Patent Abstract:
The present invention relates to a method of controlling spider mites on plants. More specifically, the invention relates to plants expressing RNAi of one or more essential genes of the spider mite, and the use of those plants to control the spider mite proliferation into pest proportions. In a preferred embodiment, the spider mite is  Tetranychus urticae.

Full Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This is a national phase entry under 35 U.S.C. §371 of international Patent Application PCT/EP2010/065311 filed on Oct. 13, 2010, published in English as International Patent Publication No. WO 2011/045333, which claims the benefit under Article 8 of the Patent Cooperation Treaty to European Patent Application Serial No. 09173040.8, filed Oct. 14, 2009. 
     
    
     TECHNICAL FIELD 
       [0002]    The present invention relates to a method to control spider mites on plants. More specifically, the invention relates to plants, expressing RNAi of one or more essential genes of the spider mite, and the use of those plants to control the spider mite proliferation into pest proportions. In a preferred embodiment, the spider mite is  Tetranychus urticae.    
       BACKGROUND 
       [0003]    Spider mites are arthropods, belonging to the subphylum of chelicerates (scorpions, horseshoe crabs, spiders, mites and ticks). The mites include different species that can be parasitic on vertebrate and invertebrate hosts, predators, or plant feeding. Within the mites, the spider mites group the web-spinning species that feed on plants. 
         [0004]    Spider mites, and particularly  T. urticae  (two-spotted spider mite) is one of the major pests in agriculture. It is extremely polyphagous and feed on over 1000 plant species. Moreover, it shows a rapid development (generation time of seven days in a hot season).  T. urticae  represent a key pest for greenhouse crops, annual field crops and many horticultural crops, such as peppers, tomatoes, potatoes, beans, corn, strawberries and roses. It is widespread all over the world, and occurs freely in nature in regions with a warm and dry climate. 
         [0005]    Spider mites cause yellow flecks on the leaf surface, and upon heavy infestation, leaves become pale, brittle and covered in webbing. This damage can cause severe reduction in yield. 
         [0006]    Spider mites are particularly important pests for vegetables. Spider mites cause significant damage to greenhouse tomato, cucumber and pepper crops. 
         [0007]    Given the short generation time and high reproduction rate of spider mites, it is expected that spider mites, with the climate change, will become one of the major pests for crops as well. Devastating effects of spider mites are already creating enormous problems for the agricultural production in Southern Europe. 
         [0008]    Spider mite control, currently, is mainly done by specific miticides, as normal insecticides have normally little effect on mites. Miticides have been disclosed, amongst others, in WO03014048 and in WO2007000098. However, miticides are polluting chemicals, and the application may not always be efficient, as spider mites are often protected by a web under the leaves. 
         [0009]    Recently, the RNA interference (RNAi) technology was developed as an attractive alternative in the control of insect pests (Gordon and Waterhouse, 2007; Baum et al., 2007; Mao et al., 2007). RNAi is based on sequence-specific gene silencing that is triggered by the presence of double-stranded RNA (dsRNA). RNAi can be used in plants, animals and insects, but the mechanism depends upon endogenous enzymes present and the efficacy depends upon the host organism used (Gordon and Waterhouse, 2007). Khila and Grbic (2007) demonstrated that dsRNA and short interfering RNA (siRNA) can be used for gene silencing in  T. urticae , by using a maternal injection protocol to deliver interfering RNAs into the maternal abdomen. This methodology has been used to silence Distal-less, a conserved gene involved in appendage specification in metazoans. 
         [0010]    However, gene silencing has never been used in pest control for spider mites. One reason is the uncertainty whether RNAi, supplied in the food, would be functional. Another reason is the lack of sequence data of spider mites, making a selection of mite-specific genes that are lethal when knocked out by RNAi impossible. 
       DISCLOSURE 
       [0011]    We sequenced and annotated the genome of  T. urticae . This effort allowed us to pinpoint a set of essential mite-specific genes without relevant plant or mammalian orthologs. From these sequences, RNAi loops were designed that were specific for one essential mite gene, without interfering with the expression in plants or in mammals. Surprisingly, we found that expressing RNAi in a plant derived from those genes, is sufficient to interfere with the spider mite&#39;s development and physiology that is feeding on this plant, resulting in death as a consequence. 
         [0012]    A first aspect of the invention is a transgenic plant expressing RNAi derived from a spider mite. Preferably, RNAi is derived from an essential gene of the spider mite. Even more preferably, the RNAi is derived from a gene-specific region (GSR) of the essential genes. A “transgenic plant” can be any plant that is, as wild-type, sensitive to spider mite infection, including, but not limited to, members of the citrus family (lemon, oranges, . . . ), grapefruit, different varieties of  Vitis , corn, as well as Solanaceae like tomatoes, cucumber, . . . and ornamental flowers. “Derived” as used here, means that the gene region that is transcribed (including the non-coding regions) is used to design the RNAi; preferably, the RNAi comprises an antisense fragment of the transcribed region. Even more preferably, it consists of an antisense region of the transcribed region. The RNAi comprises only a part of the transcribed mRNA. A “GSR” is a gene region without homology with other mite genes and without homology with the host genome, as determined according to Example 1. A GSR allows the design of RNAi that is specific for the target gene, without interfering with other mite genes or with plant or mammalian genes. An “essential gene” as used here means that the inactivation of the gene is blocking growth and/or development of the mite and may result in the death of the mite. Preferably, the essential gene is selected from the group consisting of GABA receptor gene, stem cell gene, neutralized gene, HOX gene, DEV gene, Cytochrome C gene, Hedgehog gene, NADH dehydrogenase gene, Ryanoid receptor gene, sodium channel gene, acetylcholine esterase gene, son of sevenless gene, prospero gene, acetyl choline receptor gene and distal-less gene (Dll). Preferably, the spider mite is  T. urticae . In one preferred embodiment, the RNAi is derived from the  T. urticae  distal-less gene (RNAi indicated as Tetur17g02200-SEQ ID NO:86); preferably, it is comprising the sequence between the primers as shown in  FIG. 1 . In another preferred embodiment, the RNAi is comprising a sequence selected from the group consisting of SEQ ID NOS:1-87. Even more preferred, the RNAi is comprising a sequence; even more preferably, consisting of a sequence selected from the group consisting of SEQ ID NOS:1, 2, 4, 6, 9, 14, 18, 20, 21, 22, 24, 33, 34, 35, 36, 37, 38, 39, 46, 49, 50, 63, 75, 86 and 87. Most preferably, the RNAi is comprising a sequence; even more preferably, consisting of a sequence selected from the group consisting of SEQ ID NOS:2, 18, 22, 75 and 86. 
