WO2021170868A1

WO2021170868A1 - Albino3-like gene involved in virus resistance

Info

Publication number: WO2021170868A1
Application number: PCT/EP2021/055008
Authority: WO
Inventors: Jonathan KALISVAART; Raoul Jacobus Johannes Maria FRIJTERS; Daniël Johannes Wilhelmus LUDEKING; Alwin Johannes Marinus ROOVERS
Original assignee: Rijk Zwaan Zaadteelt En Zaadhandel B.V.
Priority date: 2020-02-28
Filing date: 2021-03-01
Publication date: 2021-09-02

Abstract

The present invention relates to a modified Albino3-like gene the wildtype of which comprises SEQ ID No. 1 encoding SEQ ID No. 2, or a homologous sequence having at least 70% sequence identity to SEQ ID No. 1, which modified gene leads to resistance to a Tobamovirus, preferably to ToBRFV. The modified Albino3-like gene preferably encodes an Albino3-like protein 2 according to SEQ ID No. 6 or SEQ ID No. 7, or encodes an Albino3-like protein 2 having at least 70% sequence identity thereto. Examples of modifications comprise a C485G SNP, an A1300G SNP and/or a T1381C SNP, or a SNP on a corresponding position of a homologous sequence. The invention further relates to plants comprising the modified gene and showing resistance to a Tobamovirus.

Description

ALBIN03-LIKE GENE INVOLVED IN VIRUS RESISTANCE

The present invention relates to a modified gene which leads to resistance against Tomato Brown Rugose Fruit Virus (ToBRFV). The invention further relates to a plant comprising said modified gene, methods for producing such a plant, and methods for identification of the modified gene and selection of such a plant. The invention also relates to a marker for identification of the modified gene in a plant, and to use of said marker.

Viral diseases pose one of the major threats vegetable growers have to deal with, both in protected and open field crop cultivation. Once a crop is infected, spread of the virus can occur rapidly through hard-to-control vectors, usually insects. In addition, cultivation methods often contribute to a further spread of the virus, by sap transmission through tools and fieldworkers.

Plant viruses typically depend on their hosts for rapid replication and spread, thereby infecting that same host with the disease. Interaction between virus and host plays a large role in virus infection, in particular interactions between host and virus proteins. Common symptoms of virus infection are yellowing through leaf chlorosis or mosaic, both of which are typical photosynthesis-related symptoms. Chloroplasts are therefore regarded as a common target of plant viruses, whereby chloroplast-virus interaction appears to affect photosynthesis, resulting in a disruption of the normal chloroplast function. Viruses known to interact with the chloroplasts of their host plant are found among many different virus genera and families. The proteins affected by these viruses often belong to the chloroplast-photosynthesis related proteins (CPRPs). A number of viruses, among which several Tobamoviruses, are known to affect the photosystem II (PSII) core complex which is located in the thylakoid membrane of the chloroplasts, resulting in lower photosynthetic activity, and subsequently causing symptoms of chlorosis and mosaic. This is likely due to integration of the coat protein (CP) of the virus into the thylakoid membrane.

Numerous genes have been recognized for their involvement in virus resistance in plants. Virus resistance can be based on various mechanisms, and many different phases of plant development and plant defense pathways can be involved. However, for a large number of viruses no resistance gene has been identified yet. Especially for relatively new viruses, or viruses that are similar to others but break known resistances, there is always the challenge to identify a new source of resistance before the virus damage becomes too extensive. Newly identified resistance genes can also be an addition to the protection of crops against already known viral diseases. Viruses that result in photosynthesis-related symptoms could be defended by genes of which their proteins are known to play a role in chloroplast function.

It is an object of the present invention to provide a gene that leads to resistance against Tomato Brown Rugose Fruit Virus (ToBRFV). The present invention provides a modified Albino3-like gene the wildtype of which comprises SEQ ID No. 1 encoding SEQ ID No. 2, or comprises a homologous sequence having at least 70% sequence identity to SEQ ID No. 1, which modified gene leads to resistance against a Tobamovirus, preferably to ToBRFV, in particular to tolerance against ToBRFV.

In one embodiment, the modified Albino3-like gene has a modification that leads to a modified Albino3-like protein 2. As used herein, an Albino3-like protein 2 is a protein comprising SEQ ID No. 2, or comprising a sequence having at least 70% sequence identity to SEQ ID No. 2.

As used herein, an Albino3-like gene is a gene encoding an Albino3-like protein 2. As used herein, an Albino3-like gene is a gene comprising a wildtype CDS sequence represented by SEQ ID No. 1, or a homologous gene comprising a sequence having at least 70% sequence identity to SEQ ID No. 1; or a gene encoding an Albino3-like protein 2 comprising SEQ ID No. 2; or a gene encoding a homologous Albino3-like protein 2 comprising a sequence having at least 70% sequence identity to SEQ ID No. 2. As used herein, a gene also comprises the 5’-UTR sequence, the promoter, and the 3’-UTR sequence of that gene.

A homologous Albino3-like gene comprises a homologous sequence, which is a sequence having at least 70% sequence identity to SEQ ID No. 1, preferably at least 75%, 77%, 80%, 83%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, or 99%. A homologous Albino3-like protein 2 comprises a homologous sequence, which is a sequence having at least 70% sequence identity to SEQ ID No. 2, preferably at least 75%, 77%, 80%, 83%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, or 99%.

As used herein, sequence identity is the percentage of nucleotides or amino acids that is identical between two sequences after proper alignment of those sequences. The person skilled in the art is aware of how to align sequences, for example by using a sequence alignment tool such as BLAST^®, which can be used for both nucleotide sequences and protein sequences. To obtain the most significant result, the best possible alignment that gives the highest sequence identity score should be obtained. The percentage sequence identity is calculated through comparison over the length of the shortest sequence in the assessment, whereby in the present case a sequence represents a gene that at least comprises a start codon and a stop codon, or a complete protein encoded by such a gene.

