Methods and compositions for use in genome modification in plants

Abstract

The present disclosure is in the field of plant transformation. The disclosure provides methods for increasing the rate of site-directed integration of a sequence of interest in plants. The disclosure also provides methods for increasing the efficiency of Rhizobiales-mediated plant transformation.

1 . A method of providing a sequence of interest to the genome of a plant cell, comprising contacting the plant cell with a Rhizobiales cell capable of transforming the plant cell, wherein the Rhizobiales cell comprises at least one vector capable of forming two T-strands that are essentially complementary in at least a portion of the T-strands,
 a. wherein the at least one vector comprises a first right border DNA sequence (RB1), a second right border DNA sequence (RB2), and at least one sequence of interest, and wherein the RB1 is positioned in the vector to initiate synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5′ to 3′ end of the first T-strand; and the RB2 is positioned in the vector to initiate synthesis of a second T-strand such that the sequence of interest is in the anti-sense orientation relative to the sequence of interest in the first T-strand, and wherein the two T-strands resulting from initiation at RB1 and RB2 are essentially complementary in at least a portion of the sequence of interest; or   b. wherein the at least one vector comprises a RB1, a RB2, a sequence of interest, a first left border DNA sequence (LB1) and a second left border DNA sequence (LB2), wherein the vector is configured such that the RB1 is paired with the LB1 which are positioned in the vector to initiate (RB1) and terminate (LB1) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5′ to 3′ end of the first T-strand; and the RB2 is paired with the LB2 which are positioned in the vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the sequence of interest is in an anti-sense orientation relative to the sequence of interest in the first T-strand, and wherein the two T-strands resulting from initiation at RB1 and RB2 are essentially complementary in at least a portion of the sequence of interest; or   c. wherein the vector comprises a first sequence of interest and a second sequence of interest, wherein the first sequence of interest is essentially identical to the second sequence of interest; wherein the vector further comprises a RB1 and a LB1 which are positioned in the vector to initiate (RB1) and terminate (LB1) synthesis of a first T-strand such that the first sequence of interest is in the sense orientation from the 5′ to 3′ end of the first T-strand; and the vector further comprises a RB2 and a LB2 which are positioned in the vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the second sequence of interest is in an anti-sense orientation relative to the first sequence of interest in the first T-strand, and wherein the two T-strands resulting from initiation at RB1 and RB2 are essentially complementary in at least a portion of the first sequence of interest and the second sequence of interest; or   d. wherein the Rhizobiales cell comprises at least a first vector and a second vector, wherein each vector comprises essentially identical sequences of interest, and wherein the first vector comprises a RB1 and a LB1 which are positioned in the first vector to initiate (RB1) and terminate (LB1) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5′ to 3′ end of the first T-strand; and wherein the second vector comprises a RB2 and a LB2 which are positioned in the second vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the sequence of interest is in an anti-sense orientation relative to the sequence of interest in the first T-strand, and wherein the two T-strands resulting from initiation at RB1 and RB2 are essentially complementary in at least a portion of the sequence of interest; or   e. wherein the at least one vector comprises a first sequence of interest, a second sequence of interest different from the first sequence of interest, at least two RB DNA sequences, and one or more optional LB DNA sequences, wherein the first RB DNA sequence (RB1) and a first LB DNA sequence (LB1) are positioned in the vector to initiate (RB1) and terminate (LB1) synthesis of the first T-strand such that the first sequence of interest is in the sense orientation from the 5′ to 3′ end of the first T-strand; and the vector configuration further comprises a second RB DNA sequence (RB2) and a second LB DNA sequence (LB2) which are positioned in the vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the first sequence of interest is in an anti-sense orientation from the 5′ to 3′ end of the second T-strand, wherein the two T-strands resulting from initiation at RB1 and RB2 are essentially complementary to each other in at least a portion of the first sequence of interest, and wherein the vector configuration further comprises a third RB DNA sequence (RB3) and a third LB DNA sequence (LB3) which are positioned in the vector to initiate (RB3) and terminate (LB3) synthesis of a third T-strand such that the second sequence of interest is in the sense orientation from the 5′ to 3′ end of the third T-strand; and the vector configuration further comprises a fourth RB DNA sequence (RB4) and a fourth LB DNA sequence (LB4) which are positioned in the vector to initiate (RB4) and terminate (LB4) synthesis of a fourth T-strand such that the second sequence of interest is in an anti-sense orientation from the 5′ to 3′ end of the fourth T-strand, and the two T-strands resulting from initiation at RB3 and RB4 are essentially complementary to each other in at least a portion of the second sequence of interest.   
     