         [0013]    Although, preferably, the inactivation of the mites is obtained by expressing a single RNAi species, it is clear for the person skilled in the art that the same effect may be obtained by expressing more than one RNAi species, in order to obtain a stronger inhibition. 
         [0014]    Another aspect of the invention is a method to improve mite resistance in plants, comprising the expression of RNAi derived from spider mite. Preferably; the RNAi is derived from an essential gene from spider mite; even more preferably, the RNAi is derived from a gene-specific region (GSR) of the essential gene. Preferably, the essential gene is selected from the group consisting of GABA receptor gene, stem cell gene, neutralized gene, HOX gene, DEV gene, Cytochrome C gene, Hedgehog gene, NADH dehydrogenase gene, Ryanoid receptor gene, sodium channel gene, acetylcholine esterase gene, son of sevenless gene, prospero gene, acetyl choline receptor and distal-less gene (Dll). Preferably, the spider mite is  T. urticae . In one preferred embodiment, the RNAi is derived from the  T. urticae  distal-less gene; preferably it is comprising the sequence between the primers as shown in  FIG. 1 . In another preferred embodiment, the RNAi is derived from a sequence comprising a sequence selected from the group consisting of SEQ ID NOS:1-87. Even more preferred, the RNAi is comprising a sequence; even more preferably, consisting of a sequence selected from the group consisting of SEQ ID NOS:1, 2, 4, 6, 9, 14, 18, 20, 21, 22, 24, 33, 34, 35, 36, 37, 38, 39, 46, 49, 50, 63, 75, 86 and 87. Most preferably, the RNAi is comprising a sequence; even more preferably, consisting of a sequence selected from the group consisting of SEQ ID NOS:2, 18, 22, 75 and 86. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0015]      FIG. 1 : Sequence of the  Tetranychus urticae  distal-less gene (Dll) (SEQ ID NO:264) and the primers used (TuDII_ARBF and TuDII_ARBR) (SEQ ID NO:265 and 266, respectively). The primer regions in the distal-less sequence are underlined. The fragment in between the primers is used in the RNAi construct. The amino acid sequence is identified as SEQ ID NO:268. 
           [0016]      FIG. 2 : Construct used to express TuDll-RNAi transgene in  Arabidopsis.    
           [0017]      FIG. 3 :  Arabidopsis  plants expressing dsRNA against Tu-D11 suppress mite development. A) Northern blot analysis showing siRNAs against TuDll spider mite gene; Col is a control, not expressing the transgene. B) Effect of plant-produced TuDll-RNAi (Lines 1-5) on spider mite development. Note that number of eggs deposited on transgenic plants is lower than in the Col control. Also, the number of eggs correlates with the amount of TuDll-RNAi expressed. 
           [0018]      FIG. 4 : Plasmid map of pB-AGRIKOLA-Tetur17g02200. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Examples 
     Example 1 
     Growth Inhibition of  T. urticae  by Feeding on TuDll-RNAi Transgenic  Arabidopsis    
       [0019]    The  T. urticae  ortholog of the drosophila Dll distal-less gene was identified in the genomic sequence, using the motifs of the distal-less family (Fonseca et al., 2009). Distal-less is a transcription factor that plays an important role in neuronal development (Cobos et al., 2005). An RNAi fragment is designed on the base of its specificity (no significant homology with other  T. urticae  genes, neither with the  Arabidopsis  genome). The RNAi fragment, as well as the primers used to isolate it, is shown in  FIG. 1 . The fragment was amplified, and cloned under control of the CaMV 35S promoter, to result in the Ti-based plasmid pFGC5941 ( FIG. 2 ). The plasmid was transformed using the  Agrobacterium -mediated transformation into  Arabidopsis thaliana  (Col). The expression of the RNAi in different transformed lines was tested by Northern blot ( FIG. 3 , Panel A). Spider mites were allowed to feed on five transformed lines and a control plant. All transformed plants showed an inhibition of mite development, both of the moving stages and the number of eggs on the plant. A correlation between the expression level of RNAi and the number of eggs on the transgenic plants was found ( FIG. 3 , Panel B), proving that the expression in plants of RNAi of an essential spider mite gene is indeed an efficient way to control the pest. 
       Example 2 
     RNAi Design for Other Essential Genes 
       [0020]    From a list of candidate  Tetranychus urticae  target genes, coding sequences (CDS, from start-to-stop codons) were collected from the available predicted genes. For each of those genes, overlapping 21mer sequences were designed covering the whole CDS sequences. This was done by extracting, starting from the first nucleotide of the CDS, sub-sequences of 21 nt, with a sliding window, with steps of one nt. For each CDS from the target genes, n−20 oligos of 21 nt were designed, whereby n is the length of the CDS. 
         [0021]    Each of these 21mers was blasted (using BLASTN) against the whole  Tetranychus urticae  genome. In the case of a perfect match, an e-value of 1e−4 is obtained. To allow some mismatch the threshold was set at 0.01. The threshold was lowered to ensure that no 21mer would hit another region on the genome with a small sequence difference of 1 or 2 nt, thereby ensuring the gene specificity for the RNAi. 
         [0022]    Gene-specific regions (GSR), ideally being between 150 and 500 nt, were identified as regions for which, over the whole region, none of the consecutive 21mers derived from this region gave a hit with another sequence from the  T. urticae  (using the threshold as described above). 
         [0023]    The GSR that did meet the above conditions were subsequently blasted (BLASTN, same thresholds) against the  Arabidopsis  genome.  Arabidopsis  was chosen, as it is used as host in the proof of principle experiments. This step is to make sure that no  Arabidopsis  genes could be targeted by the RNAi constructs introduced and that might thus affect  Arabidopsis  directly; GSR can be blasted against other genomes for optimizing the RNAi in other plant hosts. 
         [0024]    All GSR that fulfilled the above criteria (SEQ ID NOS:1-85) were then used as input for primer design. The primers where designed using the OSP perl package, and as a parameter, the melting temperature was set at the 55° C. to 65° C. range in a first run (Table 1). Those targeted GSR that did not succeed in obtaining a primer pair were submitted again to the same design procedure, with slightly more relaxed primer lengths allowed (Table 2). If, with those conditions, still no primers could be designed, melting temperature range was relaxed (50° C. to 70° C.) for a third attempt (Table 3). 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 primers designed after 1 run 
               