The Albino3-like protein 2 belongs to the conserved YidC/Oxal/Alb3 membrane protein family, members of which appear to play an important role in aiding the insertion of essential other membrane proteins in membranes of bacteria (YidC), mitochondria (Oxal), and chloroplasts (Alb3). An Albino3 (Alb3) protein is encoded by a gene that is located in the nucleus, and subsequently transported to the chloroplasts. In the chloroplast, the Alb3 protein is integrated into the thylakoid membrane, the site where the light reactions of photosynthesis take place, where it appears to facilitate insertion of membrane proteins into the thylakoid membrane.

The thylakoid-integrated Alb3 protein comprises hydrophobic transmembrane segments, in which the major parts of the conserved regions of the family are found. The transmembrane segments are connected by hydrophilic stretches, which form connecting loops in the stroma of the chloroplast on one side, and in the lumen, the inner space of the thylakoid on the other side. The N- and C-terminal regions of the protein are usually also formed by such hydrophilic stretches, and protrude into the lumen or the stroma (Kuhn et al., 2003. The Alb3/Oxal/YidC protein family: membrane-localized chaperones facilitating membrane protein insertion? Trends in Cell Biology 13(10)).

The Albino3-like protein 2 of the present invention is an orthologue of the Alb3 protein and is predicted to have the same function and behavior. A prediction of the topology of the protein structure using the TMHMM transmembrane helices prediction tool, which can for example be used at services.healthtech.dtu.dk/service.php 7TMHMM-2.0, shows that four transmembrane segments are expected, the first two of which are highly significant (Figure 3).

In one embodiment, the modification in an Albino3-like gene leads to a modified Albino3-like protein 2 that has a deletion, a substitution, or an insertion of at least one amino acid when compared to SEQ ID No. 2, or in a homologous sequence thereof. In one embodiment, the modified Albino3-like protein 2 has a modification in a hydrophilic stretch of the protein that does not form a transmembrane segment. The modification in a hydrophilic stretch is preferably a modification in a stretch that forms a connecting loop between the transmembrane segments, preferably a connecting loop that is present in the thylakoid lumen.

In one embodiment, the modified Albino3-like protein 2 has a modification in the sequence that covers amino acid positions 157 to 201 of SEQ ID No. 2, or in the corresponding positions in a homologous sequence having at least 70% sequence identity to SEQ ID No. 2. The modification in a hydrophilic stretch of the protein is alternatively in the N- or C-terminal region, preferably the C-terminal region. In one embodiment, the modified Albino3-like protein 2 has a modification in the sequence that covers amino acid positions 225 to 583 of SEQ ID No. 2, preferably in the sequence from position 434 to 461, or in the corresponding positions in a homologous sequence having at least 70% sequence identity to SEQ ID No. 2.

The modification in an Albino3-like gene that leads to ToBRFV resistance will change the activity and/or function of the encoded protein. The modification in the Albino3-like gene of the invention comprises a modification resulting in an amino acid change, a modification resulting in an early stop codon, a modification resulting in a truncated protein, or a modification resulting in a frameshift. Due to the modification, the encoded protein has a changed function, a reduced function, or it is non-functional. The modification in the encoded Albino3-like protein 2 is preferably a SNP modification resulting in an amino acid change.

In one embodiment, a SNP modification resulting in an amino acid change that leads to resistance comprises a C to G SNP on position 485 (C485G) of SEQ ID No. 1, an A to G SNP on position 1300 (A1300G) of SEQ ID No. 1, or a T to C SNP on position 1381 (T 1381C) of SEQ ID No. 1 , or on any of the corresponding positions in a homologous sequence of SEQ ID No.

1. Said nucleotide changes respectively result in a T162R substitution, a K434E substitution, or a C461R substitution in SEQ ID No. 2, or an amino acid substitution at the corresponding positions of a homologous sequence of SEQ ID No. 2.

In one embodiment, the modified Albino3-like gene that leads to resistance comprises a C485G mutation as present in SEQ ID No. 3, a C485G mutation and a T1680C mutation as present in SEQ ID No. 4, or an A1300G mutation, a T1381C mutation, and a T1470C mutation as present in SEQ ID No. 5, or a mutation or combination of mutations on any of the corresponding positions in a homologous sequence having at least 70% sequence identity to SEQ ID No. 1.

In one embodiment, the modified Albino3-like gene comprises a C485G SNP and an A1300G SNP, or a C485G SNP and a T1381C SNP, or an A1300G SNP and a T1381C SNP, or a C485G SNP and an A1300G SNP and a T1381C SNP.

In a preferred embodiment, the modified Albino3-like gene comprises a C485G SNP and optionally an A1300G SNP and/or a T1381C SNP, or a SNP on a corresponding position of a homologous sequence.

In one embodiment, the modified Albino3-like gene encodes a modified Albino3- like protein 2 comprising a T162R mutation as present in SEQ ID No. 6, or a K434E and/or a C461R mutation as present in SEQ ID No. 7, or a mutation or combination of mutations on any of the corresponding positions in a homologous sequence having at least 70% identity to SEQ ID No.

2. Table 3 gives an overview of said nucleotide and resulting amino acid modifications.

In one embodiment, the modified Albino3-like gene that leads to resistance comprises SEQ ID No. 3 or a sequence having at least 70% identity thereto, or comprises SEQ ID No. 4 or a sequence having at least 70% identity thereto, or comprises SEQ ID No. 5 or a sequence having at least 70% sequence identity thereto. A sequence having at least 70% sequence identity to SEQ ID No. 3, SEQ ID No. 4, or SEQ ID No. 5 preferably comprises at least one of a C485G mutation, a T1680C mutation, an A1300G mutation, a T1381C mutation, or a T1470C mutation. As used herein, at least 70% sequence identity relates to a sequence having in order of increased preference at least 70%, 75%, 77%, 80%, 83%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, or 99% sequence identity. In one embodiment, the modified Albino3-like gene encodes a modified Albino3- like protein 2 comprising SEQ ID No. 6, or encodes a protein having at least 70% sequence identity thereto, or encodes a modified Albino3-like protein 2 comprising SEQ ID No. 7, or encodes a protein having at least 70% sequence identity thereto. A protein having at least 70% identity thereto preferably comprises at least one of a T162R mutation, a K434E mutation, or a C461R mutation. At least 70% sequence identity comprises in order of increased preference at least 70%, 75%, 77%, 80%, 83%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, or 99% sequence identity.

