wherein said mutant allele which encodes a mutant homoserine kinase protein comprising one or more amino acid substitutions compared to the wild type protein of SEQ ID NO: 1, does not comprise an amino acid substitution at amino acid 316 of SEQ ID NO: 1.
9. (canceled)
10. (canceled)
11. Seeds from which a plant according to claim 1 can be grown.
12. Lettuce leaves or heads of a plant grown from the seeds of claim 11.
13. The plant according to claim 4, wherein the different amino acid is Lysine (Lys, K).
14. A method of producing a lettuce plant comprising crossing the plant of claim 1 with a lettuce plant, and obtaining seeds from the crossing.
15. The method of claim 14, further comprising growing the seeds.
Description
FIGURES
[0046]
[0047]
DETAILED DESCRIPTION
[0048] The instant inventors surprisingly found that in cultivated lettuce (Lactuca sativa) a gene on chromosome 8 is expressed in leaf tissue and encodes a functional HSK protein, which is the ortholog of the Arabidopsis HSK gene.
[0049] Based on the previous disclosure of the lettuce HSK ortholog in WO2007/051626 (
[0050] Upon further analysis, the lettuce genome was described to contain four loci with putative HSK-like genes.
[0051] As at least two (but maybe more) HSK-like proteins appear to be present in the chloroplasts of lettuce, only mutating several genes could be expected to reduce the enzyme activity in the chloroplast sufficiently to result in an overall increase of homoserine. A similar situation had been described for the DMR6 gene in crops where two DMR6 genes were found, see e.g. WO2015193418, where resistance against the oomycete Phytophthora infestans requires downregulation of two DMR6-like genes.
[0052] It was surprisingly found that, however, no such functional redundancy exists in lettuce and that mutations of only the gene on chromosome 8 are sufficient to increase L-homoserine in leaf tissue.
TABLE-US-00001 TABLE 1 Percentage protein sequence identity, determined using pairwise protein alignments using EMBOSS-Needle (default parameters) Lettuce HSK-like Lettuce HSK-like Lettuce protein Arabidopsis protein_Chr4 protein_Chr8 of FIG. 10 of AT2G17265 (XP_023751301.1) (XP_023762184.1) WO2007/051626 Arabidopsis HSK 100% 66.4% 72.6% 66.4% protein AT2G17265 Lettuce HSK-like 100% 75.3% 99.2% protein_Chr4 (XP_023751301.1) SEQ ID NO: 3 herein Lettuce HSK-like 100% 74.7% protein_Chr8 (XP_023762184.1) SEQ ID NO: 1 herein FIG. 10 of 100% WO2007/051626
[0053] None of the mutants comprising single amino acid substitutions in the protein encoded by the gene on chromosome 4 had an in planta increase of L-homoserine (despite the fact that the single amino acid substitutions were predicted to reduce the protein activity), while three mutants comprising single amino acid substitutions in the protein on chromosome 8 had an increased in planta L-homoserine level. See Examples. These results surprisingly showed that single amino acid substitutions in the HSK-like gene on chromosome 8 are sufficient to lead to an in planta increase in L-homoserine, while the gene on chromosome 4 may remain unmutated (wild type).
[0054] A further surprising finding was that a minimum level of in planta homoserine accumulation is needed to result in the lettuce plant being resistant against Bremia lactucae. The mutant which resulted in an amount of L-homoserine of 0.38 nmol/mg FW tissue (E238K) and the mutant which resulted in an amount of 4.41 nmol/mgFW tissue (M244I) did not lead to Bremia lactucae resistance. See Examples.
[0055] Therefore, in one aspect a plant of the species Lactuca sativa is provided, which is homozygous for a mutant allele of the homoserine kinase gene, which mutant allele encodes a mutant homoserine kinase protein comprising one or more amino acid inserted, deleted or replaced (substitutions) compared to the wild type homoserine kinase protein of SEQ ID NO: 1, or compared to a wild type homoserine kinase protein comprising at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:1, or which mutant allele comprises a reduced gene expression compared to the wild type allele, said mutant allele resulting in the leaf tissue of said lettuce plant comprising an amount of L-homoserine of at least 5.0 nmol per mg fresh weight (more preferably at least 10.0, 15.0, 16.0, 17.0, 18.0 or 19.0 or more nmol per mg fresh weight), while a plant heterozygous for the mutant allele or lacking the mutant allele (i.e. homozygous for the wild type allele encoding the wild type HSK protein of e.g. SEQ ID NO: 1) does not accumulate L-homoserine in the leaf tissue.
[0056] A wild type homoserine kinase protein of SEQ ID NO: 1, or a wild type homoserine kinase protein comprising at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:1 (also referred to as wild type HSK protein variant or simply “variant of SEQ ID NO: 1”), refers to a fully functional protein, i.e. which does not have reduced enzymatic activity and converts L-homoserine into phospho-homoserine (phosphorylation). When present in the plant e.g. in homozygous form, e.g. in a normal lettuce breeding line or variety, no L-homoserine accumulates in the leaf tissue. The functionality of a homoserine kinase protein or protein variant can thus be seen in plant in vivo, either being present endogenously or overexpressed in a transgenic lettuce plant, or alternatively by in vitro assays, by e.g. measuring the consumption of ATP of the recombinantly produced protein, which is an indirect measure of enzymatic activity as ATP is used for phosphorylation of homoserine. Such in vitro assays are known in the art, e.g. described in Van Damme et al. 2009 for the Arabidopsis HSK enzyme (supra, page 2187 under “HSK Recombinant Protein Production and Enzyme assay”).
[0057] The homoserine kinase gene or allele encoding a wild type HSK protein is, accordingly, referred to as a wild type homoserine kinase gene or allele. As the coding sequence of the gene contains no intron sequences, the genomic DNA and the coding DNA (cDNA) are identical. The mRNA encoding the protein is thus also identical to the genomic DNA and cDNA except that off course the nucleoside T (thymine) is replaced by U (uracil) in the mRNA. In SEQ ID NO: 2 the genomic DNA and cDNA encoding the protein of SEQ ID NO: 1 is shown.
[0058] A plant comprising a fully functional, wild type homoserine kinase gene in homozygous form is also referred to a “wild type lettuce plant”. This is in contrast to a plant which comprises a mutant allele of the homoserine kinase gene, which mutant allele encodes a mutant homoserine kinase protein comprising one or more amino acid insertions, deletions or substitutions compared to the wild type homoserine kinase protein and wherein the mutant homoserine kinase protein has reduced enzymatic activity in vivo, resulting in an accumulation of L-homoserine in the leaf tissue, when the mutant allele is in homozygous form, as the conversion of homoserine to phospho-homoserine occurs to a lower extent than in wild type plants. When the mutant allele is in heterozygous form (i.e. the plant comprises only one copy of the mutant allele and one copy of the wild type allele, homoserine will not accumulate, as the fully functional wild type protein still carries out the conversion.
[0059] Alternatively a plant which comprises a mutant allele of the homoserine kinase gene is a plant in which the gene expression is reduced (knocked-down) compared to the wild type allele, e.g. the mutant allele comprises one or more mutations in the regulatory elements, such as the promoter sequence.
[0060] In other words, the lettuce plant according to the invention comprises a mutant homoserine kinase allele, which mutant allele either a) encodes a mutant homoserine kinase protein comprising one or more amino acid insertions, deletions or substitutions compared to the wild type homoserine kinase protein of SEQ ID NO: 1, or compared to a variant wild type homoserine kinase protein comprising at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:1, said mutant protein comprises reduced enzymatic activity compared to the wild type protein, or b) which mutant allele comprises reduced gene expression and therefore a lower amount of wild type protein, and as a result of a) or b) leads to accumulation of L-homoserine in the leaf tissue when the mutant allele is in homozygous form (while a plant homozygous for the wild type allele does not lead to homoserine accumulation in the leaves).
[0061] The reduced enzymatic activity of a mutant homoserine kinase protein or the reduced gene expression can thus be seen in a plant in vivo, when present in homozygous form in a lettuce plant (e.g. the degree of reduction of enzymatic activity or reduced gene expression will be reflected in the amount of homoserine accumulating), or alternatively by in vitro assays, by e.g. measuring the consumption of ATP of the recombinantly produced mutant HSK protein, which is an indirect measure of enzymatic activity, as ATP is used for phosphorylation of homoserine. Such in vitro assays are known in the art, e.g. described in Van Damme et al. 2009 for the Arabidopsis HSK enzyme (supra, page 2187 under “HSK Recombinant Protein Production and Enzyme assay”). Thus, the enzymatic activity of a recombinantly produced wild type homoserine kinase protein can be compared to the enzymatic activity of one or more mutant proteins and also the activity of different mutant HSK proteins can be compared. Some amino acid substitutions have a more profound effect on reducing the enzymatic activity than others, as can be seen in the Examples.
