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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.

Fungi are key players in extreme ecosystems.

(2022)

Extreme environments on Earth are typically devoid of macro life forms and are inhabited predominantly by highly adapted and specialized microorganisms. The discovery and persistence of these extremophiles provides tools to model how life arose on Earth and inform us on the limits of life. Fungi, in particular, are among the most extreme-tolerant organisms with highly versatile lifestyles and stunning ecological and morphological plasticity. Here, we overview the most notable examples of extremophilic and stress-tolerant fungi, highlighting their key roles in the functionality and balance of extreme ecosystems. The remarkable ability of fungi to tolerate and even thrive in the most extreme environments, which preclude most organisms, have reshaped current concepts regarding the limits of life on Earth.

Mouse circulating extracellular vesicles contain virus-derived siRNAs active in antiviral immunity.

(2022)

Induction and suppression of antiviral RNA interference (RNAi) has been observed in mammals during infection with at least seven distinct RNA viruses, including some that are pathogenic in humans. However, while the cell-autonomous immune response mediated by antiviral RNAi is gradually being recognized, little is known about systemic antiviral RNAi in mammals. Furthermore, extracellular vesicles (EVs) also function in viral signal spreading and host immunity. Here, we show that upon antiviral RNAi activation, virus-derived small-interfering RNAs (vsiRNAs) from Nodamura virus (NoV), Sindbis virus (SINV), and Zika virus (ZIKV) enter the murine bloodstream via EVs for systemic circulation. vsiRNAs in the EVs are biologically active, since they confer RNA-RNA homology-dependent antiviral activity in both cultured cells and infant mice. Moreover, we demonstrate that vaccination with a live-attenuated virus, rendered deficient in RNAi suppression, induces production of stably maintained vsiRNAs and confers protective immunity against virus infection in mice. This suggests that vaccination with live-attenuated VSR (viral suppressor of RNAi)-deficient mutant viruses could be a new strategy to induce immunity.

Cover page of Identification of positive and negative regulators of antiviral RNA interference in Arabidopsis thaliana.

Identification of positive and negative regulators of antiviral RNA interference in Arabidopsis thaliana.

(2022)

Virus-host coevolution often drives virus immune escape. However, it remains unknown whether natural variations of plant virus resistance are enriched in genes of RNA interference (RNAi) pathway known to confer essential antiviral defense in plants. Here, we report two genome-wide association study screens to interrogate natural variation among wild-collected Arabidopsis thaliana accessions in quantitative resistance to the endemic cucumber mosaic virus (CMV). We demonstrate that the highest-ranked gene significantly associated with resistance from both screens acts to regulate antiviral RNAi in ecotype Columbia-0. One gene, corresponding to Reduced Dormancy 5 (RDO5), enhances resistance by promoting amplification of the virus-derived small interfering RNAs (vsiRNAs). Interestingly, the second gene, designated Antiviral RNAi Regulator 1 (VIR1), dampens antiviral RNAi so its genetic inactivation by CRISPR/Cas9 editing enhances both vsiRNA production and CMV resistance. Our findings identify positive and negative regulators of the antiviral RNAi defense that may play important roles in virus-host coevolution.

Cover page of Avian-associated Aspergillus fumigatus displays broad phylogenetic distribution, no evidence for host specificity, and multiple genotypes within epizootic events.

Avian-associated Aspergillus fumigatus displays broad phylogenetic distribution, no evidence for host specificity, and multiple genotypes within epizootic events.

(2022)

Birds are highly susceptible to aspergillosis, which can manifest as a primary infection in both domestic and wild birds. Aspergillosis in wild birds causes mortalities ranging in scale from single animals to large-scale epizootic events. However, pathogenicity factors associated with aspergillosis in wild birds have not been examined. Specifically, it is unknown whether wild bird-infecting strains are host-adapted (i.e. phylogenetically related). Similarly, it is unknown whether epizootics are driven by contact with clonal strains that possess unique pathogenic or virulence properties, or by distinct and equally pathogenic strains. Here, we use a diverse collection of Aspergillus fumigatus isolates taken from aspergillosis-associated avian carcasses, representing 24 bird species from a wide geographic range, and representing individual bird mortalities as well as epizootic events. These isolates were sequenced and analyzed along with 130 phylogenetically diverse human clinical isolates to investigate the genetic diversity and phylogenetic placement of avian-associated A. fumigatus, the geographic and host distribution of avian isolates, evidence for clonal outbreaks among wild birds, and the frequency of azole resistance in avian isolates. We found that avian isolates were phylogenetically diverse, with no clear distinction from human clinical isolates, and no sign of host or geographic specificity. Avian isolates from the same epizootic events were diverse and phylogenetically distant, suggesting that avian aspergillosis is not contagious among wild birds and that outbreaks are likely driven by environmental spore loads or host comorbidities. Finally, all avian isolates were susceptible to Voriconazole and none contained the canonical azole resistance gene variants.

