Stem-regulated, plant defense promoter and uses thereof in tissue-specific expression in monocots

Abstract

The invention is directed to isolated promoters from stem-regulated, defense-inducible genes, such as JAS promoters. The promoters are useful in expression cassettes and expression vectors for the transformation of plants. Particularly, the invention provides transgenic plants of rice and sugarcane that have been modified such that expression of a heterologous coding sequence is directed by an JAS promoter and is limited to stem tissues or may be upregulated by the presence of a defense-inducing agent. The invention also discloses methods for producing the expression vectors and transgenic plants.

1 . A method of increasing formation of an expression product of an exogenous nucleic acid in a plant, the method comprising:
 contacting the plant with a defense-inducing agent,   wherein the plant comprises an expression cassette or expression vector comprising (i) the exogenous nucleic acid, (ii) a JAS promoter comprising SEQ ID No. 1 and operable to drive expression of the exogenous nucleic acid in the presence of the defense-inducing agent, and (iii) a 3′ termination sequence operably linked to the exogenous nucleic acid, and   wherein formation of the expression product of the exogenous nucleic acid is increased relative to formation of the expression product of the exogenous nucleic acid in the absence of the defense-inducing agent.   
     
     
         2 . A method according to  claim 1 , wherein the defense-inducible agent comprises methyl jasmonate, jasmonic acid, salicylic acid, or combinations thereof. 
     
     
         3 . A method according to  claim 1 , wherein the expression product alters carbon metabolism in a stem tissue of the plant when expressed. 
     
     
         4 . A method according to  claim 1 , wherein the expression product comprises an insecticide effective against at least one stem-boring insect. 
     
     
         5 . A method according to  claim 1 , wherein the expression product comprises an antimicrobial product. 
     
     
         6 . A method according to  claim 5 , wherein the antimicrobial product comprises chitinase or β-1,3-glucanase. 
     
     
         7 . A method according to  claim 1 , wherein the expression product comprises a defense elicitor peptide. 
     
     
         8 . A method according to  claim 1 , wherein the plant is a monocot. 
     
     
         9 . A method according to  claim 1 , wherein the plant is selected from the group consisting of sugarcane, a sugarcane hybrid, sorghum, a sorghum hybrid, rice, a rice hybrid, maize and a maize hybrid. 
     
     
         10 . A method according to  claim 1 , wherein the JAS promoter is further operable to drive stem-specific expression of the exogenous nucleic acid. 
     
     
         11 . A method according to  claim 10  further comprising at least partially purifying the expression product from the plant. 
     
     
         12 . A plant comprising an expression vector having:
 a JAS promoter;   an exogenous nucleic acid; and   a 3′ termination sequence,   wherein expression of the exogenous nucleic acid is stem-specific.   
     
     
         13 . A plant according to  claim 12 , wherein expression of the exogenous nucleic acid is upregulated by the presence of a defense-inducing agent. 
     
     
         14 . A plant according to  claim 12 , wherein the exogenous nucleic acid alters carbon metabolism in the plant cell when expressed or transcribed. 
     
     
         15 . A plant according to  claim 12 , wherein the exogenous nucleic acid encodes an insecticide effective against at least one stem-boring insect. 
     
     
         16 . A plant according to  claim 12 , wherein the plant is a monocot. 
     
     
         17 . A plant according to  claim 12 , wherein the plant is selected from the group consisting of: sugarcane, sorghum, rice, maize and hybrids thereof. 
     
     
         18 . A method of isolating a tissue-specific promoter in a polyploidy monocot, the method comprising:
 isolating total RNA from a first tissue on a polyploidy monocot;   preparing ds cDNA from the total RNA from the first tissue;   preparing a microarray of this cDNA;   preparing a first pool of cDNA probes from total RNA from the first tissue;   preparing a second pool of cDNA probes from total RNA from a second tissue;   probing the microarray with the first and second pools of microarray probes;   selecting cDNA that exhibits high levels of hybridization with the first pool of probes as compared to the second pool of probes, wherein this cDNA is tissue specific for the first tissue and not the second tissue.   
     
     
         19 . A method according to  claim 18 , further comprising:
 inserting the ds cDNA in multiple clones to form a cDNA library;   probing the cDNA library with at least one probe specific to the first tissue and at least one probe specific to the second tissue;   picking clones that exhibit higher levels of hybridization to the probe specific to the first tissue than to the probe specific to the second tissue; and   preparing a microarray of cDNA from the picked clones.   
     
     
         20 . A method according to  claim 18 , further comprising:
 screening a genomic library to identify at least one gene corresponding with at least one selected cDNA; and   isolating promoter regions of this gene.   
     
     
         21 . An isolated nucleic acid comprising a promoter having a sequence at least 98% identical to SEQ ID No. 1, wherein the promoter has stem-specific promoter activity or defense-inducible promoter activity. 
     
     
         22 . An isolated nucleic acid according to  claim 21  further comprising an exogenous nucleic acid, wherein the promoter is operable to drive stem-specific or defense-inducible expression or transcription of the exogenous nucleic acid. 
     
     
         23 . An isolated nucleic acid according to  claim 22 , wherein the exogenous nucleic acid comprises a transgene. 
     
     
         24 . An isolated nucleic acid according to  claim 22 , wherein the exogenous nucleic acid alters carbon metabolism in the plant cell when expressed or transcribed. 
     
     
         25 . An isolated nucleic acid according to  claim 22 , wherein the exogenous nucleic acid encodes an insecticide effective against at least one stem-boring insect. 
     
     
         26 . An isolated nucleic acid according to  claim 22 , wherein the promoter is operable to drive stem-specific expression or transcription of the exogenous nucleic acid. 
     
     
         27 . An isolated nucleic acid according to  claim 22 , wherein the promoter is operable to drive defense-inducible expression or transcription of the exogenous nucleic acid. 
     
     
         28 . An isolated nucleic acid according to  claim 21 , wherein the isolated nucleic acid is located in a plant cell. 
     
     
         29 . An isolated nucleic acid according to  claim 28 , wherein the plant cell is located in a monocot plant. 
     
     
         30 . An isolated nucleic acid according to  claim 29 , wherein the plant is selected from the group consisting of: sugarcane, a sugarcane hybrid, sorghum, a sorghum hybrid, rice, a rice hybrid, maize and a maize hybrid. 
     
     
         31 . A method for stem-specific expression of an exogenous nucleic acid in a plant, the method comprising:
 introducing an expression cassette or expression vector into the plant,   wherein the expression cassette or expression vector comprises (i) the exogenous nucleic acid, (ii) a JAS promoter comprising SEQ ID No. 1 and operable to drive expression of the exogenous nucleic acid, and (iii) a 3′ termination sequence operably linked to the exogenous nucleic acid, and
 wherein expression of the exogenous nucleic acid is stem-specific.