Plant Sciences & Plant Pathology
Permanent URI for this communityhttps://scholarworks.montana.edu/handle/1/12
The Department of Plant Sciences and Plant Pathology is part of the College of Agriculture at Montana State University in Bozeman. An exciting feature of this department is the diversity of programs in Plant Biology, Crop Science, Plant Pathology, Horticulture, Mycology, Plant Genetics and Entomology. The department offers BS, MS, and Ph.D. degree program
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Item Investigating Virus–Host Interactions in Cultured Primary Honey Bee Cells(MDPI AG, 2021-07) McMenamin, Alexander J.; Parekh, Fenali; Lawrence, Verena; Flenniken, Michelle L.Honey bee (Apis mellifera) health is impacted by viral infections at the colony, individual bee, and cellular levels. To investigate honey bee antiviral defense mechanisms at the cellular level we further developed the use of cultured primary cells, derived from either larvae or pupae, and demonstrated that these cells could be infected with a panel of viruses, including common honey bee infecting viruses (i.e., sacbrood virus (SBV) and deformed wing virus (DWV)) and an insect model virus, Flock House virus (FHV). Virus abundances were quantified over the course of infection. The production of infectious virions in cultured honey bee pupal cells was demonstrated by determining that naïve cells became infected after the transfer of deformed wing virus or Flock House virus from infected cell cultures. Initial characterization of the honey bee antiviral immune responses at the cellular level indicated that there were virus-specific responses, which included increased expression of bee antiviral protein-1 (GenBank: MF116383) in SBV-infected pupal cells and increased expression of argonaute-2 and dicer-like in FHV-infected hemocytes and pupal cells. Additional studies are required to further elucidate virus-specific honey bee antiviral defense mechanisms. The continued use of cultured primary honey bee cells for studies that involve multiple viruses will address this knowledge gap.Item Transcriptome and Small RNA Profiling of Potato Virus Y Infected Potato Cultivars, including Systemically Infected Russet Burbank(MDPI AG, 2022-03) Ross, Brian; Zidack, Nina; McDonald, Rose; Flenniken, Michelle L.Potatoes are the world’s most produced non-grain crops and an important food source for billions of people. Potatoes are susceptible to numerous pathogens that reduce yield, including Potato virus Y (PVY). Genetic resistance to PVY is a sustainable way to limit yield and quality losses due to PVY infection. Potato cultivars vary in their susceptibility to PVY and include susceptible varieties such as Russet Burbank, and resistant varieties such as Payette Russet. Although the loci and genes associated with PVY-resistance have been identified, the genes and mechanisms involved in limiting PVY during the development of systemic infections have yet to be fully elucidated. To increase our understanding of PVY infection, potato antiviral responses, and resistance, we utilized RNA sequencing to characterize the transcriptomes of two potato cultivars. Since transcriptional responses associated with the extreme resistance response occur soon after PVY contact, we analyzed the transcriptome and small RNA profile of both the PVY-resistant Payette Russet cultivar and PVY-susceptible Russet Burbank cultivar 24 hours post-inoculation. While hundreds of genes, including terpene synthase and protein kinase encoding genes, exhibited increased expression, the majority, including numerous genes involved in plant pathogen interactions, were downregulated. To gain greater understanding of the transcriptional changes that occur during the development of systemic PVY-infection, we analyzed Russet Burbank leaf samples one week and four weeks post-inoculation and identified similarities and differences, including higher expression of genes involved in chloroplast function, photosynthesis, and secondary metabolite production, and lower expression of defense response genes at those time points. Small RNA sequencing identified different populations of 21- and 24-nucleotide RNAs and revealed that the miRNA profiles in PVY-infected Russet Burbank plants were similar to those observed in other PVY-tolerant cultivars and that during systemic infection ~32% of the NLR-type disease resistance genes were targeted by 21-nt small RNAs. Analysis of alternative splicing in PVY-infected potato plants identified splice variants of several hundred genes, including isoforms that were more dominant in PVY-infected plants. The description of the PVYN-Wi-associated transcriptome and small RNA profiles of two potato cultivars described herein adds to the body