2019-20 Manna Food Security Fellows

 Tomer Orca

 The agricultural promise of microalgae has become widely recognized due to a unique feature-set, including fast-growth and the ability to cultivate in non-arable land. In this study, we exploit features of microalgal chloroplasts that make them suited for the expression of heterologous multi-component-pathways; these organelles contain a prokaryotic-like genome, organized in numerous polycistronic-units. Thus, the overarching goal of this project is to engineer a general platform for designing the expression of synthetic operons in microalgal chloroplasts. Such tool kit will allow the production of high-value products that require several genes; for example, essential micronutrients, clean energy resources and implementation of nitrogen-fixation pathways in algae. Our specific aim is to develop “orthogonal” activators of chloroplast transgenes that do not interact with endogenous systems, thus, having a minimal impact on host-growth.  We propose an innovative scheme that applies the deep knowledge of mechanisms by which pentatricopeptide repeat proteins (PPRs) activate chloroplast genes in land plants, to the practical problem of developing microalgae for industrial applications.

 
Our approach is based on the hypothesis that the mechanisms by which helical repeat RNA-binding proteins boost the expression of chloroplast genes is fundamentally similar in plants and algae. On this ground, we propose that this approach will transport readily to the model microalga Chlamydomonas reinhardtii and from there to other useful microalgae.  As a feasibility test, we will balance the production of three maturation proteins necessary for the assembly of a holo hydrogenase enzyme in C. reinhardtii; these three proteins will be co-expressed as an operon, rationally designed using our recently published Chloroplast Predicted Operon Database (Shahar et al., 2019, NAR) from the chloroplast genome, whilst a designer nucleus-encoded PPR protein will control their expression by binding its cognate cis-element found in the 5’UTR of each gene.  We will then introduce this system into a non-hydrogen producing algal species that is of high industrial significance. Should our approach succeed, it may establish new directions in sustainable energy research, and will potentially pave the way for the production of other lucrative products in algal chloroplasts.

Yalon Perelman

 The main goal of this project is to find ways to improve the welfare of the population in developing countries in a socially and environmentally sustainable manner. The purpose of the specific project, under which the research was carried out, is to help Andhra Pradesh (INDIA) vegetable farmers by increasing their economic well-being. To achieve this goal, initial collaborations between the research team(TAU)  and Tata Foundation were made for Israeli agro-tech companies interested in taking part in the project and enabling the technology to succeed in field experiments with local farmers on a small scale, at the initial stage. As a lab team, we're building a bridge of management and information, to establish activity and advancement based on data, analysis, and conclusions.

 My research deals with the adoption of technologies among small vegetable farmers in India while examining various technologies and knowledge through field experiments with local farmers in their private fields. Many farmers in the state of Andhra Pradesh in southeast India have been monitored by the study, which to my knowledge is almost nonexistent. Through this data collection, we can learn a little more about their considerations about what to grow and when to grow, what technology and knowledge are available to them and their structure of expenses and revenues during the growing season. All this in addition to trying to understand whether there are proven technologies that are not suitable for small farmers in India because of other variables in their environment and the growing chain, from planting stage to the sale stage. Such experiments enable us to adapt technologies and interventions by private or governmental entities to produce a relevant product and environment for these farmers to increase their economic well-being by increasing crops and making more efficient use of land while reducing the use of economic and environmental resources. Thus, we will be able to promote communities around the country to produce more food for themselves and their surroundings to achieve greater welfare and better environmental protection.

 Reem Abu Rass

 Tilapia are important farmed fish, serving as a global primary protein source, especially in the developing world. Since 2009, global massive losses of tilapia were identified. The Bacharach lab and co-workers identified the etiological agent for this disease as a novel virus, named ‘tilapia lake virus’ (TiLV). TiLV risks the food security of millions of people, to a level that warrant two special alerts by the Food and Agriculture Organization of the United Nations (FAO). Currently, no approved vaccine exists for TiLV. We found that TiLV expresses ten major proteins: one polymerase subunit (deduced by sequence homology), and nine proteins with no similarity to any other known proteins.

