Increase in meiotic recombination in plants by inhibiting an RECQ4 or TOP3A protein of the RTR complex

Abstract

The invention relates to a method for increasing the frequency of meiotic recombination in plants, by inhibiting the RECQ4 or TOP3A protein, especially by mutagenesis or extinction of the RECQ4 or TOP3A gene coding for said protein. The invention can be used especially in the field of plant breeding and genetic mapping.

Claims

1. A process for increasing the frequency of meiotic crossovers in a plant said process comprising: inhibiting in said plant, the expression or the function of: a protein of the RTR complex known as RECQ4; or both REQ4A and RECQ4B proteins if said plant belongs to the family of Brassicaceae and expresses two functional RECQ4 proteins, said RECQ4 protein having at least 40% sequence identity with the RECQ4 protein of SEQ ID NO: 1, and comprising a region having at least 60% sequence identity with the region which extends from positions 407 to 959 of the sequence SEQ ID NO:1; and measuring the frequency of meiotic crossovers in said plant.

2. The process of claim 1, further comprising, inhibiting in said plant, the expression or the function of at least one protein chosen from: (i) a protein hereinafter referred to as FIDG, said protein having at least 45% sequence identity with the AtFIDG protein of SEQ ID NO:46 and containing an AAA-ATPase domain and a VSP4 domain, and (ii) a protein hereinafter referred to as FANCM, said protein having at least 30% sequence identity with the AtFANCM protein of SEQ ID NO:45, and containing a DEXDc helicase domain and a HELICc helicase domain.

3. The process of claim 1, wherein the inhibition of the RECQ4 is obtained by mutagenesis of the gene encoding said protein or of its promoter.

4. The process of claim 1, wherein the inhibition of the RECQ4 is obtained by silencing the gene encoding said protein by expressing, in said plant, an interfering RNA targeting said gene.

5. The process of claim 1, further comprising, inhibiting in said plant, the expression or the function of a protein known as TOP3A, said TOP3A protein having at least 50% sequence identity with the TOP3A protein of sequence SEQ ID NO: 2.

6. The process of claim 5, wherein the inhibition of the TOP3A protein is obtained by mutagenesis of the gene encoding said protein or of its promoter.

7. The process of claim 6, wherein the mutagenesis of the TOP3A protein comprises the introduction, into an allele of the TOP3A gene, of a mutation which results in the inactivation of at least one zinc finger domain of said TOP3A protein.

8. The process of claim 5, the process further comprising the expression, in said plant, of a recombinant TOP3A protein comprising a TOPRIM domain and a Topoisomerase IA domain which have not been impaired and at least one zinc finger domain which is inactivated.

9. The process of claim 1, wherein said plant is a monocotyledonous or dicotyledonous plant of agronomical interest comprising rapeseed, sunflower, potato, corn, wheat, barley, rye, sorghum, rice, soy, bean, carrot, tomato, zucchini, bell pepper, eggplant, turnip, onion, pea, cucumber, leek, artichoke, beetroot, cabbage, cauliflower, lettuces, endive, melon, watermelon, strawberry plant, apple tree, pear tree, plum tree, poplar tree, vine, cotton, rose, or tulip.

Description

(1) The present invention will be understood more clearly by means of the further description which follows, which refers to nonlimiting examples illustrating the effect of mutations of the AtRECQ4 or AtTOP3A gene, alone or combined with mutations of the FANCM gene, on meiotic recombination and CO rate, with references to the attached drawings in which:

