Who we are and what we do?
Krasileva lab is an interdisciplinary group of people with a common interest in genomics and host-microbe interactions. Our vision is to combine the latest technologies (such as sequencing, synthetic biology, genome editing) with research to answer fundamental questions about genome evolution, eukaryotic innate immune systems.
In natural ecosystems, plants generate sufficient variation in their immune receptors to recognize diverse and rapidly evolving pathogens. However, in crops, this diversity is often lost, while the pathogens still evolve rapidly – resulting in disease epidemics and pandemics. Environmental factors and changing climate also affect biodiversity and host-pathogen interactions. We aim for research to directly address these challenges and to improve food security.
Below is a summary of the ongoing projects in the Krasileva lab.
How do organisms with innate immunity alone (no adaptive immunity) recognize rapidly evolving pathogens?
Unlike adaptive immunity, the innate immune system is ancient and utilizes shared principles across kingdoms.
Both plants and fungi successfully utilize it in the absence of an adaptive system to defend themselves against the same rapidly evolving pathogens that attack animals: viruses, bacteria, fungi, and insects. A key unresolved question is: how do new ligand-binding specificities arise in innate immune receptors? To answer these question we deploy comparative genomics to look at the evolution of innate immune receptors and signaling pathways.
Plants have an effective immune system: Plants rely on intracellular immune receptors called Nucleotide binding Leucine rich Repeat (NLR) proteins for pathogen recognition. The NLRs monitor and detect the presence of pathogen derived molecules. To uncover how plants track pathogens through their NLR immune receptors, we look at three main types of natural variations: 1) variation in copy number, 2) structural variation, 3) allelic variation.
There is extensive copy number variation in plant immune receptors: The NLR protein family varies 1000 fold in size across flowering plants, from 1 NLR in Wolffia to nearly 2,500 in wheat. The NLR protein family expansions and contractions and the evolution of downstream signaling pathways have been extensively investigated by PhD student Erin Baggs. (Baggs, Dagdas and Krasileva, 2017), (Baggs et al, 2020), (Michael et al, 2020)
Plant immune receptors evolve through targeted gene fusions: Previously, we have also characterized a new mechanism of generating new diversity in NLRs – gene fusions to other plant proteins that serve as ‘baits’ for the pathogen. Such NLRs with integrated domains (NLR-IDs) highlight plant proteins targeted by the pathogens and help to identify new sources of disease resistance. Going forward, we are interested to uncover the mechanisms driving such unique specificity of novel gene fusions. (Sarris et al 2016), (Bailey et al, 2018).
Some NLRs are hypervariable at their ligand binding sites: Our most recent work examined intra-species receptor diversity and identified hypervariable NLRs (hvNLRs). Using sequence information, we are now able to classify immune receptors of an organism into functional groups based on sequence information as well as predict ligand binding sites. This has been a collaboration with a structural biologist Daniil Prigozhin (LBNL). (Prigozhin and Krasileva, 2020)
Do fungi also have innate immune responses? Fungi also have NLR receptors, yet their role in fungal immunity has not beentested. Although fungi are ubiquitous and often are known for their detrimental or beneficial interactions with other organisms, surprisingly little is known about fungal immune system. Knowing that most eukaryotic organisms have NLR-mediated innate immunity, PhD student Grace Stark decided to investigate what is the basis of innate immunity in fungi. She is working with Neurospora crassa - Pseudomonas system. Strikingly, Pseudomonas is able to colonize and grow along fungal hyphae.
What about organisms that do not have NLRs? Although NLR receptors are present in most eukaryotes (including humans), they have been lost in some species, such as yeast. The work of Erin Baggs on NLR copy number variation led her to uncover several independent plant lineages that have lost most NLRs as well as an EDS1/PAD4 innate immune signaling pathway. How do these plants cope with infections? Erin has set up a new (unpublished) duckweed - Pseudomonas pathosystem and is addressing how duckweed that is surrounded by pathogens in its natural pond environment is capable of defending itself while missing several components of the canonical plant immune system.
Can we find new ways to boost immunity?
Rational design of new immune receptors: We are now transferring our knowledge of NLR evolution into novel strategies of engineering plant immune receptors. We propose a rational design of synthetic plant immune receptors by gene fusion process. We already demonstrated that the ability to form such gene fusions is a naturally occurring mechanism in all flowering plants. We aim to deploy this principle to generate synthetic plant immune receptors with new ‘baits’ against pathogen molecules. (Tamborski and Krasileva, 2020)
A postdoc, Janina Tamborski, already adopted wheat cell-based assay for activation of the immune response which will allow us to transiently test synthetic NLRs directly in wheat as if it was a model organism. She has rationally selected, cloned and tested natural NLR-IDs where the integrated part is a mimic of previously described effector targets and is currently working on their rational modifications.
Mutational genomics to enhance disease resistance: To complement our approaches with unbiased screens, we have conducted a field screen of EMS-mutagenized wheat and identified mutants with dominant enhanced disease resistance against a devastating fungal pathogen called wheat yellow (stripe) rust. Our goal is to identify mutations linked to resistance and clone genes with causative mutations as well as to characterize physiological pathways providing wheat with an immune boost. This project is led by our lab manager China Lunde Shaw.
Evolution of designer NLRs in a test tube: With the advancement of DNA synthesis and protein engineering, we are also embarking on new ways to screen for new NLR specificites using high-throughput screening systems. A postdoc, Lorena Parra, will be heading this project.
What are the mechanisms that allow eukaryotic genomes to introduce new sources of diversity in response to stress?
The extra chromosomal circular DNAs: A fundamental question in genome evolution is how genomes respond to stress. One of these responses is the shedding of extrachromosomal circular DNAs (eccDNAs) which have been implicated in cancer, aging, herbicide stress, and nutrient stress and are known to be ubiquitous in eukaryotes. EccDNAs can contain genes and can dramatically increase their copy number. However, while these eccDNAs have been observed in yeast, their role in the genome evolution of filamentous fungi, and especially plant pathogens, remains virtually unexplored. Fungal plant pathogens have two-speed genomes, with slowly evolving regions carrying housekeeping genes and rapidly evolving regions carrying effector genes that cause disease and genes that help the pathogens respond to environmental cues. However, the mechanisms of this rapid evolution remain poorly understood. Since the rapidly evolving regions of these genomes are rich in repeats and transposons and eccDNAs are thought to originate from similar regions, it follows that eccDNAs may be intimately linked to the rapid evolution of these pathogens under stress. EccDNAs were recently isolated from the rice blast fungus Magnaporthe oryzae and their role in response to stress by the pathogen is currently under investigation by PhD student Pierre Joubert.
We are also interested in genome evolution under stress in plants (especially, NLRs!). Get in touch with us if you are also interested in this topic or any other research described above. (Krasileva, 2019)
Our overarching research aims
- Uncover the natural history of host-microbe interactions through comparative genomics.
- Understand the evolution of innate immune systems.
- Understand how plant and fungal genomes change during domestication and adaptation to new environments.
- Engineer plant immunity using genome editing, mutagenesis and synthetic biology.
Recent talks by the lab
Ksenia - Catching the right mutation - enhanced disease resistance mutants in wheat protect against stripe rust and other pathogens, America Phytopathological Society, 2021
Ksenia - The highly variable plant immune receptors and how to catch them, iCAR, 2021
Our research is funded by