Jan Kreuze

Complete viral genome sequence and discovery of novel viruses by deep sequencing of small RNAs: A generic method for diagnosis, discovery and sequencing of viruses.

We report the first identification of novel viruses, and sequence of an entire viral genome, by a single step of high-throughput parallel sequencing of small RNAs from diseased, as well as symptomless plants. Contigs were assembled from sequenced total siRNA from plants using small sequence assembly software and could positively identify RNA, ssDNA and dsDNA reverse transcribing viruses and in one case spanned the entire genome. The results present a novel approach which cannot only identify known viral pathogens, occurring at extremely low titers, but also novel viruses, without the necessity of any prior knowledge.

Comparative and functional genomics of closteroviruses.

Abstract The largest extant RNA genomes are found in two diverse families of positive-strand RNA viruses, the animal Coronaviridae and the plant Closteroviridae. Comparative analysis of the viruses from the latter family reveals three levels of gene conservation. The most conserved gene module defines RNA replication and is shared with plant and animal viruses in the alphavirus-like superfamily. A module of five genes that function in particle assembly and transport is a hallmark of the family Closteroviridae and was likely present in the ancestor of all three closterovirus genera. This module includes a homologue of Hsp70 molecular chaperones and three diverged copies of the capsid protein gene. The remaining genes show dramatic variation in their numbers, functions, and origins among closteroviruses within and between the genera. Proteins encoded by these genes include suppressors of RNA silencing, RNAse III, papain-like proteases, the AlkB domain implicated in RNA repair, Zn-ribbon-containing protein, and a variety of proteins with no detectable homologues in the current databases. The evolutionary processes that have shaped the complex and fluid genomes of the large RNA viruses might be similar to those that have been involved in evolution of genomic complexity in other divisions of life.

Viral Class 1 RNase III Involved in Suppression of RNA Silencing

ABSTRACT Double-stranded RNA (dsRNA)-specific endonucleases belonging to RNase III classes 3 and 2 process dsRNA precursors to small interfering RNA (siRNA) or microRNA, respectively, thereby initiating and amplifying RNA silencing-based antiviral defense and gene regulation in eukaryotic cells. However, we now provide evidence that a class 1 RNase III is involved in suppression of RNA silencing. The single-stranded RNA genome of sweet potato chlorotic stunt virus (SPCSV) encodes an RNase III (RNase3) homologous to putative class 1 RNase IIIs of unknown function in rice and Arabidopsis. We show that RNase3 has dsRNA-specific endonuclease activity that enhances the RNA-silencing suppression activity of another protein (p22) encoded by SPCSV. RNase3 and p22 coexpression reduced siRNA accumulation more efficiently than p22 alone in Nicotiana benthamiana leaves expressing a strong silencing inducer (i.e., dsRNA). RNase3 did not cause intracellular silencing suppression or reduce accumulation of siRNA in the absence of p22 or enhance silencing suppression activity of a protein encoded by a heterologous virus. No other known RNA virus encodes an RNase III or uses two independent proteins cooperatively for RNA silencing suppression.

Methods in virus diagnostics: from ELISA to next generation sequencing.