[0062] As mentioned, the degree to which the enzymatic activity of the mutant homoserine kinase protein is reduced will vary, depending on the effect of the amino acid insertion, deletion or substitution on the protein. As an indication regarding whether an amino acid substitution will likely affect protein activity, various computer programs can be used, such as SIFT analysis (Sorting Intolerant from Tolerant, see www at //sift.bii.a-star.edu.sg/) or PROVEAN (www at provean.jcvi.org/index.php) can be used. However, whether the mutation has the desired effect and especially whether the enzymatic activity is sufficiently reduced to lead to a minimum desired accumulation of homoserine, e.g. at least 5.0 nmol/mg FW, always needs to be confirmed experimentally.
[0063] Interestingly, the three lettuce mutants found to lead to an increase in L-homoserine, were not in the two conserved kinase domains, but in-between the two conserved kinase domains, i.e. in-between the N terminal kinase domain and the C terminal kinase domain. The protein of SEQ ID NO: 1 contains a “GHMP kinase N terminal domain” starting at amino acid 140 and ending at amino acid 205 (Pfam domain PF00288) and a “GHMP kinase C terminal domain” starting at amino acid 268 and ending at amino acid 345 (Pfam domain PF08544).
[0064] Also, the three mutants identified resulted in very different levels of homoserine accumulation. Mutant E238K resulted in only 0.38 nmol/mgFW of L-homoserine, while the other two mutants resulted in an accumulation of L-homoserine to 4.41 (mutant M244i) and 19.46 nmol/mgFW (mutant R245K).
[0065] The three dimensional structure of the enzyme was analyzed (see Examples), and surprisingly it was found that 18 amino acids distributed across the entire protein were actually interacting with L-homoserine or with ATP. Amongst these 18 amino acids was the amino acid R245 of SEQ ID NO: 1. The three dimensional structure therefore explained why the substitution of R245 had a profound effect on enzymatic activity and resulted in a much higher L-homoserine accumulation than the other mutants, which were substitutions in amino acids which did not interact with L-homoserine or ATP.
[0066] Therefore, in one aspect a plant of the species Lactuca sativa is provided, which is homozygous for a mutant allele of the homoserine kinase gene, which mutant allele encodes a mutant homoserine kinase protein comprising one or more amino acid insertions, deletions or substitutions compared to the wild type homoserine kinase protein of SEQ ID NO: 1, or compared to a wild type homoserine kinase protein comprising at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:1, said mutant allele resulting in the leaf tissue of said lettuce plant comprising an amount of L-homoserine of at least 5.0 nmol per mg fresh weight, while a plant heterozygous for the mutant allele or lacking the mutant allele (i.e. homozygous for the wild type allele encoding the wild type HSK protein of e.g. SEQ ID NO: 1) does not accumulate L-homoserine in the leaf tissue, wherein said one or more amino acid substitutions, or deletions, are selected from the amino acids N64 (Asn at position 64 of SEQ ID NO: 1), D70 (Asp at position 70 of SEQ ID NO: 1), N118 (Asn at position 118 of SEQ ID NO: 1), C119 (Cys at position 119 of SEQ ID NO: 1), K144 (Lys at position 144 of SEQ ID NO: 1), G149 (Gly at position 149 of SEQ ID NO: 1), G151 (Gly at position 151 of SEQ ID NO: 1), L152 (Leu at position 152 of SEQ ID NO: 1), G153 (Gly at position 153 of SEQ ID NO: 1), S154 (Ser at position 154 of SEQ ID NO: 1), S155 (Ser at position 155 of SEQ ID NO: 1), S158 (Ser at position 158 of SEQ ID NO: 1), D196 (Asp at position 196 of SEQ ID NO: 1), N197 (Asn at position 197 of SEQ ID NO: 1), T241 (Thr at position 241 of SEQ ID NO: 1), R245 (Arg at position 245 of SEQ ID NO: 1), R293 (Arg at position 293 of SEQ ID NO: 1) and A320 (Ala at position 320 of SEQ ID NO: 1) being replaced by a different amino acid, or being deleted.
[0067] Other amino acid substitutions or deletions may also lead to an increase in homoserine. One way to finally find out whether a mutant will result in an increase in homoserine, and to what extent, is to generate the mutant and then to test the homoserine level. The skilled person can do this without undue burden by generating mutants and identifying lettuce plants comprising mutations which result in e.g. substitutions of particular amino acids in the protein of SEQ ID NO: 1 (or in a variant thereof), such as for example in any of the amino acids which interact with homoserine or ATP as indicated above, or amino acids near any of the 18 ‘interacting’ amino acids, such as the amino acids located 1, 2, 3 or 4 positions before or after the ‘interacting’ amino acid.
[0068] In one aspect a lettuce plant is provided comprising a mutant allele of a homoserine kinase gene, which mutant allele encodes a mutant homoserine kinase protein comprising one or more amino acid substitutions compared to the wild type homoserine kinase protein of SEQ ID NO: 1, or compared to a variant wild type homoserine kinase protein comprising at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:1, wherein the mutant homoserine kinase protein comprises one or more amino acids, selected from the group consisting of N64 (Asn at position 64 of SEQ ID NO: 1), D70 (Asp at position 70 of SEQ ID NO: 1), N118 (Asn at position 118 of SEQ ID NO: 1), C119 (Cys at position 119 of SEQ ID NO: 1), K144 (Lys at position 144 of SEQ ID NO: 1), G149 (Gly at position 149 of SEQ ID NO: 1), G151 (Gly at position 151 of SEQ ID NO: 1), L152 (Leu at position 152 of SEQ ID NO: 1), G153 (Gly at position 153 of SEQ ID NO: 1), S154 (Ser at position 154 of SEQ ID NO: 1), S155 (Ser at position 155 of SEQ ID NO: 1), S158 (Ser at position 158 of SEQ ID NO: 1), D196 (Asp at position 196 of SEQ ID NO: 1), N197 (Asn at position 197 of SEQ ID NO: 1), T241 (Thr at position 241 of SEQ ID NO: 1), R245 (Arg at position 245 of SEQ ID NO: 1), R293 (Arg at position 293 of SEQ ID NO: 1) and A320 (Ala at position 320 of SEQ ID NO: 1), being substituted by a different amino acid. Optionally, alternatively or in addition an amino acid 1, 2, 3 or 4 positions preceding or following the above amino acids may be substituted. Alternatively, one or more of the above amino acids are deleted in the mutant protein, whereby the interaction with L-homoserine or ATP will be impaired and the enzymatic activity will be reduced.
[0069] As described above, a mutant allele may either result in a HSK protein having reduced enzymatic activity or may result in a reduced gene expression of the wild type allele and thereby a reduced amount of wild type HSK protein in the tissue. It is preferred however, that the mutant HSK protein still has some enzymatic activity or that the mutant allele still has some level of gene expression, as it is believed that the lettuce plant comprising the mutant allele in homozygous form will otherwise show severe negative side effect on e.g. growth and vitality of the plant. So, neither a loss-of-function protein nor a knock-out of gene expression are preferred herein. In one aspect the mutant HSK protein has an enzymatic activity which is at least 40%, 50%, 60%, 70% or 80%, or 90% of the enzymatic activity of the wild type HSK protein, but which is less than 100% of the enzymatic activity of the wild type protein. In one aspect the mutant allele expresses at least 40%, 50%, 60%, 70%, 80% or 90% of the mRNA transcript expressed by the wild type allele, but expresses less than 100% of the mRNA transcript of the wild type allele.
[0070] Preferably the lettuce plant comprises the mutant allele in homozygous form, resulting in an accumulation of L-homoserine. Preferably at least 5.0 nmol/mg/FW L-homoserine, at least 6.0 nmol, 7.0 nmol, 8.0 nmol, 9.0 nmol, 10.0 nmol, 11.0 nmol, 12.0 nmol, 13.0 nmol, 14.0 nmol, 15.0 nmol, 16.0 nmol, 17.0 nmol, 18.0 nmol or at least 19.0 nmol accumulate in planta.
[0071] It is thus understood that the amino acid insertion, deletion or substitution does in one aspect not result in a completely inactive homoserine kinase protein and that the mutant allele is not a knock-out allele, as this is thought to have severe negative effects on the fitness of the plant and may even be lethal to the plant.