     
         2 . (canceled) 
     
     
         3 . The method of  claim 1 , wherein
 a. the RB1 and the RB2 are essentially homologous, and the LB1 and the LB2 are essentially homologous;   b. the RB1 and the RB2 are essentially homologous, and the LB1 and the LB2 are not essentially homologous;   c. the RB1 and the RB2 are not essentially homologous, and the LB1 and the LB2 are essentially homologous; or   d. the RB1 and the RB2 are not essentially homologous, and the LB1 and the LB2 are not essentially homologous.   
     
     
         4 . The method of  claim 1 , wherein
 a. at least one of the RB1 and RB2 comprise an  Agrobacterium  Ti plasmid right border consensus DNA sequence;   b. at least one of the RB1 and RB2 comprise an  Agrobacterium  Ti plasmid right border consensus DNA sequence selected from SEQ ID NO: 21 and SEQ ID NO: 22;   c. at least one of the RB1 and RB2 comprise a sequence selected from SEQ ID NOs: 1-13; or   d. at least one of the RB1 and RB2 comprise a sequence at least 80% identical to a sequence selected from SEQ ID NO:4 and SEQ ID NO:12.   
     
     
         5 . The method of  claim 1 , wherein
 a. at least one of the LB1 and LB2 comprise an  Agrobacterium  Ti plasmid left border consensus DNA sequence;   b. at least one of the LB1 and LB2 comprise an  Agrobacterium  Ti plasmid left border consensus DNA sequence selected from SEQ ID NO: 23 and SEQ ID NO: 24;   c. at least one of the LB1 and LB2 comprise a sequence selected from SEQ ID NOs: 14-20; or   d. at least one of the LB1 and LB2 comprise a sequence at least 80% identical to SEQ ID NO:19.   
     
     
         6 . (canceled) 
     
     
         7 . The method of  claim 1 , wherein the sequence of interest comprises one or more sequences selected from: a gene, a portion of a gene, an intergenic sequence, an enhancer, a promoter, an intron, an exon, a sequence encoding a transcription termination sequence, a sequence encoding a chloroplast targeting peptide, a sequence encoding a mitochondrial targeting peptide, an insulator sequence, a sequence encoding an anti-sense RNA construct, a sequence encoding a protein, a sequence encoding non-protein-coding RNA (npcRNA), a sequence encoding a recombinase, a sequence encoding a recombinase recognition site, a landing pad, an editing template, an expression cassette, a stack of two or more expression cassettes encoding transgenes, a sequence encoding a site-specific enzyme, a sequence encoding a site-specific enzyme target site, a sequence encoding a selection marker, a sequence encoding a cell factor that functions to increase DNA repair, a sequence comprising a linker or a spacer, a sequence comprising one or more restriction enzyme sites, a sequence for templated genome editing, and any combination thereof. 
     
     
         8 . The method of  claim 6 , wherein the sequence of interest further comprises at least one homology arm DNA sequence. 
     
     
         9 . The method of  claim 8 , wherein the sequence of interest comprises both a left homology arm DNA sequence and a right homology arm DNA sequence. 
     
     
         10 . The method of  claim 8 , wherein the at least one homology arm DNA sequence comprises a sequence that is at least 80% identical to a target sequence in the plant genome. 
     
     
         11 .- 12 . (canceled) 
     
     
         13 . The method of  claim 7 , wherein the site-specific enzyme is selected from a group consisting of an endonuclease, a recombinase, and a transposase. 
     
     
         14 .- 16 . (canceled) 
     
     
         17 . The method of  claim 7 , wherein the sequence of interest comprises a sequence encoding a protein involved in DNA repair, wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or any combination thereof. 
     
     
         18 . The method of  claim 1 , wherein at least part of the sequence of interest is integrated into the plant genome via homologous recombination or via non-homologous end joining, wherein the integration of at least part of the sequence of interest results in a point mutation, an insertion, a deletion, an inversion, increased transcription of an endogenous locus, decreased transcription of an endogenous locus, altered protein activity, altered RNAi products, altered RNAi target sites, altered RNAi pathway activity, increased transcription of the sequence of interest, decreased transcription of the integrated sequence of interest, or any combination thereof. 
     
