Cassava begomovirus species diversity changes during plant vegetative cycles.

Cassava is a root crop important for global food security and the third biggest source of calories on the African continent. Cassava production is threatened by Cassava mosaic disease (CMD), which is caused by a complex of single-stranded DNA viruses (family: Geminiviridae, genus: Begomovirus) that are transmitted by the sweet potato whitefly (Bemisia tabaci). Understanding the dynamics of different cassava mosaic begomovirus (CMB) species through time is important for contextualizing disease trends. Cassava plants with CMD symptoms were sampled in Lake Victoria and coastal regions of Kenya before transfer to a greenhouse setting and regular propagation. The field-collected and greenhouse samples were sequenced using Illumina short-read sequencing and analyzed on the Galaxy platform. In the field-collected samples, African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV), East African cassava mosaic Kenya virus (EACMKV), and East African cassava mosaic virus-Uganda variant (EACMV-Ug) were detected in samples from the Lake Victoria region, while EACMV and East African mosaic Zanzibar virus (EACMZV) were found in the coastal region. Many of the field-collected samples had mixed infections of EACMV and another begomovirus. After 3 years of regrowth in the greenhouse, only EACMV-like viruses were detected in all samples. The results suggest that in these samples, EACMV becomes the dominant virus through vegetative propagation in a greenhouse. This differed from whitefly transmission results. Cassava plants were inoculated with ACMV and another EACMV-like virus, East African cassava mosaic Cameroon virus (EACMCV). Only ACMV was transmitted by whiteflies from these plants to recipient plants, as indicated by sequencing reads and copy number data. These results suggest that whitefly transmission and vegetative transmission lead to different outcomes for ACMV and EACMV-like viruses.

A Transducing Bacteriophage Infecting Staphylococcus epidermidis Contributes to the Expansion of a Novel Siphovirus Genus and Implies the Genus Is Inappropriate for Phage Therapy.

The effort to discover novel phages infecting Staphylococcus epidermidis contributes to both the development of phage therapy and the expansion of genome-based phage phylogeny. Here, we report the genome of an S. epidermidis-infecting phage, Lacachita, and compare its genome with those of five other phages with high sequence identity. These phages represent a novel siphovirus genus, which was recently reported in the literature. The published member of this group was favorably evaluated as a phage therapeutic agent, but Lacachita is capable of transducing antibiotic resistance and conferring phage resistance to transduced cells. Members of this genus may be maintained within their host as extrachromosomal plasmid prophages, through stable lysogeny or pseudolysogeny. Therefore, we conclude that Lacachita may be temperate and members of this novel genus are not suitable for phage therapy. IMPORTANCE This project describes the discovery of a culturable bacteriophage infecting Staphylococcus epidermidis that is a member of a rapidly growing novel siphovirus genus. A member of this genus was recently characterized and proposed for phage therapy, as there are few phages currently available to treat S. epidermidis infections. Our data contradict this, as we show Lacachita is capable of moving DNA from one bacterium to another, and it may be capable of maintaining itself in a plasmid-like state in infected cells. These phages' putative plasmid-like extrachromosomal state appears to be due to a simplified maintenance mechanism found in true plasmids of Staphylococcus and related hosts. We suggest Lacachita and other identified members of this novel genus are not suitable for phage therapy.

Limited role of recombination in the global diversification of begomovirus DNA-B proteins.

Approximately half of the characterized begomoviruses have bipartite genomes, but the second genomic segment, the DNA-B, is understudied relative to the DNA-A, which is homologous to the entire genome of monopartite begomoviruses. We examined the evolutionary history of the two proteins encoded by the DNA-B, the genes of which make up ∼60% of the DNA-B segment, from all bipartite begomovirus species. Our dataset of 131 movement protein (MP) and nuclear shuttle protein (NSP) sequences confirmed the deep split between Old World (OW) and New World (NW) species, and showed strong support for deep, congruent branches among the OW sequences of the MP and NSP. NW sequences were much less diverse and had poor phylogenetic resolution; over half of nodes in both the NSP and MP NW clades were supported by <50% bootstrap support. This poor resolution hampered our ability to detect incongruent phylogenies between the MP and NSP datasets, and we found no statistical evidence for recombination within our MP and NSP datasets. Finally, we quantified the sequence diversity between the NW and OW proteins, showing that the NW MP has particularly low diversity, suggesting it has been subject to different evolutionary pressures than the NW NSP.

Complete Genome Sequences of Five Phietaviruses Infecting Staphylococcus aureus.

