MACUB (2021) Conference

Abstract: The Food and Agriculture Organization of the United Nations estimates that between 20 and 40 percent of crop production is lost to pests worldwide each year. Plant diseases cost the global economy approximately $220 billion in crop loses annually. This blight on crops has led to a massive decrease in food supply allowing 800 million people to suffer from hunger and starvation. Pathogens and pests are the major cause of such crop losses and account for many issues that we see in plants. Bacteria, fungi, nematodes, and oomycetes are examples of different types of pathogens that lead to such high losses. These pathogens use sophisticated molecular strategies called effector proteins to sabotage the defenses of their host. Effectors from these pathogens are secreted into the interior of plant’s cells, corrupting specific organelles such as the mitochondria, nucleus, cytoplasm, and even chloroplasts to suppress host defense and cause disease. In defense of this onslaught, plants preserve their health via their immunity response, which has been found to be a cascade of events and responses. The first phase is called Pathogen-Associated Molecular Pattern Triggered Immunity (PTI) where the plant triggers a primary immune response such as releasing reactive oxygen species to stop the pathogen. If the pathogen evades this phase, it can secrete its effectors leading to Effector Triggered Susceptibility (ETS) causing the plant to become compromised or diseased. The final phase is called Effector Triggered Immunity (ETI) where the effectors are recognized by plant resistance proteins (R Proteins) leading to cell death in the affected area. Our research focuses on effectors and their effect on plant defenses. In order to understand how the pathogen effectors interact with host defense for the purpose of causing disease, we designed and executed an experiment that would allow us to test our theories. We hypothesized that effectors from oomycete plant pathogens would interfere with the expression of host defense genes that are important in plant defense hormonal pathways such as jasmonic acid and salicylic acid. We virtually tested this theory using a simulation-based software, Plant Simulation Laboratory where we identified genes that were most critical in the host. We then confirmed our virtual results through in planta experiments where we performed qRT-PCR to determine whether the effector proteins where able to suppress the host defense genes in pathogen-treated plants. Our results show that oomycete effectors Avh73 and RxL23 were unable to target the defense genes in question and did not suppress their expression. We are currently testing other host defense genes that may be the targets of these effectors in planta.

Abstract: Bacteriophages, viruses of bacteria, are the most numerous biological particles on the planet. There are an estimated 10^31 particles, at least ten times more than the estimated number of bacteria. If true, there should be at least one phage for every bacterial species, and phages should be able to be isolated from any environment inhabited by the host. In spring of 2018, a group of students isolated two strains of Enterobacter cloacae and a bacteriophage from kitchen sponges. E. cloacae is a member of an enteric group of bacteria and also a facultative anaerobe, so we wondered if we could find bacteriophages in other environments that infect these hosts. By sampling a variety of different environmental sources, we identified novel bacteriophages that infect these strains from wastewater, brackish water in an estuary, a freshwater pond, and soil from a backyard. The diversity of these environments is consistent with the resilience of the environmental range of E. cloacae. Phage Shaolin from the kitchen sponge has a genome of 51 kb, is a tailed phage of myoviridae morphology, and capable of infecting Cronobacter muytjensii. We are continuing to characterize the isolated bacteriophages and conducting host range experiments with other related strains of bacteria. All of the bacteriophages produce a hazy plaque morphology, so we are attempting to isolate lysogens. The variety of bacteriophages found in different environments reflects the environmental range of the host, and should encourage phage hunters to think outside the toilet when trying to isolate phages that infect enteric bacteria.

