Characterization of Superoxide Dismutase from the Potato Cyst Nematode, Globodera pallida.

Potato cyst nematodes (PCNs), such as Globodera pallida and Globodera rostochiensis, are some of the most agriculturally and economically important pests of potato. Upon nematode infection, a principal component of plant defense is the generation of the reactive oxygen species (ROSs). ROSs are highly toxic molecules that cause damage to pathogens and host alike. To infect the plant, nematodes protect themselves from ROSs by activating their own antioxidant processes and ROS scavenging enzymes. One of these enzymes is a superoxide dismutase (SOD; EC 1.15.1.1), which prevents cellular damage by catalyzing conversion of the superoxide radical (O2-·) to hydrogen peroxide (H2O2) and molecular oxygen (O2). We have isolated a putatively secreted isoform of a Cu-Zn SOD (SOD-3) from G. pallida and localized the expression of this gene in the posterior region of the nematode. Furthermore, we studied the expression of the SOD-3 gene during early parasitic stages of infection (24 to 72 h) in the susceptible potato cultivar Desiree, the resistant potato cultivar Innovator, and an immune host, Solanum sisymbriifolium. The SOD-3 gene was significantly upregulated, regardless of the host type; however, the expression pattern differed between the susceptible and the resistant or immune hosts. This finding suggests that SOD-3 gene is responding to infection in plant roots differently depending on whether the nematode is experiencing a compatible or an incompatible interaction.

Initial responses of the trap-crop, Solanum sisymbriifolium, to Globodera pallida invasions.

Many researchers today are looking for mechanisms underlying plant defenses against nematodes by identifying differentially expressed genes in domesticated hosts. In order to provide a different perspective, we analyzed the root transcriptome of an undomesticated non-host species, Solanum sisymbriifolium Lamark (SSI) before and after Globodera pallida infection. Utilizing RNAseq analyses, we identified changes in the expression of 277 transcripts. Many of these genes were not annotated; however, the annotated set included peroxidases, reactive oxygen species-producing proteins, and regulators of cell death. Importantly, 60% of the nematode-responsive genes did not respond to physical damage to root tissues, or to exogenous treatments with either salicylic acid or methyl jasmonate. Based on this, we speculate that the majority of changes in SSI gene expression were promoted by either nematode effectors, pathogen-associated molecular patterns (PAMPs), or by exposure to untested endogenous signaling molecules such as ethylene, or by exposure to multiple stimuli. This study incorporates our findings into a model that accounts for part of this plant's unusual resistance to nematodes.

The effector GpRbp-1 of Globodera pallida targets a nuclear HECT E3 ubiquitin ligase to modulate gene expression in the host.

Plant-parasitic nematodes secrete effectors that manipulate plant cell morphology and physiology to achieve host invasion and establish permanent feeding sites. Effectors from the highly expanded SPRYSEC (SPRY domain with a signal peptide for secretion) family in potato cyst nematodes have been implicated in activation and suppression of plant immunity, but the mechanisms underlying these activities remain largely unexplored. To study the host mechanisms used by SPRYSEC effectors, we identified plant targets of GpRbp-1 from the potato cyst nematode Globodera pallida. Here, we show that GpRbp-1 interacts in yeast and in planta with a functional potato homologue of the Homology to E6-AP C-Terminus (HECT)-type ubiquitin E3 ligase UPL3, which is located in the nucleus. Potato lines lacking StUPL3 are not available, but the Arabidopsis mutant upl3-5 displaying a reduced UPL3 expression showed a consistently small but not significant decrease in susceptibility to cyst nematodes. We observed a major impact on the root transcriptome by the lower levels of AtUPL3 in the upl3-5 mutant, but surprisingly only in association with infections by cyst nematodes. To our knowledge, this is the first example that a HECT-type ubiquitin E3 ligase is targeted by a pathogen effector and that a member of this class of proteins specifically regulates gene expression under biotic stress conditions. Together, our data suggest that GpRbp-1 targets a specific component of the plant ubiquitination machinery to manipulate the stress response in host cells.

Transcriptome analysis of Globodera pallida from the susceptible host Solanum tuberosum or the resistant plant Solanum sisymbriifolium.