As used herein, a X000Y mutation, SNP, or substitution means that the wildtype sequence has a nucleotide or amino acid X on position 000, which is changed to nucleotide or amino acid Y in the modified sequence.

The modification in the Albino3-like gene of the invention preferably leads to resistance against ToBRFV, optionally in combination with resistance against another virus, in particular another Tobamovirus.

As used herein, resistance against ToBRFV comprises tolerance and/or field tolerance to the virus. In this application, the words “resistant” and “resistance” are used for the form of resistance that is caused by the modified gene of the invention, which in the art is named “tolerance” or “field tolerance”. Virus resistance can express itself on different levels, whereby different mechanisms are involved. When a plant is truly resistant to a virus, the infection and/or replication of the virus in the hostplant is restricted by the resistance mechanism. When a plant is tolerant to a virus, virus replication and multiplication can take place in the plant, which can for example be measured through a qPCR experiment. However, when a young plant bio-assay is performed, no or only some mild symptoms are observed. The impact of the presence of the virus on the fitness of the plant is strongly reduced as compared to the impact on a susceptible plant.

A specific form of tolerance is field tolerance. When a plant is field tolerant, the host plant is not able to limit virus replication and multiplication, and the plants will show symptoms in a bio-assay performed under controlled conditions on young plants. However, when such a plant is grown in the field under normal cultivation practices, the host is able to reduce the impact of the virus presence on the plant’s fitness, and no or limited symptoms will be seen. In addition, the yield of the crop will not be significantly reduced and will be comparable to the yield of a crop without the virus.

The present invention relates to a plant comprising a modified Albino3-like gene of the invention. The plant comprising the modified Albino3-like gene is preferably a plant of the Solanaceae family, in particular a plant of the species Solanum lycopersicum, Capsicum annuum, Solanum melongena, Capsicum frutescens, Solanum tuberosum, Petunia spp, or Nicotiana tabacum. A plant of the invention is most preferably a plant of the species Solanum lycopersicum. A plant of the invention is preferably a cultivated plant, which is non-wild and has agronomical value, and is in particular agronomically elite.

In one embodiment, the plant comprises the modified Albino3-like gene of the invention homozygously and is resistant, in particular tolerant, to a virus of the genus Tobamovirus, preferably of the species Tomato Brown Rugose Fruit Virus (ToBRFV).

ToBRFV was first described by Luria et al ((2017): A new Israeli tobamovirus isolate infects tomato plants harboring Tm-2² resistance genes. PLoS ONE 12(l):e0170429. Doi:10.1371/journal.pone.0170429). At the time of that publication, Tomato Brown Rugose Fruit Virus was still abbreviated as TBRFV, but in the meantime the commonly used abbreviation for this virus is ToBRFV, which is therefore now also used in the present application.

During research for the present invention on ToBRFV resistance in Solanum lycopersicum, several Albino 3 -like -gene polymorphisms were identified that result in ToBRFV resistance. Some of these modifications were determined in the Albino3-like genes of wild tomato species, in particular in Solanum pimpinellifolium species. When these modifications were transferred to a ToBRFV susceptible S. lycopersicum plant, the S. lycopersicum plant became resistant, in particular tolerant, to ToBRFV, demonstrating that these modifications are the cause of the resistance as claimed herein.

In a preferred embodiment, the plant of the invention is a plant of the species Solanum lycopersicum comprising a modified Albino3-like gene, which plant is resistant, in particular tolerant, to a Tobamovirus, in particular to Tomato Brown Rugose Fruit Virus (ToBRFV). The modification in an Albino3-like gene in the S. lycopersicum plant of the invention comprises a modification selected from the group consisting of a C to G SNP on position 485 of SEQ ID No. 1; an A to G SNP on position 1300 of SEQ ID No. 1; a T to C SNP on position 1381 of SEQ ID No. 1 ; and corresponding modifications in homologous sequences of SEQ ID No. 1. A certain modification in an Albino3-like gene can result in resistance of one or more categories of resistance, in particular in tolerance.

The ToBRFV resistance of the invention is determined in a test comprising a control variety known to be ToBRFV susceptible (S) to make sure that the test is performed properly. ToBRFV susceptible tomato varieties that are suitable as controls are Endeavour FI and Ramyle FI. As a resistant control a plant deposited as NCIMB 43511 or NCIMB 43512 is used. A plant of these deposits comprises a modified Albino3-like gene of the invention. NCIMB 43511 comprises an Albino3-like gene comprising SEQ ID No. 3. NCIMB 43512 comprises an Albino3- like gene comprising SEQ ID No. 4. Both Albino3-like genes of NCIMB 43511 and NCIMB 43512 encode an Albino3-like protein 2 represented by SEQ ID No. 6.

To determine resistance, seeds of the accessions to be tested are sown in standard seedling trays and seedlings are inoculated 4 weeks after sowing. Inoculum is prepared by grounding leaves of tomato plants that were infected with ToBRFV in a 0.01 M phosphate buffer (pH 7.0) mixed with celite. The seedlings are then dusted with carborundum powder prior to gently rubbing the leaf with inoculum. Resistance is scored on a scale of 0-5; the description of the scales of the scores can be found in Table 1. Observation of the symptoms on the young tomato plants in the bio-assay is done 14-21 days after inoculation (dai).

