Functional Analysis Of The Nonexpressor Of Pathogenesis-related Gene MuNPR1 From Mulberry

Plants will be affected by a variety of pathogens in the process of growth and development. To combat microbial pathogens effectively, the plant has formed a variety of defense mechanisms in the long-term survival. As for the plant disease resistance mechanism consists of different levels and ways of resistances which were crossed and overlapped together, the plant disease resistance was showed in a wide variety of forms. Typical forms of resistance include base resistance and induced resistance. Primary innate immunity, the first line of defense of plants, is achieved through a set of defined receptors, that recognize conserved microbe-associated molecular patterns, and in this way the plant can use its own special structure as well as some chemical compositions against pathogens infection. If the plants lose basic resistance to pathogens, they will show hypersensitivity to pathogens. Inducible defense is an inducible form of plant defense conferring broad-spectrum immunity to secondary infection of plant tissues above the initial infection site. NPR1s(Non-expressor of Pathogenesis-Related Genes) are a class of plant broad-spectrum disease resistance genes and play important roles in activating various plant defense responses, including expression of the pathogenesis-related genes and systemic acquired resistance. As far as we know, there is no NPR1 information reported in mulberry so far. In this study, the cDNA encoding NPR1 was isolated from mulberry and designated as MuNPR1. The characteristics the gene and its coding protein were analysed. In addition, the coding region of the MuNPR1 was inserted into the expression vector pBI121 and transformed into Arabidopsis thaliana plants and its biological functions were analysised. The results obtained in this study would provid a candidate gene for the molecular breeding for mulberry disease resistance, and laid the foundation for the research on the functions of MuNPR1 gene.The main results of this study can be summarized as follows:(1) Cloning and bioinformatics analysis of MuNPR1Based on the mulberry transcriptome sequence information, the mulberry non-expressor of pathogenesis-pelated gene was cloned using PCR and designated as MuNPR1(GenBank accession No. JX204735). The gene has an open reading frame of 1743 bp encoding a protein composed of 580 amino acids with a predicted molecular mass of 64.5 kDa and an isoelectricpoint of 5.66. It was predicted that MuPR1 c was folded into a hexahedron shape with a lot of alpha helix and extended fragments. The secondary structure of MuNPR1 was rich in alpha helix, followed by beta folded and extended fragments. The protein possesses a conservative NPR1 ankyrin repeat sequence structure and a BTB/POZ structure domains. The MuNPR1 protein was homology to Arabidopsis thaliana AtNPR1 with the identy of 51.09% indicating that MuNPR1 has similar function with Arabidopsis AtNPR1.(2) Tissue expression characteristic analysis of MuNPR1The expression pattern of MuNPR1 in the mulberry was analyzed using quantitative PCR, and the results showed that it had no specific expression in different tissues. However, its expression abundance was diverse in different tissues and organs, and the expression quantity in the flowers and barks are similar, and the expression in the leaf was lowest.(3) Biological function analysis of MuNPR1The plant expression vector of MuNPR1 was constructed, and the transgenic Arabidopsis plants of the genes were obtained successfully. qRT-PCR analysis results showed that MuNPR1 can be expressed in transgenic Arabidopsis thaliana efficiently. The efficient expression of MuNPR1 in Arabidopsis thaliana change the transgenic plants phenotypic significantly, and the transgenic plants blossom in advance and have a shorter growth period than wild type plants. The transgenic plants show strong resistance to Pst DC3000(Pseudomonas syringae pv. Tomato DC3000, Pst DC3000), indicating that MuNPR1 plays an important role in the resistance to the bacteria, and this mat be due to the deposition of callose which can obstruct the invasion of the pathogen in the transgenic plants. Experimental results from NBT and DAB staining showed that the accumulation of H2O2 and O2 – were lower in the infected leaves of transgenic plants than that of wild type plants. So the expression of MuNPR1 in Arabidopsis thaliana may help to eliminate the accumulation of H2O2 and O2– caused by pathogen infection in the infected leaves. RT-PCR analysis show that the the expression of MuNPR1 in Arabidopsis plants can not induce the expression of PR5, but the the expression of PR1 were changed not significantly without pathogen infection, however, the expression of PR1 and PR5 in the transgenic plants were significantly higher than those in wild type plant when they were infected by Pst DC3000. Therefor, the MuNPR1 gene may also be involved in the regulation of other resistant genes in the response to pathogen infection。When the transgenic Arabidopsis were subjected to drought and salt stress, the transgenic Arabidopsis plants of MuNPR1 showed less resistance to salt and drought stress compared with wild type plants. As regards the transgenic Arabidopsi plants of MuNPR1, they showedstrong resistance to salt stress but more sensitive to drought stress and pathogen infection than wild type plants. Experimental results from NBT and DAB staining showed that the accumulation of H2O2 and O2 – were higher in the leaves of transgenic plants than that in the wild type plants subjected to drought and salt stresses. So the expression of MuNPR1 in Arabidopsis thaliana may be disadvantageous to eliminate the accumulation of H2O2 and O2– in the response to abiotic stresses.In conclusion, the MuNPR1 may not only participate in plant growth and development, but also play a important role on the resistance to Pst DC3000 infection and a negative role on the resistance to drought and salt stresses.