Map-based Cloning Of Muse Gene In Arabidopsis And Bioinformatical Analysis Of Muse In Tomato

Innate immunity is an effective mechanism for plants to fight against pathogens. Plantshave evolved sophisticated network to regulate immune signal transduction during long-terminteraction with pathogens. Immune signal is turned on to resist pathogens and it is shut downwhen pathogens are defeated or there is no pathogen in the environment. And thus, the plantscan save photosynthetic energy and avoid over immune responses which are harmful to plants,like accumulation of SA and ROS. R protein mediated immune response is a key part of plantinnate immunity and it is regulated by many factors. So far, the regulation of R proteinmediated immune responses is largely unknown and firstly people should identify theregulators. The theories obtained from model plant Arabidopsis about immune signalregulation will have a positive effect in agricultural production.Arabidopsis SNC1encodes one type of R protein. Mutant snc1(suppressor of npr1-1,constitutive1) shows constitutive active immune responses even without pathogen invasion.And thus, snc1becomes an ideal tool to elucidate regulators involved in R protein mediatedimmune responses. Previously, several muse (mutant of snc1enhancing) mutants werescreened using EMS through forward genetic screen. After map-based cloning and functionstudy, we identified two negative regulators in R protein mediated immune response, MUSE5and MUSE10respectively. We also identified31Pam18s and26Pam16s related to MUSE5intomato, Arabidopsis, rice and other9plant species through bioinformatical strategies.45genes coding PHD finger domain protein related to MUSE10were searched from tomatogenome as well.According to our study, it was suggested that MUSE5as an orthologue of PAM16was alsolocalized to mitochondrial inner membrane (a subunit of import motor). The double mutantAtpam16-1Atpam16l is lethal so MUSE5/AtPAM16is essential to plant survival. It wasspeculated that MUSE5was involved in translocation of mitochondrial inner membrane, inwhich regulated immune responses negatively, resisted auto-immunity and thus protectedagainst ROS. MUSE10encoded one of PHD finger protein in Arabidopsis. It was speculatedpreliminarily that MUSE10was also a negative regulator of R protein mediated immunity,which was a newly-identified function. Previously, Pam18and Pam16(also PAM16) in yeast localize to mitochondrial inner membrane and form dimer to regulate protein translocation.Pam18and Pam16are conserved in eukaryotic species. In this study, we found that Pam18and Pam16were also highly conserved among plants. Key amino acid residues and an HPDmotif were identical except OsPam18L5. N-myristoylation sites of Pam18and casein kinaseII phosphorylation sites of Pam16were more abundant which might be key to proteinfunction. According to our prediction, some Pam18and Pam16proteins contained atransmembrane region at the N-terminal region. Sub-cellular prediction results indicated thatmany orthologues localized at mitochondria. Gene expression analyses revealed that Pam18and Pam16might play roles in senescence and abiotic stress responses.45PHD proteins intomato were conserved especially motif Cys4-His-Cys3. Some genes encode other conservedkey domains besides PHD-finger. Phylogenetic analysis of these45proteins resulted in7clusters. Most PHD finger proteins were predicted to PML body location. These PHD-fingergenes might play multiple roles in tomato based on our gene expression analysis.This study firstly elucidated MUSE5/TXR1/AtPAM16and MUSE10in Arabidopsis have anegative role in regulation of R protein SNC1mediated plant immune, which enrich theunderstanding of plant immune signal transduction. The mechanism about how MUSE5works was deeply studied and proved that mitochondrion is involved in immune responseonce again. These findings will provide a theoretical guidance in plant protection andresistance breeding practice. Through bioinformatics strategies, the related genes and proteinsof MUSE5and MUSE10were screened in tomato. The evolution history, sub-cellularlocalization, transmembrane region, and gene expression under abiotic stresses were analyzedsystematically. These findings will support gene cloning, protein function study in tomato inthe future.