The annotated whole-genome sequences of five cultured phietaviruses infecting Staphylococcus aureus are presented. They are closely related to prophages that were previously sequenced as part of S. aureus genomes.

Early detection of plant virus infection using multispectral imaging and spatial-spectral machine learning.

Cassava brown streak disease (CBSD) is an emerging viral disease that can greatly reduce cassava productivity, while causing only mild aerial symptoms that develop late in infection. Early detection of CBSD enables better crop management and intervention. Current techniques require laboratory equipment and are labour intensive and often inaccurate. We have developed a handheld active multispectral imaging (A-MSI) device combined with machine learning for early detection of CBSD in real-time. The principal benefits of A-MSI over passive MSI and conventional camera systems are improved spectral signal-to-noise ratio and temporal repeatability. Information fusion techniques further combine spectral and spatial information to reliably identify features that distinguish healthy cassava from plants with CBSD as early as 28 days post inoculation on a susceptible and a tolerant cultivar. Application of the device has the potential to increase farmers' access to healthy planting materials and reduce losses due to CBSD in Africa. It can also be adapted for sensing other biotic and abiotic stresses in real-world situations where plants are exposed to multiple pest, pathogen and environmental stresses.

An experimental strategy for preparing circular ssDNA virus genomes for next-generation sequencing.

The ability of begomoviruses to evolve rapidly threatens many crops and underscores the importance of detecting these viruses quickly and to understand their genome diversity. This study presents an improved protocol for the enhanced amplification and enrichment of begomovirus DNA for use in next generation sequencing of the viral genomes. An enhanced rolling circle amplification (RCA) method using EquiPhi29 polymerase was combined with size selection to generate a cost-effective, short-read sequencing method. This improved short-read sequencing produced at least 50 % of the reads mapping to the target viral reference genomes, African cassava mosaic virus and East African cassava mosaic virus. This study provided other insights into common misconceptions about RCA and lessons that could be learned from the sequencing of single-stranded DNA virus genomes. This protocol can be used to examine the viral DNA as it moves from host to vector, thus producing valuable information for viral DNA population studies, and would likely work well with other circular Rep-encoding ssDNA viruses (CRESS) DNA viruses.

Population diversity of cassava mosaic begomoviruses increases over the course of serial vegetative propagation.

Cassava mosaic disease (CMD) represents a serious threat to cassava, a major root crop for more than 300 million Africans. CMD is caused by single-stranded DNA begomoviruses that evolve rapidly, making it challenging to develop durable disease resistance. In addition to the evolutionary forces of mutation, recombination and reassortment, factors such as climate, agriculture practices and the presence of DNA satellites may impact viral diversity. To gain insight into the factors that alter and shape viral diversity in planta, we used high-throughput sequencing to characterize the accumulation of nucleotide diversity after inoculation of infectious clones corresponding to African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV) in the susceptible cassava landrace Kibandameno. We found that vegetative propagation had a significant effect on viral nucleotide diversity, while temperature and a satellite DNA did not have measurable impacts in our study. EACMCV diversity increased linearly with the number of vegetative propagation passages, while ACMV diversity increased for a time and then decreased in later passages. We observed a substitution bias toward C→T and G→A for mutations in the viral genomes consistent with field isolates. Non-coding regions excluding the promoter regions of genes showed the highest levels of nucleotide diversity for each genome component. Changes in the 5' intergenic region of DNA-A resembled the sequence of the cognate DNA-B sequence. The majority of nucleotide changes in coding regions were non-synonymous, most with predicted deleterious effects on protein structure, indicative of relaxed selection pressure over six vegetative passages. Overall, these results underscore the importance of knowing how cropping practices affect viral evolution and disease progression.

Interspecies Recombination Has Driven the Macroevolution of Cassava Mosaic Begomoviruses.

