Marta Vallino

Innate Nuclear Sensor IFI16 Translocates into the Cytoplasm during the Early Stage of In Vitro Human Cytomegalovirus Infection and Is Entrapped in the Egressing Virions during the Late Stage

ABSTRACT Intrinsic immune mechanisms mediated by constitutively expressed proteins termed “restriction factors” provide frontline antiviral defense. We recently demonstrated that the DNA sensor IFI16 restricts human cytomegalovirus (HCMV) replication by downregulating viral early and late but not immediate-early mRNAs and their protein expression. We show here that at an early time point during the in vitro infection of low-passage-number human embryonic lung fibroblasts, IFI16 binds to HCMV DNA. However, during a later phase following infection, IFI16 is mislocalized to the cytoplasmic virus assembly complex (AC), where it colocalizes with viral structural proteins. Indeed, upon its binding to pUL97, IFI16 undergoes phosphorylation and relocalizes to the cytoplasm of HCMV-infected cells. ESCRT (endosomal sorting complex required for transport) machinery regulates the translocation of IFI16 into the virus AC by sorting and trafficking IFI16 into multivesicular bodies (MVB), as demonstrated by the interaction of IFI16 with two MVB markers: Vps4 and TGN46. Finally, IFI16 becomes incorporated into the newly assembled virions as demonstrated by Western blotting of purified virions and electron microscopy. Together, these results suggest that HCMV has evolved mechanisms to mislocalize and hijack IFI16, trapping it within mature virions. However, the significance of this IFI16 trapping following nuclear mislocalization remains to be established. IMPORTANCE Intracellular viral DNA sensors and restriction factors are critical components of host defense, which alarm and sensitize immune system against intruding pathogens. We have recently demonstrated that the DNA sensor IFI16 restricts human cytomegalovirus (HCMV) replication by downregulating viral early and late but not immediate-early mRNAs and their protein expression. However, viruses are known to evolve numerous strategies to cope and counteract such restriction factors and neutralize the first line of host defense mechanisms. Our findings describe that during early stages of infection, IFI16 successfully recognizes HCMV DNA. However, in late stages HCMV mislocalizes IFI16 into the cytoplasmic viral assembly complex and finally entraps the protein into mature virions. We clarify here the mechanisms HCMV relies to overcome intracellular viral restriction, which provides new insights about the relevance of DNA sensors during HCMV infection.

The NSs Protein of Tomato spotted wilt virus Is Required for Persistent Infection and Transmission by Frankliniella occidentalis

ABSTRACT Tomato spotted wilt virus (TSWV) is the type member of tospoviruses (genus Tospovirus), plant-infecting viruses that cause severe damage to ornamental and vegetable crops. Tospoviruses are transmitted by thrips in the circulative propagative mode. We generated a collection of NSs-defective TSWV isolates and showed that TSWV coding for truncated NSs protein could not be transmitted by Frankliniella occidentalis. Quantitative reverse transcription (RT)-PCR and immunostaining of individual insects detected the mutant virus in second-instar larvae and adult insects, demonstrating that insects could acquire and accumulate the NSs-defective virus. Nevertheless, adults carried a significantly lower viral load, resulting in the absence of transmission. Genome sequencing and analyses of reassortant isolates showed genetic evidence of the association between the loss of competence in transmission and the mutation in the NSs coding sequence. Our findings offer new insight into the TSWV-thrips interaction and Tospovirus pathogenesis and highlight, for the first time in the Bunyaviridae family, a major role for the S segment, and specifically for the NSs protein, in virulence and efficient infection in insect vector individuals. IMPORTANCE Our work is the first to show a role for the NSs protein in virus accumulation in the insect vector in the Bunyaviridae family: demonstration was obtained for the system TSWV-F. occidentalis, arguably one of the most damaging combination for vegetable crops. Genetic evidence of the involvement of the NSs protein in vector transmission was provided with multiple approaches.

Multiple approaches for the detection and characterization of viral and plasmid symbionts from a collection of marine fungi

The number of reported mycoviruses is increasing exponentially due to the current ability to detect mycoviruses using next-generation sequencing (NGS) approaches, with a large number of viral genomes built in-silico using data from fungal transcriptome projects. We decided to screen a collection of fungi originating from a specific marine environment (associated with the seagrass Posidonia oceanica) for the presence of mycoviruses: our findings reveal a wealth of diversity among these symbionts and this complexity will require further studies to address their specific role in this ecological niche. In specific, we identified twelve new virus species belonging to nine distinct lineages: they are members of megabirnavirus, totivirus, chrysovirus, partitivirus and five still undefined clades. We showed evidence of an endogenized virus ORF, and evidence of accumulation of dsRNA from metaviridae retroviral elements. We applied different techniques for detecting the presence of mycoviruses including (i) dsRNA extraction and cDNA cloning, (ii) small and total RNA sequencing through NGS techniques, (iii) rolling circle amplification (RCA) and total DNA extraction analyses, (iv) virus purifications and electron microscopy. We tried also to critically evaluate the intrinsic value and limitations of each of these techniques. Based on the samples we could compare directly, RNAseq analysis is superior to sRNA for de novo assembly of mycoviruses. To our knowledge this is the first report on the virome of fungi isolated from marine environment. The GenBank/eMBL/DDBJ accession numbers of the sequences reported in this paper are: KT601099-KT601110; KT601114-KT601120; KT592305; KT950836-KT950841.

