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1st Objective: Identify Molecular Mechanisms of Organisms' Responses to Environmental Changes

In this Objective we will explore how environmental signals are perceived by plants and fungi, leading to transcriptional, metabolic and phenotypic changes. Our first interest looks to understand how plants detect and respond to nitrogenous nutrient signals (RG, EV and LL). We will determine the molecular mechanisms involved in the modulation of the early post-embryonic growth of Arabidopsis thaliana by N, as well as the responses during the early transition from seedling to the juvenile phase of development. Effective preparation, use, and distribution of N resources during these stages are critical to plant establishment and increased growth rate. We will address the effect of N in the context of other environmental signals (photoperiod, light or temperature) and we will analyze the interaction of these signals with endogenous hormonal pathways. Previous evidence from our group highlights the role of post-transcriptional control in N responses. We will explore the role of N-controlled mobile RNAs and their importance in N-regulated processes in plants. These efforts will allow us to test the role of RNA-based mechanisms in long-distance N regulatory networks and physiological control mechanisms.

We will also identify GRNs underlying the control of fungal physiology by light, time (circadian), and nutrients (PC, LL, RG). Although we will continue previous work mapping GRN transcriptionally controlled by the circadian rhythm in Neurospora crassa, we will focus on the phytopathogenic fungus Botrytis cinerea, an important agronomic problem worldwide. We have shown that both light and circadian regulation, modulate their virulence. Here, we will identify the molecular mechanisms underlying the interaction between light and iron homeostasis. We will also address the link between circadian components and N metabolism, as well as the circadian regulation of yet unidentified virulence factors. On the other hand, we will investigate the effect of light/time on the biocontrol capacity of Trichoderma atroviride. This will involve analyzing the extent of circadian control mechanisms in this beneficial fungus.

Finally, we want to understand the role of phenotypic diversity in adaptation to cold environments (FC, LL, RG). Yeasts are excellent model systems to dissect adaptation strategies to temperature changes. We will use various native isolates of Saccharomyces eubayanus (parental strain of the yeast hybrid used to produce lager beer) recently isolated from southern Chile. These strains have been isolated from various cold environments, which represents a great opportunity to determine the molecular basis of cryotolerance. This will allow us to also address general questions linking phenotypic variability to sequence variation and the co-evolution of cis and trans determinants. Success in this area will also allow studies of hybridization processes related to hybrid vigor.

2nd Objective: Evaluate the Effect of Environmental Cues on Interspecies Dynamics

In our 1st Objective, we follow a conventional, proven and unfailing approach for our biological subjects of interest. Here, we consider a more ambitious and realistic scenario in which organisms face environmental changes in the presence of beneficial or harmful species. One model will focus on Botrytis and Trichoderma, which have antagonistic interactions. We will examine how different environmental signals (light, N, time or temperature) modulate their interaction (resistance vs. biocontrol) and how transcriptomes reconfigure themselves when interacting under different environmental conditions. Additionally, we will compare the effect of environmental cues when Botrytis interacts with Arabidopsis versus the impact of a given cue on Arabidopsis or Botrytis separately.

It is important to highlight that we will examine how these environmental signals modify the outcome of the interaction and how the transcriptome of both organisms changes as abiotic and biotic signals occur simultaneously. A similar approach will be carried out to understand at the phenotypic and molecular level how the beneficial interaction between Trichoderma atroviride and Arabidopsis roots is modulated by the environment. Our goal is to infer GRNs that can explain the complexity of responses evoked when biotic and abiotic signals coexist. Based on the phenotypic and molecular data obtained, we will identify candidate genes that regulate the outcome of plant-fungus and fungus-fungus interactions. These genes will be tested in our 3rd Objective and their behavior in the presence of Arabidopsis will be compared. Published data have shown the existence of small RNA (sRNA) communication between Botrytis and Arabidopsis.

We will explore these observations, both in the context of Botrytis genetic variability and in relation to mechanisms of RNA mobility. We will also explore a two-way communication through RNA in a beneficial interaction using the Arabidopsis – T. atroviride system. Importantly, we will analyze the role of sRNAs in the Botrytis-Trichoderma interaction, to understand the effect of social sRNAs on fungus-fungus dynamics. Such information can help design new biocontrol strategies, providing beneficial and antagonistic fungi with personalized sRNAs.

3rd Objective: Design and development of molecular loops and analysis of transcriptional environmental memory

The implementation of adjustable orthogonal transcriptional switches allows controlled perturbation of transcriptional nodes of interest in plants or fungi, allowing to test hypotheses and models derived from the 1st and 2nd  Objectives. In addition to standard reverse genetic approaches, we will develop new synthetic controllers that process light information (optogenetic switches) or molecular compounds (nitrate signal, quorum sensing signals), for example for the design of rewired transcriptional networks or the addition of synthetic circuits as coherent/incoherent positive feedback loops.

This way, we will analyze the role of molecular loops in the integration of environmental changes in a persistent way (molecular memory) beyond what is known for circadian rhythms. As a proof of concept, we developed an optogenetic circuit that enables N. crassa to behave like a “living canvas”. Images projected onto a fungal layer, induce bioluminescent responses that faithfully recreate the original image. By connecting this optogenetic circuit with the circadian network of Neurospora, the fungus reproduces in subsequent days -circadianly- the image “seen” originally, creating an eidetic (photographic) memory (see Annexes).

This phenomenon may bring on new evidence on phased responses and cell communication, allowing us to dissect the mechanisms that modulate transcriptional memory to discrete environmental perturbations. Using a similar strategy, we will explore whether other natural (or modified) transcriptional loops show comparable temporal memory to discrete or repeated stimuli (eg. in response to transient nitrate treatments or temperature changes). How these loops interact/intersect and activate exit pathways is critical to this Objective.

4th Objective: Implementation of open source technologies and promotion of open science

iBio is committed to making a lasting impact in life sciences education, capacity building, and public engagement. Although this may seem like an extension or training activity, we consider this to be a defining objective of our Institute that will contribute substantially to furthering our 1st to 3rd research objectives.

We plan to create a platform of open-source technologies for plant/fungal systems and synthetic biology. This implies the creation of open source software, hardware and wetware resources for the efficient construction and testing of genes, new tools for fluorescent visualization in plants and fungi, among others. The arrival of customizable manufacturing tools such as 3D printers and laser cutting machines, combined with free software and easy-to-program microcontrollers, will enable low-cost self-production of scientific devices. We intend to build on and promote this “do-it-yourself” spirit that has given rise to a diverse community that designs, builds, and shares a variety of low-cost equipment around the world.

We also plan to develop tools to create, for example, open source microscopes for plants and fungi combining low-cost optics and color cameras, over-the-counter electronics, open source microcontrollers, and parametric design of 3D printed parts. In addition, we will generate tools for the rapid manufacture of DNA and an open library of genetic components (for example, fluorescent markers and inducers) for the study and engineering of biological processes of interest, such as signaling by nitrogenous nutrients or morphogenesis in the plant system Marchantia polymorpha. These resources are key and represent a comprehensive platform for research and training in synthetic and systems biology in plants and fungi. It should be noted that these resources will be shared and accessible through online repositories under open source licenses, creative commons and the OpenMTA.

Finally, we will host hands-on workshops across the country that will promote hardware fabrication, programming, parametric modeling with 3D printers, and DNA fabrication, helping to equip low-income labs and build capacity, train human capital, and develop a local community focused in open-source technologies.