Begomoviruses (family Geminiviridae, genus Begomovirus) significantly hamper crop production and threaten food security around the world. The frequent emergence of new begomovirus genotypes is facilitated by high mutation frequencies and the propensity to recombine and reassort. Homologous recombination has been especially implicated in the emergence of novel cassava mosaic begomovirus (CMB) genotypes, which cause cassava mosaic disease (CMD). Cassava (Manihot esculenta) is a staple food crop throughout Africa and an important industrial crop in Asia, two continents where production is severely constrained by CMD. The CMD species complex is comprised of 11 bipartite begomovirus species with ample distribution throughout Africa and the Indian subcontinent. While recombination is regarded as a frequent occurrence for CMBs, a revised, systematic assessment of recombination and its impact on CMB phylogeny is currently lacking. We assembled data sets of all publicly available, full-length DNA-A (n = 880) and DNA-B (n = 369) nucleotide sequences from the 11 recognized CMB species. Phylogenetic networks and complementary recombination detection methods revealed extensive recombination among the CMB sequences. Six out of the 11 species descended from unique interspecies recombination events. Estimates of recombination and mutation rates revealed that all species experience mutation more frequently than recombination, but measures of population divergence indicate that recombination is largely responsible for the genetic differences between species. Our results support that recombination has significantly impacted the CMB phylogeny and has driven speciation in the CMD species complex. IMPORTANCE Cassava mosaic disease (CMD) is a significant threat to cassava production throughout Africa and Asia. CMD is caused by a complex comprised of 11 recognized virus species exhibiting accelerated rates of evolution, driven by high frequencies of mutation and genetic exchange. Here, we present a systematic analysis of the contribution of genetic exchange to cassava mosaic virus species-level diversity. Most of these species emerged as a result of genetic exchange. This is the first study to report the significant impact of genetic exchange on speciation in a group of viruses.

Deeply Sequenced Infectious Clones of Key Cassava Begomovirus Isolates from Cameroon.

We deeply sequenced two pairs of widely used infectious clones (4 plasmids) of the bipartite begomoviruses African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV). The ACMV clones were quite divergent from published sequences. Raw reads, consensus plasmid sequences, and the infectious clones themselves are all publicly available.

Functional dissection of the ARGONAUTE7 promoter.

ARGONAUTES are the central effector proteins of RNA silencing which bind target transcripts in a small RNA-guided manner. Arabidopsis thaliana has 10 ARGONAUTE (AGO) genes, with specialized roles in RNA-directed DNA methylation, post-transcriptional gene silencing, and antiviral defense. To better understand specialization among AGO genes at the level of transcriptional regulation we tested a library of 1497 transcription factors for binding to the promoters of AGO1,AGO10, and AGO7 using yeast 1-hybrid assays. A ranked list of candidate DNA-binding TFs revealed binding of the AGO7 promoter by a number of proteins in two families: the miR156-regulated SPL family and the miR319-regulated TCP family, both of which have roles in developmental timing and leaf morphology. Possible functions for SPL and TCP binding are unclear: we showed that these binding sites are not required for the polar expression pattern of AGO7, nor for the function of AGO7 in leaf shape. Normal AGO7 transcription levels and function appear to depend instead on an adjacent 124-bp region. Progress in understanding the structure of this promoter may aid efforts to understand how the conserved AGO7-triggered TAS3 pathway functions in timing and polarity.

Raspberry Pi-powered imaging for plant phenotyping.

PREMISE OF THE STUDY: Image-based phenomics is a powerful approach to capture and quantify plant diversity. However, commercial platforms that make consistent image acquisition easy are often cost-prohibitive. To make high-throughput phenotyping methods more accessible, low-cost microcomputers and cameras can be used to acquire plant image data. METHODS AND RESULTS: We used low-cost Raspberry Pi computers and cameras to manage and capture plant image data. Detailed here are three different applications of Raspberry Pi-controlled imaging platforms for seed and shoot imaging. Images obtained from each platform were suitable for extracting quantifiable plant traits (e.g., shape, area, height, color) en masse using open-source image processing software such as PlantCV. CONCLUSIONS: This protocol describes three low-cost platforms for image acquisition that are useful for quantifying plant diversity. When coupled with open-source image processing tools, these imaging platforms provide viable low-cost solutions for incorporating high-throughput phenomics into a wide range of research programs.

PlantCV v2: Image analysis software for high-throughput plant phenotyping.

Systems for collecting image data in conjunction with computer vision techniques are a powerful tool for increasing the temporal resolution at which plant phenotypes can be measured non-destructively. Computational tools that are flexible and extendable are needed to address the diversity of plant phenotyping problems. We previously described the Plant Computer Vision (PlantCV) software package, which is an image processing toolkit for plant phenotyping analysis. The goal of the PlantCV project is to develop a set of modular, reusable, and repurposable tools for plant image analysis that are open-source and community-developed. Here we present the details and rationale for major developments in the second major release of PlantCV. In addition to overall improvements in the organization of the PlantCV project, new functionality includes a set of new image processing and normalization tools, support for analyzing images that include multiple plants, leaf segmentation, landmark identification tools for morphometrics, and modules for machine learning.

Roles and programming of Arabidopsis ARGONAUTE proteins during Turnip mosaic virus infection.