Different farming and water regimes in Italian rice fields affect arbuscular mycorrhizal fungal soil communities

Arbuscular mycorrhizal fungi (AMF) comprise one of the main components of soil microbiota in most agroecosystems. These obligate mutualistic symbionts colonize the roots of most plants, including crop plants. Many papers have indicated that different crop management practices could affect AMF communities and their root colonization. However, there is little knowledge available on the influence of conventional and low-input agriculture on root colonization and AMF molecular diversity in rice fields. Two different agroecosystems (continuous conventional high-input rice monocropping and organic farming with a five-year crop rotation) and two different water management regimes have been considered in this study. Both morphological and molecular analyses were performed. The soil mycorrhizal potential, estimated using clover trap cultures, was high and similar in the two agroecosystems. The diversity of the AMF community in the soil, calculated by means of PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) and 18S rDNA sequencing on clover trap cultures roots, was higher for the organic cultivation. The rice roots cultivated in the conventional agrosystem or under permanent flooding showed no AMF colonization, while the rice plants grown under the organic agriculture system showed typical mycorrhization patterns. Considered together, our data suggest that a high-input cropping system and conventional flooding depress AMF colonization in rice roots and that organic managements could help maintain a higher diversity of AMF communities in soil.

Imaging mycorrhizal fungal transformants that express EGFP during ericoid endosymbiosis

Ericoid endomycorrhizal fungi form intracellular associations with the epidermal root cells of plants belonging to Ericales. In natural environments, these fungi increase the ability of their host plants to colonise soils polluted with toxic metals, although the underlying mechanisms are not clearly understood. Genetic transformation is a powerful tool to study the function of specific genes involved in the interaction of symbiotic fungi with the host plants and with the environment. Here, we investigated the possibility to genetically transform an ericoid endomycorrhizal strain. A metal tolerant mycorrhizal Oidiodendron maius strain isolated from a contaminated area was chosen to develop the transformation system. Two different protocols were used: protoplasts and Agrobacterium-mediated transformation. Stable transformants were obtained with both techniques. They remained competent for mycorrhizal formation and GFP-transformed fungi were visualised in planta. This is the first report of stable transformation of an ericoid endomycorrhizal fungus. The protocol set up could represent a good starting point for the identification of genes important in the ericoid mycorrhiza formation and in the understanding of how this symbiosis is established and functions. The success in the genetic transformation of this strain will allow us to better define its potential use in bioremediation strategies.

Cu,Zn superoxide dismutase and zinc stress in the metal-tolerant ericoid mycorrhizal fungus Oidiodendron maius Zn.

The sequence encoding a superoxide dismutase (SOD) was isolated from the cDNA library of a zinc-tolerant strain of the ericoid mycorrhizal fungus Oidiodendron maius, grown under zinc-stress conditions. Sequence homology to other SODs strongly suggests that it is a copper- and zinc-containing SOD. Functional complementation assays showed that the gene confers increased tolerance to zinc and copper stress to a Cu,ZnSOD-defective yeast mutant. Monitoring of transcript and protein levels following zinc stress suggests that OmSOD1 expression is controlled at the transcriptional level. The OmSod1 protein was found both in the cell extract and in the growth medium of viable fungal cultures. This is the first characterization of an extracellular Cu,ZnSOD in a mycorrhizal fungus. In nature, the presence of OmSod1 in the extracellular environment may also extend the protective role of this enzyme to the plant symbiont. This may be of particular interest from the perspective of using mycorrhizal fungi in bioremediation programmes.

Rice flooding negatively impacts root branching and arbuscular mycorrhizal colonization, but not fungal viability.

Rice is mostly cultivated in wetlands, where arbuscular mycorrhization (AM) is reported to decrease. The mechanisms regulating such events are largely unknown. Rice uninoculated and inoculated with Rhizophagus irregularis were grown in dry and flooded conditions, allowing also for the transfer of plants from one water regime to the other. Roots were sampled at different times, from 7 to 35 d post-inoculation (dpi). The morphological and molecular parameters (root branching, aerenchyma formation, mycorrhizal colonization, AM marker gene expression) were evaluated. Root branching was more pronounced in dry conditions, and such phenotype was enhanced by the fungus. In wetlands, the colonization level was comparable till 21 dpi, when the mycorrhization then decreased, paralleled by an increase in aerenchyma. Expression of the fungal transporters was comparable under the two conditions. The root apparatus, when shifted from one water regime to the other, rapidly adapted to the new condition, revealing a marked plasticity. The reversibility of the AM rice symbiosis was also mirrored by expression changes of plant marker genes. The results demonstrate that the water regime is the driving force that regulates AM colonization under flooding conditions, by directly influencing root architecture and anatomy, but without impacting the basic AM functionality.

