| Bacterial blight is an important bacterial disease in rice cultivation.The effector protein secreted by Xanthomonas oryzae is very important for the occurrence of the disease.This paper introduces the mechanism of PthXo1 transport from two aspects,starting with rice aquaporin and rice nuclear importin protein,and clarifies the regulatory role of rice aquaporin and nuclear importin protein on PthXo1 transport in the process of rice PthXol transport from cytoplasm to rice nucleus.Gram-negative plant pathogenic bacteria mainly transfer toxic or non-toxic effector proteins to host cells through type ? secretory system,which leads to a series of resistance or susceptibility interactions.Many plant pathogens rely on secreting toxic TALE proteins into plant cells to activate the expression of host plant susceptible genes and cause plant disease.This process depends on the binding of TALE to specific elements on the promoter of the host target gene in the plant nucleus.The effector protein TALEs can be located in the nucleus of the host plant and bind to the promoter region of the target gene to play its role in transcriptional regulation.Aquaporin is a membrane channel protein that can promote the transport of water and neutral small molecules across the biofilms of most biomass.Water channel proteins in rice play an important role in the process of rice infection by Xoo.In this study,it was found that OsPIP1;3,a member of the OsPIP1 family,could affect the colonization of Xoo on rice leaves and promote the transport of TALE effector proteins.After pre-screening,it was found that there was an interaction between harpin protein and aquaporin in rice.1 The mechanism of rice aquaporin regulating effector PthXol transportAquaporin is a membrane channel protein that promotes the transport of water and neutral small molecules across biofilms.On the basis of previous studies of our group,it has been confirmed that OsPIP1;3,a member of rice aquaporin family,interacts with harpins protein hpal of Xoo.However,the specific interaction between Hpa1Xoo-OsPIP1;3 and its biological function has not been reported yet.In this paper,we determined the transport of PthXo1,the pathogenic phenotype of hpal and OsPIP1;3 on the constructed silent lines,overexpression lines and TALEN knockout mutants,and further detected the expression of Os8N3,the target gene of PthXo1,in rice leaves by fluorescence quantitative method to explore the regulatory mechanism of different OsPIP1;3 transgenic lines in resistance to Xoo paddy rice bacterial blight.The results showed that the pathogenicity of PXO99A was determined by inoculating wild type Nipponbare and OsPIP1;3 overexpression,OsPIP1;3 knockout or silencing lines to determine the pathogenicity of waterlogged disease spots.Compared with wild-type strain PXO99A,the symptoms of watery spot caused by hpal gene deletion mutants in different OsPIP1:3 transgenic lines were significantly weaker than those in the control.We used the traditional adenylate cyclase reporting system to further detect the transport process of PthXo1,the main virulence TAL effector protein of PXO99A.The translocation process of PthXol-cya fusion protein from bacterial cells to rice cytoplasm was further clarified.In this study,in addition to determining their biological functions.we further analyzed their key interaction domains,and constructed rice lines with interchangeable LE domains of OsPIP1;3 and OsPIP1;1 respectively by using rice tungro bacilliform virus mediated overexpression system.The transport of PthXol was determined on different domain replacement lines,and it was clarified that the key interaction site between Hpa1Xoo-OsPIP1;3 and OsPIP1;3 was the LE domain of OsPIP1;3.In summary,the interaction between Hpa1Xoo-OsPIP1;3 and TAL affects the transport of TAL effector protein PthXol to rice cells,which regulates the interaction between rice aquaporins and Xanthomonas oryzae,and affects the pathogenicity of Xoo.2 Mechanism of transnuclear transport of TAL effector PthXolAmong the effector proteins secreted by Xoo,a class of effectors called TAL effectors can be located in the host nucleus and transcribed by binding to the promoter region of the target gene,thus causing host disease susceptibility or resistance.In this process.the nucleocytoplasmic transport of TALEs is a key step in whether it can play a regulatory role.The rice nuclear importin proteins(16 rice nuclear importin related proteins)interacting with TAL effector PthXol were screened by yeast two-hybrid and bipartite fluorescence complementarity test.The results showed that effector PthXol could interact with rice nuclear importin proteins OsIMPala and OsIMPalb.In order to further study the biological significance of their interaction,the overexpression mutants of Oslmpa1a and OsImpa1b were constructed by using rice tungro bacilliform virus mediated overexpression system,and their pathogenicity was determined.The results showed that overexpression of OsImpa1a and OsImpa1b could promote the virulence of TAL effector PthXo1 in rice.Tobacco fragility virus system was used to silence the homologous gene NbImpa of OsImpa in tobacco.It was found that NbImpa affected the growth of tobacco and aggravated the allergic reaction caused by Xoo in non-host tobacco.It is further suggested that OsIMP?1a and OsIMP?1b play an important role in the transnuclear transport process of PthXo1.This transport process is very important for the expression of targeted susceptible genes induced by Xoo.3 Construction and Identification of Oslmpa1a and Oslmpa1b transgenic RiceIn the nuclear importin protein family of rice,it has been confirmed that two proteins of rice IMPa family can interact with TAL effector PthXo1 of Xoo.In this paper,the CRISPR-Cas9 gene editing skeleton vector was successfully constructed by CRISPR-Cas9 technology,and the mutants of Oslmpala and OsImpalb were successfully obtained.The homozygous plants of T1 generation were identified by Sanger,and the gene editing efficiency was further evaluated.During the reproductive period,compared with the wild-type rice Nipponbare,the plant of the mutant was obviously shorter,and OsIMPala and OsIMPalb of rice would affect the growth of rice,especially the plant height.The results of pathogenicity test showed that the deletion of OsImps affected the virulence of Xanthomonas to rice and the expression of OsSWEET11 the target gene of effector PthXo1.By measuring the transport of effector PthXol on the deletion mutant,it was further explained that the transport of TAL effector PthXo1 was affected by rice nuclear importin related OsIMP?1a and OsIMPalb.Rice nuclear importin protein is very important for the transport of effector PthXo1 from cytoplasm to rice nucleus.4 A study on the structural basis of the interaction between OsIMPa and PthXo1OsIMP? is an important member of the nuclear importin protein family.As a nuclear transport receptor,it plays a very important role in the process of protein entry into the nucleus.Nucleoplasmic transport is an important process in eukaryotes.On the basis of previous studies,we have confirmed the interaction between OsIMP?1a and OsIMPalb and PthXo1,but the structural mechanism of the interaction is not clear.In this paper,the fragments of OsIMP? and PthXo1 were expressed in prokaryotic cells and screened by Pull down test in vitro.The results showed that the full length of OsIMP?1a and N72 and ? IBB of OsIMP? family could interact with PthXo1 C220,and the full length of OsIMPa lb and OsIMP? 1b C76 could interact with PthXo1 C220.The screened interaction protein was co-purified by anion exchange and gel filtration chromatography,and the high purity composite protein was further obtained.Through the exploration of the crystal growth conditions of the composite protein,it was found that the condition Crystal Screen I could grow crystals,in order to explore the conditions suitable for the growth of high-quality crystals of the composite protein.In this paper,the interaction between rice aquaporin and nuclear importin protein was screened for the transport of effector PthXo1 respectively,and the regulation mechanism of Hpa1Xoo-OsPIP1;3 interaction on PthXo1 transport was clarified,and the mechanism of PthXo1 transport by rice nuclear importin protein in the process of effector PthXo1 transport from cytoplasm to rice nucleus was confirmed. |
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