Summary: To establish persistent infections in host plants, herbivorous invaders, such as root‐knot nematodes, must rely on effectors for suppressing damage‐induced jasmonate‐dependent host defenses. However, at present, the effector mechanisms targeting the biosynthesis of biologically active jasmonates to avoid adverse host responses are unknown. Using yeast two‐hybrid, in planta co‐immunoprecipitation, and mutant analyses, we identified 12‐oxophytodienoate reductase 2 (OPR2) as an important host target of the stylet‐secreted effector MiMSP32 of the root‐knot nematode Meloidogyne incognita. MiMSP32 has no informative sequence similarities with other functionally annotated genes but was selected for the discovery of novel effector mechanisms based on evidence of positive, diversifying selection. OPR2 catalyzes the conversion of a derivative of 12‐oxophytodienoate to jasmonic acid (JA) and operates parallel to 12‐oxophytodienoate reductase 3 (OPR3), which controls the main pathway in the biosynthesis of jasmonates. We show that MiMSP32 targets OPR2 to promote parasitism of M. incognita in host plants independent of OPR3‐mediated JA biosynthesis. Artificially manipulating the conversion of the 12‐oxophytodienoate by OPRs increases susceptibility to multiple unrelated plant invaders. Our study is the first to shed light on a novel effector mechanism targeting this process to regulate the susceptibility of host plants.

Most cytoplasmic-replicating negative-strand RNA viruses (NSVs) initiate genome transcription by cap snatching. The source of host mRNAs from which the cytoplasmic NSVs snatch capped-RNA leader sequences has remained elusive. Earlier reports have pointed towards cytoplasmic-RNA processing bodies (P body, PB), although several questions have remained unsolved. Here, the nucleocapsid (N) protein of plant- and animal-infecting members of the order Bunyavirales, in casu Tomato spotted wilt virus (TSWV), Rice stripe virus (RSV), Sin nombre virus (SNV), Crimean-Congo hemorrhagic fever virus (CCHFV) and Schmallenberg virus (SBV) have been expressed and localized in cells of their respective plant and animal hosts. All N proteins localized to PBs as well as stress granules (SGs), but extensively to docking stages of PB and SG. TSWV and RSV N proteins also co-localized with Ran GTPase-activating protein 2 (RanGAP2), a nucleo-cytoplasmic shuttling factor, in the perinuclear region, and partly in the nucleus when co-expressed with its WPP domain containing a nuclear-localization signal. Upon silencing of PB and SG components individually or concomitantly, replication levels of a TSWV minireplicon, as measured by the expression of a GFP reporter gene, ranged from a 30% reduction to a four-fold increase. Upon the silencing of RanGAP homologs in planta, replication of the TSWV minireplicon was reduced by 75%. During in vivo cap-donor competition experiments, TSWV used transcripts destined to PB and SG, but also functional transcripts engaged in translation. Altogether, the results implicate a more complex situation in which, besides PB, additional cytoplasmic sources are used during transcription/cap snatching of cytoplasmic-replicating and segmented NSVs.

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.

The potato immune receptor Gpa2 confers host-specific resistance to the cyst nematode Globodera pallida. When transiently expressed in Nicotiana benthamiana it triggers cell death upon recognition of the matching effector GpRBP-1. Effector-triggered immunity by Gpa2 depends on the host factor RanGAP2, which is known to regulate the nucleocytoplasmic distribution and functioning of the highly homologous potato immune receptor Rx1. However, the subcellular localisation of Gpa2 and the role of RanGAP2 in determining the subcellular localisation of Gpa2 is not yet known. Moreover, the cellular mechanisms underlying detection of the nematode effector by Gpa2 and the subsequent activation of cell death also remain unknown. Here, we co-expressed Solanum tuberosum Gpa2 fused to nuclear localisation signals and its matching effector GpRBP-1 in N. benthamiana as a model to address these questions. The results indicated that both the nuclear and cytoplasmic pools of Gpa2 contribute to effector-triggered cell death and this depends on the formation of a complex with RanGAP2, which acts as a cytoplasmic retention and stabilising factor. However, using nuclear and cytoplasmic targeting signals, we found that detection of GpRBP-1 by Gpa2 occurs specifically in the cytoplasm. Based on these results, we propose that RanGAP2 retains Gpa2 in the cytoplasm to form a pre-activation complex that aids in the detection of GpRBP-1 and the activation of immune responses in a compartment-specific manner.

