Phenolic Acid Decarboxylase Participates In The Regulatory Pathway Of Indole In Pantoea Agglomerans YS19

Bacteria evolve a lot of signal regulatory mechanisms among individuals in confronting diverse environmental stresses,of which indole-based regulation attracts more attention.As a signal molecule,indole was found to be related to the formation of multicellular aggregate structures,bacterial motility and planta colonization in our previous work.The regulatory mechanism of indole is presently the hot research direction in bacteriology.To capture the key proteins in the indole regulatory pathway is significant for understanding its regulatory mechanism.This study selected endophyte Pantoea agglomerans YS19 which can form unique multicellular aggregate structure symplasmata as the model strain.A positive mutant down-regulated by indole was screened using the mTn5 transposon mutagenesis method and GusA activity method,where the expression of the reportor gene was decreased by 34.3% when indole was added.By TAIL-PCR,the mutant gene was identified as padC,encoding phenolic acid decarboxylase(PAD)to catalyze the decarboxylation of phenolic acids.The full-length of the padC sequence is 525 bp and phylogenetic analysis showed that padC is more conservative among strains in the species of P.agglomerans.It is revealed that indole significantly inhibited the expression of padC by RT-PCR.Many physiological behaviors were changed in the padC mutant.In LB medium,the mutation of padC has no significant effect on the growth and symplasmata formation of YS19.Indole promotes the symplasmata formation in both YS19 and ?padC when the concentration of indole is less than 1.5 mM.However,the mutation of padC decreases the tolerance to phenolic acids of YS19 significantly.When exogenous ferulic acid(0.2 mM)and p-hydroxybenzonic acid(6.0 mM)was added respectively,the biomass of ?padC mutant was decreased by 30.6% and 44.7% compared with the wild strain.Besides,the mutation of padC decreases the adsorption ability of YS19 to the rice root significantly.When 0.5 mM indole was added,the adsorption capacity of mutant was only 42.6% to the wild strain.These data suggested that phenolic acid decarboxylase played an important regulatory role in confronting the environmental stresses(phenolic acid stress response)and the colonization ability,although it is not essential for the normal growth.To explore the role of phenolic acid decarboxylase in the plant-endophytes association system,gnotobiotic cultivation of rice seedling was carried out on EPA medium.Inoculation of the wild strain and ?padC improved the fresh weight of the 18-d-cultivated host rice seedlings by 30.6% and 17.1%,respectively,suggesting that the mutation of padC inhibited the growth promoting effect of YS19.Regardless of the mutation of padC,indole improved the promoting effect to rice shoot of YS19.The fresh weight of the rice shoot in the exogenous indole supplementary group increased by 18.2% and 13.7%,respectively,in comparison with none supplemented groups.The colonization ability of YS19 was also inhibited by the deletion of padC.The colonization number to rice shoot and root of ?padC mutant were 18.2% and 13.7% to the wild strain when indole was added.Meanwhile,the mutation of padC inhibited the colonization ability of YS19 to rice,when the indole was added externally,the colonization number of ?padC mutant were 54.7% and 43.1% to the wild strain.This study discussed the indole regulatory pathways from the point of interactions between endophytes and the host,which enriched our knowledges of phenolic acid decarboxylase and expanded the research ideas of indole regulation.