(2) FIG. 1: Phylogenetic tree of the RECQ4 proteins of flowering plants. The analysis was carried out on the Phylogeny.fr platform according to the following steps: the sequences were aligned with T-Coffee (v6.85) using the following methods of alignment in pairs: the ten best local alignments (Lalign_pair), an exact global alignment (slow_pair). After the alignment, the positions with spaces were eliminated from the alignment. The phylogenetic tree was reconstructed using the method of maximum probability, implemented in the PhyML v3.0 aLRT program. The sequences of the proteins were collected in the PLAZA database (http://bioinformatics.psb.ugent.be/plaza/) and the phytozome database (http://www.phytozome.net.): At: Arabidopsis thaliana. Al: Arabidopsis lyrata. Bra: Brassica rapa. Esa: Thellungiella halophila. Cp: Carica papaya. Tcacao: Theobroma cacao. Graimndii: Gossypium raimondii. VV: Vitis vinifera. Ppersica Prunus persica. RC: Ricinus communis. ME: Manihot esculenta. Solyc: Solanum lycopersicum. Eucgr: Eucalypsus grandis. Phvul: Phaseolus vulgaris. Glyma: Glycine max. OS: Oryza sativa. BD: Brachypodium distachyon. Si: Setaria italica. Zmays: Zea mays. Sbicolor: Sorghum bicolor;

(3) FIG. 2: Alignment of the TOPOISOMERASES 3a of eukaryots using T-Coffee. The conserved domains are indicated by a vertical or horizontal line. At: Arabidopsis thaliana (SEQ ID No: 2). Sc: Saccharomyces cerevisiea (SEQ ID No: 52). Os: Oryza sativa (SEQ ID No: 47). Hs: Homo sapiens (SEQ ID No: 48). Dr: Danio rerio (SEQ ID No: 49). Ce: Coenorabditis elegans (SEQ ID No: 50). Sp: Schizosaccharomyces pombe (SEQ ID No: 51). The alignment was carried out using T-Coffee with the default parameters;

(4) FIG. 3: The double mutation of RECQ4A and RECQ4B restores the formation of bivalents in the zmm mutants. Average number of bivalents per male meiocyte. Ring bivalents (black). Rod bivalents (gray). Pairs of univalents (black spots). The number of cells in metaphase I that were analyzed is indicated between parentheses;

(5) FIG. 4: The RECQ4A and RECQ4B helicases redundantly limit CO formation during meiosis. The genetic distances in four intervals, measured using lines carrying fluorescent markers, were calculated with the Perkins equation and are expressed in centiMorgans. I2a and I2b are adjacent intervals on chromosome 2, and I5c and I5d are adjacent intervals on chromosome 5;

(6) FIG. 5: The top3a-R640X mutation restores the formation of bivalents in the zmm mutants. Average number of bivalents per male meiocyte. Ring bivalents (black). Rod bivalents (gray). Pairs of univalents (black spots). The number of cells in metaphase I that were analyzed is indicated between parentheses;

(7) FIG. 6: TOP3A and FANCM independently limit CO formation during meiosis. The genetic distances in four intervals, measured using lines carrying fluorescent markers, were calculated with the Perkins equation and are expressed in centiMorgans. I2a and I2b are adjacent intervals on chromosome 2, and I5c and I5d are adjacent intervals on chromosome 5.

(8) FIG. 7: RECQ4 and FANCM independently limit CO formation during meiosis. The genetic distances in two intervals, measured using lines carrying fluorescent markers, were calculated with the Perkins equation and are expressed in centiMorgans. I2a and I2b are adjacent intervals on chromosome 2.

EXAMPLE 1

Obtaining Mutants of the RECQ4 Gene which are Suppressors of ZMM Gene Mutations

(9) Seeds of the msh4 mutant of Arabidopsis thaliana in the Landsberg eracta genetic background (cshl_GT14269; Drouaud et al., PLoS Genet., 2013, 9, e1003922; Higgins et al., Genes Dev., 2004, 18, 2557-2570) were mutated with EMS (ethyl methanesulfonate). The plants derived from the mutated seeds (M1 population, heterozygous for the EMS-induced mutations, and homozygous for the mutation of the ZMM gene) have an identical phenotype, resulting from the inactivation of the ZMM gene, which results in a strong decrease in the frequency of COs, which leads to a strong decrease in the number of bivalents, and a very large drop in fertility (semi-sterile plants), resulting in the formation of short siliques which are easily distinguished from those of the wild-type plants. The M1 plants were self-pollinated, so as to produce a population of descendants (M2 population; approximately 1000 families) potentially containing plants homozygous for the EMS-induced mutations. Plants of the M2 population having siliques longer than those of the homozygous zmm plants of the M1 generation were selected and genotyped in order to verify their homozygous status with respect to the msh4 genes. They are suppressors.

