Analyses Of Copine/BONZAI Gene Evolution In Plants And Rice OsBON1 Function In Disease Resistanse

During the whole process of growth,plants are disturbed by many environmental factors.Microbial pathogen is an important environmental factor affecting plant growth.In order to fight against microbial pathogen,plants possess a set of effective resistance to the immune system after a long period of evolution.The innate immune systems of plants mainly includes pathogen-associated molecular pattern(PAMP)triggered immunity(PTI)and effector-triggered immunity(ETI).Copine are highly conserved proteins with calcium-dependent phospholipid-binding activitives among different species.In Arabidopsis,copine proteins are shown to be regulators of plant immuity.However,it is systematically studied that the function of copine protein in Arabidopsis of the dicots.For other plants,especially monocots,the function of copine proteins have yet to be explore.We plan to present the following two steps to study the copine.The first step is that identification and analysis the copine/BONZAI proteins among evolutionarily diverse plant species and the second step is the determination of the functin of rice OsBON1 gene in disease resistance.1.Analysis of copine/BONZAI gene evolution in plantsCopine are evolutionarily conserved calcium-dependent membrane binding proteins with potentially critical biological functions.In plants,the function of these proteins has not been analyzed except for in Arabidopsis thaliana where they play critical roles in development and disease resistance.To facilitate functional studies of copine proteins in crop plants,genome-wide identification,curation,and phylogeny analysis of copine in 16 selected plant species were conducted.All the identified 32 plant copine have conserved features of in the two C2 domains(C2A and C2B)and the von Willebrand factor A(vWA)domain.Different from copine of animals and protozoa,plant copine have glycine at the second residue potentially acquiring a unique protein modification of myristoylation.Phylogenetic analysis suggests that copine was present as one copy when evolving from green algae to lower flowering plants,and duplicated before the divergence of monocots and dicots.In addition,gene expression and protein localization study of rice copine suggests both conserved and different properties of copine in dicots and monocots.This study will contribute to uncovering the role of copine genes in different plant species.2.Determine the functin of rice OsBON1 gene in disease resistance In order to further study the function of copine in plants,we cloned OsBON1 from rice TP309.The transgenic plants in TP309 with OsBON1 gene silencing and over expressing plants as well as OsBON1 fusion eGFP tags were produced.Meanwhile,the transgenic plant in NPB with OsBON1 gene silencing was also produced.Both the OsBON1 gene silencing plants in TP309 and in NPB show significant dwarf growth phenotype at seedling.The rest of the research are based on the TP309 background of transgenic plants as the experimental material.OsBON1 gene silencing plants significantly enhances rice blast disease resistance.Furthermore,OsBON1 gene silencing plants is highly resistant to Xanthomonas oryzae pv.oryzae PX099.PR gene expression and hydrogen peroxide content in OsBON1 gene silencing plants was also significantly higher than that of the wild type without inoculation treatment.There is more ROS accumulation at the M.oryzae infection sites in OsBONl silencing plants than the wild type plants.The OsBON1 silencing expression significantly delays the invasion and expasion of M.oryzae Hokul.Furthermore,OsBON1 protein accumulated at the BIC(biotrophic interfacial complex)structure when the M.oryzae infection.High temperature can significantly inhibit the dwarf phenotype and the enhanced disease resistance of OsBON1 gene silencing plant.These results indicate that OsBON1is a negative regulator of plant immunity similarly to the Arabidopsis BON1.