As used herein, a Solanum lycopersicum plant that is resistant to ToBRFV due to the presence of a modified Albinoi-like gene has a score that is 3.0 or lower than 3.0, preferably lower than 2.5, when scoring according to Table 1 is used and a bio-assay as described above is performed. In one embodiment, a plant is tolerant to ToBRFV and has a score of 2.0 or lower than 2.0, preferably a score of 1.0 or lower than 1.0. In another embodiment, a plant has field tolerance (FT) to ToBRFV, and has a score of 3.0 or lower than 3.0, preferably lower than 2.5, in a bio assay, but has a score of 2.0 or lower than 2.0 in field conditions. The test is performed with 10 plants of a certain line, and the average score is taken. The test is performed properly in case susceptible (S) controls have a score that is higher than 3.0, preferably higher than 3.5.

To determine field tolerance in field conditions tomato plants are grown under normal cultivation practices in an area where ToBRFV is known to occur, since the determination depends on natural infection. To facilitate an even distribution of the virus, once the infection is present it is actively spread throughout the crop. Infected material is picked and mixed with some water in a blender to get infectious ToBRFV juice. The juice is rubbed on the leaves of each tomato plant by hand, using thumb and index finger, when the first cluster is present and contains at least 1 or 2 open flowers. When no symptoms develop yet on the susceptible controls that should be present throughout the crop, the spreading can be repeated once or twice with fresh juice.

A plant of the invention comprises a modified Albinoi-like gene homozygously or heterozygously, i.e. a modified Albino3-like gene can be present on both chromosomes of a chromosome pair in the genome of a plant, or on only one chromosome of a chromosome pair. A plant of the invention comprises a plant of an inbred line, a hybrid, an open pollinated variety, a doubled haploid, or a plant of a segregating population.

In one embodiment, a plant of the invention is a Solanum lycopersicum plant comprising the modified Albino3-like gene as comprised in the genome of a S. lycopersicum plant representative seed of which was deposited with the NCIMB under deposit number NCIMB 43511 or NCIMB 43512.

In one embodiment, a plant of the invention is a Solanum lycopersicum plant deposited as NCIMB 43511 or NCIMB 43512, or a progeny plant thereof comprising one or more or all polymorphisms in the Albino3-like genes that are present in said deposits. In one embodiment, a plant of the invention is a Solanum lycopersicum plant comprising a modified Albinoi-like gene which encodes an Albino3-like protein 2 according to SEQ ID No. 6 or SEQ ID No. 7, or encodes a homologous protein sequence.

The virus resistance, in particular the ToBRFV resistance, in a plant of the present invention inherits in an intermediate manner. As used herein, intermediate means that a higher level of resistance is found when a modified Albinoi-like gene of the invention is homozygously present. In some embodiments, the heterozygous presence of a modified Albinoi-like gene of the invention already confers ToBRFV resistance. The ToBRFV resistance of both homozygous and heterozygous plants makes the plants more suitable for cultivation under conditions where ToBRFV is present. Therefore, both heterozygous and homozygous plants are considered to have improved agronomic characteristics. In addition, heterozygous plants can be used for development of homozygous plants through crossing and selection, which heterozygous plants therefore also form a part of this invention. In a preferred embodiment, the modified Albinoi-like gene is homozygously present in a plant.

The invention further relates to a seed that comprises a modified Albino3-like gene of the invention, which seed can grow into a plant of the invention. The invention also relates to use of said seed for the production of a plant of the invention, by growing said seed into a plant. The invention also relates to a plant part of a plant of the invention, which comprises a fruit or a seed, wherein the plant part comprises a modified Albinoi-like gene in its genome. In a preferred embodiment, the modified Albinoi-like gene is homozygously present in a seed.

The invention further relates to a method for seed production, comprising growing a plant from a seed of the invention that homozygously comprises a modified Albino3-like gene of the invention, allowing the plant to produce a fruit with seed, harvesting the fruit, and extracting those seed. Production of the seed is suitably done by selfing or by crossing with another plant that is optionally also a plant of the invention. The seed that is so produced has the capability to grow into a plant that is resistant to a virus of the genus Tobamovirus, in particular to ToBRFV. The method in particular relates to production of Solanum lycopersicum seed. In a preferred embodiment, both plants used in the crossing are a plant of the invention.

The invention further relates to hybrid seed and to a method for producing said hybrid seed, comprising crossing a first parent plant with a second parent plant and harvesting the resultant hybrid seed, wherein the first parent plant and/or the second parent plant is a plant of the invention comprising a modified Albino3-like gene of the invention. The hybrid seed is in particular seed of Solanum lycopersicum. The resulting hybrid plant that can be grown from the hybrid seed, comprising the Albino3-like gene of the invention, which hybrid plant has resistance to a virus of the genus Tobamovirus, in particular to ToBRFV, is also a plant of the invention. In a preferred embodiment, the resulting hybrid plant is a Solanum lycopersicum plant.

In a preferred embodiment, the modified Albino3-like gene is homozygously present in both parent plants of the hybrid.

The present invention relates to a method for producing a plant that is resistant to a virus of the genus Tobamovirus, in particular to ToBRFV, comprising introducing a modification in an Albino3-like gene, which modification leads to resistance. Said method comprises the introduction of a deletion, a substitution, or an insertion in the coding sequence of an Albino3-like gene. The introduction of such a modification can be done by a mutagenesis approach using a chemical compound, such as ethyl methane sulphonate (EMS); or by using physical means, such as UV-irradiation, fast neutron exposure, or other irradiation techniques.

The invention in particular relates to a method for producing a Solanum lycopersicum plant that is resistant to ToBRFV, comprising introducing a modification in an Albino3-like gene comprising SEQ ID No. 1 , or in a homologous sequence having at least 70% sequence identity thereto. The method in particular comprises the introduction of at least one of a C485G modification, an A1300G modification, and a T1381C modification in SEQ ID No. 1.

In one embodiment, the method comprises the introduction of a C485G modification and an A1300G modification. In one embodiment, the method comprises the introduction of a C485G modification and a T1381C modification. In one embodiment, the method comprises the introduction of an A1300G and a T1381C modification. In one embodiment, the method comprises the introduction of a C485G, an A1300G, and a T1381C modification.

Introduction of a modification can also be done using a more specific, targeted approach including targeted genome editing by means of homologous recombination, oligonucleotide -based mutation introduction, zinc -finger nucleases (ZFN), transcription activator like effector nucleases (TALENs) or Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems.