Abstract: Immune responses in barrier tissues are critical for protection against a host of infectious pathogens. Tissue Resident Memory T cells (TRM) are the major immune population that mediate this protection in adults by providing a potent frontline defense against previously encountered pathogens in various peripheral tissues (skin, lungs, intestines, brain, liver, salivary glands, and lymphoid organs). Despite their capacity to provide long-term enhanced protection during adulthood, we currently lack an understanding of the mechanisms of tissue-specific TRM development, maintenance, and regulation during early life that allow the formation of long-lived TRM. We performed bulk RNA-sequencing on TRM cells isolated from pediatric (0 – 10 years of age) tissue (intestine, lung, lymph nodes, and spleen) to discern the defining functional signatures and developmental stages of localized TRM subsets in early life. Gene expression analysis revealed a core TRM gene signature upregulated across all tissues, as well as tissue-specific distinctions between early life mucosal and lymphoid-derived TRM. Additionally, intestinal and lung TRM presented unique functional signatures that were dynamic over age. Intestinal TRM exhibited increasingly regulatory profiles over age, with pathway analysis revealing upregulated suppression of cell proliferation, cytotoxic molecule production, and IFNγ-mediated signaling. Importantly, we found increased expression of critical cell adhesion markers associated with TRM maturation in both lung and intestinal TRM, however intestinal TRM were characterized by a dramatic upregulation during infancy and subsequent maintenance of expression levels, whereas lung TRM upregulated maturation markers steadily over the first decade of life. We further explored gene signatures through singe-cell RNA-sequencing of infant and adult intestinal and lung tissue TRM, and identified key putative transcription factors (IKZF2, LEF1) and canonical TRM markers (ITGA1, CXCR6, LGALS1) upregulated across age. Interestingly, intestinal TRM upregulated these earlier, to a higher level, and in a greater population of cells. Taken together, these results reveal a potential biasing towards intestinal TRM development during infancy, where the bulk of antigenic challenge is faced in early life. Our analyses underscore a specialized profile of site-specific functional development in early life TRM that potentially indicates their enhanced ability to respond to the immune challenges faced in their respective tissue microenvironments.

Abstract: In recent years, the spread of resistant bacterial strain and a global pandemic led to the search for new antimicrobial agents to overcome this problem. Phthalocyanine zinc (PcZn) is a photosynthesizer that can be used in photodynamic inactivation (PDI) as it can generate reactive oxygen species (ROS) that ultimately kills the cells. This study investigates the antibacterial effect of PcZn as a PDI photosynthesizer with and without light, aerobically, and anaerobically on Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Bacillus subtilis (B. subtilis), and Staphylococcus aureus (S. aureus). Microplate assays, colony-forming unit (CFU) assay, and confocal microscopy were used to determine the antibacterial properties of PcZn. The microplate assay showed 0.012 mM PcZn inhibited bacteria in all tested conditions. The CFU results indicated that light-activated 0.2188 mM PcZn resulted in 95% or greater inhibition aerobically in all four bacteria. Furthermore, PcZn was able to inhibit bacterial growth between 9.09% to 34.98% anaerobically. In conclusion, activated PcZn could serve as an effective antibacterial agent.

Abstract: According to the USDA, approximately 40 to 50% of crops, including agronomically important crops such as corn, wheat, soybean, and cotton, are lost in the developing world due to plant disease, pests, or post-harvest losses. Plant pathogens such as bacteria, fungi, nematodes, and oomycetes infect these crops and cause crop losses in billions of dollars annually. Oomycetes such as Phytophthora infestans, P. sojae, and P. capsici, are the cause of diseases in several agronomically important crop plants such as potato, soybean, and tobacco. This loss results in the starvation of much of the world’s population. We now know that oomycetes use sophisticated molecular strategies to cause diseases. They do so by secreting proteins called effectors into the interior of host cell. In this study, we studied the relationship between oomycete effectors and several defense proteins in the host plant. We specifically investigated the relationship between the host defense protein, NPR-1, and the oomycete effector protein, RxL23. NPR-1 is a defense gene found in several plants including the model host plant, Arabidopsis thaliana. It is a critical gene that promotes the onset of Systemic Acquired Resistance or SAR in plants. NPR1 is a positive regulator of salicylic acid, a hormone that is essential for plant resistance. On the other hand, RxL23 is an oomycete effector protein which belongs to the RxLR family of secreted effector proteins. To analyze their relationship, we first performed bioinformatic characterization of both the effector and defense gene using databases such as GenBank and UniProt. Then, using a plant simulation software called PlantSimLab, we replicated the defense pathway of NPR-1 and performed virtual knockdown experiments of the genes included in this pathway. Our virtual experiments suggested that the knockdown of NPR-1 would result in the plant becoming diseased. Using this information, we predicted where the pathogen effector would target the defense pathway. To confirm our virtual results, we performed in-person experiments using qRT-PCR to determine the extent to which NPR-1 was expressed with or without pathogen infection. We concluded that the effector RxL23 was successful in inhibiting the expression of NPR-1 thereby successful in suppressing SAR in the host plant.

