Multiple Omics Analysis Of Arabidopsis Guard Cells Respond To Pseudomonas Syringae Infection

Globally,diseases caused by essential crop pathogens have a huge negative impact on agricultural production and the environment,food production and biodiversity have been severely affected.Plants have developed complex defense mechanisms to resist infection by pathogens in the long-term evolution process,and pathogens have also co-evolved strategies to circumvent defenses,understanding the mechanism of plant-pathogen interactions will help us develop disease-resistant crops and alleviating the challenges of pathogens to global food production.Numerous studies have shown that plant pathogens use stomata as the main entrance,when microbial pathogens are found,stomatal guard cells drive stomatal closure as an innate immune response by reducing turgor pressure,On the other hand,some pathogenic species have evolved virulence factors(such as coronatine)to circumvent the innate immune system and reverse stomatal closure,allowing access to the interior of the leaf to obtain nutrients that facilitate the proliferation of pathogens.Currently,the understanding of the regulatory mechanisms of plant-pathogen interactions is incomplete.Pseudomonas syringae(Pseudomonas.syringae pv tomato,Pst)is a ubiquitous gramnegative phytopathogen that can cause damage to a variety of host crops,and has become a model pathogen for studying plant immune responses.In this study,we systematically analyzed the dynamic changes of proteome and metabolome during the interaction between stomata and Pst DC3000 by using the newly reported method of Arabidopsis guard cell extraction and isolation,The specific results are as follows:(1)The Arabidopsis guard cells enriched by tearing method and mechanical disruption method were incubated with Pst DC3000 bacterial suspension respectively,and it was observed that the guard cells enriched by both methods had responsive activity,The time of the guard cells enriched by tearing method had closed stomata was slightly later,which may be related to the carrier tape,which affected the response speed of guard cells,and both guard cells were induced by Pst DC3000 to produce coronatine to reopening after 180 min of incubated with Pst DC3000.According to the experimental results,taking into account the amount of proteome and metabolome samples and subsequent processing,the proteome selected the processing samples of 30 min,60 min and 180 min by the tearing method,and the metabolome selected the mechanical crushing method of 30 min and 180 min process the sample.(2)To determine changes in the proteome of bacterial pathogens invading guard cells,label free proteomic analysis was performed on protein samples at three time points.At 30 min of Pst DC3000 treatment,31 up-regulated differentially expressed proteins(DEPs)and 72 down-regulated DEPs were screened,115 up-regulated DEPs and 54 down-regulated DEPs were screened at 60 min,and at 180 min,the number of up-regulated DEPs screened were 60,the number of down-regulated DEPs were 109.Through functional and metabolic pathway analysis of these DEPs,we obtained important candidate proteins in the process of plant-pathogen interaction,including signal transduction-related proteins,transcription factors,cell wall proteins,proteolysis-related proteins,glutathione S-Transferases,pathogenicityrelated proteins,heat shock proteins and secondary metabolism-related proteins.Validation analysis of differentially expressed proteins by q RT-PCR showed that changes in transcripts were not consistent with changes at the protein level,and proteomic analysis was important for deciphering the molecular mechanisms of plant responses to biotic stress in response to microbial pathogens at the protein level.(3)Using a broad-targeted metabolome assay,we qualitatively and quantitatively investigated the metabolites of Arabidopsis guard cells in response to Pst DC3000 infection for30 min and 180 min.16 differential metabolites were screened in 30 min,of which 10 metabolites were significantly increased and 6 were significantly decreased;13 differential metabolites were screened in 180 min,1 was significantly increased and 12 were significantly decreased.Through the metabolic pathway analysis of these metabolites,we found that the synthesis and hydrolysis of secondary metabolites may play an important role in pathogeninduced stomatal closure for 30 min,while the reduction of aminoacyl-t RNA and amino acid content in 180 min may be related to bacterial pathogens get nutrients and evade defenses.