functional-characterization-ring-type-e3-ubiquitin-ligases-vitro

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent ...

University of Idaho

In tomato, the NBARC-LRR resistance (R) protein Prf acts in concert with the Pto or Fen kinase to determine immunity against Pseudomonas syringae pv. tomato (Pst). Prf-mediated defense signaling is initiated by the recognition of two sequence-unrelated Pst-secreted effector proteins, AvrPto and AvrPtoB, by tomato Pto or Fen. Prf detects these interactions and activates signaling leading to host defense responses including localized programmed cell death (PCD) that is associated with the arrest of Pst growth. We found that Prf variants with single amino acid substitutions at D1416 in the IHD motif (isoleucine-histidine-aspartic acid) in the NBARC domain cause effector-independent PCD when transiently expressed in leaves of Nicotiana benthamiana, suggesting D1416 plays an important role in activation of Prf. The N-terminal region of Prf (NPrf) and the LRR domain are required for this autoactive Prf cell death signaling but dispensable for accumulation of the Prf(D1416V) protein. Significantly, co-expression of the Prf LRR but not NPrf, with Prf(D1416V), AvrPto/Pto, AvrPtoB/Pto, an autoactive form of Pto (Pto(Y207D)), or Fen completely suppresses PCD. However, the Prf LRR does not interfere with PCD caused by Rpi-blb1(D475V), a distinct R protein-mediated PCD signaling event, or that caused by overexpression of MAPKKKα, a protein acting downstream of Prf. Furthermore, we found the Prf(D1416V) protein is unable to accumulate in plant cells when co-expressed with the Prf LRR domain, likely explaining the cell death suppression. The mechanism for the LRR-induced degradation of Prf(D1416V) is unknown but may involve interference in the intramolecular interactions of Prf or to binding of the unattached LRR to other host proteins that are needed for Prf stability.

Plant programmed cell death caused by an autoactive form of Prf is suppressed by co-expression of the Prf LRR domain.

Plant secondary metabolites, including phenylpropanoids and carotenoids, are stress inducible, have important roles in potato physiology and influence the nutritional value of potatoes. The type and magnitude of environmental effects on tuber phytonutrients is unclear, especially under modern agricultural management that minimizes stress. Understanding factors that influence tuber secondary metabolism could facilitate production of more nutritious crops. Metabolite pools of over forty tuber phenylpropanoids and carotenoids, along with the expression of twenty structural genes, were measured in high-phenylpropanoid purple potatoes grown in environmentally diverse locations in North America (Alaska, Texas and Florida).

Differential effects of environment on potato phenylpropanoid and carotenoid expression.

The plant-specific NAC (NAM, ATAF1,2, CUC2) transcription factors play significant roles in diverse physiological processes. In this study, we determined the regulation of a stress-related tomato (Solanum lycopersicum) NAC1 (SlNAC1) transcription factor at both the transcriptional and the post-translational level. The SlNAC1 protein was found to be stable in the presence of proteasome-specific inhibitor MG132 or MG115 and ubiquitinated in plant cells, suggesting that the SlNAC1 is subject to the ubiquitin-proteasome system-mediated degradation. Deletion analysis identified a short segment of 10 amino acids (aa261-270) that was required for ubiquitin-proteasome system-mediated degradation, among which two leucine residues (L268 and L269) were critical for the protein instability of SlNAC1. Fusion of the degron (SlNAC1(191-270) ) containing these 10 amino acids to green fluorescent protein was found to be sufficient to trigger the degradation of the fusion protein. In addition, the SlNAC1 gene is strongly upregulated during Pseudomonas infection, while repression of the NAC1 ortholog in Nicotiana benthamiana resulted in enhanced susceptibility to Pseudomonas bacteria. These results suggest that rapid upregulation of the NAC1 gene resulting in more protein production is likely one of the strategies plants use to defend themselves against pathogen infection.

SlNAC1, a stress-related transcription factor, is fine-tuned on both the transcriptional and the post-translational level.

