Background: In addition to vertical transmission, organisms can also acquire genes from other distantly related species or from their extra-chromosomal elements (plasmids and viruses) via horizontal gene transfer (HGT). It has been suggested that phages represent substantial forces in prokaryotic evolution. In eukaryotes, retroviruses, which can integrate into host genome as an obligate step in their replication strategy, comprise approximately 8% of the human genome. Unlike retroviruses, few members of other virus families are known to transfer genes to host genomes. Results: Here we performed a systematic search for sequences related to circular single-stranded DNA (ssDNA) viruses in publicly available eukaryotic genome databases followed by comprehensive phylogenetic analysis. We conclude that the replication initiation protein (Rep)-related sequences of geminiviruses, nanoviruses and circoviruses have been frequently transferred to a broad range of eukaryotic species, including plants, fungi, animals and protists. Some of the transferred viral genes were conserved and expressed, suggesting that these genes have been coopted to assume cellular functions in the host genomes. We also identified geminivirus-like and parvovirus-like transposable elements in genomes of fungi and lower animals, respectively, and thereby provide direct evidence that eukaryotic transposons could derive from ssDNA viruses. Conclusions: Our discovery extends the host range of circular ssDNA viruses and sheds light on the origin and evolution of these viruses. It also suggests that ssDNA viruses act as an unforeseen source of genetic innovation in their hosts.

The bacterial pathogen Ralstonia solanacearum causes wilt disease on Arabidopsis thaliana and tomato (Solanum lycopersicum). This pathogen uses type III effectors to inhibit the plant immune system; however, how individual effectors interfere with plant immune responses, including transcriptional reprograming, remain elusive. Here, we show that the type III effector RipAB targets Arabidopsis TGACG SEQUENCE-SPECIFIC BINDING PROTEIN (TGA) transcription factors, the central regulators of plant immune gene regulation, via physical interaction in the nucleus to dampen immune responses. RipAB was required for R. solanacearum virulence on wild-type tomato and Arabidopsis but not Arabidopsis tga1 tga4 and tga2 tga5 tga6 mutants. Stable expression of RipAB in Arabidopsis suppressed the pathogen-associated molecular pattern-triggered reactive oxygen species (ROS) burst and immune gene induction as well as salicylic acid (SA) regulons including RBOHD and RBOHF, responsible for ROS production, all of which were phenocopied by the tga1 tga4 and tga2 tga5 tga6 mutants. We found that TGAs directly activate RBOHD and RBOHF expression and that RipAB inhibits this through interfering with the recruitment of RNA polymerase II. These results suggest that TGAs are the bona fide and major virulence targets of RipAB, which disrupts SA signaling by inhibiting TGA activity to achieve successful infection.

Background: Double-stranded (ds) RNA fungal viruses are typically isometric single-shelled particles that are classified into three families, Totiviridae, Partitiviridae and Chrysoviridae, the members of which possess monopartite, bipartite and quadripartite genomes, respectively. Recent findings revealed that mycovirus-related dsRNA viruses are more diverse than previously recognized. Although an increasing number of viral complete genomic sequences have become available, the evolution of these diverse dsRNA viruses remains to be clarified. This is particularly so since there is little evidence for horizontal gene transfer (HGT) among dsRNA viruses. Results: In this study, we report the molecular properties of two novel dsRNA mycoviruses that were isolated from a field strain of Sclerotinia sclerotiorum, Sunf-M: one is a large monopartite virus representing a distinct evolutionary lineage of dsRNA viruses; the other is a new member of the family Partitiviridae. Comprehensive phylogenetic analysis and genome comparison revealed that there are at least ten monopartite, three bipartite, one tripartite and three quadripartite lineages in the known dsRNA mycoviruses and that the multipartite lineages have possibly evolved from different monopartite dsRNA viruses. Moreover, we found that homologs of the S7 Domain, characteristic of members of the genus phytoreovirus in family Reoviridae are widely distributed in diverse dsRNA viral lineages, including chrysoviruses, endornaviruses and some unclassified dsRNA mycoviruses. We further provided evidence that multiple HGT events may have occurred among these dsRNA viruses from different families. Conclusions: Our study provides an insight into the phylogeny and evolution of mycovirus-related dsRNA viruses and reveals that the occurrence of HGT between different virus species and the development of multipartite genomes during evolution are important macroevolutionary mechanisms in dsRNA viruses.

