Kamal Hleibieh

An Arabidopsis Dual-Localized Pentatricopeptide Repeat Protein Interacts with Nuclear Proteins Involved in Gene Expression Regulation

This work examines a novel PPR protein that localizes to both mitochondria and nuclei in Arabidopsis. In mitochondria, it associates with polysomes, and in the nucleus, it interacts with a nucleosome assembly protein and a TCP transcription factor. This PPR protein might play a role in gene expression adjustments between mitochondria and the nucleus. Following the endosymbiotic acquisition of mitochondria by eukaryotic cells, most of the genes in this organelle were transferred to the nucleus. To maintain mitochondrial biogenesis and function, nuclear and mitochondrial genomes require regulated and coordinated expression. In plant organelles, nuclear-encoded proteins targeted to the organelles control posttranscriptional and posttranslational mechanisms. Pentatricopeptide repeat (PPR) proteins are good candidates to play such regulatory roles. Here, we identify PNM1 (for PPR protein localized to the nucleus and mitochondria 1), a novel PPR protein that is dual localized to mitochondria and nuclei in Arabidopsis thaliana, as observed by green fluorescent protein fusions and immunodetection on subcellular fractions and on histological sections. Genetic complementation showed that loss of PNM1 function in mitochondria, but not in nuclei, is lethal for the embryo. In mitochondria, it is associated with polysomes and may play a role in translation. A genetic screen in yeast identified protein partners of PNM1. These partners, the nucleosome assembly protein NAP1, and the transcription factor TCP8 interact with PNM1 in the nucleus in planta. Furthermore, TCP8 can bind the promoter of PNM1. This suggests that PNM1 might be involved in the regulation of its own gene expression in the nucleus and could thus play a role in gene expression adjustments between mitochondria and the nucleus.

The Benyvirus RNA Silencing Suppressor Is Essential for Long-Distance Movement, Requires Both Zinc-Finger and NoLS Basic Residues but Not a Nucleolar Localization for Its Silencing-Suppression Activity

The RNA silencing-suppression properties of Beet necrotic yellow vein virus (BNYVV) and Beet soil-borne mosaic virus (BSBMV) cysteine-rich p14 proteins have been investigated. Suppression of RNA silencing activities were made evident using viral infection of silenced Nicotiana benthamiana 16C, N. benthamiana agroinfiltrated with green fluorescent protein (GFP), and GF-FG hairpin triggers supplemented with viral suppressor of RNA silencing (VSR) constructs or using complementation of a silencing-suppressor-defective BNYVV virus in Chenopodium quinoa. Northern blot analyses of small-interfering RNAs (siRNAs) in agroinfiltration tests revealed reduced amounts of siRNA, especially secondary siRNA, suggesting that benyvirus VSR act downstream of the siRNA production. Using confocal laser-scanning microscopy imaging of infected protoplasts expressing functional p14 protein fused to an enhanced GFP reporter, we showed that benyvirus p14 accumulated in the nucleolus and the cytoplasm independently of other viral factors. Site-directed mutagenesis showed the importance of the nucleolar localization signal embedded in a C4 zinc-finger domain in the VSR function and intrinsic stability of the p14 protein. Conversely, RNA silencing suppression appeared independent of the nucleolar localization of the protein, and a correlation between BNYVV VSR expression and long-distance movement was established.

The P25 pathogenicity factor of Beet necrotic yellow vein virus targets the sugar beet 26S proteasome involved in the induction of a hypersensitive resistance response via interaction with an F-box protein.