[0072] In a preferred aspect, the mutant homoserine kinase protein is reduced in enzymatic activity, or the gene expression is reduced to such an extent that the amount of L-homoserine accumulating in leaf tissue of plants homozygous for the mutant allele is at least 5.0 nmol per mg fresh weight of leaf tissue, more preferably at least 6.0, 7.0 or 8.0 nmol per mg fresh weight of leaf tissue, more preferably at least 9.0 nmol or 10.0 nmol or 15.0 nmol, 16.0 nmol, 17 nmol, 18nmol, or even at least 19.0 nmol per mg fresh weight of leaf tissue.
[0073] In one aspect the mutant allele (when in homozygous form) does not result in an accumulation of L-homoserine of more than 50 or 40 nmol per mg FW, preferably not more than 30 nmol per mg FW, not more than 25.0 nmol per mg FW, or not more than 24.0 nmol, 23.0 nmol, 22.0 nmol, 21.0 nmol or 20.0 nmol per mg/FW. In particular, the homoserine level should not have a negative effect on other characteristics of the plant, such as the growth and development of the plant, yield, etc., while still providing resistance against Bremia.
[0074] In one aspect the amount of at least 5.0 nmol/mg/FW, or more (as indicated above), of L-homoserine accumulating in the leaf tissue of a plant is an average of measurements of at least two, three, four or five independent leaf samples of the plant comprising the mutant allele in homozygous form.
[0075] The skilled person can generate such mutants as described herein, e.g. by screening a mutant lettuce population (e.g. a TILLING population) for mutants in the homoserine kinase gene encoding the protein of SEQ ID NO: 1 (or a variant thereof), optionally selfing the mutants to generate homozygous plants if the plants were not yet homozygous, and analyzing the amount of homoserine in the leaf tissue of said plants. The plants comprising mutations which result in at least 5.0 nmol per milligram fresh weight of the lettuce leaf tissue (or at least another minimal amount as referred to above) can then be selected. Optionally the skilled person can then test whether these plants comprise resistance against one or more races of Bremia lactucae, using e.g. a Leaf Disc Test as described in the Examples. It is understood that, if the plant line or variety in which the mutant was generated, already contains resistance against one or more Bremia races, then the effect a mutation against the resistance cannot be tested in that genetic background and the mutation needs first to be crossed into a line which is susceptible against the Bremia race(s).
[0076] The Leaf Disc Test (LDT) described in the Examples has the advantage over other tests, such as seedling tests (also known as cotyledons test) that it provides a good correlation with field resistance against Bremia lactucae.
[0077] In one aspect the lettuce plant which is homozygous for a mutant HSK allele, as described above, comprises an amount of L-homoserine in leaf tissue of at least 5.0 nmol per mg fresh weight of leaf tissue, more preferably at least 6.0, 7.0 or 8.0 nmol per mg fresh weight of leaf tissue, more preferably at least 9.0 nmol or 10.0 nmol, 11.0 nmol, 12.0 nmol, 13.0 nmol, 14.0 nmol or 15.0 nmol or 16.0 nmol or 17.0 nmol or 18.0 nmol even at least 19 nmol per mg fresh weight of leaf tissue and is preferably resistant to Bremia lactucae, preferably at least to one or more races of Bremia lactucae, e.g. at least race BI:25. In one aspect the leaves of the plant do not show any sporulation of the Bremia lactucae race with which they were inoculated or infected, while the susceptible control plants and/or plants heterozygous for the mutant allele show sporulation when e.g. using a Bremia lactucae test Leaf Disc Test as described in the Examples. It is understood that the Bremia resistance is caused by the amount of L-homoserine accumulated in the leaf tissue, and not by any background resistance gene present in the plant.
[0078] As mentioned, the homoserine kinase gene on chromosome 4, encoding the protein of SEQ ID NO: 3, is not effective in altering homoserine kinase levels in the plant when mutated. Thus, in one aspect the plant according to the invention, comprising a mutant allele in the homoserine kinase gene on chromosome 8 in homozygous form, as described above, further comprises a wild type (non-mutated) homoserine kinase gene on chromosome 4, e.g. comprises the wild type allele of the homoserine kinase gene encoding the wild type homoserine kinase protein of SEQ ID NO: 3, or encoding another functional variant thereof, such as a protein comprising at least 85%, 90%, 95% or more sequence identity to SEQ ID NO: 3. The genomic coding sequence, which corresponds to the cDNA sequence, encoding the protein of SEQ ID NO: 3 is shown in SEQ ID NO: 4. Thus, in one aspect the lettuce plant according to the invention comprises the sequence of SEQ ID NO: 4 in its genome, preferably in homozygous form.
[0079] In a specific aspect of the invention the plant of the species Lactuca sativa which is homozygous for a mutant allele of the homoserine kinase gene, which mutant allele encodes a mutant homoserine kinase protein comprising one or more amino acid substitutions or deletions compared to the wild type protein of SEQ ID NO: 1 (or a variant thereof), comprises a change of Arginine (Arg, R) at amino acid number 245 of SEQ ID NO: 1 to a different amino acid, preferably to Lysine (Lys, K), or any other amino acid, or comprises a deletion of the Arginine at amino acid number 245 of SEQ ID NO: 1. Thus, the mutant allele results in a mutant protein, which comprises a single amino acid substitution of amino acid number 245 in SEQ ID No: 1 or comprises a deletion of one or more amino acids, whereby amino acid 245 of SEQ ID NO: 1 is deleted. In one aspect the amino acid substitution is a R245K substitution. This particular mutant lettuce plant (comprising the mutant allele in homozygous form) was found to contain L-homoserine in an amount of 19.46 nmol per mg/fresh weight of leaf tissue. The mutant also did not show any sporulation of Bremia lactucae BI:25, in the Leaf Disc Test described in the Examples, while the susceptible controls (containing only wild type HSK alleles) showed 100% sporulation.
[0080] Two different mutants, mutant E238K and (comprising a E238K substitution, i.e. Glutamic acid, Glu, at amino acid 238 being substituted by Lysine, Lys) in SEQ ID NO: 1 and mutant M244i (comprising a M244i substitution, i.e. Methionine, Met, at amino acid 244 being substituted by Isoleucine, I) in SEQ ID NO: 1 showed an increase in L-homoserine of 0.38 nmol/mg FW and 4.41 nmol/mg/FW respectively. Surprisingly, these two mutants were both susceptible to Bremia lactucae. Mutant E238K showed 100% sporulation in the Leaf Disc Test with race BI:25EU in the Examples, the same as the susceptible controls. Likewise mutant M244i was susceptible to Bremia race BI:26EU showing significant sporulation.
[0081] As mentioned, the plants according to the invention, accumulating at least 5.0 nmol/mg FW of L-homoserine (or at least 10 nmol/mg FW, etc. as described), are preferably resistant against one or more races of Bremia lactucae. Bremia lactucae comprises different races, referred to as BI:X, where X is a number from 1 to 35 for European races and a number from 1 to 9 for US races. The European races are designated with the suffix ‘EU’, while the US races are designated with the suffix ‘US’. In Europe the races BI:16-35EU are important (this designation excludes BI:19EU and BI:28EU, which are no longer existent), while BI:1-15EU are no longer important in the field. In the USA BI:1-4US have less importance, as they are no longer detected in the western USA, while BI:5-9US are important races.
[0082] Thus, in one aspect the plants according to the invention, which comprise at least 5 nmol per mg FW homoserine in their leaf tissue, comprise resistance against one or more or all races selected from BI16—35EU and BI:5-9US. Although the mutant R245K has so far only been shown to be resistant against Bremia race BI:25EU, it is expected that the mutant will likewise be resistant against the other races of Bremia lactucae, as L-homoserine is expected to negatively affect the reproduction of this pathogen.
[0083] Resistance against a race of Bremia lactucae is herein in one aspect defined as no Bremia spores being produced on the leaves or leaf parts under conditions wherein the susceptible control, e.g. the same plant lacking the mutant HSK allele, produces spores and is fully susceptible to the race. For example, in the Leaf Disc Test described in the Examples, the leaf discs of the resistant plant would show no spores (0% sporulation), while the leaf discs of the susceptible control show 100% sporulation.
[0084] Preferably the resistant lettuce plants according to the invention also show no sporulation when exposed to Bremia lactucae races in the field or in other environments, such as the glasshouses or tunnels.
[0085] Thus, in one aspect the lettuce plant comprising the mutant HSK allele in homozygous form comprises an amount of said L-homoserine in the leaf tissue which is high enough to prevent sporulation of Bremia lactucae under conditions where the susceptible control plant shows the expected sporulation. The skilled person can generate different mutants, which result in the accumulation of different amounts of L-homoserine when the allele is in homozygous form and then test whether the amount of L-homoserine is high enough to prevent sporulation of Bremia lactucae, e.g. in a Leaf Disc Test as described in the examples or another test. Importantly the susceptible control, which comprises wild type, non-mutated HSK alleles, such as variety Wendel or Cervino or others, are completely susceptible in the test and show e.g. 100% sporulation in the Leaf Disc Test (see Examples).