     
         19 . A method of providing a sequence of interest to the genome of a plant cell, comprising contacting the plant cell with a Rhizobiales cell capable of transforming the plant cell, wherein the Rhizobiales cell comprises at least one vector comprising a right border DNA sequence (RB) and a left border DNA sequence (LB) and wherein the vector comprises between the RB and LB: (i) a first sequence of interest in a sense orientation relative to the RB, (ii) a spacer, and (iii) a second sequence of interest in an anti-sense orientation relative to the RB, wherein the first sequence of interest and second sequence of interest are essentially complementary and after synthesis of the T-strand anneal to form a double-stranded DNA. 
     
     
         20 . The method of  claim 19 , wherein the first sequence of interest further comprises a first left homology arm DNA sequence and a first right homology arm DNA sequence, and the second sequence of interest further comprises a second left homology arm DNA sequence and a second right homology arm DNA sequence. 
     
     
         21 . The method of  claim 1 , wherein the Rhizobiales cell is selected from an  Agrobacterium  spp., a  Bradyrhizobium  spp., a  Mesorhizobium  spp., an  Ochrobactrum  spp., a  Phyllobacterium  spp., a  Rhizobium  spp., and a  Sinorhizobium  spp. 
     
     
         22 . (canceled) 
     
     
         23 . The method of  claim 1 , wherein the plant cell is selected from the group consisting of a corn cell, a soybean cell, a canola cell, a cotton cell, a wheat cell, or a sugarcane cell. 
     
     
         24 .- 45 . (canceled) 
     
