Mechanism Of E3 Ubiquitin Ligase OsPIE3 Interacting With Receptor-like Kinase PID2 To Regulate Rice Blast Resistance

Rice is the most important food crop in China and one of the three major food crops all over the world.Rice blast,the “cancer” of rice,is one of the three major diseases of rice,which will lead to serious yield reduction.Recently,lots of great significant progress has been made in rice blast.And the identification of rice blast resistance genes is a really meaningful work.The rice B-lectin receptor kinase,PID2,functions as a major gene in the Chinese Magnaporthe oryzae(M.oryzae)strain ZB15 resistance.Previous studies have reported that the E3 ligase OsPUB15 functions as a regulator of PID2 in blast resistance regulation.However,the molecular mechanism and the PID2-mediated downstream signaling events remains unclear.In this study,we screened the yeast library with PID2 kinase domain(PID2-KD)as a“bait”,and a U-box E3 ubiquitin ligase OsPIE3(PID2-interacting E3)was isolated.Then a series of experiments were carried out,and found that OsPIE3 played key roles in PID2-mediated rice blast resistance.The main conclusions are as follows:(1)OsPIE3 is a typical U-box PUB E3 ubiquitin ligase which has one U-box domain at the N terminal and five ARM repeats domains at the C terminal.A series of biochemical experiments such as Y2 H,GST-pull down,Bi LC,Bi FC,Co-IP and so on were used to prove that OsPIE3 and PID2 were interact with each other in vivo and in vitro.And the ARM repeat domain of OsPIE3 was necessary to mediate the OsPIE3 interaction with PID2.The interaction between OsPIE3 and PID2 was performed in the plasma membrane and nucleus in planta.(2)Subcellular localization analysis showed that OsPIE3 was mainly located in the plasma membrane and nucleus,while PID2 was located in the plasma membrane.Colocalization analysis demonstrated that OsPIE3 and PID2 were interacted in the N.benthamiana leaf cells and rice protoplast cells.OsPIE3 could affect the localization of PID2 and transfer it from the plasma membrane to nucleus.The cysteine site C230 mutation in the U-box domain and the knockout of OsPIE3 would slow down the transfer.While the Magnaporthe grisea ZB15 could enhance the interaction between OsPIE3 and PID2 and promote the transfer of PID2 from plasma membrane to nucleus.(3)OsPIE3 has E3 ubiquitin ligase activity and can be self-ubiquitination in vitro,while OsPIE3C230 S reduced the E3 ubiquitin ligase activity.OsPIE3 can also ubiquitinate PID2 in vitro by K48 polyubiquitin.The ubiquitin degradation of PID2 by OsPIE3 was in a dose-and time-dependent manner through ubiquitin-26 S proteasome system in planta,while the degradation rate of PID2 by OsPIE3C230 S was slower.We also examined PID2 accumulation by using anti-PID2 antibody and proved that PID2 accumulation was increased in the Cas9-OsPIE3 mutants and decreased in the OE-OsPIE3 mutants.These findings demonstrate that E3 ubiquitin ligase OsPIE3 specifically targets B-lectin receptor-like kinase PID2 leading to its proteasomal degradation.(4)OsPIE3 and PID2 have similar expression pattern in rice,and both of them have relatively high expression in leaf.Genetic analysis showed that Cas9-OsPIE3 mutant enhanced resistance and the expression levels of the defense marker genes PR3,PR5,PR10,and PBZ1 were significantly increased when compared to wild-type Nipponbare,while the mutants OE-OsPIE3,Cas9-PID2,Cas9-OsPIE3/PID2 showed the opposite performance.These data suggested that OsPIE3 and PID2 can regulate the rice blast resistance synergistically,and OsPIE3 acts as a negative regulator in the pathway of rice blast resistance.(5)Comapred with wild-type Nipponbare,the Cas9-OsPIE3 mutant accumulated more H2O2 after 1 d infection and fewer cell death,but the mutants OE-OsPIE3,Cas9-PID2 and Cas9-OsPIE3/PID2 were the opposite.These results showed that OsPIE3 and PID2 can alter H2O2 accumulation to trigger the early defense responses and regulate the cell death rate in rice blast resistance synergistically.Our research mainly focused on the “Mechanism of E3 Ubiquitin Ligase OsPIE3 and Receptor-like Kinase PID2 Synergistically Regulating Rice Blast Resistance” to explore.There are the following innovations in the research results:(1)OsPIE3 is a typical U-box PUB E3 ubiquitin ligase which could interact with PID2 in vivo and in vitro.OsPIE3 could also ubiquitinate PID2 in vitro and specifically target PID2 for ubiquitination through the 26 S proteasome systemin.(2)OsPIE3 could affect the localization of PID2 and transfer it from the plasma membrane to nucleus in the N.benthamiana leaf cells and rice protoplast cells.(3)OsPIE3 and PID2 can regulate the rice blast resistance synergistically,which provided a new theoretical basis for the synergistic effect of PUB-RLK in rice blast resistance.