Identification of a suitable method of inoculation for reducing background effect in mock-inoculated controls during gene expression studies in Arabidopsis thaliana.

ABSTRACT

The recent advancement in the field of transcriptome and methylome sequencing helped scientists to analyse the gene expression and epigenetic status of different genes. Several genes and their regulatory pathways have been discovered due to research into plant-microbe interactions. Previous research on plant-Agrobacterium interactions found that the method of inoculation (wounding using a syringe), resulted in altered DNA methylation of the host DNA repair gene promoters. The expression study of host defence genes revealed that the method of inoculation masked the host response to bacteria. It could be possible that these method-induced changes could interfere with various defence regulatory pathways, which otherwise would not be triggered by the bacteria alone. Hence, it would be critical to identify an appropriate method of inoculation that could provide more unambiguous interpretation of studies involving gene expression and regulation in plants under bacterial stress. The expression dynamics of two defence genes, PR1 and NPR1, under various combinations of parameters such as three different methods of inoculation, treatment with five different bacterial re-suspending solutions, and at three different post-inoculation time intervals were examined in the model plant Arabidopsis thaliana. The H2O2 and superoxide (O2-) production due to various inoculation methods and re-suspending solutions on the host was also studied. The flood inoculation method, which used sterile deionized water (SDW) to re-suspend bacteria, elicited the slightest response in mock-inoculated plants. Under this method, Agrobacterium strains carrying the GUS reporter gene were used to test bacterial infectivity. Blue sectors were found in plants infected for 24 and 48 h. PR1 and NPR1 expression were significantly altered at various time intervals after inoculation. So, for experiments involving Arabidopsis-Agrobacterium interaction with minimal background influences, such as gene expression and epigenetic analyses, the flood inoculation method using SDW as the resuspension liquid is proposed. Supplementary Information The online version contains supplementary material available at 10.1007/s12298-023-01381-x.