Introduction of a modified Albino3-like gene of the invention can also be done through introgression from a plant comprising said modified Albino3-like gene, for example from a plant that was deposited as NCIMB 43511 or NCIMB 43512, or from progeny thereof, or from another plant that is resistant to a Tobamovirus, in particular to ToBRFV, and in which a modified Albino3-like gene, in particular the modified gene of the invention, was identified. Breeding methods such as crossing and selection, backcrossing, recombinant selection, or other breeding methods that result in the transfer of a genetic sequence from a resistant plant to a susceptible plant can be used. A resistant plant can be of the same species or of a different and/or wild species. Difficulties in crossing between species can be overcome through techniques known in the art such as embryo rescue, or cis-genesis can be applied. Progeny of a deposit can be sexual or vegetative descendants of that deposit, which can be selfed and/or crossed, and can be of an FI, F2, or further generation as long as the descendants of the deposit still comprise a modified Albino3-like gene of that deposit. A plant produced by such method is also a part of the invention.

In one embodiment, a modified Albinoi-like gene is introgressed into S. lycopersicum from a plant of a wild tomato species, which is a plant of a species belonging to the genus Solanum, in particular a plant of the species S. pimpinellifolium. In another embodiment, a modified Albino3-like gene is introgressed from a S. lycopersicum plant comprising the modified Albinoi-like gene into a S. lycopersicum plant lacking a modified Albinoi-like gene.

Transgenic techniques used for transferring sequences between plants that are sexually incompatible can also be used to produce a plant of the invention, by transferring a modified Albinoi-like gene from one species to another. Techniques that can suitably be used comprise general plant transformation techniques known to the skilled person, such as the use of an Agrobacterium- mediated transformation method.

The invention also relates to a method for the production of a plant which is resistant to a Tobamovirus, in particular to ToBRFV, said method comprising: a) crossing a plant comprising a modified Albinoi-like gene of the invention with another plant; b) optionally performing one or more rounds of selfing and/or crossing a plant resulting from step a) to obtain a further generation population; c) selecting from the population resulting from the cross of step a), or from the further generation population of step b), a plant that comprises a modified Albinoi-like gene as defined herein, which plant is resistant against a Tobamovirus, in particular to ToBRFV.

In a preferred embodiment, above method relates to the production of a Solanum lycopersicum plant.

The invention also relates to a method for the production of a plant, which is resistant to a Tobamovirus, in particular to ToBRFV, said method comprising: a) crossing a first parent plant comprising a modified Albino3-like gene of the invention with a second parent plant, which is another plant not comprising a modified Albino3- like gene of the invention; b) backcrossing the plant resulting from step a) with the second parent plant for at least three generations; c) selecting from the third or higher backcross population a plant that comprises at least the modified Albino3-like gene of the first parent plant of step a).

In a preferred embodiment, the above method relates to the production of a Solanum lycopersicum plant. The invention additionally provides for a method of introducing another desired trait into a plant that is resistant to a Tobamovirus, in particular to ToBRFV, comprising: a) crossing a plant comprising a modified Albinoi-like gene of the invention with a second plant that comprises the other desired trait to produce FI progeny; b) optionally selecting in the FI for a plant that comprises the virus resistance and the other desired trait; c) crossing the optionally selected FI progeny with one of the parents for at least three generations, to produce backcross progeny; d) selecting backcross progeny comprising the virus resistance and the other desired trait; and e) optionally repeating steps c) and d) one or more times in succession to produce selected fourth or higher backcross progeny that comprises the virus resistance and the other desired trait.

In a preferred embodiment, the above method relates to the introduction of another desired trait in a ToBRFV resistant Solanum lycopersicum plant.

Optionally, selfing steps are performed after any of the crossing or backcrossing steps in above described methods. Selection of a plant comprising virus resistance and the other desired trait can alternatively be done following any crossing or selfing step of the method. The other desired trait can be selected from, but is not limited to, the following group: resistance to bacterial, fungal or viral diseases, insect or pest resistance, improved germination, plant size, plant type, improved shelf-life, water stress and heat stress tolerance, and male sterility. The invention includes a plant produced by this method and a fruit obtained therefrom.

The invention further relates to a method for the production of a plant comprising a modified Albino3-like gene of the invention, which plant is resistant to a Tobamovirus, in particular to ToBRFV, by using tissue culture or by using vegetative propagation. Said method preferably relates to the production of a Solanum lycopersicum plant.

The present invention relates to a method for identification of a plant comprising a modified Albino3-like gene of the invention, which plant is resistant to a Tobamovirus, in particular to ToBRFV, wherein the identification comprises determining the presence of a modification in the Albino3-like gene of SEQ ID No. 1 , or in a homologous sequence thereof, and analyzing if the plant comprising the modification is resistant to a Tobamovirus, in particular to ToBRFV. Said method relates to the identification of a plant of a species belonging to the Solanaceae, and in particular to the identification of a plant of a species belonging to the genus Solanum. The method preferably relates to the identification of a plant of a wild tomato species, in particular of the species Solanum pimpinellifolium, or to the identification of a plant of the species Solanum lycopersicum. Determining the presence of a modification in an Albino3-like gene comprises identification of any modification in SEQ ID No. 1 that leads to ToBRFV resistance. Determining the presence of a modification includes any of the modifications as described herein, in particular the SNP modifications C485G, A1300G, or T1381C as presented in Table 3. Determining the presence of a modification also comprises determining the presence of a silent nucleotide SNP, which does not lead to an amino acid change but can still be linked to ToBRFV resistance when it is linked to another modification in the gene that does lead to an amino acid change. In this way, the silent nucleotide SNP can be used as a marker for identification of a SNP leading to resistance. Such silent modification comprises in particular a T1470C SNP, or a T1680C SNP. The silent T1470C SNP is in particular linked to a C485G modification. The silent T1680C SNP is in particular linked to an A1300G and/or a T1381C modification. Determining the presence of a modification can be done through sequence comparison, which is known to the skilled person. Determining a modification is suitably done by using a marker that is designed to identify such modification as its sequence comprises that particular modification.