             
          
           
               
                 SEQ_ID 
                 5_PRIMER 
                 3_PRIMER 
               
               
                   
               
               
                    0_197_ 
                 ATAAAATCTCCAAGCATAGTACGAGTT (SEQ ID NO: 88) 
                 TTAACCACAGTCACTCGACCTTCA (SEQ ID NO: 89) 
               
               
                 Tetur41g00290 
                   
                   
               
               
                   
               
               
                    0_228_ 
                 No Primers could be designed with these 
                   
               
               
                 Tetur30g02230 
                 criteria 
                   
               
               
                   
               
               
                 1066_1216_ 
                 TGATTGAATTCACTTTTTCGCACAT (SEQ ID NO: 90) 
                 AAATAACTGAATCTGGCCAAGTTATTA (SEQ ID NO: 91) 
               
               
                 Tetur01g13610 
                   
                   
               
               
                   
               
               
                 1126_1276_ 
                 No Primers could be designed with these 
                   
               
               
                 Tetur19g01440 
                 criteria 
                   
               
               
                   
               
               
                  114_520_ 
                 CTAAAAATCTAATTGCAGTGGTAG (SEQ ID NO: 92) 
                 CGTTTATCTGGCAATGGAG (SEQ ID NO: 93) 
               
               
                 Tetur01g13610 
                   
                   
               
               
                   
               
               
                 1173_1324_ 
                 AATGTTTTCTTTGTGCAAGTTTCTTATC (SEQ ID NO: 94) 
                 GCTGGAAGAGTAAAATGTTTAGGT (SEQ ID NO: 95 
               
               
                 Tetur01g21600 
                   
                   
               
               
                   
               
               
                 1186_1376_ 
                 ACCTGAGAATCTTTGAGACC (SEQ ID NO: 96) 
                 ATCCTCATCACAACAACCTGAC (SEQ ID NO: 97) 
               
               
                 Tetur14g00120 
                   
                   
               
               
                   
               
               
                 1204_1399_ 
                 TAACCTCTTGATCCAGTAAAGCTTCAAT (SEQ ID NO: 98) 
                 GTTTATTAGCTGGTCGTTATGCAC (SEQ ID NO: 99) 
               
               
                 Tetur09g01840 
                   
                   
               
               
                   
               
               
                 1224_1532_ 
                 CAAGGAGGTTTCATCAGGATA (SEQ ID NO: 100) 
                 ATGAACATAATTAAAACCTGGTCTTTCG (SEQ ID NO: 101) 
               
               
                 Tetur31g00990 
                   
                   
               
               
                   
               
               
                 1236_1391_ 
                 No Primers could be designed with these 
                   
               
               
                 Tetur20g01760 
                 criteria 
                   
               
               
                   
               
               
                 1266_1490_ 
                 CTGTCGATTGAACCCTGCAT (SEQ ID NO: 102) 
                 TGTGAACATTGTTCCCATCAACAT (SEQ ID NO: 103) 
               
               
                 Tetur16g00420 
                   
                   
               
               
                   
               
               
                 1326_1516_ 
                 No Primers could be designed with these 
                   
               
               
                 Teturl9g01440 
                 criteria 
                   
               
               
                   
               
               
                 1506_1673_ 
                 TAAGCATAATAAGTTCTGATAACATCC (SEQ ID NO: 104) 
                 TCTTTGAATGTTGAGTCGGAATG (SEQ ID NO: 105) 
               
               
                 Tetur01g13610 
                   
                   
               
               
                   
               
               
                 1564_1794_ 
                 No Primers could be designed with these 
                   
               
               
                 Tetur20g01760 
                 criteria 
                   
               
               
                   
               
               
                  161_321_ 
                 CACAAACATAACTTGGCCTAAATCT (SEQ ID NO: 106) 
                 AAGATCATCGTTTAATGGTAATGTTGT (SEQ ID NO: 107) 
               
               
                 Tetur02g06230 
                   
                   
               
               
                   
               
               
                  173_391_ 
                 CCACTGTTGGTGTAAGTTGTGAAT (SEQ ID NO: 108) 
                 TTCAATCACTTGTCGATATGAGC (SEQ ID NO: 109) 
               
               
                 Tetur01g12090 
                   
                   
               
               
                   
               
               
                 1761_1957_ 
                 TGGATTGTTGATGGTTAGACTC (SEQ ID NO: 110) 
                 GCTGCTGCGGCTGCAACT (SEQ ID NO: 111) 
               