Cover page of Efficient CRISPR/Cas9-mediated genome modification of the glassy-winged sharpshooter Homalodisca vitripennis (Germar).

Efficient CRISPR/Cas9-mediated genome modification of the glassy-winged sharpshooter Homalodisca vitripennis (Germar).

(2022)

CRISPR/Cas9 technology enables the extension of genetic techniques into insect pests previously refractory to genetic analysis. We report the establishment of genetic analysis in the glassy-winged sharpshooter (GWSS), Homalodisca vitripennis, which is a significant leafhopper pest of agriculture in California. We use a novel and simple approach of embryo microinjection in situ on the host plant and obtain high frequency mutagenesis, in excess of 55%, of the cinnabar and white eye pigmentation loci. Through pair matings, we obtained 100% transmission of w and cn alleles to the G3 generation and also established that both genes are located on autosomes. Our analysis of wing phenotype revealed an unexpected discovery of the participation of pteridine pigments in wing and wing-vein coloration, indicating a role for these pigments beyond eye color. We used amplicon sequencing to examine the extent of off-target mutagenesis in adults arising from injected eggs, which was found to be negligible or non-existent. Our data show that GWSS can be easily developed as a genetic model system for the Hemiptera, enabling the study of traits that contribute to the success of invasive pests and vectors of plant pathogens. This will facilitate novel genetic control strategies.

Cover page of Understudied, underrepresented, and unknown: Methodological biases that limit detection of early diverging fungi from environmental samples.

Understudied, underrepresented, and unknown: Methodological biases that limit detection of early diverging fungi from environmental samples.

(2022)

Metabarcoding is an important tool for understanding fungal communities. The internal transcribed spacer (ITS) rDNA is the accepted fungal barcode but has known problems. The large subunit (LSU) rDNA has also been used to investigate fungal communities but available LSU metabarcoding primers were mostly designed to target Dikarya (Ascomycota + Basidiomycota) with little attention to early diverging fungi (EDF). However, evidence from multiple studies suggests that EDF comprise a large portion of unknown diversity in community sampling. Here, we investigate how DNA marker choice and methodological biases impact recovery of EDF from environmental samples. We focused on one EDF lineage, Zoopagomycota, as an example. We evaluated three primer sets (ITS1F/ITS2, LROR/LR3, and LR3 paired with new primer LR22F) to amplify and sequence a Zoopagomycota mock community and a set of 146 environmental samples with Illumina MiSeq. We compared two taxonomy assignment methods and created an LSU reference database compatible with AMPtk software. The two taxonomy assignment methods recovered strikingly different communities of fungi and EDF. Target fragment length variation exacerbated PCR amplification biases and influenced downstream taxonomic assignments, but this effect was greater for EDF than Dikarya. To improve identification of LSU amplicons we performed phylogenetic reconstruction and illustrate the advantages of this critical tool for investigating identified and unidentified sequences. Our results suggest much of the EDF community may be missed or misidentified with "standard" metabarcoding approaches and modified techniques are needed to understand the role of these taxa in a broader ecological context.

Early branching arbuscular mycorrhizal fungus Paraglomus occultum carries a small and repeat-poor genome compared to relatives in the Glomeromycotina.

(2022)

The arbuscular mycorrhizal fungi (AMFs) are obligate root symbionts in the subphylum Glomeromycotina that can benefit land plants by increasing their soil nutrient uptake in exchange for photosynthetically fixed carbon sources. To date, annotated genome data from representatives of the AMF orders Glomerales, Diversisporales and Archaeosporales have shown that these organisms have large and highly repeated genomes, and no genes to produce sugars and fatty acids. This led to the hypothesis that the most recent common ancestor (MRCA) of Glomeromycotina was fully dependent on plants for nutrition. Here, we aimed to further test this hypothesis by obtaining annotated genome data from a member of the early diverging order Paraglomerales (Paraglomus occultum). Genome analyses showed this species carries a 39.6 Mb genome and considerably fewer genes and repeats compared to most AMF relatives with annotated genomes. Consistent with phylogenies based on ribosomal genes, our phylogenetic analyses suggest P. occultum as the earliest diverged branch within Glomeromycotina. Overall, our analyses support the view that the MRCA of Glomeromycotina carried hallmarks of obligate plant biotrophy. The small genome size and content of P. occultum could either reflect adaptive reductive processes affecting some early AMF lineages, or indicate that the high gene and repeat family diversity thought to drive AMF adaptability to host and environmental change was not an ancestral feature of these prominent plant symbionts.

Cover page of A chromosomal-level reference genome of the widely utilized Coccidioides posadasii laboratory strain "Silveira".

A chromosomal-level reference genome of the widely utilized Coccidioides posadasii laboratory strain "Silveira".