P25, a Beet necrotic yellow vein virus (BNYVV) pathogenicity factor, interacts with a sugar beet protein with high homology to Arabidopsis thaliana kelch repeat containing F-box family proteins (FBK) of unknown function in yeast. FBK are members of the Skp1-Cullin-F-box (SCF) complex that mediate protein degradation. Here, we confirm this sugar beet FBK-P25 interaction in vivo and in vitro and provide evidence for in planta interaction and similar subcellular distribution in Nicotiana tabacum leaf cells. P25 even interacts with an FBK from A. thaliana, a BNYVV nonhost. FBK functional classification was possible by demonstrating the interaction with A. thaliana orthologs of Skp1-like (ASK) genes, a member of the SCF E3 ligase. By means of a yeast two-hybrid bridging assay, a direct effect of P25 on SCF-complex formation involving ASK1 protein was demonstrated. FBK transient Agrobacterium tumefaciens-mediated expression in N. benthamiana leaves induced a hypersensitive response. The full-length F-box protein consists of one F-box domain followed by two kelch repeats, which alone were unable to interact with P25 in yeast and did not lead to cell-death induction. The results support the idea that P25 is involved in virus pathogenicity in sugar beet and suggest suppression of resistance response.

Nanobody-mediated resistance to Grapevine fanleaf virus in plants.

Summary Since their discovery, single‐domain antigen‐binding fragments of camelid‐derived heavy‐chain‐only antibodies, also known as nanobodies (Nbs), have proven to be of outstanding interest as therapeutics against human diseases and pathogens including viruses, but their use against phytopathogens remains limited. Many plant viruses including Grapevine fanleaf virus (GFLV), a nematode‐transmitted icosahedral virus and causal agent of fanleaf degenerative disease, have worldwide distribution and huge burden on crop yields representing billions of US dollars of losses annually, yet solutions to combat these viruses are often limited or inefficient. Here, we identified a Nb specific to GFLV that confers strong resistance to GFLV upon stable expression in the model plant Nicotiana benthamiana and also in grapevine rootstock, the natural host of the virus. We showed that resistance was effective against a broad range of GFLV isolates independently of the inoculation method including upon nematode transmission but not against its close relative, Arabis mosaic virus. We also demonstrated that virus neutralization occurs at an early step of the virus life cycle, prior to cell‐to‐cell movement. Our findings will not only be instrumental to confer resistance to GFLV in grapevine, but more generally they pave the way for the generation of novel antiviral strategies in plants based on Nbs.

A Viral Noncoding RNA Complements a Weakened Viral RNA Silencing Suppressor and Promotes Efficient Systemic Host Infection

Systemic movement of beet necrotic yellow vein virus (BNYVV) in Beta macrocarpa depends on viral RNA3, whereas in Nicotiana benthamiana this RNA is dispensable. RNA3 contains a coremin motif of 20 nucleotides essential for the stabilization of noncoding RNA3 (ncRNA3) and for long-distance movement in Beta species. Coremin mutants that are unable to accumulate ncRNA3 also do not achieve systemic movement in Beta species. A mutant virus carrying a mutation in the p14 viral suppressor of RNA silencing (VSR), unable to move long distances, can be complemented with the ncRNA3 in the lesion phenotype, viral RNA accumulation, and systemic spread. Analyses of the BNYVV VSR mechanism of action led to the identification of the RNA-dependent RNA polymerase 6 (RDR6) pathway as a target of the virus VSR and the assignment of a VSR function to the ncRNA3.

On the interaction and localization of the beet necrotic yellow vein virus replicase.

Beet necrotic yellow vein virus (BNYVV) is a multipartite positive-strand RNA virus. BNYVV RNA-1 encodes a non-structural p237 polyprotein processed in two proteins (p150 and p66) by a cis-acting protease activity. BNYVV non-structural proteins are closely related to replication proteins of positive strand RNA viruses such as hepeviruses rather to other plant virus replicases. The p237 and dsRNA have been localized by TEM in ER structures of infected leaf cells whereas dsRNA was immunolabeled in infected protoplasts. The p150 contains domains with methyltransferase, protease, helicase and two domains of unknown function whereas p66 encompasses the RNA-dependent RNA-polymerase signature. We report the existing interactions between functional domains of the p150 and p66 proteins and the addressing of the benyvirus replicase to the endoplasmic reticulum. Yeast two-hybrid approach, colocalization with FRET-FLIM analyses and co-immunoprecipitation highlighted existing interactions that suggest the presence of a multimeric complex at the vicinity of the cellular membranous web.

