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Open Access Publications from the University of California

The goals of the Department of Microbiology and Plant Pathology are to conduct research on the basic biology of plant pathogens and microbes, to develop methods for the management of microbial diseases of plants and other organisms, to provide a quality education to our students; and be a repository of expert advice on plant diseases and microbiology to the citizens of California and the world.

Our department has its roots in the Citrus Experiment Station, which was established in Riverside in 1905. Our department is also the basis of the International Organization of Citrus Virologists (IOCV). IOCV was formed during the first international conference on citrus virus diseases held at Riverside in 1957. Although the department has maintained strength in the study of diseases of citrus, the scope has expanded to include concentrations in numerous other plant diseases as well as many sub-disciplines of microbiology. Represented among our faculty are experts in the fields of genetics, genomics, bioinformatics, molecular biology, cell biology, biochemistry, ecology, evolutionary biology, and traditional aspects of disease control. Many faculty members have close interactions with industry representatives, advisors, and policy makers throughout California and worldwide. This is critical to applied research for identifying emerging and common plant diseases and microbes, and developing innovative management programs based on ecological and epidemiological approaches.

We invite you to explore the research programs of our world-class faculty, our critical work in cooperative extension, and the graduate and undergraduate programs that we sponsor.

Cover page of Transcriptional Analysis of <i>Coccidioides immitis</i> Mycelia and Spherules by RNA Sequencing.

Transcriptional Analysis of Coccidioides immitis Mycelia and Spherules by RNA Sequencing.

(2021)

Coccidioides immitis and C. posadasii are dimorphic fungi that transform from mycelia with internal arthroconidia in the soil to a tissue form known as a spherule in mammals. This process can be recapitulated in vitro by increasing the temperature, CO2 and changing other culture conditions. In this study, we have analyzed changes in gene expression in mycelia and young and mature spherules. Genes that were highly upregulated in young spherules include a spherule surface protein and iron and copper membrane transporters. Genes that are unique to Coccidioides spp. are also overrepresented in this group, suggesting that they may be important for spherule differentiation. Enriched GO terms in young spherule upregulated genes include oxidation-reduction, response to stress and membrane proteins. Downregulated genes are enriched for transcription factors, especially helix-loop-helix and C2H2 type zinc finger domain-containing proteins, which is consistent with the dramatic change in transcriptional profile. Almost all genes that are upregulated in young spherules remain upregulated in mature spherules, but a small number of genes are differentially expressed in those two stages of spherule development. Mature spherules express more Hsp31 and amylase and less tyrosinase than young spherules. Some expression of transposons was detected and most of the differentially expressed transposons were upregulated in spherules.

Cover page of Contributions of Ubiquitin and Ubiquitination to Flaviviral Antagonism of Type I IFN.

Contributions of Ubiquitin and Ubiquitination to Flaviviral Antagonism of Type I IFN.

(2021)

Flaviviruses implement a broad range of antagonism strategies against the host antiviral response. A pivotal component of the early host response is production and signaling of type I interferon (IFN-I). Ubiquitin, a prevalent cellular protein-modifying molecule, is heavily involved in the cellular regulation of this and other immune response pathways. Viruses use ubiquitin and ubiquitin machinery to antagonize various steps of these pathways through diverse mechanisms. Here, we highlight ways in which flaviviruses use or inhibit ubiquitin to antagonize the antiviral IFN-I response.

The genome of Geosiphon pyriformis reveals ancestral traits linked to the emergence of the arbuscular mycorrhizal symbiosis.

(2021)