Unique arbuscular mycorrhizal fungal communities uncovered in date palm plantations and surrounding desert habitats of Southern Arabia

The main objective of this study was to shed light on the previously unknown arbuscular mycorrhizal fungal (AMF) communities in Southern Arabia. We explored AMF communities in two date palm (Phoenix dactylifera) plantations and the natural vegetation of their surrounding arid habitats. The plantations were managed traditionally in an oasis and according to conventional guidelines at an experimental station. Based on spore morphotyping, the AMF communities under the date palms appeared to be quite diverse at both plantations and more similar to each other than to the communities under the ruderal plant, Polygala erioptera, growing at the experimental station on the dry strip between the palm trees, and to the communities uncovered under the native vegetation (Zygophyllum hamiense, Salvadora persica, Prosopis cineraria, inter-plant area) of adjacent undisturbed arid habitat. AMF spore abundance and species richness were higher under date palms than under the ruderal and native plants. Sampling in a remote sand dune area under Heliotropium kotschyi yielded only two AMF morphospecies and only after trap culturing. Overall, 25 AMF morphospecies were detected encompassing all study habitats. Eighteen belonged to the genus Glomus including four undescribed species. Glomus sinuosum, a species typically found in undisturbed habitats, was the most frequently occurring morphospecies under the date palms. Using molecular tools, it was also found as a phylogenetic taxon associated with date palm roots. These roots were associated with nine phylogenetic taxa, among them eight from Glomus group A, but the majority could not be assigned to known morphospecies or to environmental sequences in public databases. Some phylogenetic taxa seemed to be site specific. Despite the use of group-specific primers and efficient trapping systems with a bait plant consortium, surprisingly, two of the globally most frequently found species, Glomus intraradices and Glomus mosseae, were not detected neither as phylogenetic taxa in the date palm roots nor as spores under the date palms, the intermediate ruderal plant, or the surrounding natural vegetation. The results highlight the uniqueness of AMF communities inhabiting these diverse habitats exposed to the harsh climatic conditions of Southern Arabia.

Host and non-host roots in rice: cellular and molecular approaches reveal differential responses to arbuscular mycorrhizal fungi

Oryza sativa, a model plant for Arbuscular Mycorrhizal (AM) symbiosis, has both host and non-host roots. Large lateral (LLR) and fine lateral (FLR) roots display opposite responses: LLR support AM colonization, but FLR do not. Our research aimed to study the molecular, morphological and physiological aspects related to the non-host behavior of FLR. RNA-seq analysis revealed that LLR and FLR displayed divergent expression profiles, including changes in many metabolic pathways. Compared with LLR, FLR showed down-regulation of genes instrumental for AM establishment and gibberellin signaling, and a higher expression of nutrient transporters. Consistent with the transcriptomic data, FLR had higher phosphorus content. Light and electron microscopy demonstrated that, surprisingly, in the Selenio cultivar, FLR have a two-layered cortex, which is theoretically compatible with AM colonization. According to RNA-seq, a gibberellin inhibitor treatment increased anticlinal divisions leading to a higher number of cortex cells in FLR. We propose that some of the differentially regulated genes that lead to the anatomical and physiological properties of the two root types also function as genetic factors regulating fungal colonization. The rice root apparatus offers a unique tool to study AM symbiosis, allowing direct comparisons of host and non-host roots in the same individual plant.

Gene expression of the ericoid mycorrhizal fungus Oidiodendron maius in the presence of high zinc concentrations

A heavy metal tolerant strain of the ericoid mycorrhizal species Oidiodendron maius, isolated from roots of Vaccinium myrtillus growing in soil heavily contaminated with zinc, was previously shown to tolerate high concentrations of zinc and cadmium ions in the growth medium. We have investigated the genetic basis of this fungal strain tolerance to high zinc concentrations by using an untargeted approach. From a cDNA library constructed by using mRNA from Zn-treated O. maius mycelia, 444 clones were randomly selected and 318 were sequenced. Sequence analysis identified 219 unique clones: 117 showed homology to previously identified genes, 26 matched unknown protein coding regions found in other organisms, and 76 were novel. Variation in the gene expression level after a 20-day treatment with high concentrations of Zn was monitored on 130 unigenes by reverse northern blot hybridisation. Sixteen unigenes were shown to be either up- (9) or down- (7) regulated. The putative function of these genes and their involvement in stress tolerance is discussed.