               
                 Tetur01g13860 
                   
                   
               
               
                   
               
               
                 1812_1966_ 
                 No Primers could be designed with these 
                   
               
               
                 Tetur06g02480 
                 criteria 
                   
               
               
                   
               
               
                 1821_1979_ 
                 TGATTGGCAACAATTACTCGATAT (SEQ ID NO: 112) 
                 TTTAATGTTGCTAAAAGTGGGCCCAAC (SEQ ID NO: 113) 
               
               
                 Tetur20g01760 
                   
                   
               
               
                   
               
               
                  185_411_ 
                 TGGGCTACTGATACCGAGTT (SEQ ID NO: 114) 
                 GCCTGACATAGATGGATGGGA (SEQ ID NO: 115) 
               
               
                 Tetur05g05120 
                   
                   
               
               
                   
               
               
                  200_356_ 
                 TGAGATGAGTATTTACAGGGG (SEQ ID NO: 116) 
                 TTACGTTCTTCCTCCTATTCTTCA (SEQ ID NO: 117) 
               
               
                 Tetur01g12340 
                   
                   
               
               
                   
               
               
                 2025_2185_ 
                 AATTATTGTTGTCACTAATTTCGTGTAC (SEQ ID NO: 
                 CACCATCATCAAAAAGTAAATGATTCC (SEQ ID NO: 119) 
               
               
                 Tetur23g02710 
                 118) 
                   
               
               
                   
               
               
                  210_397_ 
                 ATGGTAACCAAGTTTCAGCTAGA (SEQ ID NO: 120) 
                 CAAATCAGGTTAGCTCATACAGACA (SEQ ID NO: 121) 
               
               
                 Tetur12g05390 
                   
                   
               
               
                   
               
               
                 2129_2321_ 
                 No Primers could be designed with these 
                   
               
               
                 Tetur20g01760 
                 criteria 
                   
               
               
                   
               
               
                  226_459_ 
                 AACATAACCATAAACATCACCACC (SEQ ID NO: 122) 
                 GTGTAACTGTTGGTGATCCAGTTC (SEQ ID NO: 123) 
               
               
                 Tetur01g21600 
                   
                   
               
               
                   
               
               
                 2296_2467_ 
                 No Primers could be designed with these 
                   
               
               
                 Tetur01g13860 
                 criteria 
                   
               
               
                   
               
               
                  232_580_ 
                 CAACAAATCCATATTCAGTCAAGA (SEQ ID NO: 124) 
                 TTCAGAAGATTCAAGTTACTCATGTC (SEQ ID NO: 125) 
               
               
                 Tetur13g05360 
                   
                   
               
               
                   
               
               
                 2353_2823_ 
                 CCTGATTTTTAGTAAGCCCATAAATCC (SEQ ID NO: 126) 
                 CATTTTATAATTATTTGACTGCCTGGGT (SEQ ID NO: 127) 
               
               
                 Tetur06g02480 
                   
                   
               
               
                   
               
               
                 2371_2583_ 
                 GATAAATTTGTCCCAATAACATTCGTAA (SEQ ID NO: 
                 AATATGAAGATGATTCATCATACTCTG (SEQ ID NO: 129) 
               
               
                 Tetur23g02710 
                 128) 
                   
               
               
                   
               
               
                 2380_2694_ 
                 ATAAGCAGGAGGAGGTTGA (SEQ ID NO: 130) 
                 TTAAACGAAAAAGAAGTCGAACTGG (SEQ ID NO: 131) 
               
               
                 Tetur16g00420 
                   
                   
               
               
                   
               
               
                  409_2604_ 
                 CAGTTCAAAGTCACAATTCTCTTTACC (SEQ ID NO: 132) 
                 CAACTACTTGAATCGTTAAGAATTTTCC (SEQ ID NO: 133) 
               
               
                 Tetur19g01440 
                   
                   
               
               
                   
               
               
                  246_442_ 
                 No Primers could be designed with these 
                   
               
               
                 Tetur01g08220 
                 criteria 
                   
               
               
                   
               
               
                 2581_2750_ 
                 No Primers could be designed with these 
                   
               
               
                 Tetur01g13860 
                 criteria 
                   
               
               
                   
               
               
                 2582_2766_ 
                 No Primers could be designed with these 
                   
               
               
                 Tetur20g01760 
                 criteria 
                   
               
               
                   
               
               
                  259_421_ 
                 No Primers could be designed with these 
                   
               
               
                 Tetur07g08130 
                 criteria 
                   
               
               
                   
               
               
                 2651_2803_ 
                 CAACGATTTCTCTCTCCAACCA (SEQ ID NO: 134) 
                 TGCCAGGCAATTGACTTTGTACGA (SEQ ID NO: 135) 
               
               
                 Tetur19g01440 
                   
                   
               
               
                   
               
               
                 2685_2839_ 
                 TGTTTGACTGCCGATGAGA (SEQ ID NO: 136) 
                 TTGTTGAATGAAGAAGACGACCTTT (SEQ ID NO: 137) 
               
               
                 Tetur19g0154 
                   
                   
               
               
                   
               
               
                 2753_2877_ 
                 ATGAATGCTTTTGCCAACGG (SEQ ID NO: 138) 
                 GTTAATATTTGTTCTAGCTCTAACTAG (SEQ ID NO: 139) 
               
               
                 Tetur06g02480 
                   
                   
               
               
                   
               
               
                 2809_2985_ 
                 AATCAATTTTTTATGCTTAGGATGGAG (SEQ ID NO: 140) 
                 GAGAAATCGTTGAAACGGTCAACTT (SEQ ID NO: 141) 
               
               
                 Tetur19g01440 
                   
                   
               
               
                   
