Both Rice Aquaporin PIP1;3 And Phospholipids Regulate The Transport Of Type Ⅲ Effector PthXol And The Screening Of TALE Gene Of Xanthomonas Oryzae Pv.Oryzicola

Many gram-negative bacteria are plant or animal pathogens that use the type Ⅲsecretion system(T3SS)to transport proteins into the environment or directly into the cytoplasm of eukaryotic host cells.Type Ⅲ effector is required to interact with the recognition factor on the host cell membrane via T3SS with the help of bacterial T3 transposon protein,so as to transport from the bacterial cell to the host cytoplasm and play its toxic or non-toxic function,as shown in TALE.Rice water channel protein OsPIP1;3 has been reported in our laboratory.A TALE,namely Pthxol,is involved in the transport regulation of bacterial leaf blight.In this paper,we reported that both aquaporins and phospholipids can recognize the T3 transposon protein to regulate the transport of a T3 effector in bacterial leaf bacterial membrane.The regulatory effect of rice aquaporin on extracellular hydrogen peroxide transport to the cytoplasm was also investigated.19 TALE genes in GD41 strain of Xanthomonas oryzae pv.oryzicola(Xoc)was screened by in situ hybridization,Southern Blotting hybridization,nucleic acid dotting and other methods.TALE is not the only direct host transcriptional regulator produced by plant pathogens,but their ability to specifically transcribe and activate different host genes is unique.However,it is difficult to directly clone each TALE gene by PCR,because the pathogen contains multiple TALE genes and even different TALE genes have highly conserved repeat sequences.Therefore,the author compared several methods to screen the TALE gene of GD41 strain.Finally,19 different TALE genes were selected.By Southern Blotting hybridization and nucleic acid dotting hybridization,20 different TALE positive clones were screened from the genomic library of GD41,and 19 different TALE genes were obtained.Aquaporins are a class of Major intrinsic proteins(MIPs)that enable the transport of water and other molecules across membranes rapidly and controllably.It has been reported in our laboratory that arabidopsis water channel protein AtPIP1;4.Regulation of extracellular hydrogen peroxide transport to the cytoplasm,rice water channel protein OsPIP1;3 can regulate the transport of T3 effector.Based on this,it was speculated that rice aquaporin had an effect on the transport of hydrogen peroxide and played a regulatory role in the immune response,and OsPIP1;3 can synergistically regulate the transport of T3 effector PthXol with T3 transposon Hpa1.In addition,phosphorylated phosphatidylinositol(PI)on plant cytoplasmic membranes may also participate in plant-microbial interactions by binding to bacterial T3 transposon proteins.The T3 hydrophobic transposon protein HrpF and the hydrophilic transposon protein Hpa1 of Xoo jointly mediate the transport of TALE PthXol in Xoo strain cells to the cytoplasm of rice cells,and both are indispensable in this process.The lipid band membrane assay showed that HrpF protein could bind to PI4P located on the plasma membrane,while Hpa1 protein could not.Moreover,since HrpF relies on c-terminal sequence to bind to phospholipid receptors on the cell membrane,the obtained prokaryotic expression of HrpF key functional domain purified proteinHrpF536-803 can also bind to PI4P.The differential interaction between HrpF and PIs was confirmed by ELISA assay with specific antibody.Moreover,the binding of protein HrpF to PIs varies with the concentration of the two within a certain range.At the same time,the cya test of recombinant PXO99A strain ΔhrpFΔpthXo1/pthXo1-cya inoculated on Wormannin(inhibition of PI3K and PI4K)or LY294002(inhibition of PI3K only)inhibitor and pure water as control showed that the transfection of pthXo1 in plant cells treated with control was significantly higher than that in plant cells treated with PIKs inhibitor.however,this decrease in PthXo1 translocation is not only dependent on the interaction between HrpF and PIs,but also possible that other T3 transposon proteins can interact with PIs and jointly regulate the transport of PthXo1.Therefore,PIs in the cytoplasmic membrane of rice might recognize HrpF and promote the transport of PthXo1.