Study On The Immune Function Of Arabidopsis Lung Seven Transmembrane Receptor Family

When the plant faces the invasion of pathogenic bacteria from outside,it will respond through two molecular modes,MAMP(microorganism-related molecular pattern)and DAMP(danger-related molecular pattern)in its own immune system.MAMP is a substance produced by pathogenic bacteria,such as the bacterial flagellin flg22.DAMP is a substance produced by the plant itself,such as the danger-related peptide pep1.They are all recognized by the pattern recognition receptors PRRs on plant cell membrane and passed to the downstream,thereby amplifying the immune signal.PRRs are mainly divided into receptorlike kinases RLKs and receptor-like proteins RLPs.Most of the PRRs in plants belong to RLKs,and the number reaches more than 600.Studies have found that in plant cells,RLKs are the main source of G protein signals upstream.Typical G protein signals mediated by G protein-coupled receptors GPCRs play a very important role in animal cell signal transmission.Especially in human cells,GPCRs are the largest family of receptors,and more than 800 have been discovered so far.However,in plants,the objects and functions of typical GPCRs have not been clarified,and there are only more than 50 GPCRs candidates screened,which is far from animals.Analysis of some members of the candidates found that it has high homology and similar Lung seven transmembrane structure with orphan GPCRs in human cells named GPR107.GPR107 was discovered for the first time in human lungs,and it was found to be related to liver function regulation.The protein was initially named Lung seven transmembrane receptor 1(LUSTR1),and later confirmed as GPCRs,and was renamed GPR107.Members of plants with similar structures are called Lung seven transmembrane receptor family.Based on the fact that most of the upstream G protein signals in plants are RLKs,and the number is huge,it is speculated that RLKs in plants may play the same role as GPCRs in animals.This paper studies the model plant Arabidopsis thaliana,and explores whether the Lung seven transmembrane receptor family in Arabidopsis plays a role in plant immunity.For there has been no naming and specific research on this family before,we named the seven members found on the TAIR website as LUNG1-LUNG7.And this article clarifies the tissue expression pattern and immune-related functions of some members.(1)Tissue expression pattern of Arabidopsis Lung seven transmembrane receptor family in seedlingsIn order to explore the tissue expression pattern of the Arabidopsis Lung seven transmembrane receptor family,this study constructed the promoter sequence of this family member into the GUS stable overexpression vector.GUS reporter gene chemical staining can indicate the location of related proteins in plant tissues.In this study,LUNG1-GUS,LUNG2-GUS,LUNG4-GUS,LUNG5-GUS,LUNG6-GUS and LUNG7-GUS stable overexpression vectors were successfully constructed.LUNG1-GUS,LUNG2-GUS,LUNG4-GUS,LUNG5-GUS and LUNG6-GUS stable overexpression plants were obtained by infecting WT.Screening suitable lines for chemical staining to indicate the tissue localization of LUNG1,LUNG2,LUNG4,LUNG5 and LUNG6 in the seedlings.The results showed that LUNG1,2,4,5 and 6 were expressed in the leaves and steles.LUNG1,4,5 and6 were expressed in the hypocotyls except LUNG2,but only LUNG4 and LUNG5 were expressed in the root tip area.(2)The Arabidopsis Lung seven transmembrane receptor family single mutants have the same sensitivity to pep1 compared with WTIn order to explore whether the Lung seven transmembrane receptor family plays a role in the immune response of plants,single mutants of this family were screened for related phenotypes.Single mutants of lung1,lung4,lung5 and lung6 were purchased from the seed bank of the TAIR website,and obtained through screening.We use the typical diseaseresistant molecule pep1 in the danger-related molecular pattern(DAMP)to screen the phenotypes of single mutants.Studies have shown that pep1 treatment will shorten the root length of Arabidopsis wild-type plants WT,so we observed the changes in root length.The seedlings were germinated on 1/2MS plate for 5 days,and then transferred to pep1 plate for5 days,and the root length was statistically analyzed.Comparing the single mutant with WT,no difference in root length or any other phenotype was observed.It indicated that lung1,lung4,lung5 and lung6 single mutants had the same sensitivity to pep1 compared with WT.(3)The lung5/6 double mutant is more sensitive to pep1 compared with WTOn the basis that no immune-related phenotype was observed in the single mutant,in order to further explore whether this family is related to plant immunity,the lung1,lung4,lung5 and lung6 single mutants were hybridized,and the lung5/6 double mutant was obtained through breeding and screening.Use the same method to analyze the sensitivity changes of lung5/6.The study found that lung5/6 double mutant showed a more sensitive phenotype of root length than WT under pep1 treatment.In order to explore whether it is specifically sensitive to pep1,we observed the phenotypic changes of lung5/6 double mutant under pep2 and pep3 treatment,and found that it also showed a more sensitive phenotype of root length.To further explore whether it is a phenotype induced by pep1 treatment,the morphological changes of the root tip area of lung5/6 double mutant was observed.Studies have shown that pep1 treatment can make the WT root tip transition zone mature and differentiate in advance to form root hairs.The lung5/6 double mutant was germinated on1/2MS plate for 5 days and then transferred to pep1 plate for 24 h.Observed with the optical microscope,the morphological change of lung5/6 root tip was more severe than that of WT.In summary,it shows that lung5/6 double mutant is more sensitive than WT about pep1 treatment and plays a role in immune regulation.(4)The expression of LUNG5 in the root tip area increased in LUNG5-GUS plant after pep1 treatmentIn order to explore whether pep1 treatment affected the changes in the expression levels of LUNG5 and LUNG6,the LUNG5-GUS and LUNG6-GUS plants under pep1 treatment were observed.The LUNG5-GUS and LUNG6-GUS seedlings were germinated on 1/2MS plate for 5 days and then transferred to pep1 plate treating for 0 h,1 h,2 h,4 h,8 h and 16 h,and then photographed under an optical microscope for observation.The study found that the expression in the root tip area of the LUNG5-GUS plants increased after 8 h treatment,while the LUNG6-GUS plants did not change significantly before and after pep1 treatment.It shows that pep1 treatment can promote the expression of LUNG5 in the root tip area.(5)The more sensitive phenotype of lung5/6 double mutant root length caused by pep1 treatment requires simultaneous impaired functions of LUNG5 and LUNG6In order to further explore whether the phenotype in the lung5/6 double mutant triggered by pep1 needs the impaired function of these two genes,the LUNG6 was overexpressed in the background of lung5/6 double mutant,and OE6-lung5/6overexpression plant was obtained.The seedlings were germinated on 1/2MS plate for 5days and then transferred to pep1 plate for 4 days.The results showed that the more sensitive phenotype of root length disappeared in OE6-lung5/6 overexpression plants,showing the same root length as WT.Combining the results that lung5 and lung6 single mutants have no significant difference in root length under pep1 treatment with WT,it indicates that the more sensitive root length phenotype of lung5/6 double mutant under pep1 treatment requires the simultaneous impairment of LUNG5 and LUNG6.In summary,this article has studied the tissue expression models and the functions in plant immunity of Arabidopsis GPCRs candidate Lung seven transmembrane receptor family.It has been clarified that the simultaneous impairment of LUNG5 and LUNG6 will cause the root length more sensitive than WT under pep1 treatment.It shows that it plays a negative regulatory role in the path of pep1 regulating the shortening of root length.However,the specific regulation mechanism that causes more sensitive root length needs to be further explored and discovered.