               
               
                  281_523_ 
                 TAATGGGCAAAGGAATGGGCGA (SEQ ID NO: 142) 
                 CTTTTCAATCTTTTTGTATATACGACTC (SEQ ID NO: 143) 
               
               
                 Tetur16g02700 
                   
                   
               
               
                   
               
               
                 3048_3213_ 
                 TGAAACTAAATTATGATGGTGTCGCTT (SEQ ID NO: 144) 
                 TACATTTTTTCTGGAGCGGTTG (SEQ ID NO: 145) 
               
               
                 Tetur06g02480 
                   
                   
               
               
                   
               
               
                 3059_3244_ 
                 CAAGAGAAGCTTTTCTAACAACTA (SEQ ID NO: 146) 
                 GGTACTCATCTCTGCTCACCAA (SEQ ID NO: 147) 
               
               
                 Tetur20g01760 
                   
                   
               
               
                   
               
               
                  305_460_ 
                 TTGAACCCAATCCATCTGAATTG (SEQ ID NO: 148) 
                 TGGAGTGGCCTTAATTGGAGT (SEQ ID NO: 149) 
               
               
                 Tetur16g00420 
                   
                   
               
               
                   
               
               
                 3221_3403_ 
                 No Primers could be designed with these 
                   
               
               
                 Tetur06g02480 
                 criteria 
                   
               
               
                   
               
               
                  329_689_ 
                 AATTTGTCCACATTTTGTCGTAAAG (SEQ ID NO: 150) 
                 CAACAACTTATCACCAATAACAGCA (SEQ ID NO: 151) 
               
               
                 Tetur01g13860 
                   
                   
               
               
                   
               
               
                 3380_3547_ 
                 GTTCTAAATTTTTGAAGGCAGCTA (SEQ ID NO: 152) 
                 AAATGATTCTGTTATACCAACAGCAGT (SEQ ID NO: 153) 
               
               
                 Tetur20g01760 
                   
                   
               
               
                   
               
               
                  339_590_ 
                 GGTATAGTAATCTCGGGTCCTAA (SEQ ID NO: 154) 
                 CAAACACCAAACAATGACAATCAA (SEQ ID NO: 155) 
               
               
                 Tetur06g02480 
                   
                   
               
               
                   
               
               
                 3466_3739_ 
                 TTGTTGTTGTTGGTGAAACAGTTGC (SEQ ID NO: 156) 
                 CATTACCCACATCAACATTTATGG (SEQ ID NO: 157) 
               
               
                 Tetur19g01440 
                   
                   
               
               
                   
               
               
                  347_817_ 
                 GAGCATCGGAGGTGTCAA (SEQ ID NO: 158) 
                 GACAAAAAAAGGTTATGTTCGTGG (SEQ ID NO: 159) 
               
               
                 Tetur18g02240 
                   
                   
               
               
                   
               
               
                  365_571_ 
                 No Primers could be designed with these 
                   
               
               
                 Tetur21g03340 
                 criteria 
                   
               
               
                   
               
               
                  372_523_ 
                 CTGAAGAGTGAAATGCTGATGATCGG (SEQ ID NO: 160) 
                 CATCATCATCACCACAAGTCA (SEQ ID NO: 161) 
               
               
                 Tetur19g01540 
                   
                   
               
               
                   
               
               
                 3732_3946_ 
                 CAGAGTCAATTGGTGAACCTT (SEQ ID NO: 162) 
                 CAGGCACAGCAACATCAA (SEQ ID NO: 163) 
               
               
                 Teturl9g01540 
                   
                   
               
               
                   
               
               
                 3986_4372_ 
                 No Primers could be designed with these 
                   
               
               
                 Tetur19g01540 
                 criteria 
                   
               
               
                   
               
               
                  417_589_ 
                 CCCAACCTTTAACAAAAGAAAGCCTA (SEQ ID NO: 164) 
                 ATGCAACAACAAGCTGCTTCA (SEQ ID NO: 165) 
               
               
                 Tetur08g00500 
                   
                   
               
               
                   
               
               
                  418_692_ 
                 TCATAATCATCCTCTTCGCCA (SEQ ID NO: 166) 
                 GCATAAATAATAATCGTGATCCTTTAG (SEQ ID NO: 167) 
               
               
                 Tetur19g01440 
                   
                   
               
               
                   
               
               
                  445_650_ 
                 TGTTTCAATGTTGATTCCAATGCACT (SEQ ID NO: 168) 
                 AAAATGTACAAAATGCTAGACCTGA (SEQ ID NO: 169) 
               
               
                 Tetur31g01810 
                   
                   
               
               
                   
               
               
                 4484_4770_ 
                 AAAGTCAACAACAAGTTCTACATAAGAT (SEQ ID NO: 
                 TCTTTACAAGGAAACTCGTGATCCTG (SEQ ID NO: 171) 
               
               
                 Tetur20901760 
                 170) 
                   
               
               
                   
               
               
                  463_801_ 
                 AACATCTTTAGCCATTTGACTGGCTG (SEQ ID NO: 172) 
                 CCACGATTACAGATGGACCTGA (SEQ ID NO: 173) 
               
               
                 Tetur04g03690 
                   
                   
               
               
                   
               
               
                 4678_4905_ 
                 TTGAAGAGGAATTGAATTGCCGCAAA (SEQ ID NO: 174) 
                 ATCATCATCAAGCAGCCAC (SEQ ID NO: 175) 
               
               
                 Tetur19g01540 
                   
                   
               
               
                   
               
               
                  467_666_ 
                 TTGCCATTCAGCATATTTGACAGGAT (SEQ ID NO: 176) 
                 CTTCACCAAGAATGGCCAC (SEQ ID NO: 177) 
               
               
                 Tetur10g00660 
                   
                   
               
               
                   