[0086] In one aspect a plant of the species Lactuca sativa is provided, which is homozygous for a mutant allele of the homoserine kinase gene, which mutant allele encodes a mutant homoserine kinase protein comprising one or more amino acid insertions, deletions or substitutions compared to the wild type protein of SEQ ID NO: 1, or comprising reduced gene expression of the mutant allele, said mutant allele resulting in the leaf tissue of said lettuce plant comprising an amount of L-homoserine which is high enough to result in the absence of Bremia lactucae sporulation when the plant or plant parts are tested under conditions wherein the susceptible control shows the expected phenotype (e.g. 100% sporulation in the Leaf Disc Test).
[0087] In one aspect the lettuce plant according to the invention, which comprises a mutant allele which encodes a mutant homoserine kinase protein comprising one or more amino acid substitutions compared to the wild type protein of SEQ ID NO: 1, does not comprise an amino acid substitution at amino acid 316 of SEQ ID NO: 1. Thus, in one aspect, amino acid 316 of SEQ ID NO: 1 is a Threonine (Thr, T).
[0088] Preferably the mutation in the allele of the homoserine kinase gene described herein is induced by human intervention, such as mutagenesis (exposure to a mutagenic agent, e.g. radiation, chemical mutagens, etc.) or targeted gene modification techniques, such as CRISPR/Cas or TALENS or others. In one aspect the mutant allele is, thus, not a naturally occurring mutant allele, i.e. it is not derived from natural populations or breeding populations. The mutation in the HSK allele is, therefore, in one aspect not a naturally occurring mutation, but a mutation induced by human intervention.
[0089] In one aspect the plant comprising the mutant allele is, therefore, not exclusively obtained by an essentially biological process, such as crossing and selection, but generation of the mutant allele involved a technical step. It is understood that the mutant allele may be transferred into any other lettuce plant through crossing and selection.
[0090] Further encompassed herein are seeds from which a plant according to the invention grows, as well as any parts of a plant according to the invention, such as lettuce heads, leaves, baby leaf leaves, flowers, pollen, roots, ovaries, cells, embryos, etc.
[0091] The plant or plant parts comprise at least one copy of the mutant HSK allele in their genome, preferably two copies.
[0092] Also lettuce plants and plant parts comprising a mutant HSK allele as described herein in heterozygous form are part of the invention, as these are useful in breeding plants which comprise the mutant allele in homozygous form.
[0093] In one aspect also non-propagating cells of a lettuce plant according to the invention are provided, which comprise at least one copy, preferably two copies of the mutant HSK allele in their genome. Non-propagating cells are cells which cannot regenerate into a lettuce plant.
[0094] Also tissue- or cell cultures comprising tissues or cells of a lettuce plant according to the invention are provided herein.
[0095] Feed or food products comprising cells or tissues of a lettuce plant according to the invention are also provided herein.
[0096] The cultivated lettuce plant according to the invention may be of any type, such as butterhead, iceberg, etc. The mutant allele can easily be transferred from one lettuce plant into another by e.g. backcrossing. In this way many different lettuce types and varieties can be generated, which comprise Bremia resistance due to the presence of a mutant allele according to the invention in homozygous form.
Methods According to the Invention
[0097] In one aspect a method for generating and/or selecting a lettuce plant comprising at least 5.0 nmol L-homoserine per mg fresh weight of leaf tissue, more preferably at least 6.0, 7.0 or 8.0 nmol L-homoserine per mg fresh weight of leaf tissue, more preferably at least 9.0 nmol or 10.0 nmol or 15.0 nmol or even at least 19.0 nmol L-homoserine per mg fresh weight of leaf tissue, is provided comprising the steps: [0098] a) Providing a plurality of lettuce plants which are homozygous for a mutant allele of the homoserine kinase gene, which mutant allele encodes a mutant homoserine kinase protein comprising one or more amino acid insertions, deletions or substitutions compared to the wild type protein of SEQ ID NO: 1, or which mutant allele has reduced gene expression compared to the wild type allele, [0099] b) Analyzing the amount of L-homoserine produced in the leaf tissue of the plants, [0100] c) Identifying, and optionally selecting, a plant which comprises in its leaf tissue at least 5.0 nmol of L-homoserine per mg fresh weight of leaf tissue, more preferably at least 6.0, 7.0 or 8.0 nmol L-homoserine per mg fresh weight of leaf tissue, more preferably at least 9.0 nmol or 10.0 nmol or 15.0 nmol or even at least 19 nmol L-homoserine per mg fresh weight of leaf tissue.
[0101] In one aspect the plants of a) comprise a wild type HSK allele in homozygous form encoding a homoserine kinase protein of SEQ ID NO: 3 or a protein comprising at least 95% amino acid sequence identity to the protein of SEQ ID NO: 3.
[0102] In one aspect the plants of a) comprise SEQ ID NO: 4 in homozygous form.
[0103] The method may further comprise determining whether the identified or selected plant of step c) comprises resistance against at least one race of Bremia lactucae. Resistance can be tested by various methods, e.g. as described in the Examples or other methods. In one aspect the selected lettuce plant does not produce Bremia spores on the leaves, while the same lettuce plant, or another susceptible control lettuce plant, which only contains the wild type HSK allele and produces the wild type HSK protein of SEQ ID NO: 1 (or a variant thereof), does produce Bremia spores on the leaves.
[0104] Any plant produced by the above method is also an aspect of the invention, as are seeds from which such a plant can be grown.
[0105] Also encompassed is a method for detecting whether a lettuce plant or plant part comprises a mutant HSK allele according to the invention. This can be done in various ways. In one aspect it comprises analyzing the DNA for the presence of a mutant allele according to the invention, e.g. by detecting the mutant allele itself (e.g. Single Nucleotide Polymorphism, SNP genotyping, whereby the presence of one or two copies, or the absence of the mutant allele can be detected). The genomic coding sequence which encodes the wild type HSK protein of SEQ ID NO: 1 is provided in SEQ ID NO: 2 herein. Alternatively the presence of a mutant messenger RNA (mRNA) can be detected. The mRNA is identical to the genomic DNA of SEQ ID NO: 2, except that T is replaced by U. Alternatively, the presence of the mutant HSK protein can be detected. Alternatively, a reduced level of expression, e.g. of wild type mRNA (or cDNA) can be detected. In yet another aspect the amount of L-homoserine in the leaf tissue and/or the resistance against Bremia can be tested before or after testing whether the plant comprises a mutant HSK allele.
[0106] A method for detecting whether a lettuce plant or plant part comprises a mutant HSK allele encoding a protein of SEQ ID NO: 1 which comprises one or more amino acid insertions, deletions or substitutions comprises [0107] a) determine the presence in the plant of a mutant nucleic acid molecule comprising one or more nucleotides which are different compared to the genomic DNA of SEQ ID NO: 2 or the mRNA of SEQ ID NO: 2 and whereby the encoded protein comprises one or more amino acids which are different from SEQ ID NO: 1 or which are inserted or deleted compared to SEQ ID NO: 1; or [0108] b) determining the presence in the plant of a protein comprising one or more amino acids which are different from the protein of SEQ ID NO: 1 or which are inserted or deleted compared to SEQ ID NO: 1.
[0109] In one aspect the mutant nucleic acid molecule encodes a mutant homoserine kinase protein which comprises one or more amino acids, selected from the group consisting of: N64 (Asn at position 64 of SEQ ID NO: 1), D70 (Asp at position 70 of SEQ ID NO: 1), N118 (Asn at position 118 of SEQ ID NO: 1), C119 (Cys at position 119 of SEQ ID NO: 1), K144 (Lys at position 144 of SEQ ID NO: 1), G149 (Gly at position 149 of SEQ ID NO: 1), G151 (Gly at position 151 of SEQ ID NO: 1), L152 (Leu at position 152 of SEQ ID NO: 1), G153 (Gly at position 153 of SEQ ID NO: 1), S154 (Ser at position 154 of SEQ ID NO: 1), S155 (Ser at position 155 of SEQ ID NO: 1), S158 (Ser at position 158 of SEQ ID NO: 1), D196 (Asp at position 196 of SEQ ID NO: 1), N197 (Asn at position 197 of SEQ ID NO: 1), T241 (Thr at position 241 of SEQ ID NO: 1), R245 (Arg at position 245 of SEQ ID NO: 1), R293 (Arg at position 293 of SEQ ID NO: 1) and A320 (Ala at position 320 of SEQ ID NO: 1) being substituted by a different amino acid. In another aspect the mutant nucleic acid molecule encodes a mutant HSK protein in which one or more of the above amino acids are deleted.