(10) Two lines of mutants that are suppressors of the mutation of the AtMSH4 gene, called msh4(s)84 and msh4(s)101, are the subject of this study. The complete sequencing of the genome of these mutants showed that the corresponding mutations were located in the Atlg10930 gene encoding the RECQ4A helicase. The msh4(s)84 mutant comprises the C>T change in position TAIR 10: chr1:3652474 which introduces a stop codon at the W387 codon. The msh4(s)101 mutant comprises the C>T change in position TAIR 10: chr1:3650343 which introduces the G762D substitution. These two suppressors are allelic, demonstrating that the mutations in RECQ4A are the causal mutations of the restoration of fertility.

(11) The comparison of the sequence of the Landsberg ecotype (Gan et al., Nature, 2011, 477, 7365, 419-23) with that of the genome of the Columbia ecotype showed that their RECQ4B loci differed by a series of single-nucleotide and insertion/deletion polymorphisms, including two which introduce a premature stop codon (Q430>STOP) and a reading frame shift (Aa 501) in Landsberg. This strongly suggests that the RECQ4B gene is not functional in Landsberg.

(12) In the light of these results, the hypothesis was put forward that the recq4a mutation in Columbia was not capable of compensating for the effects of that of msh4 because of the redundancy with RECQ4B during meiosis. This would explain why the mutation of RECQ4A alone would be capable of compensating for the effects of that of msh4 in Landsberg but not Columbia.

(13) Consequently, the msh4, recq4a and recq4b mutations were combined in the Columbia genetic background. The number of bivalents in metaphase I of the msh4 recq4a-4 recq4b-2 triple mutant was compared with that of wild-type plants (Col), of zmm (shoc1/Atzip2 or msh4) plants and of msh4 recq4a-4 and msh4 recq4b-2 double mutants.

(14) The results are shown in FIG. 3. Although the wild-type plants systematically show five bivalents per meiosis, the Atmsh4 mutants show a strong decrease in the number of bivalents, and thus the appearance of univalents. In the wild-type plants, normal chromosome segregation is observed, resulting in the formation of equilibrated gametes. On the other hand, in the zmm mutants, poor chromosome segregation is observed which results from a decrease in the number of bivalents, induced by the zmm mutation, and which results in disequilibrated, non-viable gametes.

(15) Furthermore, as has been previously shown (Higgins et al., The Plant Journal, 2011, 65, 492-502), the msh4 recq4a-4 and msh4 recq4b-2 double mutants are virtually sterile, like the msh4 mutant, and have a number of metaphase I bivalents that is similar to that of msh4 (FIG. 3).

(16) Conversely, the msh4 recq4a-4 recq4b-2 triple mutant is fertile and its number of bivalents is reestablished to the level of that of the wild-type (FIG. 3). Similarly, the recq4a recq4b double mutation is also capable of restoring fertility and the formation of bivalents in another zmm mutant, shoc1/Atzip2 (FIG. 3).

(17) These results show that, in Columbia, RECQ4A and RECQ4B have redundant functions which prevent the formation of meiotic COs in the zmm mutants.

EXAMPLE 2

Influence of the Inactivation of the RECQ4 Genes on Meiotic Recombination Frequency

(18) The effects of the recq4 mutations on meiotic recombination were then measured in a wild-type plant (Columbia), i.e. in the presence of functional ZMMs. The meiotic recombination frequency was measured by analyzing tetrads using lines labeled with a fluorescent label, as described by Berchowitz & Copenhaver, Nat. Protoc., 2008, 3, 41-50. The genetic distance was measured on four different genetic intervals, two adjacent intervals on chromosome 2 (12a and 12b) and two adjacent intervals on chromosome 5 (intervals 15c and 15d). The results are shown in FIG. 4.

(19) In the recq4a and recq4b single mutants, the genetic distances were not significantly different than the wild-type for all the intervals tested (p>5%). Conversely, the genetic distances were considerably increased in the recq4a recq4b double mutant (p<10.sup.9). This increase was by a mean factor of 6.2 with a deviation of 4.6 to 8.6.

