Principles of Carbon Catabolite Repression in the Rice Blast Fungus: Tps1, Nmr1-3, and a MATE–Family Pump Regulate Glucose Metabolism during Infection

Understanding the genetic pathways that regulate how pathogenic fungi respond to their environment is paramount to developing effective mitigation strategies against disease. Carbon catabolite repression (CCR) is a global regulatory mechanism found in a wide range of microbial organisms that ensures the preferential utilization of glucose over less favourable carbon sources, but little is known about the components of CCR in filamentous fungi. Here we report three new mediators of CCR in the devastating rice blast fungus Magnaporthe oryzae: the sugar sensor Tps1, the Nmr1-3 inhibitor proteins, and the multidrug and toxin extrusion (MATE)–family pump, Mdt1. Using simple plate tests coupled with transcriptional analysis, we show that Tps1, in response to glucose-6-phosphate sensing, triggers CCR via the inactivation of Nmr1-3. In addition, by dissecting the CCR pathway using Agrobacterium tumefaciens-mediated mutagenesis, we also show that Mdt1 is an additional and previously unknown regulator of glucose metabolism. Mdt1 regulates glucose assimilation downstream of Tps1 and is necessary for nutrient utilization, sporulation, and pathogenicity. This is the first functional characterization of a MATE–family protein in filamentous fungi and the first description of a MATE protein in genetic regulation or plant pathogenicity. Perturbing CCR in Δtps1 and MDT1 disruption strains thus results in physiological defects that impact pathogenesis, possibly through the early expression of cell wall–degrading enzymes. Taken together, the importance of discovering three new regulators of carbon metabolism lies in understanding how M. oryzae and other pathogenic fungi respond to nutrient availability and control development during infection

Comparative genomics, transcriptomics, and physiology distinguish symbiotic from free-living \u3ci\u3eChlorella\u3c/i\u3e strains

Most animal–microbe symbiotic interactions must be advantageous to the host and provide nutritional benefits to the endosymbiont. When the host provides nutrients, it can gain the capacity to control the interaction, promote self-growth, and increase its fitness. Chlorella-like green algae engage in symbiotic relationships with certain protozoans, a partnership that significantly impacts the physiology of both organisms. Consequently, it is often challenging to grow axenic Chlorella cultures after isolation from the host because they are nutrient fastidious and often susceptible to virus infection. We hypothesize that the establishment of a symbiotic relationship resulted in natural selection for nutritional and metabolic traits that differentiate symbiotic algae from their free-living counterparts. Here, we compare metabolic capabilities of 5 symbiotic and 4 free-living Chlorella strains by determining growth levels on combinations of nitrogen and carbon sources. Data analysis by hierarchical clustering revealed clear separation of the symbiotic and free-living Chlorella into two distinct clades. Symbiotic algae did not grow on nitrate but did grow on two symbiont-specific amino acids (Asn and Ser) on which the free-living strains did not grow. The use of these amino acids was exclusively affected by the presence/absence of Ca2+ in the medium, and the differences were magnified if galactose was provided rather than sucrose or glucose. In addition, Chlorella variabilis NC64A genomic and differential expression analysis confirmed the presence of abundant amino acid transporter protein motifs, some of which were expressed constitutively both axenically and within the host. Significantly, all 5 symbiotic strains exhibited similar physiological phenotypes even though they were isolated as symbionts from different host organisms. Such similarities indicate a parallel coevolution of shared metabolic pathways across multiple independent symbiotic events. Collectively, our results suggest that physiological changes drive the Chlorella symbiotic phenotype and contribute to their natural fitness. Includes Supplementary materials

Comparative genomics, transcriptomics, and physiology distinguish symbiotic from free-living \u3ci\u3eChlorella\u3c/i\u3e strains

Most animal–microbe symbiotic interactions must be advantageous to the host and provide nutritional benefits to the endosymbiont. When the host provides nutrients, it can gain the capacity to control the interaction, promote self-growth, and increase its fitness. Chlorella-like green algae engage in symbiotic relationships with certain protozoans, a partnership that significantly impacts the physiology of both organisms. Consequently, it is often challenging to grow axenic Chlorella cultures after isolation from the host because they are nutrient fastidious and often susceptible to virus infection. We hypothesize that the establishment of a symbiotic relationship resulted in natural selection for nutritional and metabolic traits that differentiate symbiotic algae from their free-living counterparts. Here, we compare metabolic capabilities of 5 symbiotic and 4 free-living Chlorella strains by determining growth levels on combinations of nitrogen and carbon sources. Data analysis by hierarchical clustering revealed clear separation of the symbiotic and free-living Chlorella into two distinct clades. Symbiotic algae did not grow on nitrate but did grow on two symbiont-specific amino acids (Asn and Ser) on which the free-living strains did not grow. The use of these amino acids was exclusively affected by the presence/absence of Ca2+ in the medium, and the differences were magnified if galactose was provided rather than sucrose or glucose. In addition, Chlorella variabilis NC64A genomic and differential expression analysis confirmed the presence of abundant amino acid transporter protein motifs, some of which were expressed constitutively both axenically and within the host. Significantly, all 5 symbiotic strains exhibited similar physiological phenotypes even though they were isolated as symbionts from different host organisms. Such similarities indicate a parallel coevolution of shared metabolic pathways across multiple independent symbiotic events. Collectively, our results suggest that physiological changes drive the Chlorella symbiotic phenotype and contribute to their natural fitness. Includes Supplementary materials