Arbuscular mycorrhizal fungi (AMF) (subphylum Glomeromycotina)1 are among the most prominent symbionts and form the Arbuscular Mycorrhizal symbiosis (AMS) with over 70% of known land plants.2,3 AMS allows plants to efficiently acquire poorly soluble soil nutrients4 and AMF to receive photosynthetically fixed carbohydrates. This plant-fungus symbiosis dates back more than 400 million years5 and is thought to be one of the key innovations that allowed the colonization of lands by plants.6 Genomic and genetic analyses of diverse plant species started to reveal the molecular mechanisms that allowed the evolution of this symbiosis on the host side, but how and when AMS abilities emerged in AMF remain elusive. Comparative phylogenomics could be used to understand the evolution of AMS.7,8 However, the availability of genome data covering basal AMF phylogenetic nodes (Archaeosporales, Paraglomerales) is presently based on fragmentary protein coding datasets.9Geosiphon pyriformis (Archaeosporales) is the only fungus known to produce endosymbiosis with nitrogen-fixing cyanobacteria (Nostoc punctiforme) presumably representing the ancestral AMF state.10-12 Unlike other AMF, it forms long fungal cells ("bladders") that enclose cyanobacteria. Once in the bladder, the cyanobacteria are photosynthetically active and fix nitrogen, receiving inorganic nutrients and water from the fungus. Arguably, G. pyriformis represents an ideal candidate to investigate the origin of AMS and the emergence of a unique endosymbiosis. Here, we aimed to advance knowledge in these questions by sequencing the genome of G. pyriformis, using a re-discovered isolate.

Cover page of Balancing Positive and Negative Selection: <i>In Vivo</i> Evolution of Candida lusitaniae <i>MRR1</i>.

Balancing Positive and Negative Selection: In Vivo Evolution of Candida lusitaniae MRR1.

(2021)

The evolution of pathogens in response to selective pressures present during chronic infections can influence their persistence and virulence and the outcomes of antimicrobial therapy. Because subpopulations within an infection can be spatially separated and the host environment can fluctuate, an appreciation of the pathways under selection may be most easily revealed through the analysis of numerous isolates from single infections. Here, we continued our analysis of a set of clonally derived Clavispora (Candida) lusitaniae isolates from a single chronic lung infection with a striking enrichment in the number of alleles of MRR1 Genetic and genomic analyses found evidence for repeated acquisition of gain-of-function mutations that conferred constitutive Mrr1 activity. In the same population, there were multiple alleles with both gain-of-function mutations and secondary suppressor mutations that either attenuated or abolished the constitutive activity, suggesting the presence of counteracting selective pressures. Our studies demonstrated trade-offs between high Mrr1 activity, which confers resistance to the antifungal fluconazole, host factors, and bacterial products through its regulation of MDR1, and resistance to hydrogen peroxide, a reactive oxygen species produced in the neutrophilic environment associated with this infection. This inverse correlation between high Mrr1 activity and hydrogen peroxide resistance was observed in multiple Candida species and in serially collected populations from this individual over 3 years. These data lead us to propose that dynamic or variable selective pressures can be reflected in population genomics and that these dynamics can complicate the drug resistance profile of the population.IMPORTANCE Understanding microbial evolution within patients is critical for managing chronic infections and understanding host-pathogen interactions. Here, our analysis of multiple MRR1 alleles in isolates from a single Clavispora (Candida) lusitaniae infection revealed the selection for both high and low Mrr1 activity. Our studies reveal trade-offs between high Mrr1 activity, which confers resistance to the commonly used antifungal fluconazole, host antimicrobial peptides, and bacterial products, and resistance to hydrogen peroxide. This work suggests that spatial or temporal differences within chronic infections can support a large amount of dynamic and parallel evolution and that Mrr1 activity is under both positive and negative selective pressure to balance different traits that are important for microbial survival.

Cover page of Datura genome reveals duplications of psychoactive alkaloid biosynthetic genes and high mutation rate following tissue culture

Datura genome reveals duplications of psychoactive alkaloid biosynthetic genes and high mutation rate following tissue culture

(2021)

Background

Datura stramonium (Jimsonweed) is a medicinally and pharmaceutically important plant in the nightshade family (Solanaceae) known for its production of various toxic, hallucinogenic, and therapeutic tropane alkaloids. Recently, we published a tissue-culture based transformation protocol for D. stramonium that enables more thorough functional genomics studies of this plant. However, the tissue culture process can lead to undesirable phenotypic and genomic consequences independent of the transgene used. Here, we have assembled and annotated a draft genome of D. stramonium with a focus on tropane alkaloid biosynthetic genes. We then use mRNA sequencing and genome resequencing of transformants to characterize changes following tissue culture.

