Functional Domains Of Harpin_Xooc From Xanthomonas Oryzae Pv.oryzicola

Harpinxooc is a protein encoded by the hrf2 gene of Xanthomonas oryzae pv.oryzicola. Application of the protein to several plants induces hypersensitive response (HR) and disease resistance (DE) and enhances plant growth (EPG). To determine whether any functional domains in harpinxooc are responsible for the diverse functions of the protein, 13 truncated harpinxooc fragments were generated by PCR-based mutations of hrf2 and recombinant production of the mutation-derived gene fragments. These fragments differentially induced HR, DR and EPG . Thus harpinxooc confers its different effects on plants through particular domains, this suggests that genetic modification of the gene may greatly improve biological activity of the protein.1. Generation of truncated harpinxooc fragmentsModifying the hrf2 gene of the bacterial stain RS105 using three methods produced thirteen truncated harpinxooc fragments. Direct amplification of the bacterial DNA by PCR using specific primers produced 10 fragments of the gene. To ligate R1 and R3, the Rl and R3 containing nucleotides recognized specific restriction enzymes formed the fragment Rl+3. In the third method, the fragments R(mutant) was obtained by directed replacement of the code for thereonine(T), which may result in the substitution of C by T at the site 45 of harpinxooc- In addition, to delete the two motifs of R(G), which is a characteristics of harpins, in harpinxooc, the gene fragment R(G) was produced by assembling Head, Mid and hrf2 which cover some portions of hrf2. The 13 fragments of hrf2 were transferred separately into E. coli, after coning in the vector pET30a (+), which contains two promoters inducible by IPTG . Incubation of the recombinant E. coli strains in the presence of IPTG resulted in the production of truncated harpinxooc fragments. 2L-T-SDS-PAGE show that desired proteins were produced for the truncated fragments Rl+2, Rl, RAC, RBD, RBC, RCD, R1+3,R (G) and R (mutant) because they appeared on the gel. However , no suspected bands forR2+3,R2, Rp,R3.2. Differential induction of HR and pathogen defense by truncated harpinxooc fragmentsThe 13 truncated harpinxooc fragments were tested for functions in inducing the HR and pathogen resistance in tobacco and Arabidopsis. Results from the assay suggested that some of the fragments primarily acted as functional domains for the induction of the HR or pathogen defense. The domain for the HR contains 11-50 amino acids (AA) portion of harpinxooc . However, the cysteine at the site 45 and the two GGG-GG motifs in the middle and N-terminal is not required for HR induction. Interestingly, the 13 fragments induced resistance to tobacco mosaic virus in tobacco, while the fragments RAC, RCD, R2, Rp, R (G) were more effective. Furthermore, RAC, RCD induced resistance to Pseudomonas. syringae. pv. tomato DC3000. Consistently, treatment with R2+3 or Rp caused expression of the HR-marker gene hsr203J and several other genes that encode regulators and effectors involved in defense signal transduction pathways in tobacco and Arabidopsis. In contrast, treatment with R2+3 did not induce expression of these genes. Intriguingly treatment of both plants with Rp, Rp induces expression of the signaling-regulatory genes and effector genes, but it did not induce expression of Hsr203J. These results suggest that the functional domains for the HR and pathogen defense act consistently for the phenotypes and molecular response.3. Differential promotion of plant growth by truncated harpinXooc fragmentsThe author determined whether the 13 truncated harpinxooc fragments enhance plant growth. After treating tomato plants and seed treatment, both aerial portions and roots of plants grown better than the control plants treated with proteins extracted from pET30a (+). Treatment with these fragments resulted in an increase of chlorophyll contents in leaves, which is consistent with the enhancement of plant growth. Moreover, treatments with RCD, R2+3, Rl+3 and R (G) and intact harpinxooc, induced expression of several effector genes th...