Functional Analysis Of HSP MoSsb1-MoSsz1-MoZuo1 Complex And Interacting Proteins Of SNARE MoSec22 In Pathogenicity Of Magnaporthe Oryzae

Rice Blast,resulting from Magnapo rthe oryzae,is one of the three major diseases in rice.It happens all around the world and causes various degrees of crop failure,which creats a severe threatening to the safety production of grain crop such as rice.At present,the main means to prevent and control of Rice Blast is to choose resistant cultivars,supplemented with chemicals simultaneously.Due to the variability of physiological races,the resistant cultivars loss its disease-resistance after a period of time,which makes controlling the Rice Blast a big challenge.The completion of the whole genome sequencing of M.oryzae in 2005 lays a important foundation,of exploring the molecular mechanisms of pathogenesis-related genes by means of molecular biology.Here are two main parts.one is the identification of three ribosome-associated chaperones.We analyze their function and the mechanism of action in pathogenesis;the other one is the screening and identification of the binding proteins of SNARE protein MoSec22 studied in our laboratory before for further exploration of their mechanisms.Chaperones are a kind of proteins that can bind and stabilize the unstable conformation of other protein,and promote the folding of nascent polypeptide chains,the degradation and assemble of polymers and the transmembrane transport of organelle proteins by controlled binding and release.The biggest family of chaperones consists of heat shock proteins(Hsp).MoMkkl has been studied by our laboratory as an important component of cell wall integrity(CWI)Mitogen-activated protein(MAP)kinases cascade.To further explore its mechanism,we looked for the binding proteins of MoMkkl by co-immunoprecipitation(co-IP)and identified MoSsbl,a member of Hsp70 family.We found MoSsbl form a complex with other members of heat shock proteins:a Hsp70 MoSszl and a Hsp40 MoZuol.This complex binds to the ribosomes and helps the folding and assemble of nascent polypeptide chains.We obtained MoSSB1,MoSSZl and MoZUO1 mutants and found that compared with the wild-type strain Guyl1,the AMosszl,ΔMossz1 and AMozuol mutants showed a significantly reduced vegetative growth and conidiation,and most of conidia were abnormal.The pathogenicity of the three mutants on rice and barley leaves was significantly reduced and we could hardly find any lesions.The three mutants showed retarded appressorium formation and aberrant turgor.In addition,MoSsbl,MoSszl and MoZuol change the resistance to the osmotic stress inducer and affect the perithecia and asci development.Furthermore,MoSsbl,MoSszl and MoZuol are all involved in the regulation of the CWI MAP kinase pathway by modulating MoMkkl biosynthesis,which indicates that MoMkkl is a potential client of MoSsbl.Our studies reveal the conserved functional mechanisms of Hsp proteins in fungi and provide new insights into how Hsp70 and Hsp40 chaperone proteins affect signal transduction pathways to impact the pathogenicity of the blast fungus.SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor)proteins are a kind of proteins functioning in vesicular trafficking,which play important roles in endocytosis and secretion.SNARE protein MoSec22 has been studied in our laboratory and are critical for the aerial hyphal growth,asexual development,endocytosis,CWI and pathogenicity of M.oryzae.But the mechanisms by which MoSec22 regulates these biological processes were unclear.To further explore the mechanisms of MoSec22 in the pathogenesis of M.oryzae,we screened their binding proteins by co-IP and yeast two-hybrid.Eventually,a plasma membrane ATPase MoPmal was identified as an interacting-protein of MoSec22.The main function of plasma membrane ATPase is to use the energy released by intracellular ATP across proton transport out of the cell.The electrochemical gradient is generated to maintain the cell membrane H+,which provides energy for a series of secondary transporters and channel protein,and lets them achieve cross membrane transport of a variety of nutrients and ions.Key players in fungal pH control are V-ATPases and the P-type proton pump Pmal.Here,we found MoPmal is required for normal functions,including vegetative growth,conidiogenesis and full pathogenicity.Importantly,we found that MoPmal co-localizes with plasma membrane and critical for maintain intercellular pH.In addition,MoPmal as a protein cargo transported by SNARE protein MoSec22,and then move on to the Golgi apparatus.After passing the Golgi,MoPmal is delivered by SNARE proteins MoSsol and MoSso2 and then arrives at the plasma membrane.