(20) These results show that RECQ4A and RECQ4B play a major redundant role in limiting CO formation during meiosis and constitute the most powerful anti-meiotic CO genetic factors identified to date. Furthermore, this demonstrates that having more than six times more COs than the wild-type does not have deleterious effects on the integrity of the chromosomes, the equilibrated segregation of the chromosomes, the completion of meiosis, and fertility.

EXAMPLE 3

Obtaining Mutants of the TOP3A Gene that are Suppressors of ZMM Gene Mutations

(21) A screening similar to that carried out for msh4 (example 1) was carried out in order to search for suppressors of the zmm mutant hei10 (Chelysheva et al., PLoS Genet., 2012, 8, e1002799). Among 1000 mutagenized lines derived from the hei10-2 mutant, a suppressor, hei10(s)61, exhibiting fertility and a number of bivalents greater than the hei10 mutant, was isolated (FIG. 5). The cloning of the mutation on the basis of the genetic map defined an interval of 2.4 Mb on chromosome 5, between the positions Tair10_chr5:23.819.915 and Tair10_chr5:26.497.664. The complete sequencing of the genome of hei10(s)61 identified a nonsense mutation inside this interval, in the At5g63920 gene which encodes TOPOISOMERASE 3 alpha (TOP3A).

(22) This gene appears to be a good candidate given that TOP3A and Sgs1 belong to the RTR complex, a conserved complex which is essential for maintaining the integrity of the genome (Mankouri and Hickson, Trends Biochem Sci., 2007, 32, 538-46).

(23) In order to test whether this mutation was the cause of the phenotype, hei10(s)61 was transformed with a 10 kb genomic clone containing TOP3A. All the transformants exhibited the phenotype of hei10 (24 independent lines), were sterile and had 1.8 bivalents0.75 in metaphase I (mean of three independent lines; FIG. 5). This demonstrates that the mutation in TOP3A was in fact the causal mutation that restored the fertility and the formation of bivalents in the hei10(s)61 suppressor. This shows that AtTOP3A prevents the formation of additional COs in the hei10 context. The hei10(s)61 mutation changes Arg640 into a stop codon (hereinafter referred to as top3a-R640X). Consequently, top3a-R640X encodes a protein which has intact TOPRIM and Topoisomerase domains, but which is truncated, removing its final 286 amino acids which contain the two predicted zinc finger domains (FIG. 2).

(24) The increase in the number of bivalents and the restoration of fertility observed in the hei10 top3a-R640X mutant were also observed in an msh5 top3a-R640X double mutant (4.20.9 bivalents per meiosis; FIG. 5), showing that the suppression observed was not specific to the hei10 genetic background.

(25) The hei10-2 top3a-2 and hei10-2 top3a-R640X plants differ in terms of both their somatic phenotype and their meiotic phenotype. Like a top3a-2 single mutant, hei10-2 top3a-2 shows stunted growth and complete sterility, contrasting with the normal growth and the fertility of hei10-2 top3a-R640X. In meiosis, the hei10 top3a-2 double mutant was indistinguishable from the top3a-2 single mutant, with aberrant structures in metaphase I and a massive fragmentation in anaphase I. In hei10-2 top3a-R640X, the bivalents observed (4.2 per cell, compared with 1.7 in hei10-2, FIG. 6) segregate in anaphase I without chromosome fragmentation. The top3a-2 phenotype showed that TOP3A is essential for preventing a meiotic catastrophe, whereas top3a-R640X appears to be a separation of function resulting in the restoration of COs in the hei10 genetic background, but keeping its efficient repair activity. This suggests that TOP3A has a double function during meiosis, since it is essential both for resolving meiotic recombination intermediates and also for limiting CO formation.

(26) In the hei10-2 top3a-R640X/top3a-2 plants, no fragmentation was observed, showing that one copy of TOP3AR640X is sufficient to repair the recombination intermediates. The number of bivalents in this genetic background is even higher than in the hei10 top3a-R640X double mutant (4.90.1 compared with 4.21.1 bivalents, T-test, FIG. 5), suggesting that the TOP3A-R640X protein could conserve the anti-CO activity.