Three-year survey of abundance, prevalence and genetic diversity of chlorovirus populations in a small urban lake

Inland water environments cover about 2.5 percent of our planet and harbor huge numbers of known and still unknown microorganisms. In this report, we examined water samples for the abundance, prevalence, and genetic diversity of a group of infectious viruses (chloroviruses) that infect symbiotic chlorella-like green algae. Samples were collected on a weekly basis for a period of 24 to 36 months from a recreational freshwater lake in Lincoln, Nebraska, and assayed for infectious viruses by plaque assay. The numbers of infectious virus particles were both host- and site-dependent. The consistent fluctuations in numbers of viruses suggest their impact as key factors in shaping microbial community structures in the water surface. Even in low-viral-abundance months, infectious chlorovirus populations were maintained, suggesting either that the viruses are very stable or that there is ongoing viral production in natural hosts. Includes supplementary figure

Identification of a Chlorovirus PBCV-1 Protein Involved in Degrading the Host Cell Wall during Virus Infection

Chloroviruses are unusual among viruses infecting eukaryotic organisms in that they must, like bacteriophages, penetrate a rigid cell wall to initiate infection. Chlorovirus PBCV-1 infects its host, Chlorella variabilis NC64A by specifically binding to and degrading the cell wall of the host at the point of contact by a virus-packaged enzyme(s). However, PBCV-1 does not use any of the five previously characterized virus-encoded polysaccharide degrading enzymes to digest the Chlorella host cell wall during virus entry because none of the enzymes are packaged in the virion. A search for another PBCV-1-encoded and virion-associated protein identified protein A561L. The fourth domain of A561L is a 242 amino acid C-terminal domain, named A561LD4, with cell wall degrading activity. An A561LD4 homolog was present in all 52 genomically sequenced chloroviruses, infecting four different algal hosts. A561LD4 degraded the cell walls of all four chlorovirus hosts, as well as several non-host Chlorella spp. Thus, A561LD4 was not cell-type specific. Finally, we discovered that exposure of highly purified PBCV-1 virions to A561LD4 increased the specific infectivity of PBCV-1 from about 25–30% of the particles forming plaques to almost 50%. We attribute this increase to removal of residual host receptor that attached to newly replicated viruses in the cell lysates

Characterization of a New Chlorovirus Type with Permissive and Non-permissive Features on Phylogenetically Related Algal Strains

A previous report indicated that prototype chlorovirus PBCV-1 replicated in two Chlorella variabilis algal strains, NC64A and Syngen 2-3, that are ex-endosymbionts isolated from the protozoan Paramecium bursaria. Surprisingly, plaque-forming viruses on Syngen 2-3 lawns were often higher than on NC64A lawns from indigenous water samples. These differences led to the discovery of viruses that exclusively replicate in Syngen 2-3 cells, named Only Syngen (OSy) viruses. OSy-NE5, the prototype virus for the proposed new species, had a linear dsDNA genome of 327 kb with 44-nucleotide-long, incompletely base-paired, covalently closed hairpin ends. Each hairpin structure was followed by an identical 2,612 base-paired inverted sequence after which the DNA sequence diverged. OSy-NE5 encoded 357 predicted CDSs and 13 tRNAs. Interestingly, OSy-NE5 attached to and initiated infection in NC64A cells but infectious progeny viruses were not produced; thus OSy-NE5 replication in NC64A is blocked at some later stage of replication

Mental Disorders and Level of Resilience in Eight High-Altitude Cities of Peru during the Second Pandemic Wave: A Multicenter Population-Based Study

