Characterization And Functional Analyses Of Plasma Membrane Intrinsic Protein Family In Rice

Plant plasma membrane intrinsic proteins（PIPs）belong to a subgroup of aquaporins（AQPs）family,function as transporters of water and/or many other substances across cell membranes,and regulate many plant physiological processes such as cell metabolism,growth and development,the resistance to biotic and abiotic stresses.Recent study shows that,At PIP1;4 mediates transport of hydrogen peroxide（H₂O₂）and involves in resistance to pathogen in Arabidopsis.To understand the function of OsPIPs family in rice,the characteristics and biological functions of 11 OsPIPs were studied.The main results are as follows:1.Bioinformatic analysis indicated that there were 13 highly conserved OsPIPs in rice.However,only 11 full-length of the OsPIPs were cloned by PCR for further characteristic and functional study.2.Spatial-temporal expression pattern of OsPIPs in various rice tissues/organs was investigated.OsPIP2;6 was mainly expressed in the stem of vegetative period and the root of reproductive stage,OsPIP2;7 was mainly expressed in leaf and OsPIP2;8 was specifically expressed in germinating seeds,while eight other OsPIPs were preferentially expressed in the root,and the expression of these OsPIPs increased gradually in vegetative stage and then decreased gradually in reproductive stage.3.The response of these OsPIPs to Magnaporthe oryzae（M.oryzae）and chitin treatment were investigated.OsPIP2;2,OsPIP2;3 and OsPIP2;7 were slightly up-regulated after chitin treatment,while OsPIP2;4 was down-regulated slightly.The expression of OsPIP1;3,OsPIP2;4,OsPIP2;5,OsPIP2;6,OsPIP2;7 and OsPIP2;8 were up-regulated after inoculation,and those of OsPIP1;1,OsPIP1;2 and OsPIP2;2 were down-regulated after inoculation with M.oryzae.4.The response of OsPIPs to pathogenic bacteria was investigated by inoculation with Xanthomonas oryzae pv.oryzae（Xoo.）.The expression of OsPIP2;4,OsPIP2;5,OsPIP2;6,OsPIP2;7 and OsPIP2;8 were up-regulated after inoculation,while OsPIP1;1,OsPIP2;3,OsPIP2;4 and OsPIP2;8 were down-regulated after Xoo.inoculation.5.Three OsPIP1s were localized in the endoplasmic reticulum（ER）of rice cell,while eight OsPIP2s were localized both in the ER and plasma membrane（PM）.6.Y2H and Bi FC assays showed that the interaction among OsPIPs were diverse and selective.Three OsPIP1s interacted with each other in the ER,OsPIP1s also interacted with OsPIP2s in the PM,while not all members of the eight OsPIP2s interacted with each other.7.OsPIP2;2,OsPIP2;3,OsPIP2;4,OsPIP2;5,OsPIP2;6 and OsPIP2;7 facilitated the transport of H₂O₂in yeast,especially OsPIP2;6 with strong H₂O₂-transporting capacity.8.To study the biological function of OsPIPs,11 individual OsPIPs knockout lines were generated by CRISPR/Cas9-based gene editing system.Germination experiments showed that the germination of rice seeds was inhibited in OsPIP1;2-KO,OsPIP2;1-KO and OsPIP2;4-KO lines.Ten OsPIPs except OsPIP2;1 participated in rice growth and development at seedling stage.Nine knockout lines of OsPIPs except OsPIP1;3-KO and OsPIP2;4-KO exhibited lower rice yield,especially knockout of OsPIP2;6 significantly reduced the seed setting rate and grain yield.9.Pathogen inoculation assays showed that:knockout of OsPIP1;1 enhanced the resistance of rice to M.oryzae,while knockout of OsPIP2;6 or OsPIP2;8 reduced the resistance to M.oryzae.OsPIP1;2-KO,OsPIP1;3-KO,OsPIP2;1-KO or OsPIP2;6-KO lines became resistant to Xoo.compared with wild type plants,while OsPIP2;3-KO became susceptible to Xoo..10.Except OsPIP2;8,ten OsPIPs knockout lines failed in the tolerance of drought stress.In summary,the properties of OsPIPs family such as gene expression patterns,protein structure,subcellular localization and biological functions were systematically analyzed in this study.OsPIPs exhibit conserved structure and biological functions as plant AQPs,OsPIPs form homodimers or heterologous dimers that participate in rice growth and development,and the response to biotic and abiotic stress.This study reveals the molecular basis of the synergistic effect of OsPIPs in plant growth and development and the role in pathogen resistance and drought tolerance.Meanwhile,the important genetic materials constructed not only be useful for further elucidation of the molecular mechanism of OsPIPs’function,but also lay down a good research approach and vision for material construction for rice disease resistance breeding.