The identification of TiLV envelope protein(s) and their cognate receptor(s), is crucial for the development of an efficient vaccine that elicits neutralizing antibodies, breaking envelope-receptor interactions. Our bioinformatics analyses hint that the protein product of Open Reading Frame 5 (Protein5) is the envelope protein, since it has putative motifs, resembling a signal peptide, N-glycosylation sites, transmembrane domains and a RGD sequence (known to bind cellular integrins, exposed from the cell surface).

One of my research goals is to experimentally evaluate if Protein5 is indeed the envelope protein of TiLV and to investigate the envelope-receptor interactions. Using protease-sensitivity assays and immunoblotting, we demonstrated that Protein5 is exposed from TiLV virions’ surface. Next, we will try to inhibit TiLV infection by using RGD-containing peptides and a variety of new monoclonal antibodies against Protein5 (recently developed in our lab). Inhibition of TiLV infection will also be tested in fish cells lacking specific integrins (using CRISPR/Cas9 technology), as these proteins may serve as receptors for the virus.
TiLV is expected to encode a Nucleoprotein (NP) that should be expressed to high levels and coat its RNA genome. Thus, development of antibodies against TiLV’s NP should assist TiLV-detection by immunoassays. Using genetic screens for TiLV RNA-binding proteins I have identified a NP candidate and currently, am developing antibodies against it.

Altogether, these analyses should identify TiLV’s envelope-receptor interactions and nucleoproteins, and greatly assist the development of efficient means of detection and vaccination, to combat this emerging pathogen.

 Svetlana Tsizin

 Liquid Chromatography – Mass Spectrometry (LC-MS) is one of the most common and most accurate instrumentation techniques used today in the field of food safety and security. These ambient ionization instruments, however, suffer from some limitations that are absent in the new kind of LC-MS system with in-vacuum electron ionization (EI) that was developed in the research group of Prof. Aviv Amirav.  
One familiar with the GC-MS technique that is vastly used in the field of food safety and security as well, knows to appreciate NIST library-base identification of the compounds found in a chromatogram. This kind of accurate identification remains to be unreachable for the world of ambient ionization LC-MS where compounds can be considered identified only when an extremely expensive high resolution MS (HRMS) is used. Even then though the molecules’ structure remains a mystery.

In the past several years I work, under the supervision of Prof. Aviv Amirav, on the development of a novel EI-LC-MS system with supersonic molecular beams (SMB) that enables the use of the NIST library for the LC-MS analysis. In other words, the beauty of the accurate library based identification with names and structures, often even up to isomer level, can now be done in the world of LC-MS. Moreover, the system has many benefits due to the provision of a Cold EI mass spectra.

Cold EI is EI of vibrationally cold molecules in supersonic molecular beams that results in enhanced molecular ions which are often weak or missing in standard EI. Yet, due to the remaining fragmentation pattern, it is fully compatible with the NIST library and enables identification of LC eluting compound with names and structures. This kind of identification is not amenable even for HRMS when ambient ion sources are used. EI-LC-MS-SMB instrument is based on a pneumatic spray formation and its vaporization at ambient pressure, while the ionization is made in vacuum. This way it can also facilitate faster LC-MS analysis through the elimination of ion suppression effects that plague commercial LC-MS instruments. Thus, bringing EI to LC-MS is highly valuable for the field of food safety and security.

 Lee Bar-on

 The concepts of a complete plant “Internet of Things” with direct data collection
from the plant, is a novel approach. Extensive plant research is available. However, study of plants in terms of electronics and electrical conduction mechanisms are not well defined. Here would like to establish an improved understanding of the electronic conduction within the plant and deploy it for sensing and communication applications. Using this new approach, we seek to establish whether a measurable electrical change, will allow detection of biological and physiological changes within the plant. These findings will be valuable for improving technology for precision agriculture.

 My research is aimed at finding new methods to directly measure plants and define electronic behavior in order to implement functional plant sensor and improve sensor technology available for plants.
These electronic measures will allow better understanding in electrical terms and promote sensor device implementation. All these, will of course, be beneficial for food security and closer monitoring of food crops.