of knowledge regarding differential outcomes of infection for specific PVY strain and host cultivar pairs, which will help further understanding of the mechanisms governing genetic resistance and/or virus-limiting responses in potato plants.Item Longitudinal monitoring of honey bee colonies reveals dynamic nature of virus abundance and indicates a negative impact of Lake Sinai virus 2 on colony health(Public Library of Science, 2020-09) Faurot-Daniels, Cayley; Glenny, William; Daughenbaugh, Katie F.; McMenamin, Alexander J.; Burkle, Laura A.; Flenniken, Michelle L.Honey bees (Apis mellifera) are important pollinators of plants, including those that produce nut, fruit, and vegetable crops. Therefore, high annual losses of managed honey bee colonies in the United States and many other countries threaten global agriculture. Honey bee colony deaths have been associated with multiple abiotic and biotic factors, including pathogens, but the impact of virus infections on honey bee colony population size and survival are not well understood. To further investigate seasonal patterns of pathogen presence and abundance and the impact of viruses on honey bee colony health, commercially managed colonies involved in the 2016 California almond pollination event were monitored for one year. At each sample date, colony health and pathogen burden were assessed. Data from this 50-colony cohort study illustrate the dynamic nature of honey bee colony health and the temporal patterns of virus infection. Black queen cell virus, deformed wing virus, sacbrood virus, and the Lake Sinai viruses were the most readily detected viruses in honey bee samples obtained throughout the year. Analyses of virus prevalence and abundance revealed pathogen-specific trends including the overall increase in deformed wing virus abundance from summer to fall, while the levels of Lake Sinai virus 2 (LSV2) decreased over the same time period. Though virus prevalence and abundance varied in individual colonies, analyses of the overall trends reveal correlation with sample date. Total virus abundance increased from November 2015 (post-honey harvest) to the end of the almond pollination event in March 2016, which coincides with spring increase in colony population size. Peak total virus abundance occurred in late fall (August and October 2016), which correlated with the time period when the majority of colonies died. Honey bee colonies with larger populations harbored less LSV2 than weaker colonies with smaller populations, suggesting an inverse relationship between colony health and LSV2 abundance. Together, data from this and other longitudinal studies at the colony level are forming a better understanding of the impact of viruses on honey bee colony losses.Item Longitudinal monitoring of honey bee colonies reveals dynamic nature of virus abundance and indicates a negative impact of Lake Sinai virus 2 on colony health(2020-09) Faurot-Daniels, Cayley; Glenny, William; Daughenbaugh, Katie F.; McMenamin, Alexander J.; Burkle, Laura A.; Flenniken, Michelle L.Honey bees (Apis mellifera) are important pollinators of plants, including those that produce nut, fruit, and vegetable crops. Therefore, high annual losses of managed honey bee colonies in the United States and many other countries threaten global agriculture. Honey bee colony deaths have been associated with multiple abiotic and biotic factors, including pathogens, but the impact of virus infections on honey bee colony population size and survival are not well understood. To further investigate seasonal patterns of pathogen presence and abundance and the impact of viruses on honey bee colony health, commercially managed colonies involved in the 2016 California almond pollination event were monitored for one year. At each sample date, colony health and pathogen burden were assessed. Data from this 50-colony cohort study illustrate the dynamic nature of honey bee colony health and the temporal patterns of virus infection. Black queen cell virus, deformed wing virus, sacbrood virus, and the Lake Sinai viruses were the most readily detected viruses in honey bee samples obtained throughout the year. Analyses of virus prevalence and abundance revealed pathogen-specific trends including the overall increase in deformed wing virus abundance from summer to fall, while the levels of Lake Sinai virus 2 (LSV2) decreased over the same time period. Though virus prevalence and abundance varied in individual colonies, analyses of the overall trends reveal correlation with sample date. Total virus abundance increased from November 2015 (post-honey harvest) to the end of the almond pollination event in March 2016, which coincides with spring increase in colony population size. Peak total virus abundance