The present invention further relates to a method for selection of a plant, which is resistant to a Tobamovirus, in particular to ToBRFV, the method comprising identification of a modified Albino3-like gene of the invention in a plant, and subsequently selecting said plant as a plant which is resistant to a Tobamovirus, in particular to ToBRFV. Optionally, the virus resistance can be confirmed by performing a bio-assay as described in Example 1. The selected plant obtained by such method is also a part of this invention.

The invention also relates to a method for selecting a ToBRFV resistant plant of a plant of the Solanaceae, in particular a species belonging to the genus Solanum. The species of the genus Solanum is preferably a wild tomato relative, most preferably of the species Solanum pimpinellifolium. The method comprises identifying the presence of a modification in an Albino3- like gene, optionally testing the plant for ToBRFV resistance, and selecting a plant that comprises said modification as a ToBRFV resistant plant. The modification in the Albino3-like gene comprises a C485G SNP, an A1300G SNP, a T1381C SNP, a T1470C SNP, or a T1680C SNP when compared to SEQ ID No. 1 or a SNP on a corresponding position in a homologous sequence thereof. The plant thus selected can be used as a source to introduce the modified gene into another plant, such as a Solanum lycopersicum plant, thereby introducing ToBRFV resistance.

The invention in particular relates to a method for selecting a ToBRFV resistant Solanum lycopersicum plant, the method comprising identifying the presence of a modification in an Albino3-like gene, optionally testing the plant for ToBRFV resistance, and selecting a plant that comprises said modification as a ToBRFV resistant plant. The modification in the Albino3-like gene comprises at least one of a C485G SNP, an A1300G SNP, a T1381C SNP, a T1470C SNP, or a T1680C SNP when compared to SEQ ID No. 1 or to a homologous sequence. The invention also relates to propagation material suitable for producing a plant of the invention, wherein the propagation material is suitable for sexual reproduction, and is in particular selected from a microspore, pollen, an ovary, an ovule, an embryo sac, or an egg cell, or is suitable for vegetative reproduction, and is in particular selected from a cutting, a root, a stem cell, or a protoplast, or is suitable for tissue culture of regenerable cells, and is in particular selected from a leaf, pollen, an embryo, a cotyledon, a hypocotyl, a meristematic cell, a root, a root tip, an anther, a flower, a seed and a stem, and wherein the plant produced from the propagation material comprises the modified Albinoi-like gene of the invention that provides resistance to a Tobamovirus, in particular to ToBRFV. A plant of the invention may be used as a source of the propagation material. A tissue culture comprising regenerable cells also forms a part of this invention.

The invention further relates to a cell of a plant of the invention. Such a cell may either be in isolated form or a part of the complete plant or parts thereof and still forms a cell of the invention, because such a cell comprises the modified Albinoi-like gene of the invention. Each cell of a plant of the invention carries the modified Albinoi-like gene of the invention. A cell of the invention may also be a regenerable cell that can regenerate into a new plant of the invention.

The invention further relates to plant tissue of a plant of the invention, which comprises the modified Albinoi-like gene of the invention. The tissue can be undifferentiated tissue or already differentiated tissue. Undifferentiated tissue is for example a stem tip, an anther, a petal, or pollen, and can be used in micropropagation to obtain new plantlets that are grown into new plants of the invention. The tissue can also be grown from a cell of the invention.

The invention moreover relates to progeny of a plant, a cell, a tissue, or a seed of the invention, which progeny comprises the modified Albinoi-like gene of the invention. Such progeny can in itself be a plant, a cell, a tissue, or a seed. The progeny can in particular be progeny of a plant of the invention deposited under NCIMB number 43511 or NCIMB 43512. As used herein, progeny comprises the first and all further descendants from a cross with a plant of the invention, wherein a cross comprises a cross with itself or a cross with another plant, and wherein a descendant that is determined to be progeny comprises a modified Albinoi-like gene of the invention. Descendants can be obtained through selfing and/or further crossing of the deposit. Progeny also encompasses material that is obtained by vegetative propagation or another form of multiplication.

The invention also relates to a marker for the identification of a modified Albinoi- like gene in a plant, which marker comprises any of the modifications in an Albinoi-like gene as described herein and can thereby identify said modifications. A marker of the invention is in particular a marker comprising, and thereby suitable for identifying, a SNP modification, i.e. a polymorphism, C485G, T1680C, A1300G, T1381C, or T1470C when compared to SEQ ID No. 1 or to a homologous sequence thereof.

The invention also relates to the use of a marker for identification of a modified Albinoi-like gene. The invention further relates to the use of a marker, in particular a marker as described herein, for identification of a modified Albino3-like gene that leads to ToBRFV resistance in a Solanum lycopersicum plant and/or for selection of a Solanum lycopersicum plant comprising a modified Albino3-like gene that leads to ToBRFV resistance. The invention also relates to the use of a marker for identification of a modified Albino3-like gene that leads to ToBRFV resistance in a plant of the genus Solanum, in particular a plant of a wild tomato relative, preferably in a plant of the species Solanum pimpinellifolium. The invention also relates to selection of a plant identified by a marker as described herein and to the plant thus selected.

The present invention will be further illustrated in the Examples that follow and that are for illustration purposes only. The Examples are not intended to limit the invention in any way. In the Examples and in the application reference is made to the following figures.

FIGURES

Figure 1 - CDS sequence of SEQ ID No. 1 (the wildtype Albino3-like gene of Solanum lycopersicum), SEQ ID No. 3 ( Albino3-like gene comprising a C485G mutation), SEQ ID No. 4 (Albino3-like gene comprising a C485G mutation and a T1680C mutation), and SEQ ID No. 5 ( Albino3-like gene comprising an A1300G mutation, a T1381C mutation and a T1470C mutation).