Sarah Ohayon

Fish spillover represent individuals exported from well-established Marine Nature Reserves (MNR) to unprotected areas and contribute to local fishery resource.  Documenting spillover magnitude and distance is important for understanding the necessary spacing of MNR networks and to quantify the benefits of MNR to surrounding habitats which is important to fishers and other stakeholders. To date, empirical studies on spillover used mainly underwater visual censuses, fish tagging or experimental fishing as methods to quantify fish export from MNR. These methods are time consuming and expensive meaning they are not suited as a routine monitoring tool.  Recently, hydroacoustic is emerging in marine conservation science, as a major monitoring tool to asses various ecological processes with conservation value. I implement, for the first time, hydroacoustic methods to study spillover from Yam Rosh Hanikra, an old and well enforced MNR located on the northern Israeli coastline.

The acoustic survey is designed to detect a gradual change in fish density or biomass from within the MNR to fished areas around it. In my research I aim to study in depth the process of fish spillover and to develop hydroacoustic as a fast, cheap and reliable monitoring tool for understanding the beneficial impact of MNR on marine ecosystems. According to the Food and Agriculture Organization (FAO), Ten percent of the world's population depends on fisheries for their livelihoods, and 4.3 billion people are reliant on fish for 15 percent of their animal protein intake. In recent decades, marine resources are declining dramatically as a result of over fishing, habitat destruction, climate change and pollution. Marine Nature Reserves (MNR) are considered an efficient tools in marine biodiversity conservation, leading to a significant increase in size, biomass, density and diversity of fish and invertebrates within their boundaries. 

In addition to their conservation goals, MNR are recognized as an effective fishery management tool, leading to a recovery of fish stocks and increasing fish yields beyond their boundaries through a process called spillover.  While fish community response to protection is well documented, empirical studies documenting the extend and distance of spillover from MNR borders are relatively limited. One of the reasons to the short of field studies is the challenge to measure spillover with the current methodological approach. Developing a new method to measure fish spillover will improve our ability to quantify the direct contribution of MNR to fishery resources.

 Shai Ran Sapir

 Listeria monocytogenes (Lm) is a foodborne human pathogen that is found in a wide variety of growth habitats such as soil, vegetables, fruits, meat, and water. Lm is the causative agent of listeriosis, a life-threatening disease in immune-compromised patients, pregnant women, elderly and undernourishment. The risk management of Lm contaminated food demands strict conditions, especially because Lm can survive and multiply in household fridge temperatures.

Most Lm pathogenic strains carry bacteriophages in their chromosomes, as prophages. These prophages are dormant, but upon stress conditions can switch into lytic production, producing infective virions that are released via bacterial lysis. Since Lm co-evolved with its prophages, the latter are well adapted to the different niches and lifestyles of Lm. These include extracellular niches as a saprophyte and intracellular niches in the mammalian host as a pathogen. When Lm lives as a saprophyte in soil or water, the prophages are classically activated under stress, lyse the bacteria and release new virions that are capable of  infecting neighbouring cells. However, when Lm infects mammalian cells, the prophages gene expression is blocked and the lytic pathway is halted (i.e., virion production and bacterial lysis do not occur). These observations suggest that there is a strong selection against virion production in the intracellular environment and a positive selection for bacteria-phage cooperation, which supports mutual survival in the mammalian niche.

My research aims to study the relationship between Lm pathogenic strains and their inhabiting prophages in the mammalian environment. I specifically ask, How prophages are coopted to the intracellular life style of their host?  And how prophages affect the virulence of Lm strains? Furthermore, using this knowledge I plan to use these prophages as a tool to kill/lyse Lm bacteria under desired conditions. Preliminary data already suggested that prophages significantly affect the pathogenicity of their host, and demonstrate a novel mechanism of bacteria-phage interaction. We foresee that these studies will open new approaches for  industrial and clinical applications.

 Chaya Mushka Fridman

 Vibrio parahaemolyticus (Vpara) is a marine bacterial pathogen that is a leading cause of acute seafood-borne gastroenteritis; it is also a major cause of Acute Hepatopancreatic Necrosis Disease (AHPND), a devastating disease that annihilates shrimp farms in Asia and America. Vpara use toxin delivery machines called type III secretion systems to manipulate eukaryotic hosts and cause disease in mammals, while they use a plasmid-acquired binary toxin to cause AHPND. Importantly, pathogenic Vpara isolates also harbor a toxin secretion apparatus called type VI secretion system 1 (T6SS1). T6SSs are found in many Gram-negative bacteria. They deliver toxins, called effectors, directly into neighboring bacterial or eukaryotic cells. Effectors may mediate either antibacterial toxicity, anti-eukaryotic toxicity, or both. Therefore, T6SSs may play a role in interbacterial competition and virulence. Notably, antibacterial T6SS effectors appear in bicistronic operons adjacent to a cognate immunity gene that protects against self-intoxication.

