Endoglucanase gene promoter upregulated by nematodes

Abstract

The present invention provides a nucleic acid construct comprising a cyst and root knot nematode responsive promoter, preferably the Nicotiana Ntcel7 promoter or promoters that hybridize thereto, operatively associated with a heterologous nucleic acid segment that encodes a product disruptive of nematode attack. Plants and plant cells using the same and methods of use thereof are also disclosed.

1 . A nucleic acid construct which comprises, in the 5′ to 3′ direction: 
 (a) a cyst and root knot nematode responsive promoter selected from the group consisting of: (i) nucleic acid encoding the  Nicotiana  Ntcel7 promoter according to SEQ ID NO:9, and (ii) nucleic acid sequences which hybridize to nucleic acid having a sequence of (i) above or the complement thereof under conditions represented by a wash stringency of 0.3M NaCl, 0.03M sodium citrate, 0.1% SDS at 60° C., and which encodes a cyst and/or root knot nematode responsive promoter; and    (b) a heterologous nucleic acid positioned downstream from said cyst and root knot nematode promoter and operatively associated therewith, said heterologous nucleic acid encoding a product disruptive of nematode attack.    
     
     
         2 . A nucleic acid construct according to  claim 1 , wherein said promoter is the  Nicotiana  Ntcel7 promoter of SEQ ID NO: 9.  
     
     
         3 . A nucleic acid construct according to  claim 1 , wherein said heterologous nucleic acid segment is a gene coding for an insecticidal protein.  
     
     
         4 . A nucleic acid construct according to  claim 4 , wherein said heterologous nucleic acid segment is a gene coding for a  Bacillus thuringiensis  crystal protein toxic to insects.  
     
     
         5 . A nucleic acid construct according to  claim 1 , wherein said heterologous nucleic acid segment is a gene coding for a product toxic to said plant cell.  
     
     
         6 . A nucleic acid construct according to  claim 1 , wherein said construct comprises a plasmid.  
     
     
         7 . A plant cell transformed with a nucleic acid construct according to  claim 1 .  
     
     
         8 . A method of making a transformed plant, comprising regenerating a plant from a plant cell according to  claim 7 .  
     
     
         9 . An  Agrobacterium tumefaciens  cell containing a nucleic acid construct according to  claim 1 , and wherein said nucleic acid construct comprises a Ti plasmid.  
     
     
         10 . A method of making a transformed plant, comprising infecting a plant cell with an  Agrobacterium tumefaciens  according to  claim 9  to produce a transformed plant cell, and then regenerating a plant from said transformed plant cell.  
     
     
         11 . A microparticle carrying a nucleic acid construct according to  claim 1 , wherein said microparticle is suitable for the ballistic transformation of a plant cell.  
     
     
         12 . A method of making a transformed plant, comprising propelling a microparticle according to  claim 11  into a plant cell to produce a transformed plant cell, and then regenerating a plant from said transformed plant cell.  
     
     
         13 . A plant cell protoplast comprising a nucleic acid construct according to  claim 1 .  
     
     
         14 . A method of making a transformed plant, comprising regenerating a plant from a plant cell protoplast according to  claim 13 .  
     
     
         15 . A transformed plant comprising transformed plant cells, said transformed plant cells containing a heterologous nucleic acid construct according to  claim 1 .  
     
     
         16 . A transformed plant according to  claim 15 , wherein said plant is a dicot.  
     
     
         17 . A transformed plant according to  claim 15 , wherein said plant is a monocot.  
     
     
         18 . A transformed plant according to  claim 15 , wherein said plant is a tobacco ( Nicotiana tabacum ) plant.  
     
     
         19 . Plant seed that germinates into a plant of  claim 15 .  
     
     
         20 . A method of controlling cyst and root knot nematodes, comprising the steps of: 
 (a) providing a cyst and root knot nematode-responsive promoter selected from the group consisting of: (i) nucleic acid encoding the  Nicotiana  Ntcel7 promoter of SEQ ID NO:9, and (ii) nucleic acid sequences which hybridize to nucleic acid having a sequence of (i) above, or the complement thereof, under conditions represented by a wash stringency of 0.3M NaCl, 0.03M sodium citrate, 0.1% SDS at 60° C., and which encodes a cyst and root knot nematode-responsive promoter;    (b) preparing a construct by combining said promoter with a further heterologous region which codes for a product disruptive of nematode attack, and    (c) transforming plants with the construct to obtain plants which are cyst and root knot nematode resistant.    
     
     
         21 . A method according to  claim 20 , wherein said plants are monocots.  
     
     
         22 . A method according to  claim 20 , wherein said plants are dicots.  
     
     
         23 . A plant produced by the method of  claim 20 .  
     
     
         24 . An isolated nucleic acid comprising a cyst and root knot nematode responsive promoter selected from the group consisting of: 
 (i) nucleic acid encoding the  Nicotiana  Ntcel7 promoter according to SEQ ID NO:9, and    (ii) nucleic acids which hybridize to nucleic acids having a sequence of (i) above, or the complement thereof, under conditions represented by a wash stringency of 0.3M NaCl, 0.03M sodium citrate, 0.1% SDS at 60° C., and which encodes a cyst and/or root knot nematode responsive promoter.    
     
     
         25 . An isolated nucleic acid according to  claim 24  comprising the  Nicotiana  Ntcel7 promoter of SEQ ID NO: 9.