The Identification Of Differentially Expressed Proteins Induced By Rice Blast Fungus&Preparation Of Polyclonal OsAGO18 Antibody

To defend against phytopathogens,the plants have evolved a two-branched innate immune system.The first branch is pathogen-associate molecular patterns(PAMPs)-triggered immunity(PTI),which is activated by plant cell-surface pattern recognition receptors(PRRs).The second immunity is from intracellular components and is called effector-triggered immunity(ETI),which depends on the resistance gene encoding protein to defense the infection.We use the Magnaporthe grisea strain Guyl 1 and 2539 to infect rice cultivar Nipponbare.We extracted the total protein and plasma membrane proteins,and used a quantitative proteomics approach with iTRAQ LC-MS/MS technologies to discover the key proteins in PTI and ETI.About 43.35%plasma membranes was enrichment by using a dextran T-500 and polyethylene glycol 3350,6.4%(w/w)for PM isolation system.In this work,we have found 124 upregulated and 41 down-regulated plasma proteins,and 173 upregulated and 49 down-regulated total proteins,in which 21 upregulated and 5 down-regulated proteins show agreement in both the PM and total proteins dataset.After infected by M.oryzae for 24h and 96h,there are 22 and 33 proteins were induced by Guyll or 2539.More upregulated proteins were induced by 2539 than Guyll,and as the infection continue advanced,the number of proteins also increased.There are 6 upregulated proteins were identified in 4 treatments,some of which prossess functions that were related to resistance such as,protein serine/threonine kinase activity,ATP/nucleotide binding,metal-binding,transportor,receptor etc.These could act as PRRs,which are localized on the plasma membrane and able to recognize pathogens in the early defense responses.Rice resistance to evolutionarily diverse viruses requires Argonaute18(AGO 18).Unpublished data from our laboratory show that AGO 18 is also required in rice blast resistance.The antiviral function of AGO 18 depends on its activity to sequester microRNA168(miR168)to alleviate repression of rice AGO1 essential for antiviral RNAi.In order to reveal the function of OsAG018 in the study of blast resistance,we produced recombinant protein AGO 18639 by using tobacco transient expression.There are 300?g AGO 18639 antigen has been purified to prepare polyclonal AGO 18 antibody.The antibody will be used to study the role of AGO 18 playing in disease resistance.