We recently identified a defense-related tomato (Solanum lycopersicum) NAC (NAM, ATAF1,2, CUC2) transcription factor, NAC1, that is subjected to ubiquitin-proteasome system-dependent degradation in plant cells. In this study, we identified a tomato ubiquitin ligase (termed SEVEN IN ABSENTIA3; SINA3) that ubiquitinates NAC1, promoting its degradation. We conducted coimmunoprecipitation and bimolecular fluorescence complementation to determine that SINA3 specifically interacts with the NAC1 transcription factor in the nucleus. Moreover, we found that SINA3 ubiquitinates NAC1 in vitro and promotes NAC1 degradation via polyubiquitination in vivo, indicating that SINA3 is a ubiquitin ligase that ubiquitinates NAC1, promoting its degradation. Our real-time PCR analysis indicated that, in contrast to our previous finding that NAC1 mRNA abundance increases upon Pseudomonas infection, the SINA3 mRNA abundance decreases in response to Pseudomonas infection. Moreover, using Agrobacterium-mediated transient expression, we found that overexpression of SINA3 interferes with the hypersensitive response cell death triggered by multiple plant resistance proteins. These results suggest that SINA3 ubiquitinates a defense-related NAC transcription factor for degradation and plays a negative role in defense signaling.

The ubiquitin ligase SEVEN IN ABSENTIA (SINA) ubiquitinates a defense-related NAC transcription factor and is involved in defense signaling.

The introduction of high-throughput sequencing technologies has made transcriptome analyses of plant-pathogen interactions almost routine. Nevertheless, it is still challenging to obtain RNA from populations made up of two species. An RNA extraction method that worked well on free-living  failed when applied to isolated  J2 larva. Furthermore, alternative protocols that extracted RNA from free-living J2 larva produced less satisfactory results once the animals entered their hosts' roots. We have compared several extraction procedures to ascertain whether a single protocol was capable of recovering high-quality, high-molecular-weight RNA from newly hatched J2 larva as well as from larva embedded in roots of both potatoes ( L. cv. Desiree) and a very distantly related species, . Although it was possible to recover large amounts of RNA from J2 larvae using Proteinase K treatments, this protocol failed to yield high-quality nematode RNA from infected roots. By comparison, mechanical disruption procedures yielded lower amounts of RNA from infected roots, but what was recovered was of higher quality. We conclude that different extraction protocols need to be developed to sample mixed populations of organisms.

Assessment of  RNA Extracted from  Roots.

Sedentary endoparasitic cyst nematodes form a feeding structure in plant roots, called a syncytium. Syncytium formation involves extensive transcriptional modifications, which leads to cell modifications such as increased cytoplasmic streaming, enlarged nuclei, increased numbers of organelles, and replacement of a central vacuole by many small vacuoles. When whole root RNA is isolated and analyzed, transcript changes manifested in the infected plant cells are overshadowed by gene expression from cells of the entire root system. Use of microaspiration allows isolation of the content of nematode infected cells from a heterogeneous cell population. However, one challenge with this method is identifying the nematode infected cells under the microscope at early stages of infection. This problem was addressed by staining nematode juveniles with a fluorescent dye prior to infection so that the infected cells could be located and microaspirated.

Microaspiration of  root cells at early stages of infection by .

, also known as "Litchi Tomato" or "Sticky Nightshade," is an undomesticated and poorly researched plant related to potato and tomato. Unlike the latter species,  induces eggs of the cyst nematode, , 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  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  a bigger competitive advantage than the latter did.

Assessment of an Organ-Specific  Transcriptome of the Nematode Trap-Crop, .

In the United States, potato cyst nematodes Globodera rostochiensis and G. pallida are quarantined pests. A new cyst nematode species, Globodera ellingtonae, discovered in Oregon and Idaho, reproduces well on potato but is not currently a quarantine pest. Identifying resistance to all three Globodera spp. would provide a valuable management tool. Thirteen breeding clones and nine cultivars were evaluated in Oregon, Idaho, and New York laboratories where the nematode populations are maintained. Minitubers or tissue culture plants were planted into pots and inoculated with eggs in replicated experiments. Results indicated that five entries were partially resistant or resistant to all three species, while another five were resistant or partially resistant to G. rostochiensis and G. ellingtonae. Resistance to G. rostochiensis pathotypes Ro1 and Ro4 is controlled by the H1 gene and this study suggests that H1 may confer resistance to G. ellingtonae as well. Observed resistance to G. pallida was lower relative to the levels of resistance observed for G. rostochiensis and G. ellingtonae. Germplasm with G. pallida or G. ellingtonae resistance will be used in hybridizations to develop russet-skinned cultivars with long tubers which represent the predominant market class in western U.S. production, and to further explore the basis of potato resistance to Globodera spp.