Ectoparasitic Nematode Vectors of Grapevine Viruses

Nematodes of the genera Longidorus, Paralongidorus, and Xiphinema in the family Longidoridae can parasitize grapevine roots. These ectoparasitic soil-borne nematodes live in proximity to the rhizosphere and use a stylet to feed on root cells. During the feeding process, some nematodes are able to acquire and transmit nepoviruses, the causal agents of grapevine degeneration disease. The association between nematode vectors and the virus(es) they transmit is specific. This specificity is linked to the nature, site, and mechanism of virus retention. Here, we present and discuss major features of the interaction between nematodes and viruses with regard to the biology, ecology, and life cycle of the nematodes. In addition, we discuss how reverse genetics in combination with virus structural data have recently provided insights into the specificity of the transmission process through the identification of a cavity-like domain on the virion surface that could be recognized by a ligand within the nematode feeding apparatus. Finally, we offer some perspectives on future research to further advance nematode-virus interactions.

Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

Double-stranded RNA (dsRNA) plays essential functions in many biological processes, including the activation of innate immune responses and RNA interference. dsRNA also represents the genetic entity of some viruses and is a hallmark of infections by positive-sense single-stranded RNA viruses. Methods for detecting dsRNA rely essentially on immunological approaches and their use is often limited to in vitro applications, although recent developments have allowed the visualization of dsRNA in vivo. Here, we report the sensitive and rapid detection of long dsRNA both in vitro and in vivo using the dsRNA binding domain of the B2 protein from Flock house virus. In vitro, we adapted the system for the detection of dsRNA either enzymatically by northwestern blotting or by direct fluorescence labeling on fixed samples. In vivo, we produced stable transgenic Nicotiana benthamiana lines allowing the visualization of dsRNA by fluorescence microscopy. Using these techniques, we were able to discriminate healthy and positive-sense single-stranded RNA virus-infected material in plants and insect cells. In N. benthamiana, our system proved to be very potent for the spatio-temporal visualization of replicative RNA intermediates of a broad range of positive-sense RNA viruses, including high- vs. low-copy number viruses.

Phloem-Triggered Virus-Induced Gene Silencing Using a Recombinant Polerovirus

The phloem-limited poleroviruses infect Arabidopsis thaliana without causing noticeable disease symptoms. In order to facilitate visual infection identification, we developed virus-induced gene silencing (VIGS) vectors derived from Turnip yellows virus (TuYV). Short sequences from the host gene AtCHLI1 required for chlorophyll biosynthesis [42 nucleotides in sense or antisense orientation or as an inverted-repeat (IR), or an 81 nucleotide sense fragment] were inserted into the 3′ non-coding region of the TuYV genome to screen for the most efficient and robust silencing vector. All recombinant viruses produced a clear vein chlorosis phenotype on infected Arabidopsis plants due to the expression inhibition of the AtCHLI1 gene. The introduction of a sense-oriented sequence into TuYV genome resulted in a virus exhibiting a more sustainable chlorosis than the virus containing an IR of the same length. This observation was correlated with a higher stability of the sense sequence insertion in the viral genome. In order to evaluate the impact of the TuYV silencing suppressor P0 in the VIGS mechanism a P0 knock-out mutation was introduced into the recombinant TuYV viruses. They induced a similar but milder vein clearing phenotype due to lower viral accumulation. This indicates that P0 does not hinder the performances of the TuYV silencing effect and confirms that in the viral infection context, P0 has no major impact on the production, propagation and action of the short distance silencing signal in phloem cells. Finally, we showed that TuYV can be used to strongly silence the phloem specific AtRTM1 gene. The TuYV-derived VIGS vectors therefore represent powerful tools to easily detect and monitor TuYV in infected plants and conduct functional analysis of phloem-restricted genes. Moreover this example indicates the potential of poleroviruses for use in functional genomic studies of agronomic plants.