     
         46 . A method of providing a sequence of interest to a plant genome, comprising contacting at least one plant cell on a co-culture medium for at least 2 days, with at least one Rhizobiales cell capable of transforming the plant cell, wherein the Rhizobiales cell comprises at least one vector capable of forming two essentially complementary T-strands, wherein,
 a. the at least one vector comprises a first right border (RB1) DNA sequence, a second right border DNA sequence (RB2), and at least one sequence of interest, and wherein the RB1 is positioned in the vector to initiate synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5′ to 3′ end of the first T-strand; and the RB2 is positioned in the vector to initiate synthesis of a second T-strand such that the sequence of interest is in the anti-sense orientation relative to the sequence of interest in the first T-strand, and wherein the sequence of interest in the two T-strands resulting from initiation at RB1 and RB2 are essentially complementary in at least a portion of the sequence of interest; or   b. the at least one vector disclosed herein comprises a RB1, a RB2, and a sequence of interest, and further comprises a first left border DNA sequence (LB1) and a second left border DNA sequence (LB2), and wherein the vector is configured such that the RB1 is paired with the LB1 which are positioned in the vector to initiate (RB1) and terminate (LB1) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5′ to 3′ end of the first T-strand; and the RB2 is paired with the LB2 which are positioned in the vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the sequence of interest is in an anti-sense orientation relative to the sequence of interest in the first T-strand, and wherein the sequence of interest in the two T-strands resulting from initiation at RB1 and RB2 are essentially complementary in at least a portion of the sequence of interest; or   c. the at least one vector comprises a first sequence of interest and a second sequence of interest, where the first sequence of interest is essentially identical to the second sequence of interest; and the vector configuration further comprises a RB1 with aLB1 which are positioned in the vector to initiate (RB1) and terminate (LB1) synthesis of a first T-strand such that the first sequence of interest is in the sense orientation from the 5′ to 3′ end of the first T-strand; and the vector further comprises a RB2 and a LB2 which are positioned in the vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the second sequence of interest is in an anti-sense orientation relative to the first sequence of interest in the first T-strand, and wherein the sequence of interest in the two T-strands resulting from initiation at RB1 and RB2 are essentially complementary in at least a portion of the first sequence of interest and the second sequence of interest; or   d. the Rhizobiales cell comprises at least a first vector and a second vector, wherein each vector comprises essentially identical sequences of interest, and wherein the first vector configuration comprises a RB1 and a LB1 which are positioned in the first vector to initiate (RB1) and terminate (LB1) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5′ to 3′ end of the first T-strand; and wherein the second vector configuration comprises a RB2 and a LB2 which are positioned in the second vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the sequence of interest is in an anti-sense orientation relative to the sequence of interest in the first T-strand, and wherein the sequence of interest in the two T-strands resulting from initiation at RB1 and RB2 are essentially complementary in at least a portion of the sequence of interest; or   e. the at least one vector comprises a first sequence of interest, a second sequence of interest different from the first sequence of interest, at least two RB DNA sequences, and one or more optional LB DNA sequences, wherein the first RB DNA sequence (RB1) and a first LB DNA sequence (LB1) are positioned in the vector to initiate (RB1) and terminate (LB1) synthesis of the first T-strand such that the first sequence of interest is in the sense orientation from the 5′ to 3′ end of the first T-strand; and the vector configuration further comprises a second RB DNA sequence (RB2) and a second LB DNA sequence (LB2) which are positioned in the vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the first sequence of interest is in an anti-sense orientation from the 5′ to 3′ end of the second T-strand, wherein the two T-strands resulting from initiation at RB1 and RB2 are essentially complementary to each other in at least a portion of the first sequence of interest, and wherein the vector configuration further comprises a third RB DNA sequence (RB3) and a third LB DNA sequence (LB3) which are positioned in the vector to initiate (RB3) and terminate (LB3) synthesis of a third T-strand such that the second sequence of interest is in the sense orientation from the 5′ to 3′ end of the third T-strand; and the vector configuration further comprises a fourth RB DNA sequence (RB4) and a fourth LB DNA sequence (LB4) which are positioned in the vector to initiate (RB4) and terminate (LB4) synthesis of a fourth T-strand such that the second sequence of interest is in an anti-sense orientation from the 5′ to 3′ end of the fourth T-strand, and the two T-strands resulting from initiation at RB3 and RB4 are essentially complementary to each other in at least a portion of the second sequence of interest; or   f. the at least one Rhizobiales cell comprises a first Rhizobiales cell and a second Rhizobiales cell, wherein each Rhizobiales cell contains at least one of two vectors, wherein each vector comprises an essentially identical sequence of interest, and where the first vector comprises a RB1, and wherein the RB1 is positioned in the vector to initiate synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5′ to 3′ end of the first T-strand; and the second vector comprises a RB2 which is positioned in the vector to initiate synthesis of a second T-strand such that the sequence of interest is in the anti-sense orientation relative to the sequence of interest in the first T-strand, and wherein the two T-strands resulting from initiation at RB1 and RB2 are essentially complementary in at least a portion of the sequence of interest; or   g. the at least one Rhizobiales cell comprises a first Rhizobiales cell and a second Rhizobiales cell, wherein each Rhizobiales cell contains at least one of two vectors, wherein each vector comprises an essentially identical sequence of interest, and where the first vector comprises a RB1 and a LB1 which are positioned in the first vector to initiate (RB1) and terminate (LB1) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5′ to 3′ end of the first T-strand; and the second vector comprises a RB2 and a LB2 which are positioned in the second vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the sequence of interest is in an anti-sense orientation from the 5′ to 3′ end of the second T-strand, and wherein the sequence of interest in the two T-strands resulting from initiation at RB1 and RB2 are essentially complementary to each other.   
     
     
         47 . The method of  claim 46 , wherein the contacting comprises co-culturing the plant cell with the Rhizobiales cell for at least 3 days. 
     
     
         48 . The method of  claim 46 , wherein the Rhizobiales cell is selected from the group consisting of an  Agrobacterium  spp. cell, a  Bradyrhizobium  spp. cell, a  Mesorhizobium  spp. cell, an  Ochrobactrum  spp. cell, a  Phyllobacterium  spp. cell, a  Rhizobium  spp. cell, and a  Sinorhizobium  spp. cell. 
     
     
         49 . (canceled) 
     
     
         50 . The method of  claim 46 , wherein the plant cell is selected from the group consisting of a corn immature embryo cell, a corn mature embryo cell, a corn seed cell, a soybean immature embryo cell, a soybean mature embryo cell, a soybean seed cell, a canola immature embryo cell, a canola mature embryo cell, a canola seed cell, a cotton immature embryo cell, a cotton mature embryo cell, a cotton seed cell, a wheat immature embryo cell, a wheat mature embryo cell, a wheat seed cell, a sugarcane immature embryo cell, a sugarcane mature embryo cell, and a sugarcane seed cell. 
     
     
         51 . (canceled)