In eukaryotes, ARGONAUTE proteins (AGOs) associate with microRNAs (miRNAs), short interfering RNAs (siRNAs), and other classes of small RNAs to regulate target RNA or target loci. Viral infection in plants induces a potent and highly specific antiviral RNA silencing response characterized by the formation of virus-derived siRNAs. Arabidopsis thaliana has ten AGO genes of which AGO1, AGO2, and AGO7 have been shown to play roles in antiviral defense. A genetic analysis was used to identify and characterize the roles of AGO proteins in antiviral defense against Turnip mosaic virus (TuMV) in Arabidopsis. AGO1, AGO2 and AGO10 promoted anti-TuMV defense in a modular way in various organs, with AGO2 providing a prominent antiviral role in leaves. AGO5, AGO7 and AGO10 had minor effects in leaves. AGO1 and AGO10 had overlapping antiviral functions in inflorescence tissues after systemic movement of the virus, although the roles of AGO1 and AGO10 accounted for only a minor amount of the overall antiviral activity. By combining AGO protein immunoprecipitation with high-throughput sequencing of associated small RNAs, AGO2, AGO10, and to a lesser extent AGO1 were shown to associate with siRNAs derived from silencing suppressor (HC-Pro)-deficient TuMV-AS9, but not with siRNAs derived from wild-type TuMV. Co-immunoprecipitation and small RNA sequencing revealed that viral siRNAs broadly associated with wild-type HC-Pro during TuMV infection. These results support the hypothesis that suppression of antiviral silencing during TuMV infection, at least in part, occurs through sequestration of virus-derived siRNAs away from antiviral AGO proteins by HC-Pro. These findings indicate that distinct AGO proteins function as antiviral modules, and provide a molecular explanation for the silencing suppressor activity of HC-Pro.

Phytophthora have distinct endogenous small RNA populations that include short interfering and microRNAs.

In eukaryotes, RNA silencing pathways utilize 20-30-nucleotide small RNAs to regulate gene expression, specify and maintain chromatin structure, and repress viruses and mobile genetic elements. RNA silencing was likely present in the common ancestor of modern eukaryotes, but most research has focused on plant and animal RNA silencing systems. Phytophthora species belong to a phylogenetically distinct group of economically important plant pathogens that cause billions of dollars in yield losses annually as well as ecologically devastating outbreaks. We analyzed the small RNA-generating components of the genomes of P. infestans, P. sojae and P. ramorum using bioinformatics, genetic, phylogenetic and high-throughput sequencing-based methods. Each species produces two distinct populations of small RNAs that are predominantly 21- or 25-nucleotides long. The 25-nucleotide small RNAs were primarily derived from loci encoding transposable elements and we propose that these small RNAs define a pathway of short-interfering RNAs that silence repetitive genetic elements. The 21-nucleotide small RNAs were primarily derived from inverted repeats, including a novel microRNA family that is conserved among the three species, and several gene families, including Crinkler effectors and type III fibronectins. The Phytophthora microRNA is predicted to target a family of amino acid/auxin permeases, and we propose that 21-nucleotide small RNAs function at the post-transcriptional level. The functional significance of microRNA-guided regulation of amino acid/auxin permeases and the association of 21-nucleotide small RNAs with Crinkler effectors remains unclear, but this work provides a framework for testing the role of small RNAs in Phytophthora biology and pathogenesis in future work.

BOBBER1 is a noncanonical Arabidopsis small heat shock protein required for both development and thermotolerance.

Plants have evolved a range of cellular responses to maintain developmental homeostasis and to survive over a range of temperatures. Here, we describe the in vivo and in vitro functions of BOBBER1 (BOB1), a NudC domain containing Arabidopsis (Arabidopsis thaliana) small heat shock protein. BOB1 is an essential gene required for the normal partitioning and patterning of the apical domain of the Arabidopsis embryo. Because BOB1 loss-of-function mutants are embryo lethal, we used a partial loss-of-function allele (bob1-3) to demonstrate that BOB1 is required for organismal thermotolerance and postembryonic development. Recombinant BOB1 protein functions as a molecular chaperone and prevents the aggregation of a model protein substrate in vitro. In plants, BOB1 is cytoplasmic at basal temperatures, but forms heat shock granules containing canonical small heat shock proteins at high temperatures. In addition to thermotolerance defects, bob1-3 exhibits pleiotropic development defects during all phases of development. bob1-3 phenotypes include decreased rates of shoot and root growth as well as patterning defects in leaves, flowers, and inflorescence meristems. Most eukaryotic chaperones play important roles in protein folding either during protein synthesis or during cellular responses to denaturing stress. Our results provide, to our knowledge, the first evidence of a plant small heat shock protein that has both developmental and thermotolerance functions and may play a role in both of these folding networks.