[0110] In one aspect nucleotide 734 of SEQ ID NO: 2 is changed from a guanine into an adenine, whereby the codon ‘agg’ (Arginine) is changed into codon ‘aag’ (Lysine). Thus, in one aspect the nucleic acid molecule comprising an adenine at nucleotide 734 of SEQ ID NO: 2 is detected. For example SNP genotyping can be used to distinguish between the presence of two copies of the wild type allele of SEQ ID NO: 2, one copy of the wild type allele (comprising SEQ ID NO: 2) and one copy of the mutant allele of SEQ ID NO: 2 (comprising an adenine at nucleotide 734 of SEQ ID NO: 2) or two copies of the mutant allele in the genome of the plant or plant part or plant cell.
[0111] Thus, in one aspect the lettuce plant of the invention comprises in its genome SEQ ID NO: 2, wherein the codon 733 to 735 (agg) comprises a nucleotide substitution, e.g. nucleotide 734 is changed into a different nucleotide, e.g. adenine. In one aspect a mutation in this codon can be used to detect the mutant allele.
[0112] Also provided is a method of growing a plant of the invention in an area where Bremia lactucae can occur and harvesting the leaves of the plant.
[0113] Likewise the mutant proteins and mutant nucleotide sequences described herein are provided, e.g. in isolated form.
[0114] Further, the use of a mutant HSK allele for detecting a lettuce plant or for breeding a lettuce plant which accumulates L-homoserine in the leaf tissue is provided herein.
TABLE-US-00002 SEQUENCES HSK protein on chromosome 8 >SEQ ID NO: 1 MAICHHHQPSFTIPSSFPFTTNLSNKSQLHLPSSFRCNLSVTTNLEPEPV YTAVKSFAPATVANLGPGFDFLGCAVDGIGDYVTLKIDPQVHPGEVSITE ITGTGNSANKLSKNPIWNCAGIAAISVMKMLNIRSVGLSLSLEKGLPLGS GLGSSAASAAAAAIAVNEIFGGKLPALDLVLAGLESEAKVSGYHADNIAP AIMGGFVLVRSYDPLELIPLQFPVDKNLYFVLVNPEFEAPTKKMRAALPK EITMSHHVWNSSQAGALVAAVLQGDLKGFGKALSSDKIVEPRRAPLIPGM DAVKKAALEAGAYGCTISGAGPTAVAVTDNEEKGREIGEKMVEAFMAEGN LKAVAMVKQLDRVGARLVSSISR genomic DNA, cDNA encoding SEQ ID NO: 1 >SEQ ID NO: 2 ATGGCGATTTGTCATCACCATCAACCTTCATTCACCATCCCTTCTTCTTT CCCATTCACTACTAATCTTTCAAACAAATCCCAACTTCATCTCCCATCGT CTTTCCGCTGCAATCTATCCGTCACTACAAATCTCGAACCCGAACCCGTT TACACCGCCGTCAAGTCATTCGCCCCCGCCACCGTAGCCAACCTCGGCCC TGGGTTTGACTTTCTCGGTTGCGCAGTCGACGGGATCGGAGACTATGTCA CCCTCAAAATCGACCCCCAAGTTCACCCTGGCGAGGTCTCAATCACCGAA ATCACCGGAACCGGCAACTCCGCCAATAAGCTCAGCAAAAACCCTATCTG GAATTGCGCTGGGATTGCTGCCATTTCTGTCATGAAGATGCTCAACATCC GATCCGTCGGCCTCTCTCTATCTCTAGAAAAGGGTCTCCCCCTCGGAAGC GGTCTCGGTTCCAGCGCCGCTAGTGCCGCCGCCGCGGCAATCGCCGTTAA TGAGATTTTTGGTGGAAAGTTACCTGCATTGGATTTAGTCCTCGCAGGGC TTGAATCGGAAGCTAAAGTATCCGGATACCACGCTGATAACATTGCGCCG GCAATCATGGGTGGTTTCGTTCTCGTTCGGAGCTACGATCCTTTAGAGCT GATTCCGTTGCAGTTTCCGGTCGACAAAAACCTCTATTTCGTCTTGGTGA ATCCGGAATTCGAAGCGCCGACGAAGAAGATGAGGGCGGCGTTACCAAAA GAGATAACAATGTCGCACCATGTATGGAACAGTAGTCAAGCAGGTGCCTT GGTGGCGGCGGTGTTGCAGGGGGATTTGAAGGGGTTTGGAAAGGCGTTGT CTTCTGATAAGATAGTGGAACCGAGGAGGGCGCCATTGATTCCGGGAATG GATGCTGTGAAGAAGGCTGCACTTGAGGCAGGGGCTTATGGGTGTACGAT CAGTGGAGCAGGGCCAACTGCGGTGGCTGTTACAGATAACGAGGAAAAAG GGAGGGAGATTGGGGAGAAGATGGTGGAAGCTTTCATGGCGGAAGGAAAT TTGAAAGCTGTGGCTATGGTGAAGCAATTGGACAGAGTTGGTGCTAGACT TGTTAGTAGCATTTCCAGATAA HSK like protein on chromosome 4 >SEQ ID NO: 3 MAIRHYQPPFASTSSSISSTDLFKPPKLHLSSSVRCNISVASKLEPEPHP VFTSVKSFAPATVANLGPGFDFLGCAIDGIGDYVTLTVDPQVQPGRLSIA EINGVDKSSKRLSRNPLWNCAGIAAISVMKMLKIRSVGLSLSINTCLPLR GGLGSSAASAAAAAVAVNEIFGGKLQDSDLILAGLEAEAKLSGYHADNIA PAIMGGFVLIRSYDPLELISLKFPPEKNLFFVLVNPEFQAQTKKMRAVLP TEITMSDHVWNCSQAAALVAGVLQGDLVGFGKALSSDRIVEPRRAPLLPG MEDVKKAAMEAGAYGCTISGSGPTVVAVTDDEDRGREIGEKMVEAFVEKG KLKALAMVKKLDRVGARVISRISSQ genomic DNA, cDNA encoding SEQ ID NO: 3 >SEQ ID NO: 4 ATGGCAATTCGCCATTATCAACCTCCATTCGCCTCCACTTCTTCTTCTAT CTCTAGTACAGATTTATTCAAACCCCCTAAACTTCATCTTTCATCGTCTG TCCGGTGCAACATCTCCGTCGCTTCCAAACTGGAACCCGAACCTCATCCA GTTTTCACCTCCGTTAAGTCATTCGCCCCCGCCACCGTAGCCAACCTCGG GCCTGGTTTCGACTTCCTCGGCTGCGCAATCGACGGCATCGGAGATTACG TTACCCTCACAGTCGACCCCCAAGTCCAACCCGGCAGATTATCAATTGCA GAAATCAACGGCGTTGACAAGTCTTCCAAGAGGCTCAGCAGAAACCCTCT ATGGAATTGCGCCGGAATTGCTGCAATCTCCGTCATGAAGATGCTCAAGA TCCGATCCGTTGGCCTCTCTTTATCCATCAATACATGTCTCCCCCTTCGA GGCGGCCTAGGCTCCAGCGCCGCTAGCGCTGCCGCCGCCGCCGTTGCGGT TAATGAGATTTTCGGAGGGAAGTTACAGGATTCCGATTTGATACTCGCGG GGCTCGAAGCTGAAGCGAAGTTATCCGGTTATCACGCCGATAACATTGCT CCGGCGATCATGGGCGGGTTTGTGTTGATCAGAAGCTACGATCCATTAGA GTTGATCTCCTTGAAGTTTCCACCGGAAAAGAATCTGTTTTTCGTGTTGG TGAATCCTGAATTCCAAGCACAAACGAAGAAGATGAGGGCGGTTCTACCG ACGGAGATAACAATGTCGGATCATGTATGGAATTGTAGTCAGGCGGCAGC GTTGGTGGCAGGCGTATTGCAGGGGGATTTGGTGGGGTTTGGGAAGGCAT TGTCATCGGATAGAATTGTGGAGCCACGGCGGGCGCCATTGCTTCCGGGG ATGGAAGATGTGAAGAAGGCAGCAATGGAAGCAGGGGCATATGGGTGTAC GATAAGTGGGTCAGGGCCGACGGTGGTGGCGGTGACGGATGATGAAGATA GAGGGAGGGAGATCGGGGAGAAGATGGTGGAAGCTTTTGTGGAGAAGGGA AAGTTGAAAGCTTTGGCTATGGTGAAGAAACTGGACAGAGTTGGTGCTAG AGTTATCAGTCGTATCTCCAGCCAATGA
EXAMPLES
Identification of HSK-Like Genes in Lettuce
[0115] To identify the lettuce orthologs of the Arabidopsis DMR1 gene, the Arabidopsis DMR1 (Homoserine Kinase, HSK) protein sequence (At2g17265) was aligned (tblastn alignment) to the translated lettuce genome V8 using the blast tool on the Lettuce Genome Research website of UCDavis Genome Centre. This led to the identification of two lettuce genes with amino acid sequence identity to the Arabidopsis protein. See Table 1 in the specification.