Figure 2 - protein sequences of SEQ ID No. 2 (the wildtype Albino3-like protein 2 encoded by SEQ ID No. 1), SEQ ID No. 6 (Albino3-like protein 2 encoded by SEQ ID No. 3 and SEQ ID No. 4), and SEQ ID No. 7 (Albino34ike protein 2 encoded by SEQ ID No. 5).

Figure 3 - prediction of the transmembrane sections of the Albino3-like protein 2 that are integrated into the thylakoid membrane of a chloroplast.

DEPOSIT

Seed of tomato Solanum lycopersicum comprising a modified Albino3-like gene of the invention was deposited with NCIMB Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, UK on 7 November, 2019, under deposit accession numbers NCIMB 43511 (comprising SEQ ID No. 3) and NCIMB 43512 (comprising SEQ ID No. 4). EXAMPLES

EXAMPLE 1

Bio-assay for ToBRFV resistance in Solanum lycopersicum

S. lycopersicum lines having a modification in an Albinoi-like gene were observed in a ToBRFV bio-assay. As resistant controls two internal S. pimpinellifolium sources, GNL.3951 and GNL.3919, were included. As susceptible controls Endeavour FI and Ramyle FI were used.

Seeds of the accessions to be tested were sown in standard seedling trays and 10 seedlings per accession were inoculated 4 weeks after sowing. Inoculum was prepared by grounding leaves of tomato plants that were infected with ToBRFV in a 0.01 M phosphate buffer (pH 7.0) mixed with celite. Plants were dusted with carborundum powder prior to gently rubbing the leaf with inoculum. Scoring of the symptoms was done according to Table 1 at 19 days after inoculation.

Table 1: scoring scales ToBRFV resistance

Results of the bio-assay are presented in Table 2; the average score of the 10 inoculated seedlings is given. T0313 is a cross between a line with the Albinoi-like gene having SEQ ID No. 3 homozygously and a line with the Albinoi-like gene having SEQ ID No. 4 homozygously. Table 2: ToBRFV bio-assay scores

EXAMPLE 2

Identification of modifications in Albino 3 -like genes that lead to ToBRFV resistance.

Various internally developed Solanum lycopersicum populations that segregated for ToBRFV resistance were fine-mapped to a small region on chromosome 11 that contained only four potential genes, which were likely to contribute to the ToBRFV resistance. Whole genome sequences were available in-house for the backgrounds of the resistant and susceptible lines that were used in the development of these populations. Therefore, a SNP-calling approach for the region was done, which means unique polymorphisms in the region were identified by comparing the sequences to each other and determining the differences. Among the genes in the region of interest was an Albino3-like gene, which had various polymorphisms between susceptible and resistant material. Different resistant lines were observed to have different polymorphisms. Some polymorphisms that were observed were silent, meaning they did not result in an amino acid change, for example a T1470C SNP, or a T1680C SNP. A number of the other polymorphisms resulted in non-conservative amino acid changes, which polymorphisms are presented in Table 3.

The Albino3-like protein 2 is a protein that is incorporated in the thylakoid membrane of a chloroplast of a plant. It comprises both hydrophobic stretches, that get embedded in the membrane, and hydrophilic stretches that protrude out of the membrane in the thylakoid lumen or the chloroplast stroma. (Figure 3). It was observed that the modifications presented in Table 3 that were found to result in resistance were all present in a hydrophilic stretch of the Albino3-like 2 protein.

Table 3: Certain SNP modifications correlating with ToBRFV resistance in S. lycopersicum

Through analysis of the correlation in segregation of phenotypes and genotypes it was determined that a modification in an Albino3-like gene resulting in a modified Albino3-like protein 2 was the cause of the ToBRFV resistance of the resistant Solanum lycopersicum plants.

EXAMPLE 3

Modification of an Albino 3 -like gene to obtain resistance to a Tobamovirus, in particular to ToBRFV

Modifications are introduced in seed of a plant of interest in which resistance to a Tobamovirus is needed, such as ToBRFV, ToMV, or TMV. The modification is introduced through mutagenesis, such as an EMS treatment, through radiation means, or through a specific targeted approach, such as CRISPR. When a non-targeted approach such as EMS is used, this is combined with an identification technique such as TILLING. In this way, both for mutagenesis as well as a targeted modification means, a modification in an Albino3-like gene can be generated and identified. The skilled person is familiar with these means for introducing modifications into the genome of a plant of interest.

Modified seed is then germinated and plants are grown, which are crossed or selfed to generate M2 seed. Subsequently a plant screen is performed to identify the modifications in an Albino3-like gene, based on comparison to the wildtype sequence of the one or more Albino3-like genes of that species. For Solanum lycopersicum for example, comparison to SEQ ID No. 1 can be done. The skilled person is familiar with TILLING to identify mutations in specific genes (McCallum et. al. (2000) Nature Biotechnology, 18: 455-457), and with techniques for identifying nucleotide changes such as DNA sequencing, amongst others.

Plants with a modified Albino3-like gene are homozygous or made homozygous by selfing, crossing, or the use of doubled haploid techniques which are familiar to the skilled person. Plants identified and selected on the basis of a modification in an Albino3-like gene can then be tested for resistance to a Tobamovirus, for example resistance to ToBRFV, ToMV, or TMV. A plant that is produced, identified and selected in this way is confirmed to have their virus resistance as a result from one or more modifications in the Albino3-like gene.

Claims

1. A modified Albino 3 -like gene the wildtype of which comprises SEQ ID No. 1 encoding SEQ ID No. 2, or a homologous sequence having at least 70% sequence identity to SEQ ID No. 1, which modified gene leads to resistance to a Tobamovirus, preferably to ToBRFV.

2. A modified Albino3-like gene as claimed in claim 1, which comprises a modification that leads to a modification in a hydrophilic stretch of the encoded Albino3-like protein 2.

3. A modified Albino3-like gene as claimed in claim 2, wherein the hydrophilic stretch of the encoded protein is present in the sequence that covers positions 157-201 of SEQ ID No. 2, or is present in the sequence that covers positions 225-583 of SEQ ID No. 2.