COVID-19 has led us to take preventive measures, such as social isolation, to reduce the high transmissibility of the disease. This could have affected the mental health of various population groups and the development of resilience as a mitigator. A cross-sectional analytical study was conducted with 700 participants from eight cities. The dependent variables were depression, anxiety, and post-traumatic stress disorder (PTSD). The independent variable was resilience. Generalized logistic regressions were used to identify the associations between the variables. The population consisted mostly of university students (65.0%); the rest of the population was distributed among workers of public or private institutions, housewives, and others (35.0%). High prevalences of anxiety (72.7%), depression (64.1%), and PTSD (15.1%) were found, as well as a median (interquartile range) resilience score of 24 points was determined. Factors associated with a high prevalence of PTSD were having lost employment and having a family member who died from COVID-19. For depression, associated factors were severe food insecurity and hypersomnia. For anxiety, associated factors were were having a deceased family member with COVID-19 and mild food insecurity. Our results show that, during the pandemic, the general population had a higher prevalence of mental disorders. In addition, anxiety was the most prevalent of the dependent variables. Special attention should be paid to the factors influencing the development of mental disorders and mental health prevention and promotion programs should be established

Impacto científico y temático de la Rev. Cuerpo Méd. HNAAA: un análisis bibliométrico, 2011-2020

Introduction: Peru has several journals about biomedical areas, but a few serial publications are visible and indexing in different global indexing media. Objective: To analyze the main publication characteristics from Revista del Cuerpo Médico Hospital Nacional Almanzor Aguinaga Asenjo (Chiclayo) based on bibliometric methods. Methods: Descriptive study that analyzes 587 articles published by the journal from 2011 to 2020, based on bibliometric methods and indicators: citations, scientific impact and co-word analysis. Results: Original articles were the highest types of document published (60%; X̅=66). Published research articles on general medicine received more impact in previous years than the recents. Most frequent terms analyzed were studies about patients from hospitals in Chiclayo city (Lambayeque) with covid-19 clinical cases.  Conclusion: Revista del Cuerpo Médico HNAAA is emerging as an important scholarly publication to do visible local, national, and international production due to health subject coverage into medical specialties.Introducción: El Perú posee un gran número revistas de áreas biomédicas, pero solo algunas han logrado visibilizar e indizar sus publicaciones en diferentes medios de indización de alcance global. Objetivo: Analizar las características de publicación de la Revista del Cuerpo Médico Hospital Nacional Almanzor Aguinaga Asenjo (Chiclayo) a partir de indicadores bibliométricos. Métodos: Estudio descriptivo que analiza 587 registros bibliográficos de los artículos publicados en la revista durante 2011-2020, a partir de indicadores de impacto científico y análisis de términos. Resultados: La mayor producción de artículos corresponde a originales (60%; X̅=66). Los artículos publicados sobre medicina general obtuvieron más impacto en años anteriores. Los temas más frecuentes fueron estudios sobre pacientes de hospitales de Chiclayo, Lambayeque con casos de covid-19. Conclusiones: La Revista del Cuerpo Médico HNAAA se perfila como una publicación seriada de importancia para visibilizar la producción nacional y local por la cobertura temática en especialidades médicas de relevancia regional que publica

GATA-Family Transcription Factors in \u3ci\u3eMagnaporthe oryzae\u3c/i\u3e

The filamentous fungus, Magnaporthe oryzae, responsible for blast rice disease, destroys around 10-30% of the rice crop annually. Infection begins when the specialized infection structure, the appressorium, generates enormous internal turgor pressure through the accumulation of glycerol. This turgor acts on a penetration peg emerging at the base of the cell, causing it to breach the leaf surface allowing its infection. The enzyme trehalose-6- phosphate synthase (Tps1) is a central regulator of the transition from appressorium development to infectious hyphal growth. In the first chapter we show that initiation of rice blast disease requires a regulatory mechanism involving an NADPH sensor protein, Tps1, a set of NADP-dependent transcriptional inhibitors and the nonconsuming interconversion of NADPH and NADP acting as signal transducer. The Tps1-dependent Nmr inhibitor proteins regulate the activity of GATA-family transcription factors that are involved in several essential aspects of the life style of the fungus. Thus, We elucidated that the GATA transcription factor Asd4 is essential for pathogenicity and is a positive regulator of appressorium development and sporulation, in contrast to the GATA transcription factor Pas1 that is a negative regulator of appressorium development and sporulation. In chapter two, we set out to understand the specific role of the GATA transcription factor Asd4. We show that Asd4 controls the activation of the Tor kinase pathway, which is essential for virulence and integrates important processes such as regulation of transcription, translation, ribosome biogenesis, trafficking and regulation of Golgi, secondary metabolism, autophagy and nitrogen metabolism. This mechanism is essential to the initiation of infection-associated autophagy during plant infection and during in planta invasion. In summary, in this study we characterized two essential regulators, Tps1 and Asd4, in M. oryzae, and we believe that understanding the regulatory mechanisms that allow the fungus to cause disease may provide new means to control the rice blast pathogen and play an important role in ensuring global food security in the future. Adviser: Richard A. Wilso