               
               
                   46_199_ 
                 TTGTTGTGGTTGTCGTTATAACCT (SEQ ID NO: 178) 
                 GCGATTTAACCACACTTTTCCT (SEQ ID NO: 179) 
               
               
                 Tetur14g00860 
                   
                   
               
               
                   
               
               
                 4755_5024_ 
                 TCCTCTTCATCGTCACCGAAACA (SEQ ID NO: 180) 
                 ACCACAACCATCACATTGAAC (SEQ ID NO: 181) 
               
               
                 Tetur01g13860 
                   
                   
               
               
                   
               
               
                   47_255_ 
                 AAGGTAAGAGTTGAAAACAAATCCAAG (SEQ ID NO: 182) 
                 AGATGATGCAGAAAGACAAACTCAG (SEQ ID NO: 183) 
               
               
                 Tetur26g02710 
                   
                   
               
               
                   
               
               
                 *494_599_ 
                 TACTCCACTAGAGTTATATCATGAGTCT (SEQ ID NO: 
                 AATGGACGATGAACTGGTTAAATT (SEQ ID NO: 185) 
               
               
                 Tetur01g08060 
                 184) 
                   
               
               
                   
               
               
                   50_206_ 
                 No Primers could be designed with these 
                   
               
               
                 Tetur01g21600 
                 criteria 
                   
               
               
                   
               
               
                  518_697_ 
                 ACCAATAAACATTTCCTTGTGGTG (SEQ ID NO: 186) 
                 CGAGAAATTTTTGGCTCGTGAT (SEQ ID NO: 187) 
               
               
                 Tetur01g07940 
                   
                   
               
               
                   
               
               
                  545_715_ 
                 CAAATTTACACTCTCGAGCGCGAGTT (SEQ ID NO: 188) 
                 TTTGCTGGTTGTTGTTCCTAAAGCAT (SEQ ID NO: 189) 
               
               
                 Tetur30g02230 
                   
                   
               
               
                   
               
               
                 5574_6004_ 
                 AAATCATTAATGGTAAGCCTTCAC (SEQ ID NO: 190) 
                 AAACGAGAAAAGGCAACTAAATTGG (SEQ ID NO: 191) 
               
               
                 Tetur20g01760 
                   
                   
               
               
                   
               
               
                  566_774_ 
                 No Primers could be designed with these 
                   
               
               
                 Tetur07g01500 
                 criteria 
                   
               
               
                   
               
               
                  588_759_ 
                 No Primers could be designed with these 
                   
               
               
                 Tetur07g05390 
                 criteria 
                   
               
               
                   
               
               
                    5_168_ 
                 ACAAGTGATTGAATTGAATCGACAAA (SEQ ID NO: 192) 
                 CAATGTGAACCAAAACACCTCT (SEQ ID NO: 193) 
               
               
                 Tetur01g12090 
                   
                   
               
               
                   
               
               
                 6075_6322_ 
                 No Primers could be designed with these 
                   
               
               
                 Tetur20g01760 
                 criteria 
                   
               
               
                   
               
               
                  643_815_ 
                 TATTTTTTTGCCTCGGGCTGAGGT (SEQ ID NO: 194) 
                 ATCGTTATGATGATGAATTGGGTA (SEQ ID NO: 195) 
               
               
                 Tetur13g05360 
                   
                   
               
               
                   
               
               
                  653_806_ 
                 No Primers could be designed with these 
                   
               
               
                 Tetur19g01540 
                 criteria 
                   
               
               
                   
               
               
                  694_948_ 
                 TTTACCTTTACGGGGAACCAA (SEQ ID NO: 196) 
                 ATGTGGACAAATTTATGAACGAATCGCT (SEQ ID NO: 197) 
               
               
                 Tetur01g13860 
                   
                   
               
               
                   
               
               
                  701_937_ 
                 TCATTCGATTGGTAATGAATCGTATCT (SEQ ID NO: 198) 
                 TGGTTTACCTTGTGATCAACTTAATCT (SEQ ID NO: 199) 
               
               
                 Tetur21g03340 
                   
                   
               
               
                   
               
               
                  719_896_ 
                 No Primers could be designed with these 
                   
               
               
                 Tetur01g12340 
                 criteria 
                   
               
               
                   
               
               
                 *747_1103_ 
                 CGAGTCGAGGTTGACCCACAG (SEQ ID NO: 200) 
                 ATTTTTGTCTCCATTAACTATCGTGTTG (SEQ ID NO: 201) 
               
               
                 Tetur18g02240 
                   
                   
               
               
                   
               
               
                  747_966_ 
                 TCTTCTTTGTTGTTTCTTATTGGG (SEQ ID NO: 202) 
                 CAATACAATGAACAAGAAATTGCAGAT (SEQ ID NO: 203) 
               
               
                 Tetur30g02230 
                   
                   
               
               
                   
               
               
                  748_1010_ 
                 TAAACTGGAGTGGTTCGCCGTA (SEQ ID NO: 204) 
                 CTCAACAGCAGCAACATGAT (SEQ ID NO: 205) 
               
               
                 Tetur16g02700 
                   
                   
               
               
                   
               
               
                  751_910_ 
                 AAATTTTGGTGAATTCATATTCAGACTG (SEQ ID NO: 
                 ATGGAAAAATCTTTGAGGTTAAACATGC (SEQ ID NO: 207) 
               
               
                 Tetur31g01810 
                 206) 
                   
               
               
                   
               
               
                  762_1003_ 
                 CACCTTTAACTCCTACTGGAA (SEQ ID NO: 208) 
                 GGTTTAATGGATGACATTTATCAATGG (SEQ ID NO: 209) 
               
               
                 Tetur07g08130 
                   
                   
               
               
                   
               
               
                  764_938_ 
                 No Primers could be designed with these 
                   
               
               
