Comparative Transcriptome Analysis Reveals the Specific Activation of Defense Pathways Against Globodera pallida in Gpa2 Resistant Potato Roots.

Cyst nematodes are considered a dominant threat to yield for a wide range of major food crops. Current control strategies are mainly dependent on crop rotation and the use of resistant cultivars. Various crops exhibit single dominant resistance (R) genes that are able to activate effective host-specific resistance to certain cyst nematode species and/or populations. An example is the potato R gene Gpa2, which confers resistance against the potato cyst nematode (PCN), Globodera pallida population D383. Activation of Gpa2 results in a delayed resistance response, which is characterized by a layer of necrotic cells formed around the developing nematode feeding structure. However, knowledge about the Gpa2-induced defense pathways is still lacking. Here, we uncover the transcriptional changes and gene expression network induced upon Gpa2 activation in potato roots infected with G. pallida. To this end, in vitro-grown Gpa2-resistant potato roots were infected with the avirulent population D383 and virulent population Rookmaker. Infected root segments were harvested at 3 and 6 dpi and sent for RNA sequencing. Comparative transcriptomics revealed a total of 1,743 differentially expressed genes (DEGs) upon nematode infection, of which 559 DEGs were specifically regulated in response to D383 infection. D383-specific DEGs associated with Gpa2-mediated defense mainly relates to calcium-binding activity, salicylic acid (SA) biosynthesis, and systemic acquired resistance (SAR). These data reveal that cyst nematode resistance in potato roots depends on conserved downstream signaling pathways involved in plant immunity, which are also known to contribute to R genes-mediated resistance against other pathogens with different lifestyles.

GLYCINE-RICH RNA-BINDING PROTEIN 7 potentiates effector-triggered immunity through an RNA recognition motif.

The activity of intracellular plant nucleotide-binding leucine-rich repeat (NB-LRR) immune receptors is fine-tuned by interactions between the receptors and their partners. Identifying NB-LRR interacting proteins is therefore crucial to advance our understanding of how these receptors function. A co-immunoprecipitation/mass spectrometry screening was performed in Nicotiana benthamiana to identify host proteins associated with the resistance protein Gpa2, a CC-NB-LRR immune receptor conferring resistance against the potato cyst nematode Globodera pallida. A combination of biochemical, cellular, and functional assays was used to assess the role of a candidate interactor in defense. A N. benthamiana homolog of the GLYCINE-RICH RNA-BINDING PROTEIN7 (NbGRP7) protein was prioritized as a Gpa2-interacting protein for further investigations. NbGRP7 also associates in planta with the homologous Rx1 receptor, which confers immunity to Potato Virus X. We show that NbGRP7 positively regulates extreme resistance by Rx1 and cell death by Gpa2. Mutating the NbGRP7 RNA recognition motif (RRM) compromises its role in Rx1-mediated defense. Strikingly, ectopic NbGRP7 expression is likely to impact the steady-state levels of Rx1, which relies on an intact RRM. Our findings illustrate that NbGRP7 is a pro-immune component in effector-triggered immunity by regulating Gpa2/Rx1 function at a posttranscriptional level.

Members of the ribosomal protein S6 (RPS6) family act as pro-viral factor for tomato spotted wilt orthotospovirus infectivity in Nicotiana benthamiana.

To identify host factors for tomato spotted wilt orthotospovirus (TSWV), a virus-induced gene silencing (VIGS) screen using tobacco rattle virus (TRV) was performed on Nicotiana benthamiana for TSWV susceptibility. To rule out any negative effect on the plants' performance due to a double viral infection, the method was optimized to allow screening of hundreds of clones in a standardized fashion. To normalize the results obtained in and between experiments, a set of controls was developed to evaluate in a consist manner both VIGS efficacy and the level of TSWV resistance. Using this method, 4532 random clones of an N. benthamiana cDNA library were tested, resulting in five TRV clones that provided nearly complete resistance against TSWV. Here we report on one of these clones, of which the insert targets a small gene family coding for the ribosomal protein S6 (RPS6) that is part of the 40S ribosomal subunit. This RPS6 family is represented by three gene clades in the genome of Solanaceae family members, which were jointly important for TSWV susceptibility. Interestingly, RPS6 is a known host factor implicated in the replication of different plant RNA viruses, including the negative-stranded TSWV and the positive-stranded potato virus X.