Summary: Glycosylation is an important aspect for many biopharmaceuticals, including vaccines against parasitic helminths. Plants, especially Nicotiana benthamiana , have proven to be excellent production hosts for biopharmaceuticals with tailor‐made glycosylation. If desired, galactosylation can be introduced on biopharmaceuticals through co‐expression of the appropriate glycosyltransferase. However, achieving homogenous glycoforms with terminal galactose residues remains difficult as native N. benthamiana β‐galactosidases (NbBGALs) truncate these glycans. Recently, the first NbBGAL has been identified, but a knockout line was insufficient to achieve near complete galactosylation, suggesting that other enzymes could have similar activity. In this study, we selected 10 NbBGALs for further investigation into subcellular localization, in vitro and in vivo activity against β1,4‐linked galactose on N‐glycans and β1,3‐linked galactose on O‐glycans. We show that NbBGAL3B is localized in the apoplast and has similar specificity for β1,4‐linked galactose on N‐glycans as the previously identified NbBGAL1. In contrast, none of the selected NbBGALs cleaved β1,3‐linked galactose from O‐glycans besides BGAL1. In addition, we provide a novel strategy to achieve near complete galactosylation on galactosidase‐prone glycoproteins by using the protective capacity of the Lewis X motif and subsequent removal of the antennary fucose residues. Taken together, our results provide a broad view of the ability of NbBGALs to cleave galactoses and have identified NbBGAL3B as the second major contributor of undesired β‐galactosidase activity while engineering N‐glycans. This work lays the foundation for generating knockout lines that are devoid of undesired NbBGALs and therefore do not hamper the production of recombinant glycoproteins with galactose‐extended glycans.

Plant-parasitic nematodes (PPN) cause yield losses to multiple important crop species and counteractive measures are only partially successful. Recently the first viruses that infect PPN were discovered, presenting new research opportunities to understand and halt PPN. A critical step in this process is to identify how PPN respond to viruses. Here, two PPN species, the potato cyst nematode Globodera rostochiensis and the beet cyst nematode Heterodera schachtii, were used to study PPN-virus interactions. Viral discovery based on bioinformatic analyses indicated three putative viral genomes present in G. rostochiensis (a Toti-like, Xinzhou-like, and Picorna-like virus) and five putative viral genomes in H. schachtii (a Bunya-like, Tobamo-like, Picorna-like, and two Nyami-like viruses). A genome composition analysis and RT-PCR data confirmed that these viruses infect the nematodes and not their host plants. Subsequently, antiviral RNAi activity was detected in both H. schachtii and G. rostochiensis using small RNA sequencing. For G. rostochiensis two distinct genotypes were investigated. These strongly differed in their RNAi response: Ro5-line 22 produced roughly 40 times more antiviral viRNAs than Ro1-line 19. Our measurement of an active antiviral response represents the first indication of antiviral activity in PPN. Author summary: Plant-parasitic nematodes (PPN) are key agricultural pests and although some PPN viruses have been discovered recently, it is still unknown if and how PPN respond to viral infection. Here we find three viruses in the potato cyst nematode ( Globodera rostochiensis ) and five viruses in the beet cyst nematode ( Heterodera schachtii ). Of these viruses, six are newly discovered. Genome and molecular analyses confirm that all found viruses specifically infect nematodes and not their host plants. By measuring small RNA profiles, it was confirmed that PPN react to viral infection by means of RNAi, except for a weak response against two negatively stranded Nyami-like viruses. Moreover, distinct RNAi profiles were found for two potato cyst nematode lines, giving the first insight into which genes might be involved in RNAi in PPN. Together, this research provides the first proof of an active antiviral response in PPN. Thereby this finding opens new opportunities for biological control and gene editing tools.