Results

Our draft assembly conforms to the expected 2 gigabasepair haploid genome size of this plant and achieved a BUSCO score of 94.7% complete, single-copy genes. The repetitive content of the genome is 61%, with Gypsy-type retrotransposons accounting for half of this. Our gene annotation estimates the number of protein-coding genes at 52,149 and shows evidence of duplications in two key alkaloid biosynthetic genes, tropinone reductase I and hyoscyamine 6 β-hydroxylase. Following tissue culture, we detected only 186 differentially expressed genes, but were unable to correlate these changes in expression with either polymorphisms from resequencing or positional effects of transposons.

Conclusions

We have assembled, annotated, and characterized the first draft genome for this important model plant species. Using this resource, we show duplications of genes leading to the synthesis of the medicinally important alkaloid, scopolamine. Our results also demonstrate that following tissue culture, mutation rates of transformed plants are quite high (1.16 × 10- 3 mutations per site), but do not have a drastic impact on gene expression.

Cover page of Pre-Cambrian roots of novel Antarctic cryptoendolithic bacterial lineages.

Pre-Cambrian roots of novel Antarctic cryptoendolithic bacterial lineages.

(2021)

Background

Cryptoendolithic communities are microbial ecosystems dwelling inside porous rocks that are able to persist at the edge of the biological potential for life in the ice-free areas of the Antarctic desert. These regions include the McMurdo Dry Valleys, often accounted as the closest terrestrial counterpart of the Martian environment and thought to be devoid of life until the discovery of these cryptic life-forms. Despite their interest as a model for the early colonization by living organisms of terrestrial ecosystems and for adaptation to extreme conditions of stress, little is known about the evolution, diversity, and genetic makeup of bacterial species that reside in these environments. Using the Illumina Novaseq platform, we generated the first metagenomes from rocks collected in Continental Antarctica over a distance of about 350 km along an altitudinal transect from 834 up to 3100 m above sea level (a.s.l.).

Results

A total of 497 draft bacterial genome sequences were assembled and clustered into 269 candidate species that lack a representative genome in public databases. Actinobacteria represent the most abundant phylum, followed by Chloroflexi and Proteobacteria. The "Candidatus Jiangella antarctica" has been recorded across all samples, suggesting a high adaptation and specialization of this species to the harshest Antarctic desert environment. The majority of these new species belong to monophyletic bacterial clades that diverged from related taxa in a range from 1.2 billion to 410 Ma and are functionally distinct from known related taxa.

Conclusions

Our findings significantly increase the repertoire of genomic data for several taxa and, to date, represent the first example of bacterial genomes recovered from endolithic communities. Their ancient origin seems to not be related to the geological history of the continent, rather they may represent evolutionary remnants of pristine clades that evolved across the Tonian glaciation. These unique genomic resources will underpin future studies on the structure, evolution, and function of these ecosystems at the edge of life. Video abstract.

Cover page of Culture-Dependent and Amplicon Sequencing Approaches Reveal Diversity and Distribution of Black Fungi in Antarctic Cryptoendolithic Communities.

Culture-Dependent and Amplicon Sequencing Approaches Reveal Diversity and Distribution of Black Fungi in Antarctic Cryptoendolithic Communities.

(2021)

In the harshest environmental conditions of the Antarctic desert, normally incompatible with active life, microbes are adapted to exploit the cryptoendolithic habitat (i.e., pore spaces of rocks) and represent the predominant life-forms. In the rocky niche, microbes take advantage of the thermal buffering, physical stability, protection against UV radiation, excessive solar radiation, and water retention-of paramount importance in one of the driest environments on Earth. In this work, high-throughput sequencing and culture-dependent approaches have been combined, for the first time, to untangle the diversity and distribution of black fungi in the Antarctic cryptoendolithic microbial communities, hosting some of the most extreme-tolerant microorganisms. Rock samples were collected in a vast area, along an altitudinal gradient and opposite sun exposure-known to influence microbial diversity-with the aim to compare and integrate results gained with the two approaches. Among black fungi, Friedmanniomyces endolithicus was confirmed as the most abundant taxon. Despite the much stronger power of the high-throughput sequencing, several species were not retrieved with DNA sequencing and were detectable by cultivation only. We conclude that both culture-dependent and -independent analyses are needed for a complete overview of black fungi diversity. The reason why some species remain undetectable with molecular methods are speculated upon. The effect of environmental parameters such as sun exposure on relative abundance was clearer if based on the wider biodiversity detected with the molecular approach.

