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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).

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Genomics of Pathogen-pathogen interactions during co-infection

Host-pathogen interactions are often viewed as a conflict between two organisms: the pathogen thrives if it can exploit its host, and the host thrives if it can fend off infection. This restricted view, however, overlooks the fact that organisms in nature are exposed to many pathogens simultaneously, which can lead to co-infection by multiple pathogen strains or species within each host [1].

Interactions between co-infecting pathogens can profoundly alter the ability of individual pathogens to invade and exploit a host, with consequences for disease progression and host fitness. For example, we have previously found that pathogen strains lacking key virulence genes proliferate better during mixed infections with more aggressive strains [3]. In general, interactions among pathogens range from antagonistic to beneficial: microbes not only compete for resources but can also mount synergistic strategies to subvert host defenses and better exploit host resources [1,2]. How pathogens maximize their performance during co-infections, and the genes underlying these strategies, is largely unknown.

We are studying co-infections using a genetic model plant species, Arabidopsis thaliana, and one of its most abundant bacterial pathogens, Pseudomonas viridiflava. Recent ecological genomics work has revealed that co-infection is remarkably common in this system: most individual Arabidopsis plants in natural settings harbor multiple virulent P. viridflava strains within their leaves [4]. Our work uses high throughput assays to answer the following questions in this system:

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
  • Do pathogen strains differ in performance depending on which other strains are present during co-infections? Our earliest results suggest this is overwhelmingly the case (Figure 1). We are therefore pursuing the genetic basis of these microbe-microbe interactions.
  • What genes, and combinations of alleles, determine co-infection outcomes? This knowledge will help us understand pathogen ecology and evolution. But it also has practical applications: it could improve our ability to model, predict, and prevent disease outbreaks in natural populations.

One of the main advantages of the Arabidopsis-Pseudomonas study system is its amenity to large-scale experiments (Figure 2). Hundreds of plants can quickly be inoculated using pipetting robot, infection outcomes (pathogen abundance) can be monitored by high-throughput DNA sequencing, and new statistical approaches in two-organism genome-wide association mapping [5] are poised to identify combinations of alleles in the pathogens’ genomes with beneficial or deleterious effects on their performance. Although this project focuses on a single plant and pathogen species, we hope to generate conceptual and statistical insights relevant to ecological, evolutionary, and biomedical models of host-enemy interactions.

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.

References

[1] Tollenaere, Charlotte, et al. “Evolutionary and epidemiological implications of multiple infection in plants.” Trends in Plant Science (2016).

[2] Abdullah, Araz S., et al. “Host–multi-pathogen warfare: pathogen interactions in co-infected plants.” Frontiers in Plant Science (2017).

[3] Barrett, Luke G., et al. “Cheating, trade‐offs and the evolution of aggressiveness in a natural pathogen population.” Ecology Letters (2011).

[4] Karasov, Talia L., et al. “Arabidopsis thaliana and Pseudomonas pathogens exhibit stable associations over evolutionary timescales.” Cell Host & Microbe (2018).

[5] Wang, Miaoyan, et al. “Two-way mixed-effects methods for joint association analysis using both host and pathogen genomes.” Proceedings of the National Academy of Sciences (2018).

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Local adaptation and the accessory genome in an endemic plant-pathogen

infected crop cultivars from the ongoing adaptation experiment

 

ABSTRACT

Genetic variation is fodder for evolution, and microbial plant-pathogens have it in spades. The Pseudomonas syringae genome is characterized by many rare “accessory” genes that co-occur with “core” genes found in all individuals. In fact, accessory genes outnumber core genes 2:1, even though accessory genes are not essential for survival. Moreover, there is tremendous variation in the gene content of P. syringae; isolates from different crop species, for example, differ in gene content by ~32% (Karasov et al. 2017). Whether these strain-specific genes have adaptive potential remains unknown; they may simply be a consequence of high rates of mutation and lateral gene transfer, even if purifying selection to remove deleterious variants is strong. Another, not mutually exclusive possibility is that accessory genes are maintained by positive selection as pathogens adapt to alternative hosts. Indeed, local adaptation has been hypothesized to explain the presence of rare alleles in P. syringae, which causes major agricultural loss in multiple crop species each year. To address these hypotheses, I have paired a set of P. syringae isolates with their original hosts of isolation. I first test for local adaptation by comparing the in planta fitness of each isolate in its own, and in each other’s, native host. Next, I ask to what degree strain-specific genes influence adaptive patterns by using Tn-seq to track the in planta gene frequencies of each pathogen over the course of infection in each host. From this combination of experiments, we will learn to what extent host ecology influences genome evolution and virulence in P. syringae; this is important not only to inform our understanding of the selective process, but also to fields concerned with the emergence and spread of infectious disease.

P. syringae transposon mutants!

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Evolution of pathogenicity and intraspecific interactions in Pseudomonas syringae

The high selective pressures involved in the “arms race” between plants and their pathogens drives rapid evolution of genes involved in immunity on the host side and virulence on the pathogen side (Alcázar et al., 2011). However, plants are not typically infected by individual pathogens: they interact with a community of inter- and intraspecifically diverse microbes that also experience competitive pressures from one another. How these interactions among microbes affect their ability to cause disease and how the host plant influences the microbial community it harbors remain open questions for investigation.

Researchers have observed that P. syringae is a common natural pathogen of A. thaliana and that resistance to P. syringae infection varies among different A. thaliana accessions (Jakob et al., 2002). Recent work has shown that  P. syringae strains isolated from A. thaliana leaf tissue are not only genetically diverse but also differ in their degree of virulence: many isolates harbor a polymorphism in the type three secretion system (T3SS), losing the ability to cause disease (Barrett et al., 2011; Kniskern et al., 2011). Such strains show increased growth in plant tissue when co-inoculated with other P. syringae isolates harboring an intact T3SS. This result suggests a model where non-pathogenic strains engage in “cheating” through taking advantage of the nutrients released from host cells infected by pathogenic strains (Barrett et al., 2011).

Works cited

Alcázar, R., Reymond, M., Schmitz, G., and de Meaux, J. (2011). Genetic and evolutionary perspectives on the interplay between plant immunity and development. Curr. Opin. Plant Biol. 14, 378–384.

Barrett, L.G., Bell, T., Dwyer, G., and Bergelson, J. (2011). Cheating, trade-offs and the evolution of aggressiveness in a natural pathogen population. Ecol. Lett. 14, 1149–1157.

Jakob, K., Goss, E.M., Araki, H., Van, T., Kreitman, M., and Bergelson, J. (2002). Pseudomonas viridiflava and P. syringae–natural pathogens of Arabidopsis thaliana. Mol. Plant Microbe Interact. 15, 1195–1203.

Kniskern, J.M., Barrett, L.G., and Bergelson, J. (2011). Maladaptation in wild populations of the generalist plant pathogen Pseudomonas syringae. Evolution 65, 818–830.