occurred in late fall (August and October 2016), which correlated with the time period when the majority of colonies died. Honey bee colonies with larger populations harbored less LSV2 than weaker colonies with smaller populations, suggesting an inverse relationship between colony health and LSV2 abundance. Together, data from this and other longitudinal studies at the colony level are forming a better understanding of the impact of viruses on honey bee colony losses.Item Honey Bee and Bumble Bee Antiviral Defense(2018-08) McMenamin, Alexander J.; Daughenbaugh, Katie F.; Parekh, Fenali; Pizzorno, Marie C.; Flenniken, Michelle L.Bees are important plant pollinators in both natural and agricultural ecosystems. Managed and wild bees have experienced high average annual colony losses, population declines, and local extinctions in many geographic regions. Multiple factors, including virus infections, impact bee health and longevity. The majority of bee-infecting viruses are positive-sense single-stranded RNA viruses. Bee-infecting viruses often cause asymptomatic infections but may also cause paralysis, deformity or death. The severity of infection is governed by bee host immune responses and influenced by additional biotic and abiotic factors. Herein, we highlight studies that have contributed to the current understanding of antiviral defense in bees, including the Western honey bee (Apis mellifera), the Eastern honey bee (Apis cerana) and bumble bee species (Bombus spp.). Bee antiviral defense mechanisms include RNA interference (RNAi), endocytosis, melanization, encapsulation, autophagy and conserved immune pathways including Jak/STAT (Janus kinase/signal transducer and activator of transcription), JNK (c-Jun N-terminal kinase), MAPK (mitogen-activated protein kinases) and the NF-κB mediated Toll and Imd (immune deficiency) pathways. Studies in Dipteran insects, including the model organism Drosophila melanogaster and pathogen-transmitting mosquitos, provide the framework for understanding bee antiviral defense. However, there are notable differences such as the more prominent role of a non-sequence specific, dsRNA-triggered, virus limiting response in honey bees and bumble bees. This virus-limiting response in bees is akin to pathways in a range of organisms including other invertebrates (i.e., oysters, shrimp and sand flies), as well as the mammalian interferon response. Current and future research aimed at elucidating bee antiviral defense mechanisms may lead to development of strategies that mitigate bee losses, while expanding our understanding of insect antiviral defense and the potential evolutionary relationship between sociality and immune function.Item Antiviral Defense in Invertebrates(2018-08) Flenniken, Michelle L.Invertebrate organisms include vectors of human viruses (mosquitoes, sand flies), model organisms (fruit fly), insect pollinators (honey bees and bumble bees), plant virus vectors (aphids), and commercially valuable aquatic species (oysters and shrimp) that play important roles in shaping ecosystems throughout the world. Like all organisms, invertebrates are infected by viruses and have, in turn, evolved strategies to limit virus infection. There are some fundamental similarities in host defense mechanisms, including the host recognition of non-self, pathogen-associated molecular patterns (e.g., viral dsRNA) that in turn stimulate the activation of host proteins, and expression of genes required to restrict virus replication, as well as unique aspects of specific host–virus interactions that are a result of co-evolution. Invertebrate antiviral defense mechanisms include canonical immune signaling cascades (e.g., Jak/STAT, Toll, Imd), heat shock responses, apoptosis, and dsRNA-triggered responses including the sequence-specific RNA interference mechanism and a less well characterized, non-sequence-specific dsRNA mediated response.Item Temporal Analysis of the Honey Bee Microbiome Reveals Four Novel Viruses and Seasonal Prevalence of Known Viruses, Nosema and Crithidia(2011-06) Runckel, Charles; Flenniken, Michelle L.; Engel, Juan C.; Ruby, J. Graham; Ganem, Donald; Andino, Raul; DeRisi, Joseph L.Honey bees (Apis mellifera) play a critical role in global food production as pollinators of numerous crops. Recently, honey bee populations in the United States, Canada, and Europe have suffered an unexplained increase in annual losses due to a phenomenon known as Colony Collapse Disorder (CCD). Epidemiological analysis of CCD is confounded by a relative dearth of bee pathogen field studies. To identify what constitutes an abnormal pathophysiological condition in a honey bee colony, it is critical to have characterized the spectrum of exogenous infectious agents in healthy