Our lab previously found the Vpara T6SS1 plays a role in interbacterial competition and provides Vpara with a fitness advantage under marine conditions by delivering antibacterial effectors into its neighbor competitors. Therefore, the T6SS1 plays a major role in Vpara ability to thrive in the environment. Previous reports revealed that different Vpara isolates carry diverse and dynamic T6SS1 effector repertoires. The identities of T6SS1 effectors in most Vpara isolates remain unknown.

To identify potential novel T6SS1 effectors, we utilized a comparative genomics approach. Using this methodology, we identified genes that are genetically linked to the presence of T6SS1 in Vpara genomes and can therefore encode effectors of this system. In my research, I will investigate these effector candidates, validate their T6SS1-mediated delivery, determine whether they mediate antibacterial or anti-eukaryotic activities, and decipher their mechanisms of action.

 Michale Goldberger

 Commercial fertilizer is added to agricultural fields to increase crop yield in the form of nitrate and phosphate. High consumption ratio of nitrogen, phosphorus and potassium (NPK) from fertilizer compared to the desirable ratio results in unused nutrients discharged to the water. Our research is focused on developing a cheap sensor and networks devices for low cost detection of nitrates in water. Currently, there are the following sensors for nitrates in water:
1. Optical sensors, which measure transmission or reflection of different wavelengths by the water.
2. Electrochemical sensors – ion sensitive electrochemical field effect transistors (ISFET) or an electrode based sensor whose electrical properties change due to interaction with the chemical compound we are detecting.

We propose developing a sensor to measure nitrogen-polluted water, using a microfluidic chip integrated with the sensing element and interfacing with the internet to send information about contamination to the cloud, where customers and/or government agencies can continuously monitor the water. In parallel to developing our own sensors, we will also explore commercially available sensors. Although they are usually expensive and in some cases not reliable, they will be used to build the network “from the water to the cloud” allowing us to run simple algorithms and optimize information quality (i.e. improve signal to noise ratio, data compression, preprocessing and use of metadata).

The sensor will be developed using standard microfabrication techniques and processed at the TAU Nano Science and Technology facilities. It will benefit from existing knowledge about thin film technology that exist at TAU at large and our group in particular. Additionally, the Shacham-Diamond lab at TAU has developed a similar cheap and simple sensor for detection of a specific chemical in plants. We will use the technological basis of that work– an analog potentiostat front end (AFE), analog to digital converter (ADC), and single board computer (SBC)– as a starting point for our nitrogen detector as well. The Mamane lab has expertise in analysis and detection of nitrate and experience in experimental design both in the lab and in the field.

Naama Wagner

The reserch will develop machine-learning algorithm for predicting type III effectors. Specifically, my goals in the first year will be to (1) develop machine-learning for type III effector prediction that utilizes numerous novel features that have never been used before for the same task. For example, knowledge regarding the level of expression for each ORF during infection is highly informative to predict novel effectors and I thus plan to integrate NGS expression data. I will also apply hidden Markov models to capture the motifs that allow a host protein to be recognized by type III secretion complex, which will further improve effector identification.

In parallel, we will develop a user-friendly web-server, such that other researchers can use our algorithm to predict type III effectors on their data;  (2) We will utilize our machine-learning algorithm on a large number of pathogenic bacteria, including various Acidovorax strains as well as additional pathogens (Edwardsiella anguillarum, E. tarda, E. ictaluri and Xanthomonas arboricola). We aim to identify core effectors, which are shared across many Acidovorax strains. (3) Together with the lab of Prof. Burdman (Hebrew University, Israel), we will then screen for host resistant strains against these core effectors, and detect specific resistance proteins combining genetics and genomics approaches.