Despite the seemingly continuous development of newer and ever more elaborate methods for detecting and identifying viruses, very few of these new methods get adopted for routine use in testing laboratories, often despite the many and varied claimed advantages they possess. To understand why the rate of uptake of new technologies is so low, requires a strong understanding of what makes a good routine diagnostic tool to begin. This can be done by looking at the two most successfully established plant virus detection methods: enzyme-linked immunosorbant assay (ELISA) and more recently introduced real-time polymerase chain reaction (PCR). By examining the characteristics of this pair of technologies, it becomes clear that they share many benefits, such as an industry standard format and high levels of repeatability and reproducibility. These combine to make methods that are accessible to testing labs, which are easy to establish and robust in their use, even with new and inexperienced users. Hence, to ensure the establishment of new techniques it is necessary to not only provide benefits not found with ELISA or real-time PCR, but also to provide a platform that is easy to establish and use. In plant virus diagnostics, recent developments can be clustered into three core areas: (1) techniques that can be performed in the field or resource poor locations (e.g., loop-mediated isothermal amplification LAMP); (2) multiplex methods that are able to detect many viruses in a single test (e.g., Luminex bead arrays); and (3) methods suited to virus discovery (e.g., next generation sequencing, NGS). Field based methods are not new, with Lateral Flow Devices (LFDs) for the detection being available for a number of years now. However, the widespread uptake of this technology remains poor. LAMP does offer significant advantages over LFDs, in terms of sensitivity and generic application, but still faces challenges in terms of establishment. It is likely that the main barrier to the uptake of field-based technologies is behavioural influences, rather than specific concerns about the performance of the technologies themselves. To overcome this, a new relationship will need to develop between centralised testing laboratories offering services and those requiring tests; a relationship which is currently in its infancy. Looking further into the future, virus discovery and multiplex methods seem to converge as NGS becomes ever cheaper, easier to perform and can provide high levels of multiplexing without the use of virus specific reagents. So ultimately the key challenge from a routine testing lab perspective will not be one of investment in platforms-which could even be outsourced to commercial sequencing services-but one of having the skills and expertise to analyse the large datasets generated and their subsequent interpretation. In conclusion, only time will tell which of the next-generation of methods currently in development will become the routine diagnostics of the future. This will be determined through a combination of factors. And while the technology itself will have to offer performance advantages over existing methods in order to supplant them, it is likely to be human factors e.g., the behaviours of end users, laboratories and policy makers, the availability of appropriate expertise, that ultimately determine which ones become established. Hence factors cannot be ignored and early engagement with diagnostic stakeholders is essential.

The genome of cultivated sweet potato contains Agrobacterium T-DNAs with expressed genes: An example of a naturally transgenic food crop

Significance We communicate the rather remarkable observation that among 291 tested accessions of cultivated sweet potato, all contain one or more transfer DNA (T-DNA) sequences. These sequences, which are shown to be expressed in a cultivated sweet potato clone (“Huachano”) that was analyzed in detail, suggest that an Agrobacterium infection occurred in evolutionary times. One of the T-DNAs is apparently present in all cultivated sweet potato clones, but not in the crop’s closely related wild relatives, suggesting the T-DNA provided a trait or traits that were selected for during domestication. This finding draws attention to the importance of plant–microbe interactions, and given that this crop has been eaten for millennia, it may change the paradigm governing the “unnatural” status of transgenic crops. Agrobacterium rhizogenes and Agrobacterium tumefaciens are plant pathogenic bacteria capable of transferring DNA fragments [transfer DNA (T-DNA)] bearing functional genes into the host plant genome. This naturally occurring mechanism has been adapted by plant biotechnologists to develop genetically modified crops that today are grown on more than 10% of the world’s arable land, although their use can result in considerable controversy. While assembling small interfering RNAs, or siRNAs, of sweet potato plants for metagenomic analysis, sequences homologous to T-DNA sequences from Agrobacterium spp. were discovered. Simple and quantitative PCR, Southern blotting, genome walking, and bacterial artificial chromosome library screening and sequencing unambiguously demonstrated that two different T-DNA regions (IbT-DNA1 and IbT-DNA2) are present in the cultivated sweet potato (Ipomoea batatas [L.] Lam.) genome and that these foreign genes are expressed at detectable levels in different tissues of the sweet potato plant. IbT-DNA1 was found to contain four open reading frames (ORFs) homologous to the tryptophan-2-monooxygenase (iaaM), indole-3-acetamide hydrolase (iaaH), C-protein (C-prot), and agrocinopine synthase (Acs) genes of Agrobacterium spp. IbT-DNA1 was detected in all 291 cultigens examined, but not in close wild relatives. IbT-DNA2 contained at least five ORFs with significant homology to the ORF14, ORF17n, rooting locus (Rol)B/RolC, ORF13, and ORF18/ORF17n genes of A. rhizogenes. IbT-DNA2 was detected in 45 of 217 genotypes that included both cultivated and wild species. Our finding, that sweet potato is naturally transgenic while being a widely and traditionally consumed food crop, could affect the current consumer distrust of the safety of transgenic food crops.

