Cloning And Characterization Of The Root Knot Nematode Resistant Genes From Tomato And Pepper

Plant parasitic root knot nematodes (Meloidogyne spp.) are important obligate, sedentary endoparasites of many crop species and cause major economic damage to agriculture production around the world which are responsible for the annual 125 billion dollars losses. In the four plant infection pathogens, the damage attributed to parasitic nematodes is more than that of bacteria and viruses but only lower than that of fungi. Current control of root-knot nematodes is deficient. Chemical nematicides have traditionally been widely used in the field, but they have undesirable health and environment risks due to their toxicity and persistence. Potentially, the use of resistance cultivars is illustrated to be the most efficient, durable and environmentally safe control measure to retard invasion by the root-knot nematodes.This research was to clone the root knot nematode resistance genes SlMi and CaMi from tomato and pepper, analyze the gene structures and expression profiling, and identify their functions. Then these genes were transformed into the nematode susceptible tomato by Agrobacterium-mediated transformation and we obtained the tomato transgenic plants which confer resistance to the root knot nematodes. The main results were as follows:1. The gDNA of tomato root knot nematode resistance gene SlMi was cloned based on the published sequence (AF039682) of Mi. The full length of gDNA of SlMi is 5345bp with two introns. The SIMi cDNA is 3964 bp long, containing a full length open reading frame (ORF) of 3774bp encoding 1257 amino acids.2. 24 RGAs with complete open reading frames (ORF) were obtained from pepper genomic DNA of PR 205 by PCR with degenerated primers designed according to the NBS-LRR conserved domains. Comparison of putative amino acid sequences of these 24 RGAs with the reported genes showed that they have 31-44% homologous with I2C-1, 32-53% with prf, 26-34% with RPM1, 43-95% with Hero and 39-99% with Mi. It is interested that p20 has 99% identity with Mi and it is probable one part of the root knot nematode resistance gene.3. By use of homologous sequence method, a root knot nematode resistance gene CaMi was cloned from resistant pepper and the sequence data have been submitted to the DDBJ/EMBL/GenBank databases under accession number DQ465824. The gDNA of CaMi is 5355 bp long with two introns. The full-length cDNA of CaMi was 3986bp long, which included the putative transcription start site, a potential open reading frame of 3774bp, a 5'-untranslated region (5'-UTR) of 86bp and a 3'-untranslated region (3'-UTR) of 108bp, including the poly(A) tail. The potential open reading frame encoded for 1257 amino acid residues and contained a potential leucine zipper and a heptad repeat motif. The predicted nucleotide binding site (NBS) domain was comprised of the kinase-la (P loop), kinase-2 and kinase-3a motif. At the C terminus, it contained a leucine rich repeat (LRR) domain. The two introns are 1297 and 72 nucleotides in length respectively. The genomic DNA sequence of SlMi and CaMi gene is 98% identical and the deduced amino acid is 99% identical. However, the intron structures have a striking dissimilarity between these two genes. Intron 1 and intron 2 are both shorter in CaMi (1297 and 72bp) than in Mi-1.2 (1306 and 75bp), with the sequence identities of 92% and 80%, respectively.4. RT-PCR analysis of the resistant tomato showed that SlMi was highly expressed in the roots and leaves, weakly expressed in the stems, and was undetectable in the flowers and fruits. Analysis of expression profiling of CaMi in resistant pepper showed it was highly expressed in roots, leaves, and flowers and expressed at a lower level in stems and was not detectable in fruits.5. Southern blot analysis indicated that the gene corresponding to CaMi might be present as three copies in the genome of resistant pepper.6. The sense and RNAi vector of SIMi gene and the sense vector of CaMi gene were constructed. Subsequently the sense vectors of SIMi and CaMi were transferred into the nematode susceptible tomato cultivars ZS5 and JIA 8 and the RNAi vector of SIMi was transferred into the nematode resistant tomato RN-1 using the Agrobacterium-mediated transformation. PCR and Southern blot analysis indicated that the T-DNA region was inserted into the tomato genomic with 1 to 7 copies.7. RT-PCR analysis indicated that the expression level of most target genes were higher in sense transgenic plants and lower t in RNAi transgenic plants than that in the control.8. Nematode assays showed that the resistance to root-knot nematodes was significantly improved in some sense transgenic plants compared to untransformed susceptible control. And some of the RNAi transgenic plants lost their resistance to root knot nematodes.9. Paraffin section analysis indicated that the root ultrastructure was remarkablely changed after root-knot nematode invading. In the root of RNAi transgenic plants, the nematode moved in the root and induced several giant-cells and produced many egg masses. But in the root of sense SlMi transgenic plants it elicits hypersensitive response (HR) and produced cell death around the nematode after nematodes penetrates the root.