Tag Archives: infection assay

Epigenetics of trans-generational defense Induction

Some of the best evidence for environmentally induced epigenetic inheritance comes from studies of pathogen infection in A. thaliana. When infected by the common laboratory strain of the bacterial pathogen Pseudomonas syringae (DC3000), A. thaliana plants undergo extensive DNA methylation changes that regulate defense gene expression. Furthermore, some of these induced methylation changes can be transmitted to offspring, trans-generationally ‘priming’ offspring for more effective defense responses when they encounter similar pathogens.

However, plants in nature are typically subject to simultaneous infection by pathogens that induce different defense responses. The defense systems activated by different pathogens may even antagonize each other via hormonal crosstalk. The effects of such co-infection on DNA methylation patterns and trans-generational defense priming remain entirely unexplored, as does the extent of host genetic variation for these epigenetic responses.

To address these issues, we generated A. thaliana lineages with different histories of bacterial infection across generations. This framework enables several key determinations, including the specific DNA methylation changes that are induced in parents by single- versus co-infection, which of these changes are inherited by offspring, and how inherited methylation changes influence offspring defense responses when offspring are infected. To date, we have characterized the genome-wide DNA methylomes of the founding (parental) plants of these lineages, which were infected by the natural bacterial pathogens Pseudomonas syringae (Michigan strain NP29.1A) and P. viridiflava (Michigan strain RMX3.1B),separately and in combination (i.e., co-infection).

Co-infections with pathogenic and beneficial bacteria

Our preliminary experimental data reveals that coinfection has exceptionally strong impacts on pathogen performance in Arabidopsis. To test for these effects, we conducted infections consisting of a luciferase-labeled focal strain of P. viridiflava that was separately co-inoculated with each of 60 randomly chosen strains from the P. syringae complex. At 36 hours post-inoculation, luciferase activity (i.e., photon counts) in each infected plant was measured to quantify abundance of the focal strain. This was replicated in triplicate for two different focal strains. Four aspects of these results are favorable for the proposed work:

  • First, the effects of coinfection on pathogen performance are large. The mean abundance of both focal strains differed by two orders of magnitude between the most and least favorable coinfection combinations.
  • Second, these effects are highly consistent and dwarf experimental noise. The identity of the coinfecting strain explained over 70% of the variance in focal strain abundance in our experiments (linear mixed model, abundance ~ coinfecting strain + batch effect covariates), and this effect was statistically significant (P < 1e-16).
  • Third, our data indicate the potential for both costs and benefits to coinfection, depending on the identity of the coinfecting strains. In 40% of the pairwise coinfection combinations, the focal strain grew to a higher abundance than when singly inoculated; conversely, in 60% of cases, its abundance decreased relative to single infections.
  • Fourth, the two focal strains differed in how their abundance was affected by the coinfecting strains (aforementioned linear mixed model; focal strain x coinfecting strain interaction, P < 0.001). This underscores the importance of accounting for genotype x genotype interactions, as we propose to do, when predicting infection outcomes.
Figure 1. Experimental system for plant growth and infections. Above, gnotobiotic Arabidopsis in plant growth (MS) media in 24-well microplates. Below, from left: a lightly, moderately, and heavily diseased plant, 36 hours after infection with P. viridiflava strains differing in pathogenicity
Figure 2. Pairwise co-infections strongly shape pathogen performance in gnotobiotic Arabidopsis. The abundance of two luciferase-tagged strains of P. viridiflava (strain p13.G4, “b”; strain p25.A12, “c”) were measured 36 hours after co-inoculation with each of 60 different strains from the P. syringae complex, whose phylogenetic relationship is shown in “a”. Abundances of the two focal strains in each pairwise co-infection are expressed relative to their abundance in single infections. Effects on focal strain abundance are expressed as log10 units of photon counts/second.