Elimination of antiviral defense by viral RNase III

Sweet potato (Ipomoea batatas) is an important subsistence and famine reserve crop grown in developing countries where Sweet potato chlorotic stunt virus (SPCSV; Closteroviridae), a single-stranded RNA (ssRNA) crinivirus, synergizes unrelated viruses in co-infected sweet potato plants. The most severe disease and yield losses are caused by co-infection with SPCSV and a potyvirus, Sweet potato feathery mottle virus (SPFMV; Potyviridae). Potyviruses synergize unrelated viruses by suppression of RNA silencing with the P1/HC-Pro polyprotein; however, the SPCSV-SPFMV synergism is unusual in that the potyvirus is the beneficiary. Our data show that transformation of an SPFMV-resistant sweet potato variety with the double-stranded RNA (dsRNA)-specific class 1 RNA endoribonuclease III (RNase3) of SPCSV broke down resistance to SPFMV, leading to high accumulation of SPFMV antigen and severe disease symptoms similar to the synergism in plants co-infected with SPCSV and SPFMV. RNase3-transgenic sweet potatoes also accumulated higher concentrations of 2 other unrelated viruses and developed more severe symptoms than non-transgenic plants. In leaves, RNase3 suppressed ssRNA-induced gene silencing (RNAi) in an endonuclease activity-dependent manner. It cleaved synthetic double-stranded small interfering RNAs (siRNAs) of 21, 22, and 24 bp in vitro to products of approximately 14 bp that are inactive in RNAi. It also affected total siRNA isolated from SPFMV-infected sweet potato plants, suggesting a viral mechanism for suppression of RNAi by cleavage of siRNA. Results implicate RNase3 in suppression of antiviral defense in sweet potato plants and reveal RNase3 as a protein that mediates viral synergism with several unrelated viruses, a function previously described only for P1/HC-Pro.

Self-excision of the antibiotic resistance gene nptII using a heat inducible Cre-loxP system from transgenic potato

Resistance to antibiotics mediated by selectable marker genes remains a powerful selection tool for transgenic event production. However, regulatory agencies and consumer concerns favor these to be eliminated from food crops. Several excision systems exist but none have been optimized or shown to be functional for clonally propagated crops. The excision of the nptII gene conferring resistance to kanamycin has been achieved here using a gene construct based on a heat-inducible cre gene producing a recombinase that eliminates cre and nptII genes flanked by two loxP sites. First-generation regenerants with the Cre-loxP system were obtained by selection on kanamycin media. Following a heat treatment, second generation regenerants were screened for excision by PCR using nptII, cre, and T-DNA borders primers. Excision efficiency appeared to be at 4.7% depending on the heat treatment. The footprint of the excision was shown by sequencing between T-DNA borders to correspond to a perfect recombination event. Selectable marker-free sprouts were also obtained from tubers of transgenic events when submitted to similar heat treatment at 4% frequency. Spontaneous excision was not observed out of 196 regenerants from untreated transgenic explants. Biosafety concerns are minimized because the expression of cre gene driven by the hsp70 promoter of Drosophilamelanogaster was remarkably low even under heat activation and no functional loxP site were found in published Solanum sequence database. A new plant transformation vector pCIP54/55 was developed including a multiple cloning site and the self-excision system which should be a useful tool not only for marker genes in potato but for any gene or sequence removal in any plant.

Sweetpotato Viruses: 15 Years of Progress on Understanding and Managing Complex Diseases

Sweetpotato is a member of the morning glory family that is thought to have originated in Central or South America but also has a secondary center of diversity in the southwest Pacific islands. It is grown in all tropical and subtropical areas of the world and consistently ranks among the 10 most important food crops worldwide on the basis of dry weight produced, yielding about 130 million metric tons per year on about 9 million hectares. Sweetpotato is an important crop for food security. It has been relied on as a source of calories in many circumstances. Vines and/or storage roots can be used for direct human consumption or animal feed. Growing awareness of health benefits attributed to sweetpotato has stimulated renewed interest in the crop. Orange-fleshed cultivars, a source of vitamin A, were introduced to developing countries with hope that they would replace the white-flesh varieties and help alleviate vitamin A deficiencies. In East Africa, sweetpotato virus disease, which is caused by the synergistic interaction of the whitefly-transmitted crinivirus and the aphid-transmitted potyvirus, can cause losses of 80 to 90% in many high-yielding genotypes. During the past 15 years, as molecular methods have been adopted, much has been learned about the composition of the sweetpotato virus complexes, the effects of virus diseases on production systems, the biology of the virus–plant interaction, and management approaches to sweetpotato virus diseases. This article is intended to summarize what has been learned since earlier reviews, integrate knowledge gleaned from experiences in tropical and temperate production systems, and suggest courses of action to develop sustainable management programs for these diseases.