hives over time. We conducted a prospective study of a large scale migratory bee keeping operation using high-frequency sampling paired with comprehensive molecular detection methods, including a custom microarray, qPCR, and ultra deep sequencing. We established seasonal incidence and abundance of known viruses, Nosema sp., Crithidia mellificae, and bacteria. Ultra deep sequence analysis further identified four novel RNA viruses, two of which were the most abundant observed components of the honey bee microbiome (∼1011 viruses per honey bee). Our results demonstrate episodic viral incidence and distinct pathogen patterns between summer and winter time-points. Peak infection of common honey bee viruses and Nosema occurred in the summer, whereas levels of the trypanosomatid Crithidia mellificae and Lake Sinai virus 2, a novel virus, peaked in January.Item Non-specific dsRNA-Mediated Innate Immune Response in the Honey Bee(2013-10) Flenniken, Michelle L.; Andino, RaulHoney bees are essential pollinators of numerous agricultural crops. Since 2006, honey bee populations have suffered considerable annual losses that are partially attributed to Colony Collapse Disorder (CCD). CCD is an unexplained phenomenon that correlates with elevated incidence of pathogens, including RNA viruses. Honey bees are eusocial insects that live in colonies of genetically related individuals that work in concert to gather and store nutrients. Their social organization provides numerous benefits, but also facilitates pathogen transmission between individuals. To investigate honey bee antiviral defense mechanisms, we developed an RNA virus infection model and discovered that administration of dsRNA, regardless of sequence, reduced virus infection. Our results suggest that dsRNA, a viral pathogen associated molecular pattern (PAMP), triggers an antiviral response that controls virus infection in honey bees.Item A draft genome of the honey bee trypanosomatid parasite Crithidia mellificae(2014-04) Runckel, Charles; DeRisi, Joseph; Flenniken, Michelle L.Since 2006, honey bee colonies in North America and Europe have experienced increased annual mortality. These losses correlate with increased pathogen incidence and abundance, though no single etiologic agent has been identified. Crithidia mellificae is a unicellular eukaryotic honey bee parasite that has been associated with colony losses in the USA and Belgium. C. mellificae is a member of the family Trypanosomatidae, which primarily includes other insect-infecting species (e.g., the bumble bee pathogen Crithidia bombi), as well as species that infect both invertebrate and vertebrate hosts including human pathogens (e.g.,Trypanosoma cruzi, T. brucei, and Leishmania spp.). To better characterize C. mellificae, we sequenced the genome and transcriptome of strain SF, which was isolated and cultured in 2010. The 32 megabase draft genome, presented herein, shares a high degree of conservation with the related species Leishmania major. We estimate that C. mellificae encodes over 8,300 genes, the majority of which are orthologs of genes encoded by L. major and other Leishmania or Trypanosoma species. Genes unique to C. mellificae, including those of possible bacterial origin, were annotated based on function and include genes putatively involved in carbohydrate metabolism. This draft genome will facilitate additional investigations of the impact of C. mellificae infection on honey bee health and provide insight into the evolution of this unique family.Item Honey Bee Infecting “Plant Virus” with Implications on Honey Bee Colony Health(2014-02) Flenniken, Michelle L.Honey bees are eusocial insects that are commercially managed to provide pollination services for agricultural crops. Recent increased losses of honey bee colonies (averaging 32% annually since 2006) are associated with the incidence and abundance of pathogens. In their study in mBio, J. L. Li et al. [mBio 5(1):e00898-13, 2014, doi:10.1128/mBio.00898-13] share their discovery that a plant virus, tobacco ring spot virus (TRSV), replicates in honey bees and that the prevalence of this virus was high in weak colonies. Their findings increase our understanding of the role of viruses in honey bee colony losses and underscore the importance of surveying for new and/or emerging viruses in honey bees. Furthermore, their findings will pique the interest of virologists and biologists across all disciplines. The discovery that a plant virus (TRSV) replicates, spreads, and negatively affects the health of an insect host will lead to additional studies on the mechanisms of host-specific adaptation and the role of cross-kingdom infections in the transmission of this virus.
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