4. A modified Albino 3 -like gene as claimed in any of the claims 1-3, which comprises a C485G SNP and/or an A1300G SNP, and/or a T1381C SNP, or a SNP on a corresponding position of a homologous sequence.

5. A modified Albino3-like gene as claimed in claim 4, which comprises a C485G SNP and an A1300G SNP, or a C485G SNP and a T1381C SNP, or an A1300G SNP and a T1381C SNP, or a C485G SNP and an A1300G SNP and a T1381C SNP, or a combination of SNPs on corresponding positions of a homologous sequence.

6. A modified Albino3-like gene as claimed in claim 4, which comprises a C485G SNP and optionally an A1300G SNP and/or a T1381C SNP, or a SNP on a corresponding position of a homologous sequence.

7. A modified Albino 3 -like gene as claimed in any of the claims 1-6, which modified gene encodes an Albino3-like protein 2 according to SEQ ID No. 6 or SEQ ID No. 7, or encodes an Albino3-like protein 2 having at least 70% sequence identity thereto.

8. Plant comprising a modified Albino3-like gene as defined in any of the claims 1- 7.

9. Plant as claimed in claim 8, which is resistant to a Tobamovirus, preferably to

ToBRFV.

10. Plant as claimed in claim 8 or 9, which is a plant of the family Solanaceae, preferably a plant of the species Solanum lycopersicum.

11. A Solanum lycopersicum plant as claimed in claim 10, wherein the modified Albino3-like gene is as comprised in the genome of a Solanum lycopersicum plant representative seed of which was deposited with the NCIMB under deposit number NCIMB 43511 or NCIMB 43512.

12. A Solanum lycopersicum plant as claimed in claim 10, wherein the modified Albino3-like gene encodes an Albino3-like protein 2 according to SEQ ID No. 6 or SEQ ID No. 7.

13. Seed, comprising a modified Albino3-like gene as defined in any of the claims 1-7.

14. Marker for the identification of a modified Albino3-like gene, wherein the marker detects a modification selected from the group consisting of:

- a C to G SNP on position 485 of SEQ ID No. 1,

- an A to G SNP on position 1300 of SEQ ID No. 1,

- a T to C SNP on position 1381 of SEQ ID No. 1,

- a T to C SNP on position 1470 of SEQ ID No. 1, and

- a T to C SNP on position 1680 of SEQ ID No. 1, or wherein the marker detects a modification on a corresponding position of a homologous sequence having at least 70% sequence identity to SEQ ID No. 1.

15. Use of a marker as claimed in claim 14 for identification of a modified Albinoi-like gene that leads to ToBRFV resistance in a Solanum lycopersicum plant and/or for selection of a ToBRFV resistant Solanum lycopersicum plant.

16. Use of a marker as claimed in claim 14 for identification of a modified Albino3-like gene that leads to ToBRFV resistance in a plant of the genus Solanum, preferably in a plant of the species Solanum pimpinellifolium.

17. Method for producing a ToBRFV resistant Solanum lycopersicum plant comprising introducing a modification in an Albino3-like gene, wherein the Albino3-like gene comprising the modification is as defined in any of the claims 1-7.

18. Method as claimed in claim 17, wherein the modification comprises at least one of a C485G SNP, an A1300G SNP, and a T1381C SNP.

19. Method for selecting a ToBRFV resistant Solanum lycopersicum plant, comprising identifying the presence of a modification in an Albino3-like gene, optionally testing the plant for ToBRFV resistance, and selecting a plant that comprises said modification as a ToBRFV resistant plant.

20. Method as claimed in claim 19, wherein the modification to be identified comprises at least one of a C485G SNP, an A1300G SNP, a T1381C SNP, a T1470C SNP, and a T1680C SNP.

21. Method as claimed in claim 19 or 20, wherein the identification is performed by using a marker as defined in claim 14.

22. Method for the production of a plant which is resistant to a Tobamovirus, preferably to ToBRFV, said method comprising: a) crossing a first parent plant as claimed in any of the claims 8-12, with a second parent plant; b) optionally performing one or more rounds of selfing and/or crossing of the plant resulting from the cross in step a) to obtain a further generation population; c) selecting from the plant resulting from the cross in step a), or from the further generation population of step b), a plant that comprises a modified Albinoi-like gene, wherein the selected plant is resistant to a Tobamovirus, preferably to ToBRFV.

23. Method for the production of a Solanum lycopersicum plant which is resistant to a Tobamovirus, preferably to ToBRFV, said method comprising: a) crossing a first parent plant as claimed in any of the claims 10-12, with a second parent plant; b) optionally performing one or more rounds of selfing and/or crossing of the plant resulting from the cross in step a) to obtain a further generation population; c) selecting from the plant resulting from the cross in step a), or from the further generation population of step b), a plant that comprises a modified Albinoi-like gene, wherein the selected plant is resistant to a Tobamovirus, preferably to ToBRFV.

24. Method as claimed in claim 22 or 23, wherein the second parent plant also comprises a modified Albinoi-like gene.

25. Method as claimed in any of the claims 22-24, wherein selection of a plant comprising a modification in an Albinoi-like gene is performed by using a marker as claimed in claim 14.

26. Method as claimed in any of the claim 22-24, wherein a plant which is resistant to ToBRFV is phenotypically selected, in particular by using a bio-assay for ToBRFV resistance.

27. Method as claimed in any of the claims 22-26, wherein the plant as claimed in any of the claims 8-12 is a plant grown from seed deposited under NCIMB accession number 43511 or NCIMB 43512, or a progeny plant thereof.

28. Method for the production of hybrid seed comprising crossing a first parent plant with a second parent plant and harvesting the resultant hybrid seed, wherein the first parent plant and/or the second parent plant is a plant comprising a modified Albinoi-like gene as claimed in any of the claims 1-7, and wherein the presence of said modified Albinoi-like gene leads to ToBRFV resistance in a plant that is grown from the hybrid seed.

29. Method as claimed in claim 28, wherein the hybrid seed is seed of the species Solanum lycopersicum.