                 Tetur07g05390 
                 criteria 
                   
               
               
                   
               
               
                  819_1066_ 
                 CTTCCAACACTTGACGAG (SEQ ID NO: 210) 
                 AATAAACATACAAACCGTGAGCC (SEQ ID NO: 211) 
               
               
                 Tetur06g02480 
                   
                   
               
               
                   
               
               
                  868_1056_ 
                 No Primers could be designed with these 
                   
               
               
                 Tetur14g00860 
                 criteria 
                   
               
               
                   
               
               
                  943_1154_ 
                 TAAAGATCACCGGTTGTCTTGTA (SEQ ID NO: 212) 
                 TTGGTGTTGGTGGCTCGT (SEQ ID NO: 213) 
               
               
                 Tetur07g05390 
                   
                   
               
               
                   
               
               
                  944_1108_ 
                 CAAATTCAACATTTTCGGCCATC (SEQ ID NO: 214) 
                 TAAGCCATTAATTAGTGAGAAAGACAT (SEQ ID NO: 215) 
               
               
                 Tetur19g01440 
                   
                   
               
               
                   
               
               
                   94_564_ 
                 TACTTGGTGCACTTGTAACAATACGG (SEQ ID NO: 216) 
                 TAACCACAGGCGATATGAG (SEQ ID NO: 217) 
               
               
                 Tetur01g08060 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 primers designed after two runs 
               
             
          
           
               
                 SEQ_ID 
                 5_PRIMER 
                 3_PRIMER 
               
               
                   
               
               
                    0_228_ 
                 ATTTTTGTTTTCAAAGATATCGTGGATACAGG (SEQ ID NO: 
                 AGTGAATTTTGGCTCATCTCAG (SEQ ID NO: 219) 
               
               
                 Tetur30g02230 
                 218) 
                   
               
               
                   
               
               
                 1126_1276_ 
                 ATTTTGGTAAAATATACTTGGCAGAAAGA (SEQ ID NO: 220) 
                 AAGTATTTGAAAAATATACCCTTGATATG (SEQ ID 
               
               
                 Tetur19g01440 
                   
                 NO: 221) 
               
               
                   
               
               
                 1236_1391_ 
                 GCACCAACACTGAAATAACCCCAAA (SEQ ID NO: 222) 
                 AATGATAATCCAATTGACTTCAAATTAGGAC (SEQ ID 
               
               
                 Tetur20g01760 
                   
                 NO: 223) 
               
               
                   
               
               
                 1326_1516_ 
                 TTTTGTTCAACATATTTCTTTTGTTTTTACTC (SEQ ID NO: 
                 TATTTTGATTACATGAAGTTACTGATGAGCC (SEQ ID 
               
               
                 Tetur19g01440 
                 224) 
                 NO: 225) 
               
               
                   
               
               
                 1564_1794_ 
                 TACATTTTCGTAGATTAGTTCAACATTAAC (SEQ ID NO: 
                 TATTAGAAACGGAAGCTTTCCAG (SEQ ID NO: 227) 
               
               
                 Tetur20g01760 
                 226) 
                   
               
               
                   
               
               
                 1812_1966_ 
                 ATTGTTTTTGGTTATGGAGGAATCG (SEQ ID NO: 228) 
                 TATTTACCTTTATTCCATGGAAGATTTTT (SEQ ID NO: 
               
               
                 Tetur06g02480 
                   
                 229) 
               
               
                   
               
               
                 2129_2321_ 
                 GCAGAATCAGTTTCACTAGGATTTTTTCCCA (SEQ ID NO: 
                 GAAAATGATAATGACATTAACAACTTCAG (SEQ ID NO: 
               
               
                 Tetur20g01760 
                 230) 
                 231) 
               
               
                   
               
               
                 2296_2467_ 
                 ATTGGGATAAAAGTGAATTTGTAATTGATTG (SEQ ID NO: 
                 CATCATCTTCTTCCACCTC (SEQ ID NO: 233) 
               
               
                 Tetur01g13860 
                 232) 
                   
               
               
                   
               
               
                  246_442_ 
                 TACTGTTATTATTGTTAGGTTGATTGGCGG (SEQ ID NO: 
                 ACCAATAATAATGGTAGTCTTTATTCAAGT (SEQ ID NO: 
               
               
                 Tetur01g08220 
                 234) 
                 235) 
               
               
                   
               
               
                 2581_2750_ 
                 AGAAACATTTTCATTCTAATGAAAGGTTC (SEQ ID NO: 236) 
                 ATACTGAAGACATCGTCAAGAAGG (SEQ ID NO: 237) 
               
               
                 Tetur01g13860 
                   
                   
               
               
                   
               
               
                 2582_2766_ 
                 TTTAAGTAAATCTTGAACACAACTTCTTAAAC (SEQ ID NO: 
                 TGCCAAGAATATAACCGCTG (SEQ ID NO: 239) 
               
               
                 Tetur20g01760 
                 238) 
                   
               
               
                   
               
               
                  259_421_ 
                 GAGTATATGTTTTATATTCCATCAGTTTT (SEQ ID NO: 240) 
                 AGCCTCATGAAAAAGTGATCCAA (SEQ ID NO: 241) 
               
               
                 Tetur07g08130 
                   
                   
               
               
                   
               
               
                 3221_3403_ 
                 TATCATCAGGTAAATGTGAGGTAGT (SEQ ID NO: 242) 
                 TTTAGTTTCATATTCACGACGTATTTATC (SEQ ID NO: 
               
               
                 Tetur06g02480 
                   
                 243) 
               
               
                   
               
               
                  365_571_ 
                 No Primers could be designed with these 
                   
               
               
                 Tetur21g03340 
                 criteria 
                   
               
               
                   
               