Genomic insights into the host specific adaptation of the Pneumocystis genus.

(2021)

Pneumocystis jirovecii, the fungal agent of human Pneumocystis pneumonia, is closely related to macaque Pneumocystis. Little is known about other Pneumocystis species in distantly related mammals, none of which are capable of establishing infection in humans. The molecular basis of host specificity in Pneumocystis remains unknown as experiments are limited due to an inability to culture any species in vitro. To explore Pneumocystis evolutionary adaptations, we have sequenced the genomes of species infecting macaques, rabbits, dogs and rats and compared them to available genomes of species infecting humans, mice and rats. Complete whole genome sequence data enables analysis and robust phylogeny, identification of important genetic features of the host adaptation, and estimation of speciation timing relative to the rise of their mammalian hosts. Our data reveals insights into the evolution of P. jirovecii, the sole member of the genus able to infect humans.

Cover page of Identification and phylogenetic analysis of RNA binding domain abundant in apicomplexans or RAP proteins.

Identification and phylogenetic analysis of RNA binding domain abundant in apicomplexans or RAP proteins.

(2021)

The RNA binding domain abundant in apicomplexans (RAP) is a protein domain identified in a diverse group of proteins, called RAP proteins, many of which have been shown to be involved in RNA binding. To understand the expansion and potential function of the RAP proteins, we conducted a hidden Markov model based screen among the proteomes of 54 eukaryotes, 17 bacteria and 12 archaea. We demonstrated that the domain is present in closely and distantly related organisms with particular expansions in Alveolata and Chlorophyta, and are not unique to Apicomplexa as previously believed. All RAP proteins identified can be decomposed into two parts. In the N-terminal region, the presence of variable helical repeats seems to participate in the specific targeting of diverse RNAs, while the RAP domain is mostly identified in the C-terminal region and is highly conserved across the different phylogenetic groups studied. Several conserved residues defining the signature motif could be crucial to ensure the function(s) of the RAP proteins. Modelling of RAP domains in apicomplexan parasites confirmed an ⍺/β structure of a restriction endonuclease-like fold. The phylogenetic trees generated from multiple alignment of RAP domains and full-length proteins from various distantly related eukaryotes indicated a complex evolutionary history of this family. We further discuss these results to assess the potential function of this protein family in apicomplexan parasites.

Cover page of Genome-scale phylogenetic analyses confirm Olpidium as the closest living zoosporic fungus to the non-flagellated, terrestrial fungi.

Genome-scale phylogenetic analyses confirm Olpidium as the closest living zoosporic fungus to the non-flagellated, terrestrial fungi.

(2021)

The zoosporic obligate endoparasites, Olpidium, hold a pivotal position to the reconstruction of the flagellum loss in fungi, one of the key morphological transitions associated with the colonization of land by the early fungi. We generated genome and transcriptome data from non-axenic zoospores of Olpidium bornovanus and used a metagenome approach to extract phylogenetically informative fungal markers. Our phylogenetic reconstruction strongly supported Olpidium as the closest zoosporic relative of the non-flagellated terrestrial fungi. Super-alignment analyses resolved Olpidium as sister to the non-flagellated terrestrial fungi, whereas a super-tree approach recovered different placements of Olpidium, but without strong support. Further investigations detected little conflicting signal among the sampled markers but revealed a potential polytomy in early fungal evolution associated with the branching order among Olpidium, Zoopagomycota and Mucoromycota. The branches defining the evolutionary relationships of these lineages were characterized by short branch lengths and low phylogenetic content and received equivocal support for alternative phylogenetic hypotheses from individual markers. These nodes were marked by important morphological innovations, including the transition to hyphal growth and the loss of flagellum, which enabled early fungi to explore new niches and resulted in rapid and temporally concurrent Precambrian diversifications of the ancestors of several phyla of fungi.