A transcriptome analysis of G. pallida juveniles collected from S. tuberosum or S. sisymbriifolium 24 h post infestation was performed to provide insights into the parasitic process of this nematode. A total of 41 G. pallida genes were found to be significantly differentially expressed when parasitizing the two plant species. Among this set, 12 were overexpressed when G. pallida was parasitizing S. tuberosum and 29 were overexpressed when parasitizing S. sisymbriifolium. Out of the 12 genes, three code for secretory proteins; one is homologous to effector gene Rbp-4, the second is an uncharacterized protein with a signal peptide sequence, and the third is an ortholog of a Globodera rostochiensis effector belonging to the 1106 effector family. Other overexpressed genes from G. pallida when parasitizing S. tuberosum were either unknown, associated with a stress or defense response, or associated with sex differentiation. Effector genes namely Eng-1, Cathepsin S-like cysteine protease, cellulase, and two unknown genes with secretory characteristics were over expressed when G. pallida was parasitizing S. sisymbriifolium relative to expression from S. tuberosum. Our findings provide insight into gene regulation of G. pallida while infecting either the trap crop S. sisymbriifolium or the susceptible host, S. tuberosum.

The potato cyst nematode effector RHA1B is a ubiquitin ligase and uses two distinct mechanisms to suppress plant immune signaling.

Plant pathogens, such as bacteria, fungi, oomycetes and nematodes, rely on wide range of virulent effectors delivered into host cells to suppress plant immunity. Although phytobacterial effectors have been intensively investigated, little is known about the function of effectors of plant-parasitic nematodes, such as Globodera pallida, a cyst nematode responsible for vast losses in the potato and tomato industries. Here, we demonstrate using in vivo and in vitro ubiquitination assays the potato cyst nematode (Globodera pallida) effector RHA1B is an E3 ubiquitin ligase that employs multiple host plant E2 ubiquitin conjugation enzymes to catalyze ubiquitination. RHA1B was able to suppress effector-triggered immunity (ETI), as manifested by suppression of hypersensitive response (HR) mediated by a broad range of nucleotide-binding leucine-rich repeat (NB-LRR) immune receptors, presumably via E3-dependent degradation of the NB-LRR receptors. RHA1B also blocked the flg22-triggered expression of Acre31 and WRKY22, marker genes of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), but this did not require the E3 activity of RHA1B. Moreover, transgenic potato overexpressing the RHA1B transgene exhibited enhanced susceptibility to G. pallida. Thus, our data suggest RHA1B facilitates nematode parasitism not only by triggering degradation of NB-LRR immune receptors to block ETI signaling but also by suppressing PTI signaling via an as yet unknown E3-independent mechanism.

Assessment of an Organ-Specific de Novo Transcriptome of the Nematode Trap-Crop, Solanum sisymbriifolium.

Solanum sisymbriifolium, also known as "Litchi Tomato" or "Sticky Nightshade," is an undomesticated and poorly researched plant related to potato and tomato. Unlike the latter species, S. sisymbriifolium induces eggs of the cyst nematode, Globodera pallida, to hatch and migrate into its roots, but then arrests further nematode maturation. In order to provide researchers with a partial blueprint of its genetic make-up so that the mechanism of this response might be identified, we used single molecule real time (SMRT) sequencing to compile a high quality de novo transcriptome of 41,189 unigenes drawn from individually sequenced bud, root, stem, and leaf RNA populations. Functional annotation and BUSCO analysis showed that this transcriptome was surprisingly complete, even though it represented genes expressed at a single time point. By sequencing the 4 organ libraries separately, we found we could get a reliable snapshot of transcript distributions in each organ. A divergent site analysis of the merged transcriptome indicated that this species might have undergone a recent genome duplication and re-diploidization. Further analysis indicated that the plant then retained a disproportionate number of genes associated with photosynthesis and amino acid metabolism in comparison to genes with characteristics of R-proteins or involved in secondary metabolism. The former processes may have given S. sisymbriifolium a bigger competitive advantage than the latter did.

Host-mediated phosphorylation of type III effector AvrPto promotes Pseudomonas virulence and avirulence in tomato.

The AvrPto protein from Pseudomonas syringae pv tomato is delivered into plant cells by the bacterial type III secretion system, where it either promotes host susceptibility or, in tomato plants expressing the Pto kinase, elicits disease resistance. Using two-dimensional gel electrophoresis, we obtained evidence that AvrPto is phosphorylated when expressed in plant leaves. In vitro phosphorylation of AvrPto by plant extracts occurs independently of Pto and is due to a kinase activity that is conserved in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), and Arabidopsis thaliana. Three Ser residues clustered in the C-terminal 18 amino acids of AvrPto were identified in vitro as putative phosphorylation sites, and one site at S149 was directly confirmed as an in vivo phosphorylation site by mass spectrometry. Substitution of Ala for S149 significantly decreased the ability of AvrPto to enhance disease symptoms and promote growth of P. s. tomato in susceptible tomato leaves. In addition, S149A significantly decreased the avirulence activity of AvrPto in resistant tomato plants. Our observations support a model in which AvrPto has evolved to mimic a substrate of a highly conserved plant kinase to enhance its virulence activity. Furthermore, residues of AvrPto that promote virulence are also monitored by plant defenses.