(27) Next, the effect of the top3a-R640X mutation in plants having a functional HERO was analyzed. In agreement with the results obtained in the hei10 context, the top3a-R640X plants show no somatic abnormality and are fertile [seeds per fruit: wild-type=614 (n=40), top3a-R640X=613 (n=40)]. The meiosis of the top3a-R640X mutant is virtually indistinguishable from that of the wild-type, with no observation of fragmentation.

(28) However, a low frequency of univalents was observed in the top3a-R640X single mutant (0.3 per cell, FIG. 5), whereas univalents are not observed in the wild-type. This suggests that the obligatory CO is slightly affected in the top3a-R640X mutant. The top3a-R640X/top3a-2 plants exhibit no developmental abnormality, are fertile, and no univalent was observed (FIG. 5).

EXAMPLE 4

Influence of the Mutation of the TOP3A Gene on Meiotic Recombination Frequency

(29) The meiotic recombination frequency of the top3a-R640X mutant was then analyzed by analyzing tetrads using lines labeled with a fluorescent label, in four different genetic intervals, as described in example 2 (FIG. 6). In all the intervals tested, the genetic distances were increased in comparison with the wild-type (P<10.sup.5). This increase was, on average, by a factor of 1.5, with a deviation of 1.3 to 1.8. This shows that TOP3A has an anti-CO activity not only in an hei10 context, but also in plants that are wild-type for ZMMs.

EXAMPLE 5

Influence of the Combined Inactivation of the TOP3A and FANCM Gene on Meiotic Recombination Frequency

(30) The FANCM helicase was the first anti-meiotic CO gene described in Arabidopsis (PCT application WO 2013/038376; Crismani et al., Science, 336, 6088, 1588-90). In order to test whether TOP3A and FANCM act in the same path, the recombination frequency was measured in the top3a-R640X fancm-1 double mutant (FIG. 6). In the four intervals tested, it was observed that the genetic distances were significantly greater in the top3a-R640X fancm-1 double mutant than in the two single mutants (p<10-5). The increase in CO in the double mutant, in comparison with the wild-type, was on average by a factor of 4.9, with a deviation of 3.5 to 5.6. The effects of the two mutations on CO formation are cumulative, demonstrating that FANCM and TOP3A act on two parallel pathways to limit CO formation during meiosis. Interestingly, the top3a-R640X fancm-1 double mutant has a normal growth, has wild-type meiosis and is completely fertile [seeds per fruit: wild-type=614 (n=40), top3a-R640X fancm-1=652 (n=40)]. This confirms that having five times more COs than the wild-type does not have deleterious immediate effects on the integrity of the chromosomes, the equilibrated segregation of the chromosomes, completion of meiosis, and fertility.

EXAMPLE 6

Influence of the Combined Inactivation of the FANCM and RECQ4 Genes on Meiotic Recombination Frequency

(31) The FANCM helicase was the first anti-meiotic CO gene described in Arabidopsis (PCT application WO 2013/038376; Crismani et al., Science, 336, 6088, 1588-90). In order to test whether RECQ4 and FANCM act on the same pathway, the recombination frequency was measured in the recq4a recq4b fancm-1 triple mutant (FIG. 7). In the two intervals tested, it was observed that the genetic distances were significantly greater in the recq4a recq4b fancm-1 triple mutant than in the recq4a recq4b double mutant (p<0.01) and the fancm-1 single mutant (p<10.sup.6). The increase in CO in the triple mutant, in comparison with the wild-type, was on average by a factor of 9. The effects of the mutations of the recq4 and fancm genes on CO formation are cumulative, demonstrating that FANCM and RECQ4 act in parallel to limit CO formation during meiosis. Interestingly, the recq4a recq4b fancm-1 triple mutant has a normal growth, has wild-type meiosis (n=450) and is completely fertile [seeds per fruit: wild-type=53.74.8 (n=40), recq4a recq4b fancm-1=49.25.3 (n=40)]. This confirms that having nine times more COs than the wild-type has no deleterious immediate effects on the integrity of the chromosomes, the equilibrated segregation of the chromosomes, completion of meiosis, and fertility.