| Plants have evolved a complex immune system to cope with pathogen attack,including basal resistance activated at the site of infection and systemic acquired resistance(SAR)activated in distal bacteria-free tissues.Understanding the molecular mechanism of plant basal resistance and SAR is helpful to the improvement of crop.In the present work,isobaric labels and tandem mass tag(TMT)proteomics,metabonomics,DIA phosphoproteomics and reverse genetics were used to identify basal resistance-and SAR-associated genes,to understand the resistance mechanism of Arabidopsis in defense against the infection of Pseudomonas syringae.Numerous phosphorylated proteins related with SAR were identified by DIA phosphoproteomics,including the proteins in receptor-like protein kinase family,calcium-dependent protein kinase family,mitogen-activated protein kinase family,WRKY transcriptional factor family and MYB transcriptional factor family.A large number of metabolites associated with basal resistance and SAR in Arabidopsis were identified based on UPLC-MS/MS,an effective approach of metabolomics.Among them,multiple phenolic compounds and amino acids were found to be involved in establishment of SAR.TMT proteomics data showed that accumulation of most differentially expressed proteins associated with photosynthesis and carbon metabolism was increased in Psm inducing systemic leaves,whereas the accumulation of all the detected differentially expressed proteins associated with nitrogen metabolism was decreased in Psm inducing systemic leaves.Physiological experiments confirmed that enhanced photosynthesis,enhanced carbon metabolism and suppressed nitrogen metabolism were associated with establishment of SAR.Parallel reaction monitoring/mass spectrometry(PRM)was used to verify proteomics results in protein level.The results of PRM indicated that the data of proteomics analysis was stable and reliable.However,comparison of RT-q PCR data,proteomics data in current study and the published transcriptomics data showed that there is a low correlation between m RNA abundance and protein accumulation,indicating it is necessary to study the mechanism of biotic stress at the protein level.Numerous proteins were identified as potential components associated with basal resistance and SAR,and they could be categorized into the families of lipid transfer protein,receptor-like kinase and GA-stimulated protein,respectively.Among them,VSR6,RGP1,AT3G19010 and COX2 could be induced both in Psm-inoculated leaves and Psm-induced systemic leaves,indicating these four proteins were probably involved in basal resistance and SAR.To further identified the proteins associated with basal resistance and SAR,the proteomics data were compared with published transcriptomics data,and the result showed that 62 genes could be induced in infected leaves by avirulent and virulent P.syringae both in protein and transcription levels.In systemic leaves,three genes were found to be upregulated by avirulent and virulent P.syringae at protein and transcription levels,including VSR6,CYK8,and AT3G62370.These genes may be functionally related to basal immunity and(or)SAR.Comparison with the data of transcriptomics analysis showed that expression of VSR6,CYK8,and AT3G62370 was compromised or lost in sid2 and ald1 mutants,suggesting that they might function downstream of SA-and Pip-mediated anti-bacterial signaling pathways during SAR.To test the reliability of the proteomics data,T-DNA insertion mutants of LTPG13 and LTP5 were selected to further evaluate the roles of these two genes in plant immunity.The results showed that LTPG13 and LTP5 were involved both in basal resistance and SAR of Arabidopsis.According to the comparison results of proteomics data and transcriptomics data,phosphatidyl-inositol-anchored lipid transfer protein LTPG13 with a more clear expression pattern was chosen in subsequent in-depth functional verification experiments.The results of laser confocal microscopy showed that LTPG13-GFP was localized on the plasma membrane.RT-q PCR and GUS staining showed that LTPG13 could be induced by Psm both in local infected leaves and distal SAR-inducing systemic leaves,indicating LTPG13 is associated with basal resistance and SAR.In addition,GUS staining also showed that LTPG13 displayed a higher expression level in flowers,suggesting LTPG13 may participate in reproductive growth process of Arabidopsis.The essential role of LTPG13 in basal immunity and SAR was confirmed by RT-q PCR analysis to pathogenesis-related gene PR1,phenotypic observation,and statistical analysis to the counting data of bacteria colony.To further elucidate the effects of LTPG13 on basal resistance and systemic immunity,transcriptome sequencing was performed and the results showed that LTPG13 could regulate the expression of multiple resistant genes,including multiple transcriptional factor genes,Pip biosynthesis-related genes,SA biosynthesis-related genes,and EARLI1 subfamily genes.By RT-q PCR and content determination of SA and Pip,it was found that LTPG13 could regulate the biosynthesis of SA and Pip in pathogen-infected leaves,and regulate the biosynthesis of Pip in SAR-inducing systemic leaves,indicating LTPG13 acts in the upstream of SA and Pip signaling pathways.Consistent with this,exogenous Pip and SA could partially recover the bacterial resistance of ltpg13 mutant.Although the results of RT-q PCR and GUS staining showed that exogenous Pip and SA could induce the expression of LTPG13 in Psm ES4326 inoculated leaves and SAR-inducing systemic leaves of wild-type Col-0 plants,LTPG13 could not be activated by P.syringae in infected leaves and systemic leaves of the mutants deficient in biosynthesis of SA and Pip,indicating endogenous SA and Pip can also regulate the transcription of LTPG13,and LTPG13 and SA/Pip can form a positive feedback regulation loop in Psm ES4326 inoculated leaves,LTPG13 and Pip can form a positive feedback regulation loop in SAR-inducing systemic leaves.The long-distance SAR signal substances synthesized in pathogen-infected leaves can be transported to systemic uninfected leaves mainly via phloem.It suggests that the petiole exudate of the leaves infected by pathogens contains the long-distance SAR signal substances.In the present work,it was found that the petiole exudate of the wild-type Col-0 leaves infected by P.syringae could induce normal anti-bacterial response in ltpg13 mutant,indicating LTPG13 is unrelated to reception of the long-distance SAR signals.On the contrary,the petiole exudate of ltpg13 leaves infected by P.syringae was unable to induce the normal resistance in wild-type Col-0 plants,suggesting that LTPG13 was involved in production and transmission of the mobile signals during SAR.Additionally,the petiole exudate of ltpg13 leaves infected by P.syringae could inhibit the expression of multiple resistant genes in wild-type Arabidopsis plants,and made Col-0 plants more susceptible to P.syringae.Consistent with this,the transcriptomics data showed that expression of numerous resistant genes was suppressed in P.syringae infected leaves and uninfected systemic leaves of ltpg13,indicating that mutation of LTPG13 resulted in production of inhibitory factors.It is possible that these inhibitory factors can repress the expression of resistant genes.Taken together,these results indicate that LTPG13 plays an important role in basal resistance and SAR.In conclusion,multiple phenolic compounds and amino acids were found to be related to the establishment of SAR by metobonomics technology.Photosynthesis and carbon metabolism were enhanced in systemic leaves along with the establishment of SAR,whereas nitrogen metabolism was suppressed.Numerous proteins associated with basal resistance and SAR were identified in the present work,which provides the important informations for screening of the genes related to resistance against biotic stress.In addition,differentially expressed protein LTPG13 was found to play crucial functions in basal resistance and in establishment of SAR. |
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