[0116] Based on the homology of both proteins to the Arabidopsis HSK gene, both proteins are considered to be HSK-like proteins. Since in Arabidopsis HSK is encoded by only one gene, the presence of two genes in lettuce was surprising and therefore we analyzed gene expression to determine whether both genes are active.
Gene Expression of the HSK-Like Genes in Lettuce
[0117] Expression was determined by analyzing RNA seq reads from Next Gen Seq sequence analyses from NCBI databases in an in-house Genome browser (JBrowse). In general, the number of reads, used as a measure for gene activity, is comparable for both genes in leaves of lettuce. This strongly suggests that both HSK-like genes are translated into mRNA and may therefore contribute to the HSK-activity in leaves. Therefore, we decided to screen for mutations in both genes to determine whether mutations in either one of the genes or in both genes can lead to increased homoserine level and Bremia lactucae resistance.
Generation of EMS Mutants and Screening of Mutants in Lettuce HSK-Like Genes
[0118] Lettuce mutants were generated by incubating lettuce seeds in a solution of EMS in water for several hours. After washing with water, the seeds were sown in soil and allowed to flower and self pollinate to set M2 seed (Mutant 2.sup.nd generation seed). M2 seeds were collected per plant and a small fraction of the seeds were used for DNA isolation. The DNA representing M2 seeds families was used to amplify fragments of the homoserine kinase-like genes. These PCR fragments were sequenced to identify possible mutations. The identified mutations were evaluated using SIFT analysis (Sorting Intolerant From Tolerant) which is a program that predicts whether an amino acid substitution may affect protein function so that users can prioritize substitutions for further study.
[0119] The mutants were tested for Bremia resistance and in parallel the homoserine level was measured twice in young leaves. See below.
Homoserine Measurements
Amino Acid Extraction
[0120] Leaf tissue was frozen in liquid nitrogen and ground in Eppendorf tubes using Eppendorf micropestles. Aliquots of 10-50 mg fresh weight (FW) were taken in new Eppendorf tubes and weighed. Amino acids were extracted with 750 μl 80% ethanol (twice), supernatants were pooled in a new tube. Amino acid extracts were lyophilized using a freeze dryer and dissolved in 150 μl 80% ethanol. Additional aliquots of 10-50 mg FW were taken and dried o/n at 95° C. to determine the FW/DW ratio per sample.
Amino Acid Detection
[0121] An EZfaast kit (Phenomenex) was used to clean-up and derivatize concentrated amino acid extracts, according to the manufacturer's instructions. Shortly, this includes adding 100 μl 0.2 mM Norvaline as an internal standard to each of the samples, solid phase extraction using a cation-exchange resin filled sorbent tip, derivatization producing chloroformate derivatives of amino and carboxylic acid groups and lastly liquid-liquid extraction using a combination of organic solvents. The supplied Amino Acid Standard mixture was mixed in a 1:1 ratio with 200nmol/mL solution of L-Homoserine (Sigma-Aldrich). To create an amino acid calibration curve, 25 μl, 50 μl 100 μl and 200 μl of the obtained amino acid standard mixture including homoserine was extracted and derivatized using the EZfaast kit as described above. Amino acids were separated by gas chromatography—flame ionization detection (GC-FID) on an Agilent Technologies 7890A GC system, using an Agilent Technologies 7683B series injector. A Phenomenex ZB-AAA, 10m×250 μm Zebron Amino Acid column was used on the GC-FID system. Parameters for GC-FID are as described in the EZfaast kit manual.
Amino Acid Quantification
[0122] Amino Acid peaks were called based on retention time, using Agilent Chemstation software. Total peak areas per amino acid per sample were normalized using the peak are of the internal standard (Norvaline). Calibration curves were constructed based on the normalized peak areas of the amino acid mixture, measured in four concentrations as described above. Obtained calibration curves were used to calculate the amino acid amounts in the samples in nmol.
TABLE-US-00003 TABLE A-1 Homoserine measurements for plants comprising mutations in HSK gene encoding SEQ ID NO: 1 (experiment 1) SIFT Homoserine Homoserine Bremia Amino Acid prediction levels in nmol per levels in nmol per resistance substitution in on protein Zygosity of mg Fresh Weight mg Dry Weight Bl: 25 (see Plant SEQ ID NO: 1 activity mutant allele (nmol/mgFW) (nmol/mgDW) tests below) CERVINO_M-312H2 V233M Not tolerated Heterozygous 0.00 0.0 No data CERVINO_M-312H2 V233M Not tolerated Homozygous 0.00 0.0 No data WENDEL_M-1418H1 P223L Not tolerated Homozygous 0.00 0.0 No WENDEL_M-1418H1 P223L Not tolerated Homozygous 0.00 0.0 No WENDEL_M-1503H1 E238K Not tolerated Heterozygous 0.00 0.0 No WENDEL_M-1503H1 E238K Not tolerated Homozygous 0.38 6.4 No WENDEL_M-1848H1 R245K Not tolerated Heterozygous 0.00 0.0 No WENDEL_M-1848H1 R245K Not tolerated Homozygous 19.46 302.3 Yes Cervino Wild type 0.00 0.0 No Wendel Wild type 0.00 0.0 No Bremia susceptible Wild type 0.00 0.0 No control Bremia resistant Wild type 0.00 0.0 yes control
TABLE-US-00004 TABLE A-2 Homoserine measurements for plants comprising mutations in HSK gene encoding SEQ ID NO: 1 (experiment 2) SIFT Homoserine Bremia Amino Acid prediction levels in nmol per resistance substitution in on protein Zygosity of mg Fresh Weight Bl: 26 (data Plant SEQ ID NO: 1 activity mutant allele (nmol/mgFW) not shown) WENDEL_M-1108H2 M244I tolerated Homozygous 4.41 No WENDEL_M-589H2 P219S tolerated homozygous −0.01 No Cervino Wild type 0.00 No Wendel Wild type 0.00 No
[0123] From the above it is clear that mutations in the HSK gene encoding SEQ ID NO: 1 only lead to Bremia resistance if a minimal amount of L-homoserine accumulates in the leaves, as the mutants having an amount of 4.41 nmol/mg FW, or less, did not result in Bremia resistance, while the mutant having an amount of 19.46 nmol/mgFW of L-homoserine in the leaves was resistant against Bremia lactucae.
TABLE-US-00005 TABLE B-1 Homoserine measurements for plants comprising mutations in HSK gene encoding SEQ ID NO: 3 (experiment 1) SIFT Homoserine Homoserine Bremia Amino Acid prediction levels in nmol per levels in nmol per resistance substitution in on protein Zygosity of mg Fresh Weight mg Dry Weight Bl: 25 (see Plant SEQ ID NO: 3 activity mutant allele (nmol/mgFW) (nmol/mgDW) tests below) CERVINO_M-712H1 D82N Not tolerated Homozygous 0.00 0.0 No CERVINO_M-712H1 D82N Not tolerated Homozygous 0.00 0.0 No CERVINO_M-748H2 V278M Not tolerated Homozygous −0.01 −0.1 No CERVINO_M-748H2 V278M Not tolerated Homozygous 0.00 0.0 No CERVINO_M-787H2 P292S Not tolerated Homozygous 0.00 0.0 No CERVINO_M-787H2 P292S Not tolerated Homozygous 0.00 0.0 No CERVINO_M-863H1 G104D Not tolerated Homozygous 0.00 0.0 No CERVINO_M-863H1 G104D Not tolerated Homozygous 0.00 0.0 No CERVINO_M-972H2 P250L Not tolerated Homozygous 0.00 0.0 No CERVINO_M-972H2 P250L Not tolerated Homozygous 0.00 0.0 No CERVINO_M-1049H2 S127F Not tolerated Heterozygous 0.00 0.0 No CERVINO_M-1049H2 S127F Not tolerated Homozygous 0.00 0.0 No CERVINO_M-1473H1 V278M Not tolerated Homozygous 0.00 0.0 No CERVINO_M-1473H1 V278M Not tolerated Homozygous 0.00 0.0 No WENDEL_M-195H2 P236S Not tolerated Homozygous −0.01 −0.1 No WENDEL_M-195H2 P236S Not tolerated Homozygous 0.00 0.0 No WENDEL_M-518H1 G81R Tolerated Homozygous 0.00 0.0 No WENDEL_M-518H1 G81R Tolerated Homozygous 0.00 0.0 No WENDEL_M-1121H1 T62I Not tolerated Homozygous 0.00 −0.1 No WENDEL_M-1121H1 T62I Not tolerated Homozygous 0.00 0.0 No WENDEL_M-1121H2 T62I Not tolerated Homozygous 0.00 0.0 No WENDEL_M-1121H2 T62I Not tolerated Homozygous 0.00 0.0 No Cervino Wild type 0.00 0.0 No Wendel Wild type 0.00 0.0 No Bremia susceptible Wild type 0.00 0.0 No control Bremia resistant Wild type 0.00 0.0 yes control
TABLE-US-00006 TABLE B-2 Homoserine measurements for plants comprising mutations in HSK gene encoding SEQ ID NO: 3 (experiment 2) SIFT Homoserine Bremia Bremia Amino Acid prediction levels in nmol per resistance resistance substitution in on protein Zygosity of mg Fresh Weight Bl: 21 (data Bl: 26 (data Plant SEQ ID NO: 3 activity mutant allele (nmol/mgFW) not shown) not shown) CERVINO_M-1049H2 S127F Not tolerated homozygous 0.00 No No CERVINO_M-611H2 C262Y tolerated homozygous −0.01 No No CERVINO_M-1H2 P116S tolerated homozygous −0.01 No No CERVINO_M-726H2 P68S tolerated homozygous 0.00 No No CERVINO_M-732H2 G122R tolerated homozygous 0.0 No No CERVINO_M-1595H1 C75Y tolerated homozygous 0.0 No No Cervino Wild type 0.00 No No Wendel Wild type 0.00 Yes* No *Wendel contains the resistance gene R18 conferring resistance against Bl: 1-16, 19, 21, 23 and 32EU. From the above it can be seen that none of the mutations in the gene encoding SEQ ID NO: 3 result in an increase in L-homoserine.