Abstract: Antibiotic resistance has been a growing issue of concern, especially post-pandemic. Therefore, a natural antibacterial alternative is in urgent demand. Carvacrol, the primary component in Oregano and other essential oils, is reported to exhibit antimicrobial activity on a broad range of bacterial species. The aim of this study is to evaluate the antibacterial activity against Gram-negative Pseudomonas fluorescens (P. fluorescens) and Gram-positive Staphylococcus epidermidis (S. epidermidis). P. fluorescens can be used as a surrogate for Pseudomonas aeruginosa (P. aeruginosa) which causes many nosocomial diseases including respiratory tract, urinary tract, and gastrointestinal infections while S. epidermidis is a bacterium that causes endocarditis, dermatitis, as well as other pathogenic diseases harmful to human life. Growth analysis results indicated that the 0.50% and 1% carvacrol showed significant inhibition of P. fluorescens and S. epidermidis. Colony-forming unit (CFU) assay results indicated that bacterial viability reduced when treated with 1% carvacrol. Microscopic observation was also carried out to explore the potential mechanism of the carvacrol. In conclusion, the preliminary results suggested that carvacrol could be used as a potential broad-spectrum antibacterial agent.

Abstract: Diseased crops have impacted many lives worldwide. For example, it has brought great limitations on food supply and economic deficits to the agricultural industry. To help our cause, we must increase our knowledge on plant pathogens; and the molecular strategies they use to cause disease in plants. These invaders are known for causing infection in plant host cells by increasing susceptibility. The most common infectious organisms known to cause disease in plants include fungi, oomycetes, bacteria, viruses, viroid, phytoplasmas, protozoa, nematodes, and parasitic plants. Although, plants have evolved an inmate immune system that enables them to recognize these invaders and their molecular patterns through the use of membrane receptors (PRRs and PAMPs)pathogens continue to evolve their mechanisms. This initial recognition, results in a PTI (pattern-triggered immunity) response. Which provides a broad level of basic disease resistance against a variety of microbes. To counteract the negative effects of PTI, pathogens have developed effector molecules, these are proteins which are delivered to the host apoplast or into host cells to effectively suppress immunity and cause infection. In turn, plants have developed a second type of intracellular receptors in the form of pathogen resistance proteins. These PR proteins can then trigger an ETI(effector-triggered immunity) response in the presence of an effector protein; allowing for the plant to fully recover or self- distrust. This response is accomplished by the use of ROS, or through the biosynthesis of plant hormones, such as ET (ethylene) and JA (jasmonic acid ). Effector proteins are known to suppress plant host immunity through targeting defense pathways and their critical genes, such as pathogenesis and antimicrobial related proteins. The main objective is to identify a critical gene known to activate ETI response in plants, and to conclude where in the signaling cascade might the effector protein target the host to increase susceptibility. This hypothesis was tested through the plant simulation software (PlantSimLab), which allowed for the re-construction and modeling of specific defense pathways(ET/JA pathway) found in plants such as (Arabidopsis thaliana). In addition, several knockout experiments were tested to model and identify those critical genes in the defense pathways. The results indicate that effector proteins can in fact disrupt and suppress plant immunity by targeting critical defense genes involved in the ETI response, such as PDF1.2 (DEFENSIN).