To decipher the mechanism of pathogenicity in Sclerotinia sclerotiorum, a pathogenicity-defective mutant, Sunf-MT6, was isolated from a T-DNA insertional library. Sunf-MT6 could not form compound appressorium and failed to induce lesions on leaves of rapeseed though it could produce more oxalic acid than the wild-type strain. However, it could enter into host tissues via wounds and cause typical necrotic lesions. Furthermore, Sunf-MT6 produced fewer but larger sclerotia than the wild-type strain Sunf-M. A gene, named Ss-caf1, was disrupted by T-DNA insertion in Sunf-MT6. Gene complementation and knockdown experiments confirmed that the disruption of Ss-caf1 was responsible for the phenotypic changes of Sunf-MT6. Ss-caf1 encodes a secretory protein with a putative Ca²⁺-binding EF-hand motif. High expression levels of Ss-caf1 were observed at an early stage of compound appressorium formation and in immature sclerotia. Expression of Ss-caf1 without signal peptides in Nicotiana benthamiana via Tobacco rattle virus-based vectors elicited cell death. These results suggest that Ss-caf1 plays an important role in compound appressorium formation and sclerotial development of S. sclerotiorum. In addition, Ss-Caf1 has the potential to interact with certain host proteins or unknown substances in host cells, resulting in subsequent host cell death.

Background: Oxytropis ochrocephala Bunge, an indigenous locoweed species in China, poses great threats to livestock on grasslands. There is a need for further genetic study in the plants per se, for understanding the basis of its acclimation mechanism in various unfavorable environmental conditions and to implement effective control measures. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is the most commonly used method for gene expression analysis. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable reference genes is required. The aim of this study was to select the most stable reference genes for transcriptional analysis in O. ochrocephala. Results: We selected 12 candidate reference genes, 18S ribosomal RNA (18S RNA), actin2/7 (ACT7), β-actin (ACTB), actin101 (ACT101), actin11 (ACT11), β-tubulin (TUB), α-tubulin (TUA), glyceraldehyde-3-phosphate dehydrogenase-1 (GAPDH1), GAPDH2, metallothionein-like protein (MET), fructose-bisphosphate aldolase (FBA) and histone H3 (HIS), from the transcriptome datasets of O. ochrocephala and determined the suitability by analyzing their expression levels when exposed to a range of abiotic stress conditions. By employing software packages including geNorm, NormFinder and BestKeeper, HIS, ACT7, and ACT101 were assessed as the most suitable set for normalization in all samples. When normalized with the most stable reference genes, the expression patterns of the three target genes were in accordance with those in the transcriptome data, indicating that the reference genes selected in this study are suitable. Conclusions: The study provided appropriate reference genes for accurate normalization in qRT-PCR analysis in O. ochrocephala and emphasized the importance of validating reference genes for gene expression analysis under specific experimental condition. The usage of inappropriate reference gene would cause misinterpretation.

Background: Sclerotinia sclerotiorumis a notorious plant fungal pathogen which spreads across the world. Hypovirulence is a phenomenon where the virulence of fungal pathogens is decreased, even lost, due to mycovirus infection. The potential of hypoviruses for biological control of the chestnut blight fungus (Cryphonectria parasitica) has attracted much interest, and has led to discovery of new hypovirulent strains in other fungi.Results: A hypovirulent strain, strain XG36-1, was isolated from a typical lesion on the stem of rapeseed (Brassica napus) caused bySclerotinia sclerotiorum. Strain XG36-1 grew on PDA very slowly (average 2.5 ± 0.1 mm/d) with sectoring, and developed abnormal colony morphology with few sclerotia. Unlike health strains (such as wildtype strain XG-13), it was unable to induce lesions on detached leaves of rapeseed. Sclerotia of strain XG36-1 produced apothecia rarely. A sexual progeny test showed that the phenotypes of all 104 sexual progeny were not different from wildtype strain XG-13 which shows normal phenotype ofS. sclerotiorum, and protoplast regeneration tests showed that 25.5% of the regenerants of strain XG36-1 were recovered fully. Furthermore, the hypovirulence and its associated traits could be transmitted to XG36-1A34^R, a hygromycin-resistance gene labelled sexual progeny of strain XG36-1, by hyphal anastomosis. Transmission electron microscope (TEM) observation showed that the cytoplasm of strain XG36-1 was destroyed and granulated; the membranes of nuclei and mitochondria were disintegrated; and mitochondrial cristae were cavitated. Viral particles (about 40 nm) in hyphae of strain XG36-1, but not in its sexual progeny and wildtype strain XG-13, could be observed with TEM, and several virus-like particles were uniquely enveloped by single layer membrane in the cells of strain XG36-1. Furthermore, the viral particles could be co-transmitted with the hypovirulence traits through hyphal anastomosis.Conclusion: Hypovirulence and its associated traits of strain XG36-1 could be mediated by a fungal virus. Currently, we could not know the characteristic of this virus, but it likely represent a new type of mycovirus inS. sclerotiorum, and possibly in fungi.