Bremia lactucae Resistance Test—“Leaf Disk Test” (LDT)
Material and Solutions
[0124] Blotting paper: 255×435 mm (original size); Grade 358, 135 g/m.sup.2; Munktell & Filtrak GmbH, Germany.
[0125] Black plastic boxes: 460×310×80 mm.
[0126] Plastic bags: LPDE transparent bags; 400×600 mm; 0.05 mm thickness; Paardekooper Verpakkingen B.V., Netherlands.
[0127] Plastic jars: Straight sample container with screw cap (PS clear); 33×70 mm (int.Ø×h); 60 ml capacity; VWR, Netherlands.
[0128] Cheese cloth: Cotton cheesecloth; C. van't Riet Zuiveltechnologie, Netherlands.
[0129] Spraying equipment: 1 liter mechanical pump sprayer with maximum operating pressure of 3 bar (Gloria type 89 Profiline; GLORIA Haus-und Gartengeräte GmbH, Germany) fitted with a brass spray lance/extension and nozzle (GLORIA Haus-und Gartengeräte GmbH, Germany).
[0130] Nutrient Solution: The Nutrient Solution consists of 1× Hoagland No 2 basal salt mixture supplemented with 50 mg/L Iprodione. The Nutrient Solution is prepared by re-suspending 1.63 g of Hoagland's No 2 basal salt mixture (See Appendix 02 for exact composition) (Caisson Labs, US) and 10 ml of 5 g/L Iprodione laboratory stock into 1 L of tap water. The Nutrient Solution is made the same morning as the leaf disk test will be inoculated.
TABLE-US-00007 APPENDIX 02 Ingredients Hoagland's No2 Basal salt mixture. Components mg/L Ammonium Phosphate, Monobasic (NH4H2PO4) 115.03 Boric Acid (H3BO3) 2.86 Calcium Nitrate, Tetrahydrate (Ca(NO3)2•4H2O) 656.40 Cupric Sulfate, Pentahydrate (CuSO4•5H2O) 0.08 Ferric Tartrate (C12Fe2H12O8) 5.32 Magnesium Sulfate, Anhydrous (MgSO4) 240.76 Manganese Chloride, Tetrahydrate (MnCl2•4H2O) 1.81 Molybdenum Trioxide (MoO3) 0.02 Potassium Nitrate (KNO3) 606.60 Zinc Nitrate, Hexahydrate (Zn(NO3)2•6H2O) 0.22
Trial Design
[0131] Seeds are sown on Day 1 (See Appendix 01 for planning). Material to be tested will be sown together with plants that are known to be resistant or susceptible for the isolates that are used in the test.
[0132] The disease test is performed on healthy looking 4 weeks old plants in the 6.sup.th to 8.sup.th leaf stage.
[0133] Plants that do not look healthy are discarded.
[0134] For each pathogen race/plant combination, at least two leaf disks are tested in separate boxes. In total, a minimum of 4 leaf disks are tested per plant.
[0135] In each black box, one leaf disk is placed per plant in the grid drawn on the blotting paper. The grid will be filled with leaf disks corresponding with the printed grid. Among the leaf disks in a box there will be leaf disks of the susceptible and resistant control plants at fixed positions.
[0136] For example in the test with race BI:25EU, the resistant control used was variety Balesta, and the susceptible control used was variety Cervino and/or Wendel. In the test with race BI: 26EU, NunDM17 was used as resistant control and Cervino and/or Wendel as susceptible control. In the test with race BI:21 Cervino was used as susceptible control, while Wendel and/or NunDm17 were used as resistant varieties. Other varieties with known resistance or susceptibility to Bremia races can be equally used, such as those described in the UPOV test guidelines for lettuce, on the world wide web at uov.int/edocs/tgdocs/en/tg013.pdf.
TABLE-US-00008 APPENDIX 01 B. lactucae leaf disk test experiment planning. Week Day post inoculation Event 1 1 Sowing of plants to test 4 28 (i) Inoculum preparation (ii) Inoculation of leaf disks 6 39 Intermediate scoring (11 dpi) 6 42 Final scoring (14 dpi)
Preparing Inoculum
[0137] The preparation of B. lactucae inocula needed for the trial inoculation begins on Day 28. Make sure to wear gloves whenever handling B. lactucae. The preparation goes as follow:
[0138] The plastic jar containing the relevant B. lactucae working stocks is taken out of the −20° C. freezer and approximately 50 ml of cold tap water is added to the jar (the jar is left standing for a while after this step). Make sure the water is cold by having the tap open for a while before taking any water. The jar is vigorously shaken a few times to loosen the spores into the water. A workable size is cut off a “cheese cloth” using scissors. The content of the jar is then filtered through a layer of wet-made “cheese cloth”, into a clean measuring cup (usually size 200 ml). Squeeze the cloth after you have softly poured the content of the jar onto the cheese cloth.
[0139] The spore concentration of the inoculum is measured using a hemocytometer (Bürker Türk). Where necessary, the spore concentration is adjusted to reach 1×10.sup.5 spores ml.sup.−1. Note that in cases where the concentration is too low, the content of an additional jar of working stock should be used to reach the desired concentration.
[0140] Once prepared, the spore solution is dispensed into a 1 liter mechanical pump sprayer. Two jars of working stock is usually enough to prepare a spore solution for inoculating 6 boxes of leaf disks.
Trial Inoculation
[0141] Three sheets of blotting papers are stacked on top of each other. The top sheet has either several lines drawn on it using a ruler and a pencil to form a grid used to designate the position of the leaf disks or a printed grid. The stack of blotting papers are placed at the bottom of a black plastic box.
[0142] The stack of blotting papers is humidified using the Nutrient Solution (see above).
[0143] One or two leaves are removed from each plant to be tested. Leaf disks of 17 mm in diameter are punched out of the leaves. The leaf punch is laid upside down on the top sheet of blotting paper in the square that corresponds with the printed lay-out and a record is made when the actual lay-out differs from the printed lay-out (e.g. plant is not available).
[0144] When all leaf disks are in place, the prepared inoculum solution is sprayed evenly over the leaf discs to the point of solution “run off”.
[0145] Following inoculation, the black boxes are placed inside clear plastic bags (bags are closed with pegs). From that point on, the boxes will remain inside closed bags throughout the test (except of course for scorings).
[0146] The bagged boxes are moved to the climate cells (12/12 hours day/night cycle using standard fluorescent light bulbs; light intensity is 70 μmol/m.sup.2/s (PAR); temperature is 15° C. constant. Daylight period is from 20.00 μm until 08.00 am. Directly after inoculation a dark period of approximately 24 hours is applied by covering the inoculated trays with thick light impermeable sheets. The boxes are kept in the climate cells until the end of the experiment.