               
                 3986_4372_ 
                 No Primers could be designed with these 
                   
               
               
                 Tetur19g01540 
                 criteria 
                   
               
               
                   
               
               
                   50_206_ 
                 GATGTTTCTTCATAAACTTGAATGGTTGCT (SEQ ID NO: 
                 AAATGAAAAATTATACGGATATGTCCAAGGAG (SEQ ID 
               
               
                 Tetur01g21600 
                 244) 
                 NO: 245) 
               
               
                   
               
               
                  566_774_ 
                 No Primers could be designed with these 
                   
               
               
                 Tetur07g01500 
                 criteria 
                   
               
               
                   
               
               
                  588_759_ 
                 No Primers could be designed with these 
                   
               
               
                 Tetur07g05390 
                 criteria 
                   
               
               
                   
               
               
                 6075_6322_ 
                 CAATAATCTTTTTACAGATAACGTCATTT (SEQ ID NO: 246) 
                 CTGAAATTTGGTGCTCAAATCGT (SEQ ID NO: 247) 
               
               
                 Tetur20g01760 
                   
                   
               
               
                   
               
               
                  653_806_ 
                 TTACAGCTAATATTGTTCTCTTTGTATTG (SEQ ID NO: 248) 
                 GTCACCATCATCTAGTTACGCCCTACCA (SEQ ID NO: 
               
               
                 Tetur19g01540 
                   
                 249) 
               
               
                   
               
               
                  719_896_ 
                 TAAACAGGAGAAATGGTGACATTTAT (SEQ ID NO: 250) 
                 AGAAAAATTTATTTATCGTCTCGAATTAAAC (SEQ ID 
               
               
                 Tetur01g12340 
                   
                 NO: 251) 
               
               
                   
               
               
                  764_938_ 
                 CCACCAACACCAACGGAT (SEQ ID NO: 252) 
                 TGAAGCTTTTTTCAAACTTTTCTATTACT (SEQ ID NO: 
               
               
                 Tetur07g05390 
                   
                 253) 
               
               
                   
               
               
                  868_1056_ 
                 TTCACTTTTAGGTTGCTGTGG (SEQ ID NO: 254) 
                 TTCAATCACATCATTACAATGTTAAAACACG (SEQ ID 
               
               
                 Tetur14g00860 
                   
                 NO: 255) 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 primers designed after 3 runs 
               
             
          
           
               
                 SEQ_ID 
                 5_PRIMER 
                 3_PRIMER 
               
               
                   
               
               
                  365_571_ 
                 TATTAACAATATTATTAACATTGGTAGGA (SEQ ID NO: 
                 GCAACATTGGAATACCAT (SEQ ID NO: 257) 
               
               
                 Tetur21g03340 
                 256) 
                   
               
               
                   
               
               
                 3986_4372_ 
                 CTGCCGCTGCTGCAGCCG (SEQ ID NO: 258) 
                 TGACTTGAGTGATTTAGCAAGTGA (SEQ ID NO: 259) 
               
               
                 Tetur19g01540 
                   
                   
               
               
                   
               
               
                  566_774_ 
                 GTTGGTCACTTTGAAAATACGA (SEQ ID NO: 260) 
                 TAATGCTAATATATTTTTTGTGATACT (SEQ ID NO: 261) 
               
               
                 Tetur07g01500 
                   
                   
               
               
                   
               
               
                  588_759_ 
                 GAAAAAAGCTTCAGCAAAGT (SEQ ID NO: 262) 
                 TCTAATATTTGTGTTTATATATCATCAT (SEQ ID NO: 263) 
               
               
                 Tetur07g05390 
               
               
                   
               
             
          
         
       
     
       Example 3 
     Expression of RNAi in Plants 
       [0025]    Similar to the RNAi distal-less construct, RNAi constructs of the other essential genes are placed under control of the CaMV 35 S promoter, in pB-Agrikola. The plasmid map of pB Agrikola (carrying the RNAi construct of Tetur17g02200-SEQ ID NO:86) is given in  FIG. 4 ; the sequence of the plasmid is given in SEQ ID NO:267. In a similar way, constructs were made for the RNAi of SEQ ID NOS:2, 18, 22 and 75. The resulting constructs were agro-infiltrated into  Arabidopis . RNAi expression is checked by Northern blot. RNAi positive lines are further cultivated to be used in a feeding test. 
       Example 4 
     Feeding Tests with  T. urticae    
       [0026]      Arabidopsis  plants expressing dsRNA from the selected genes are used in spider mite food tests, and the effect on mite development is measured, as described in Example 1. A reduction in living mites, as well in eggs, on the plants is obtained. 
       REFERENCES 
       [0000]    
       
         Baum J. A., T. Bogaert, W. Clinton, G. R. Heck, P. Feldmann, O. Ilagan, S. Johnson, G. Plaetinck, T. Munyikwa, M. Pleau, T. Vaughn and J. Roberts (2007). Control of coleopteran insect pests through RNA interference.  Nature Biotech.  25:1322-1326. 
         Cobos I., V. Broccoli, and J. L. Rubenstein (2005). The vertebrate ortholog of Aristaless is regulated by Dlx genes in the developing forebrain.  J. Comp. Neurol.  483:292-303. 
         Fonseca N. A., C. P. Vieira, and J. Vieira (2009). Gene classification based on amino acid motifs and residues: the DLX (distal-less) test case.  PLoS One,  4:e5748. 
         Gordon K. H. J and P. M. Waterhouse (2007). RNAi for insect-proof plants.  Nature Biotech.  25:1231-1232. 
         Mao Y. B., W. J. Cai, J. W. Wang, G. J. Hong, X. Y. Tao, L. J. Wang, Y. P. Huang, and X. Y. Chen (2007). Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol.  Nat. Biotechnol.  25:1307-1313.

Technology Classification (CPC): 2