An Isoform of the Eukaryotic Translation Elongation Factor 1A (eEF1a) Acts as a Pro-Viral Factor Required for Tomato Spotted Wilt Virus Disease in Nicotiana benthamiana.

The tripartite genome of the negative-stranded RNA virus Tomato spotted wilt orthotospovirus (TSWV) is assembled, together with two viral proteins, the nucleocapsid protein and the RNA-dependent RNA polymerase, into infectious ribonucleoprotein complexes (RNPs). These two viral proteins are, together, essential for viral replication and transcription, yet our knowledge on the host factors supporting these two processes remains limited. To fill this knowledge gap, the protein composition of viral RNPs collected from TSWV-infected Nicotiana benthamiana plants, and of those collected from a reconstituted TSWV replicon system in the yeast Saccharomyces cerevisiae, was analysed. RNPs obtained from infected plant material were enriched for plant proteins implicated in (i) sugar and phosphate transport and (ii) responses to cellular stress. In contrast, the yeast-derived viral RNPs primarily contained proteins implicated in RNA processing and ribosome biogenesis. The latter suggests that, in yeast, the translational machinery is recruited to these viral RNPs. To examine whether one of these cellular proteins is important for a TSWV infection, the corresponding N. benthamiana genes were targeted for virus-induced gene silencing, and these plants were subsequently challenged with TSWV. This approach revealed four host factors that are important for systemic spread of TSWV and disease symptom development.

On the role of dauer in the adaptation of nematodes to a parasitic lifestyle.

Nematodes are presumably the most abundant Metazoa on Earth, and can even be found in some of the most hostile environments of our planet. Various types of hypobiosis evolved to adapt their life cycles to such harsh environmental conditions. The five most distal major clades of the phylum Nematoda (Clades 8-12), formerly referred to as the Secernentea, contain many economically relevant parasitic nematodes. In this group, a special type of hypobiosis, dauer, has evolved. The dauer signalling pathway, which culminates in the biosynthesis of dafachronic acid (DA), is intensively studied in the free-living nematode Caenorhabditis elegans, and it has been hypothesized that the dauer stage may have been a prerequisite for the evolution of a wide range of parasitic lifestyles among other nematode species. Biosynthesis of DA is not specific for hypobiosis, but if it results in exit of the hypobiotic state, it is one of the main criteria to define certain behaviour as dauer. Within Clades 9 and 10, the involvement of DA has been validated experimentally, and dauer is therefore generally accepted to occur in those clades. However, for other clades, such as Clade 12, this has hardly been explored. In this review, we provide clarity on the nomenclature associated with hypobiosis and dauer across different nematological subfields. We discuss evidence for dauer-like stages in Clades 8 to 12 and support this with a meta-analysis of available genomic data. Furthermore, we discuss indications for a simplified dauer signalling pathway in parasitic nematodes. Finally, we zoom in on the host cues that induce exit from the hypobiotic stage and introduce two hypotheses on how these signals might feed into the dauer signalling pathway for plant-parasitic nematodes. With this work, we contribute to the deeper understanding of the molecular mechanisms underlying hypobiosis in parasitic nematodes. Based on this, novel strategies for the control of parasitic nematodes can be developed.

A functional investigation of the suppression of CpG and UpA dinucleotide frequencies in plant RNA virus genomes.