Data Collection and Analysis
[0147] For each test, two scorings are done. An intermediate scoring is done at 11 dpi (days post inoculation). A final scoring is done at 14 dpi. At each time point two scores are taken: a) the percentage of leaf disc surface area displaying Bremia lactucae sporulation, on a scale of 0% to 100% and b) the intensity/severity of the sporulation on the surface area which displays spores. The intensity/severity is scored on a scale of 0-100 (the score given is the average severity of the area with sporulation).
Results:
[0148] The Tables below show the results of resistance tests using Bremia lactucae strain BI:25EU (European strain). The TILLING populations used do not contain resistance against this race. Only the results of the final scoring are shown.
[0149] In one aspect a plant is considered resistant against a Bremia race, such as BI:25EU, when the score is 0% sporulation. The resistant control should have a score of 0% sporulation. The susceptible control should have a score of 100% sporulation.
TABLE-US-00009 TABLE A Resistance of mutants comprising an amino acid substitution in SEQ ID NO: 1 % surface Intensity of % surface Intensity of area showing sporulation on area showing sporulation on Amino Acid sporulating surface area sporulating surface area Overall Conclusion substitution in Zygosity of (0-100%) (scale 0-100) (0-100%) (scale 0-100) S = susceptible Plant SEQ ID NO: 1 mutant allele Repetition 1 Repetition 2 R = resistant Susceptible control susceptible 100 100 100 100 S Susceptible control susceptible 100 100 100 100 S Resistant control resistant 0 0 0 0 R Susceptible control susceptible 100 100 100 100 S WENDEL_M-1418H1 P223L Heterozygous 100 100 80 100 S WENDEL_M-1418H1 P223L Homozygous 100 100 100 100 WENDEL_M-1418H1 P223L Homozygous 75 100 100 100 WENDEL_M-1418H1 P223L Homozygous 80 100 100 100 WENDEL_M-1503H1 E238K Heterozygous 100 100 100 100 S WENDEL_M-1503H1 E238K Heterozygous 100 100 100 100 WENDEL_M-1503H1 E238K Heterozygous 50 100 100 100 WENDEL_M-1503H1 E238K Heterozygous 100 100 100 100 WENDEL_M-1503H1 E238K Homozygous 100 100 100 100 WENDEL_M-1848H1 R245K Heterozygous 100 100 80 100 S WENDEL_M-1848H1 R245K Homozygous 0 0 R Susceptible control susceptible 100 100 100 100 S Resistant control resistant 0 0 0 0 R Resistant control resistant 0 0 0 0 R Susceptible control susceptible 100 100 100 100 S
[0150] For empty datapoints, no data were available
TABLE-US-00010 TABLE B Resistance of mutants comprising an amino acid substitution in SEQ ID NO: 3 % surface Intensity of % surface Intensity of area showing sporulation on area showing sporulation on Overall Conclusion Amino Acid sporulating surface area sporulating surface area for plant genotype substitution in Zygosity of (0-100%) (scale 0-100) (0-100%) (scale 0-100) S = susceptible Plant SEQ ID NO: 3 mutant allele Repetition 1 Repetition 2 R = resistant Susceptible control 100 100 100 100 S Resistant control 0 0 0 0 R CERVINO_M-712H1 D82N Homozygous 80 100 50 100 S CERVINO_M-712H1 D82N Homozygous 75 100 100 100 CERVINO_M-712H1 D82N Homozygous 70 100 100 75 CERVINO_M-712H1 D82N Homozygous 100 75 75 100 CERVINO_M-712H1 D82N Homozygous 75 100 90 100 CERVINO_M-748H2 V278M Heterozygous 100 100 S CERVINO_M-748H2 V278M Homozygous 100 100 100 100 CERVINO_M-748H2 V278M Homozygous 80 100 100 75 CERVINO_M-748H2 V278M Homozygous 100 100 80 100 CERVINO_M-748H2 V278M Homozygous 100 100 100 100 CERVINO_M-787H2 P292S Heterozygous 100 100 80 100 S CERVINO_M-787H2 P292S Heterozygous 75 75 100 75 CERVINO_M-787H2 P292S Homozygous 75 100 100 100 CERVINO_M-787H2 P292S Homozygous 75 100 75 75 CERVINO_M-787H2 P292S Homozygous 90 100 100 100 Susceptible control susceptible 100 100 100 100 S CERVINO_M-863H1 G104D Homozygous 100 75 40 100 S CERVINO_M-863H1 G104D Homozygous 50 100 100 100 CERVINO_M-863H1 G104D Homozygous 50 100 75 100 CERVINO_M-863H1 G104D Homozygous 75 100 CERVINO_M-863H1 G104D Homozygous 80 75 75 50 CERVINO_M-972H2 P250L Homozygous 90 100 100 100 S CERVINO_M-972H2 P250L Homozygous 100 100 75 100 CERVINO_M-972H2 P250L Homozygous 100 100 75 100 CERVINO_M-972H2 P250L Homozygous 10 25 100 75 CERVINO_M-972H2 P250L Homozygous 100 100 100 100 CERVINO_M-972H2 P250L Heterozygous 60 100 50 100 Susceptible control susceptible 100 100 100 100 S CERVINO_M-1049H2 S127F Heterozygous 100 75 100 100 S CERVINO_M-1049H2 S127F Heterozygous 100 100 75 75 CERVINO_M-1049H2 S127F Homozygous 50 100 25 75 CERVINO_M-972H2 P250L Heterozygous 25 75 100 100 S CERVINO_M-972H2 P250L Heterozygous 80 100 50 100 Resistant control resistant 0 0 0 0 R Susceptible control susceptible 100 100 100 100 S Susceptible control susceptible 100 100 100 100 S Resistant control resistant 0 0 0 0 R Resistant control resistant 0 0 0 0 R Susceptible control susceptible 100 100 100 100 S CERVINO_M-1473H1 V278M Homozygous 100 100 10 100 S CERVINO_M-1473H1 V278M Homozygous 100 100 100 75 CERVINO_M-1473H1 V278M Homozygous 100 100 100 100 CERVINO_M-1473H1 V278M Homozygous 80 100 50 75 CERVINO_M-1473H1 V278M Homozygous 100 75 100 100 WENDEL_M-195H2 P236S Homozygous 100 100 100 100 S WENDEL_M-195H2 P236S Heterozygous 100 100 100 100 WENDEL_M-195H2 P236S Heterozygous 50 100 50 100 WENDEL_M-195H2 P236S Heterozygous 100 100 100 100 WENDEL_M-195H2 P236S Homozygous 100 100 100 100 Susceptible control susceptible 100 100 100 100 S WENDEL_M-518H1 G81R Heterozygous 100 100 100 100 S WENDEL_M-518H1 G81R Heterozygous 100 100 WENDEL_M-518H1 G81R Homozygous 100 100 100 100 WENDEL_M-518H1 G81R Homozygous 100 100 100 100 WENDEL_M-518H1 G81R Homozygous 100 100 100 100 WENDEL_M-1121H1 T62I Heterozygous 100 100 100 100 S WENDEL_M-1121H1 T62I Heterozygous 100 100 100 100 WENDEL_M-1121H1 T62I Heterozygous 100 100 50 100 WENDEL_M-1121H1 T62I Homozygous 100 100 100 100 WENDEL_M-1121H1 T62I Homozygous 100 100 100 100 WENDEL_M-1121H1 T62I Homozygous 100 100 100 75 WENDEL_M-1121H1 T62I Homozygous 25 100 100 100 WENDEL_M-1121H1 T62I Homozygous 100 100 100 100 WENDEL_M-1121H2 T62I Heterozygous 100 100 100 100 WENDEL_M-1121H2 T62I Homozygous 100 100 100 100 WENDEL_M-1121H2 T62I Heterozygous 100 100 100 50 WENDEL_M-1121H2 T62I Homozygous 100 100 100 100 WENDEL_M-1121H2 T62I Homozygous 80 100 100 100 Susceptible control susceptible 100 100 100 100 S Resistant control resistant 0 0 0 0 R
[0151] For empty datapoints, no data were available
Three-Dimensional Structure Analysis of the HSK Proteins
[0152] In order to find out why amino acid R245 of SEQ ID NO: 1 led to such a high accumulation of L-homoserine in planta, while the other mutants did not, the Arabidopsis HSK amino acid sequence was BLASTed against the PDB database (Protein Data Bank). This identified the Crystal structure of the homoserine kinase protein 1H72 (PDB ID 1H72). Alignment of the Arabidopsis HSK protein with the HSK Crystal structure (PDB ID 1H72, Crystal structure of Homoserine Kinase complexed with homoserine) lead to the identification of 18 amino acid residues, which interact with L-homoserine or with ATP (Adenosine tri-phosphate), as shown in