miRNA396 AND GROWTH REGULATING FACTORS FOR CYST NEMATODE TOLERANCE IN PLANTS

Abstract

The present invention presents methods to alter the genetic composition of crop plants susceptible to nematode infection to improve tolerance to the same. Methods and compositions for modulating key pathways involved in the syncytial event of nematode infection and for preventing the cascade of differential gene expression caused by the same as disclosed. Applicants have found that the microRNA miR396 acts as a master switch of syncytial gene expression changes in plants after infection, and further that miR396 and certain growth regulating transcription factors (GRF) are connected through feedback interaction in syncytium initiation and maintenance.

1 . A method for improving nematode tolerance in plants comprising:
 a) introducing into a plant cell a polynucleotide sequence encoding a plant miRNA396 and/or GRF protein with a miRNA396 binding site operably linked to a promoter functional in said plant cell;   b) regenerating a genetically modified plant from the plant cell; whereby a genetically modified plant demonstrates improved tolerance to nematode infection compared to a plant without such modification.   
     
     
         2 . The method of  claim 1 , wherein said plant miRNA396 is selected from the group consisting of: miRNA396a, miRNA396b, miRNA396c, and miRNA396e. 
     
     
         3 . The method of  claim 1 , wherein said plant GRF protein is selected from the group consisting of: GRF 1, GRF2, GRF3, GRF 4, GRF 7, GRF8, GRF9, GRF13, GRF15, and GRF16. 
     
     
         4 . The method of  claim 1 , wherein the plant cell is from a plant selected from the group consisting of: Potato, tomato,  Phaseolus  spp., sugarbeet, peas,  Vicia faba , sugar cane, eggplant, peppers, tobacco, wheat, rice, sorghum, barley, oat, lawn grass, rye, soybean, canola,  Brassica , sunflower, maize, sorghum, alfalfa, cotton, millet, peanut and cacao. 
     
     
         5 . The method of  claim 1  wherein said miRNA396 is from  Arabidopsis.    
     
     
         6 . The method of  claim 1  wherein said GRF is from  Arabidopsis.    
     
     
         7 . The method of  claim 1  wherein said miRNA396 is from  Glycine max.    
     
     
         8 . The method of  claim 1  wherein said GRF is from  Glycine max.    
     
     
         9 . A method of improving nematode tolerance in a plant comprising:
 modulating the miRNA396-GRF interaction in said plant.   
     
     
         10 . The method of  claim 6  further comprising introducing a plant miRNA396 nucleic acid into the plant. 
     
     
         11 . The method of  claim 6  further comprising increasing the expression of one or more plant GRF nucleic acids in the plant. 
     
     
         12 . A method for making a genetically modified plant with improved nematode tolerance comprising:
 a) transforming a cell with an expression cassette comprising an isolated nucleic acid which encodes a miRNA396 operably linked to a promoter sequence functional in a plant cell   b) regenerating a genetically modified plant from the cell; and   c) selecting for a genetically modified plant with improved nematode tolerance when compared to a non-modified plant.   
     
     
         13 . A plant made by the method of  claim 9 . 
     
     
         14 . A method for making a genetically modified plant with improved nematode tolerance comprising:
 a) transforming a cell with an expression cassette comprising an isolated nucleic acid which encodes a GRF protein with a miRNA396 binding site operably linked to a promoter sequence functional in a plant cell   b) regenerating a genetically modified plant from the cell; and   c) selecting for a genetically modified plant with improved nematode tolerance when compared to a non-modified plant.   
     
     
         15 . A plant made by the method of  claim 14 . 
     
     
         16 . A genetically modified plant, with improved tolerance to nematode infection compared to a plant without such modification, said plant comprising:
 a heterologous nucleotide sequence encoding a plant miRNA396 and/or a GRF protein with a miRNA396 binding site, operably linked to a promoter functional in a said plant.   
     
     
         17 . Seed of the plant of  claim 16 . 
     
     
         18 . A method for making a genetically modified plant with improved nematode tolerance comprising:
 a) transforming a cell with an expression cassette comprising an isolated nucleic acid which encodes a GRF protein with a miRNA396 binding site (SEQ ID NO:28) or an miRNA396 (SEQ ID NO:27) operably linked to a promoter sequence functional in a plant cell, and   regenerating a genetically modified plant from the plant cell; whereby a genetically modified plant demonstrates improved tolerance to nematode infection compared to a plant without such modification.   
     
     
         19 . A method for conferring or improving nematode resistance of a plant, said method comprising:
 stably introducing into the genome of a plant, at least one nucleotide construct comprising an miRNA396 and/or GRF nucleic acid molecule operably linked to a promoter that drives expression in a plant cell, wherein said nucleic acid molecule encodes a product that modulates the miRNA396-GRF interaction upon nematode infection.   
     
     
         20 . A method of breeding for improved nematode tolerance in plants comprising:
 measuring the amount or activity of an miRNA396 or GRF protein or nucleic acid present in one or more plant cells, and   selecting a plant with higher levels of activity or amounts of miRNA396 or GRF for breeding.   
     
     
         21 . A method of identifying a marker for improved nematode tolerance in plants comprising:
 screening an endogenous miRNA396 or GRF nucleotide sequence for a polymorphism that is associated with higher levels of expression or activity and   selecting plants for breeding based upon the presence of absence of said polymorphism.