Frequencies of CpG and UpA dinucleotides in most plant RNA virus genomes show degrees of suppression comparable to those of vertebrate RNA viruses. While pathways that target CpG and UpAs in HIV-1 and echovirus 7 genomes and restrict their replication have been partly characterised, whether an analogous process drives dinucleotide underrepresentation in plant viruses remains undetermined. We examined replication phenotypes of compositionally modified mutants of potato virus Y (PVY) in which CpG or UpA frequencies were maximised in non-structural genes (including helicase and polymerase encoding domains) while retaining protein coding. PYV mutants with increased CpG dinucleotide frequencies showed a dose-dependent reduction in systemic spread and pathogenicity and up to 1000-fold attenuated replication kinetics in distal sites on agroinfiltration of tobacco plants (Nicotiana benthamiana). Even more extraordinarily, comparably modified UpA-high mutants displayed no pathology and over a million-fold reduction in replication. Tobacco plants with knockdown of RDP6 displayed similar attenuation of CpG- and UpA-high mutants suggesting that restriction occurred independently of the plant siRNA antiviral responses. Despite the evolutionary gulf between plant and vertebrate genomes and encoded antiviral strategies, these findings point towards the existence of novel virus restriction pathways in plants functionally analogous to innate defence components in vertebrate cells.

Host-specific accumulation and temperature effects on the generation of dimeric viral RNA species derived from the S-RNA of members of the Tospovirus genus.

Polygonum ringspot virus (PolRSV) is a recently characterized Tospovirus reported in Italy. Northern blot analyses of PolRSV infections in Nicotiana benthamiana and tomato plants showed that a viral RNA species with nearly twice the length of the Small genomic RNA (S-RNA) accumulated abundantly in the former host, but was not detected in the latter. Additional assays confirmed that biogenesis of this novel RNA species was common to all PolRSV isolates tested and also to an isolate of Tomato spotted wilt virus (TSWV). Given its size, we hypothesized that the novel RNA species was a dimer molecule and we confirmed this hypothesis by RNA sequencing (RNAseq) analysis and reverse transcription (RT)-PCR of putative predicted dimer junction sites in RNA extracts of N. benthamiana challenged with PolRSV isolates Plg6 and Plg13/2. We also confirmed that these molecules are derived from head-to-tail dimers and often contain deletions at their junction sites. We named these novel molecules imperfect dimer RNAs (IMPD-RNAs). PolRSV IMPD-RNAs systemic accumulation in a range of host plants was restricted to N. benthamiana and Nicotiana occidentalis. Notably, IMPD-RNAs accumulation was modulated by temperature and their generation was restricted to late stages of systemic infection (12 days post-inoculation) in N. benthamiana. Differently from all other PolRSV isolates used in this study, Plg13/2 generated more IMPD-RNAs coupled with low amounts of genomic S-RNA and maintained them even at 18 °C, besides having lost the ability to infect tomato plants. This is the first characterization of S-RNA dimers for Tospovirus, and of occurrence of dimers of genomic segments at the whole organism level for Bunyaviridae.

An RNA-dependent RNA polymerase gene of a distinct Brazilian tospovirus.

The tospoviral RNA-dependent RNA polymerases (RdRp), or L proteins, perform several conserved functions during virus replication in host cells. In this study, an L segment sequence of 9,040 bp from a new tospovirus (family Bunyaviridae) naturally infecting bean (Phaseolus vulgaris L.) plants was characterized. It encodes the largest RdRp gene known yet for this genus, with deduced 2932aa and a molecular mass of approximately 336 kDa. A Lysine-rich C-terminal extension was found, which apart from our isolate, was only recognized in another recently discovered tospovirus infecting Fabaceae, Soybean vein necrosis associated virus (SVNaV). Due to its distinct biological features and L protein-based phylogenetic analysis showing an almost equidistant position in comparison to Eurasian and American Tospovirus groups, as well as the clustering with SVNaV, we suggest the tentative name Bean necrotic mosaic virus for this unique isolate.