(2022)

Coccidioidomycosis is a common fungal disease that is endemic to arid and semi-arid regions of both American continents. Coccidioides immitis and Coccidioides posadasii are the etiological agents of the disease, also known as Valley Fever. For several decades, the C. posadasii strain Silveira has been used widely in vaccine studies, is the source strain for production of diagnostic antigens, and is a widely used experimental strain for functional studies. In 2009, the genome was sequenced using Sanger sequencing technology, and a draft assembly and annotation were made available. In this study, the genome of the Silveira strain was sequenced using single molecule real-time sequencing PacBio technology, assembled into chromosomal-level contigs, genotyped, and the genome was reannotated using sophisticated and curated in silico tools. This high-quality genome sequencing effort has improved our understanding of chromosomal structure, gene set annotation, and lays the groundwork for identification of structural variants (e.g. transversions, translocations, and copy number variants), assessment of gene gain and loss, and comparison of transposable elements in future phylogenetic and population genomics studies.

Rocks support a distinctive and consistent mycobiome across contrasting dry regions of Earth.

(2022)

Rock-dwelling fungi play critical ecological roles in drylands, including soil formation and nutrient cycling; however, we know very little about the identity, function and environmental preferences of these important organisms, and the mere existence of a consistent rock mycobiome across diverse arid regions of the planet remains undetermined. To address this knowledge gap, we conducted a meta-analysis of rock fungi and spatially associated soil communities, surveyed across 28 unique sites spanning four major biogeographic regions (North America, Arctic, Maritime and Continental Antarctica) including contrasting climates, from cold and hot deserts to semiarid drylands. We show that rocks support a consistent and unique mycobiome that was different from that found in surrounding soils. Lichenized fungi from class Lecanoromycetes were consistently indicative of rocks across contrasting regions, together with ascomycetous representatives of black fungi in Arthoniomycetes, Dothideomycetes and Eurotiomycetes. In addition, compared with soil, rocks had a lower proportion of saprobes and plant symbiotic fungi. The main drivers structuring rock fungi distribution were spatial distance and, to a larger extent, climatic factors regulating moisture and temperature (i.e. mean annual temperature and mean annual precipitation), suggesting that these paramount and unique communities might be particularly sensitive to increases in temperature and desertification.

Decoding Transcription Regulatory Mechanisms Associated with Coccidioides immitis Phase Transition Using Total RNA.

(2022)

New or emerging infectious diseases are commonly caused by pathogens that cannot be readily manipulated or studied under common laboratory conditions. These limitations hinder standard experimental approaches and our abilities to define the fundamental molecular mechanisms underlying pathogenesis. The advance of capped small RNA sequencing (csRNA-seq) now enables genome-wide mapping of actively initiated transcripts from genes and other regulatory transcribed start regions (TSRs) such as enhancers at a precise moment from total RNA. As RNA is nonpathogenic and can be readily isolated from inactivated infectious samples, csRNA-seq can detect acute changes in gene regulation within or in response to a pathogen with remarkable sensitivity under common laboratory conditions. Studying valley fever (coccidioidomycosis), an emerging endemic fungal infection that increasingly impacts livestock, pet, and human health, we show how csRNA-seq can unravel transcriptional programs driving pathogenesis. Performing csRNA-seq on RNA isolated from different stages of the valley fever pathogen Coccidioides immitis revealed alternative promoter usage, connected cis-regulatory domains, and a WOPR family transcription factor, which are known regulators of virulence in other fungi, as being critical for pathogenic growth. We further demonstrate that a C. immitis WOPR homologue, CIMG_02671, activates transcription in a WOPR motif-dependent manner. Collectively, these findings provide novel insights into valley fever pathogenesis and provide a proof of principle for csRNA-seq as a powerful means to determine the genes, regulatory mechanisms, and transcription factors that control the pathogenesis of highly infectious agents. IMPORTANCE Infectious pathogens like airborne viruses or fungal spores are difficult to study; they require high-containment facilities, special equipment, and expertise. As such, establishing approaches such as genome editing or other means to identify the factors and mechanisms underlying caused diseases, and, thus, promising drug targets, is costly and time-intensive. These obstacles particularly hinder the analysis of new, emerging, or rare infectious diseases. We recently developed a method termed capped small RNA sequencing (csRNA-seq) that enables capturing acute changes in active gene expression from total RNA. Prior to csRNA-seq, such an analysis was possible only by using living cells or nuclei, in which pathogens are highly infectious. The process of RNA purification, however, inactivates pathogens and thus enables the analysis of gene expression during disease progression under standard laboratory conditions. As a proof of principle, here, we use csRNA-seq to unravel the gene regulatory programs and factors likely critical for the pathogenesis of valley fever, an emerging endemic fungal infection that increasingly impacts livestock, pet, and human health.