The Effect Of Signal Peptide-like On HpaXm Protein Of Xanthonamas Mechanism And Analysis Of HpaXm Transport Pathway

Harpin protein is encoded by the hypersensitive response and pathogenicity gene(hrp)of Gram-negative bacteria in the pathogen-plant interaction process,it is a class of proteins which can induce non-host plants to produce hypersensitive response(HR),and are pathogenic to host plants.At present,the detailed mechanism of action of harpin protein in the interaction between pathogens and plants is not clear.In previous reports,harpin protein can be secreted by the type III secretion system(T3SS),but in the previous work,we worked on the first 15 amino acids(LP)at the N-terminus of hpaXm from the pathogen Xanthomonas citri subsp.malvacearum(Xm)are similar signal peptides.It is speculated that hpaXm may be secreted by othersecretion systems beyond T3SS.Therefore,this paper is divided into two parts.The first part is to study the effect of LP on hpaXm secretion,localization,interaction,HR stimulation,disease resistance,plant growth and heat tolerance;the second part is on pathogens and plants during the interaction,the whole process of hpaXm from pathogen cells to plant cells was studied.The main research results obtained are as follows:1.In order to explore the effect of LP on the secretion of hpaXm,we used a genetic analysis method based on the requirement of yeast cells for invertase to confirm that LP has no secretion ability,and LP-deficient hpaXm(hpaXmALP)still has secretion ability.In addition,the secretion ability of hpaXm as a reported functional fragment was verified.The results showed that hpaXml6-59 could be secreted,hpaXm60-133 could not be secreted.Theses indicatied that the fragment affecting hpaXm secretion may be located between 16 to 59 amino acids;By comparing the difference between hpaXm and hpaXmALP with respect to the extracellular secretion,using immunocolloidal gold technology,Xm and ALP were respectively infected on cotton leaves,and it was found that hpaXmALP can still be secreted from Xm to cotton cells.Through statistical analysis of the number of colloidal gold particles in single cells of cotton after each treatment,it was found that Xm was significantly(P<0.05)greater than ALP.It shows that although hpaXm can be secreted after LP deletion,the absence of LP reduces the secretion ability of hpaXm.By comparing the difference between hpaXm and hpaXmALP in terms of localization in host cells,using Agrobacterium-mediated transient expression technology,it was found that hpaXm and hpaXmALP can both still be located in the nucleus,cytoplasm and chloroplast of cotton cells.It was found that hpaXm could be located on the cell wall,cytoplasm,nucleus,and chloroplast,but hpaXm could not be located on the cell wall when LP was missing.It shows that LP determines the positioning on the cell wall.Using Pull-down technology,mass spectrometry identification technology and yeast two-hybrid technology,six hpaXm interacting proteins were screened,which were m3(KJB79067.1,fructose-1,6-diphosphate aldolase),m5(KHG27361.1,plant aldehyde ketone reductase),m25(ABC73623.1,optical system ?),m27(AGU09699.1,Allene oxide cyclase),m29(KHG25151.1,universal stress protein)and m33(KHG04311.1,60S ribosomal L4 subunit).The yeast two-hybrid technique was used to verify the interaction between hpaXmALP and the above 6 proteins.The results showed that hpaXmALP and the above 6 proteins could interact with each other.This indicates that LP may not affect hpaXm of the interaction with the above 6 proteins.By comparing the difference between hpaXm and hpaXmALP with respect to the ability to stimulate HR,induce defense response,and promote plant growth,it was found that hpaXm and hpaXmALP stimulate HR is equivalent to the induction of defense response,but significantly(P<0.05)reduces hpaXm to promote plant growth ability.It shows that LP does not affect tthe ability to stimulate HR and induce defense responses,but reduced ability to promote plant growth.By comparing the ability to stimulate tobacco HR between hpaXm and hpaXmALP after different high temperature treatments.It was found that LP has no effect on the heat resistance of hpaXm.2.Using the principle of homologous recombination,the T3SS deletion mutant(AT3SS)was obtained by knocking out the hrcV gene in Xm,and the T2SS deletion mutant(AT2SS)was obtained by knocking out the xpsL gene in Xm.And by detecting the secretion of typical pathogenic biochemical factors in different secretion systems,to verify whether the secretion system is successfully closed,the results show that AT3SS and AT2SS are successfully constructed,and Xm is the same as other Xanthomonas,T2SS and T3SS are the main secretory pathway of pathogenic biochemical factors.3.Using immunocolloidal gold technology to infect Xm,?T2SS,AT3SS separately on cotton leaves,it was found that the presence of colloidal gold particles in the cotton cells infected by each bacterial cell,indicating that hpaXm remains can be secreted from after ?T2SS or ?T3SS.It showed that in addition to T3SS,hpaXm also has other secretion methods.Through statistical analysis of the number of colloidal gold particles in individual cotton cells after each treatment,it was found that Xm was significantly(P<0.05)greater than ?T3SS or ?T2SS.It indicated that not only T3SS exists in the secretion mode of hpaXm,but also T2SS and other ways may be secreted out of the cell.4.When detecting the location of hpaXm after each mutant infects cotton,it was found that hpaXm can be located on the vesicles of cotton cells,which implies that one of the ways that hpaXm enters cotton cells may be endocytosis of plants.Through monitoring the entire process of fluorescently labeled hpaXm being endocytosed from the extracellular into the suspension cells of model plant tobacco,it was found that hpaXm can be endocytosed into tobacco cells in a strict time and energy-dependent manner,combined with hpaXm in vesicles positioning confirmed that hpaXm can be endocytosed into tobacco cells.5.In order to explore the effect of T3SS,T2SS and hpaXm on Xm metabolism,UHPLC-Q-TOF/MS technology was used to detect the relationship between ?T3SS,?T2SS and the ?hpaXm(?hpa).The results of KEGG enrichment analysis of differential metabolites show that compared with Xm,the differential metabolites in ?T3SS,?T2SS and ?hpa are significantly(P<0.01)enriched on the ABC transporter pathway,and ?T3SS,?T2SS and ?hpa The differential metabolites in the sample were significantly(P<0.05)enriched in multiple metabolic pathways,indicating that hpaXm is closely related to the secretory pathways T2SS and T3SS.