The OsAGO2–OsNAC300–OsNAP module regulates leaf senescence in rice

Leaves play a crucial role in the growth and development of rice(Oryza sativa) as sites for the production of photosynthesis.Early leaf senescence leads to substantial drops in rice yields.Whether and how DNA methylation regulates gene expression and affects leaf senescence remains elusive.Here,we demonstrate that mutations in rice ARGONAUTE 2(OsAGO2)lead to premature leaf senescence,with chloroplasts in Osago2 having lower chlorophyll content and an abnormal thylakoid structure compared with those from wild-type plants.We show that OsAGO2 associates with a 24-nt micro RNA and binds to the promoter region of Os NAC300,which causes DNA methylation and suppressed expression of Os NAC300.Overexpressing Os NAC300 causes the similar premature leaf senescence as Osago2 mutants and knocking out Os NAC300 in the Osago2mutant background suppresses the early senescence of Osago2 mutants.Based on yeast one-hybrid,dual-luciferase,and electrophoresis mobility shift assays,we propose that Os NAC300 directly regulates transcription of the key rice aging gene NAC-like,activated by APETALA3/PISTILLATA(Os NAP)to control leaf senescence.Our results unravel a previously unknown epigenetic regulatory mechanism underlying leaf senescence in which OsAGO2–Os NAC300–Os NAP acts as a key regulatory module of leaf senescence to maintain leaf function.

An OsPRMT5-OsAGO2/miR1875-OsHXK1 module regulates rice immunity to blast disease

Rice ARGONAUTE2(OsAGO2)is a core component of the rice RNA-induced silencing complex(RISC),which is repressed by Magnaporthe oryzae(M.oryzae)infection.Whether and how OsAGO2-mediated gene silencing plays a role in rice blast resistance and which sRNAs participate in this process are unknown.Our results indicate that OsAGO2 is a key immune player that manipulates rice defense responses against blast disease.OsAGO2 associates with the 24-nt miR1875 and binds to the promoter region of HEXOKINASE1(OsHXK1),which causes DNA methylation and leads to gene silencing.Our multiple genetic evidence showed that,without M.oryzae infection,OsAGO2/miR1875 RISC promoted OsHXK1 promoter DNA methylation and OsHXK1 silencing;after M.oryzae infection,the reduced OsAGO2/miR1875 led to a relatively activated OsHXK1 expression.OsHXK1 acts as a positive regulator of blast disease resistance that OsHXK1-OE rice exhibited enhanced resistance,whereas Cas9-Oshxk1 rice showed reduced resistance against M.oryzae infection.OsHXK1 may function through its sugar sensor activity as glucose induced defense-related gene expression and reactive oxygen species(ROS)accumulation in Nipponbare and OsHXK1-OE but not in Cas9-Oshxk1 rice.OsAGO2 itself is delicately regulated by OsPRMT5,which senses M.oryzae infection and attenuates OsAGO2-mediated gene silencing through OsAGO2 arginine methylation.Our study reveals an OsPRMT5-OsAGO2/miR1875-OsHXK1 regulatory module that fine tunes the rice defense response to blast disease.

水稻Argonaute 2蛋白的原核表达与多克隆抗体制备

为了制备水稻Argonaute 2 （AGO2）的多克隆抗体,该研究采用RT PCR扩增OsAGO2蛋白165～401 aa片段和440～570 aa片段的编码序列,并构建了2个原核表达载体.诱导表达重组蛋白后注射家兔,制备了相应的多克隆抗体,最后利用Western blot初步分析水稻AGO2蛋白的表达模式.结果表明：成功构建2个表达载体,通过诱导获得了分子量约为30 kD和23 kD的重组蛋白.其中,以440～570 aa片段为抗原所制备的多克隆抗体免疫印记效果较好.Western blot表明在水稻花药、愈伤组织及小穗中检测到OsAGO2表达.该研究为进一步深入探讨水稻OsAGO2基因的特性与功能奠定了基础.