Complete Genome Sequence and Analyses of the Subgenomic RNAs of Sweet Potato Chlorotic Stunt Virus Reveal Several New Features for the Genus Crinivirus

ABSTRACT The complete nucleotide sequences of genomic RNA1 (9,407 nucleotides [nt]) and RNA2 (8,223 nt) of Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae) were determined, revealing that SPCSV possesses the second largest identified positive-strand single-stranded RNA genome among plant viruses after Citrus tristeza virus. RNA1 contains two overlapping open reading frames (ORFs) that encode the replication module, consisting of the putative papain-like cysteine proteinase, methyltransferase, helicase, and polymerase domains. RNA2 contains the Closteroviridae hallmark gene array represented by a heat shock protein homologue (Hsp70h), a protein of 50 to 60 kDa depending on the virus, the major coat protein, and a divergent copy of the coat protein. This grouping resembles the genome organization of Lettuce infectious yellows virus (LIYV), the only other crinivirus for which the whole genomic sequence is available. However, in striking contrast to LIYV, the two genomic RNAs of SPCSV contained nearly identical 208-nt-long 3′ terminal sequences, and the ORF for a putative small hydrophobic protein present in LIYV RNA2 was found at a novel position in SPCSV RNA1. Furthermore, unlike any other plant or animal virus, SPCSV carried an ORF for a putative RNase III-like protein (ORF2 on RNA1). Several subgenomic RNAs (sgRNAs) were detected in SPCSV-infected plants, indicating that the sgRNAs formed from RNA1 accumulated earlier in infection than those of RNA2. The 5′ ends of seven sgRNAs were cloned and sequenced by an approach that provided compelling evidence that the sgRNAs are capped in infected plants, a novel finding for members of the Closteroviridae.

RNAi-mediated resistance to diverse isolates belonging to two virus species involved in Cassava brown streak disease

Cassava brown streak disease (CBSD) is emerging as one of the most important viral diseases of cassava (Manihot esculenta) and is considered today as the biggest threat to cassava cultivation in East Africa. The disease is caused by isolates of at least two phylogenetically distinct species of single-stranded RNA viruses belonging to the family Potyviridae, genus Ipomovirus. The two species are present predominantly in the coastal lowland [Cassava brown streak virus (CBSV); Tanzania and Mozambique] and highland [Cassava brown streak Uganda virus (CBSUV); Lake Victoria Basin, Uganda, Kenya and Malawi] in East Africa. In this study, we demonstrate that CBSD can be efficiently controlled using RNA interference (RNAi). Three RNAi constructs targeting the highland species were generated, consisting of the full-length (FL; 894 nucleotides), 397-nucleotide N-terminal and 491-nucleotide C-terminal portions of the coat protein (CP) gene of a Ugandan isolate of CBSUV (CBSUV-[UG:Nam:04]), and expressed constitutively in Nicotiana benthamiana. After challenge with CBSUV-[UG:Nam:04], plants homozygous for FL-CP showed the highest resistance, followed by the N-terminal and C-terminal lines with similar resistance. In the case of FL, approximately 85% of the transgenic plant lines produced were completely resistant. Some transgenic lines were also challenged with six distinct isolates representing both species: CBSV and CBSUV. In addition to nearly complete resistance to the homologous virus, two FL plant lines showed 100% resistance and two C-terminal lines expressed 50-100% resistance, whereas the N-terminal lines succumbed to the nonhomologous CBSV isolates. Northern blotting revealed a positive correlation between the level of transgene-specific small interfering RNAs detected in transgenic plants and the level of virus resistance. This is the first demonstration of RNAi-mediated resistance to CBSD and protection across very distant isolates (more than 25% in nucleotide sequence) belonging to two different species: Cassava brown streak virus and Cassava brown streak Uganda virus.