Resistance of Potato Breeding Clones and Cultivars to Three Species of Potato Cyst Nematode.

Potato is an important source of food in South Korea, and viruses represent a significant threat to sustainable and profitable potato production. However, information about viruses affecting the potato crop in South Korea is limited. In 2017, potato plants of five cultivars exhibiting foliar mosaic, crinkling, and mottle were collected in two seed potato production areas, in Gangwon-do and Jeollabuk-do Provinces, and subjected to virus testing and characterization. Potato virus Y (PVY) was found associated with mosaic symptoms, and samples were characterized using reverse transcription polymerase chain reaction (RT-PCR) and whole genome sequencing. All analyzed PVY-positive samples were found to represent the same recombinant PVY strain: PVY. Three PVY isolates were subjected to whole genome sequencing using overlapping RT-PCR fragments and Sanger methodology, and all three were confirmed to represent strain PVY after a recombination analysis of the complete genomes. In phylogenetic analysis, the three South Korean isolates were placed most closely to several PVY isolates reported from Japan and Vietnam, suggesting a common source of infection. This is the first report and complete molecular characterization of a PVY strain present in the country, and because this strain induces tuber necrotic ringspot disease in susceptible cultivars of potato, appropriate management tools need to be implemented to mitigate potential tuber quality losses.

Identification and Molecular Characterization of Recombinant Potato Virus Y (PVY) in Potato from South Korea, PVY Strain.

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.

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

The potato cyst nematodes (PCNs)  and  are internationally recognized quarantine pests. Although not widely distributed in either the United States or Canada, both are present and are regulated by the national plant protection organizations (NPPOs) of each country.  was first discovered in New York in the 1940s, and  was first detected in a limited area of Idaho in 2006. In Canada,  and  were first detected in Newfoundland in 1962 and 1977, respectively, and further detections of  occurred in British Columbia and Québec, most recently in 2006. Adherence to a stringent NPPO-agreed-upon phytosanitary program has prevented the spread of PCNs to other potato-growing areas in both countries. The successful research and regulatory PCN programs in both countries rely on a network of state, federal, university, and private industry cooperatorspursuing a common goal of containment, management/eradication, and regulation. The regulatory and research efforts of these collaborative groups spanning from the 1940s to the present are highlighted in this review.

Current Status of Potato Cyst Nematodes in North America.

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.

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

'Candidatus Liberibacter solanacearum' (Lso) is a plant pathogen affecting Solanaceae and Apiaceae in different parts of the world. 'Candidatus Liberibacter solanacearum' is a gram negative, fastidious α-proteobacterium that is vectored by different psyllid species. Plant pathogenic bacteria are known for interfering with the host physiology and/or defense mechanisms, often by secreting bacterial effectors. Effector proteins are critical for virulence therefore the identification of effectors could help with disease management. In this study we characterized the Sec-translocon dependent Lso protein, Lso-HPE1 (hypothetical protein effector1). We compared this protein sequence in the different Lso haplotypes. We predicted the signal peptide and validated its function using Escherichia coli's alkaline phosphatase fusion assay. Agrobacterium tumefaciens-mediated transient expression in Nicotiana benthamiana demonstrated that Lso-HPE1 from Lso haplotypes A and B were able to inhibit the induction of cell death in plants. We also compared gene expression of the Lso-HPE1- transcripts in Lso haplotypes A and B in tomato and in the vector, Bactericera cockerelli. This work validates the identification of a Sec-translocon dependent Lso protein possibly involved in suppression of plant cell death.

Article	Year
Plant programmed cell death caused by an autoactive form of Prf is suppressed by co-expression of the Prf LRR domain. Molecular plant\| PubMed ID: 22451646	2012
Differential effects of environment on potato phenylpropanoid and carotenoid expression. BMC plant biology\| PubMed ID: 22429339	2012
SlNAC1, a stress-related transcription factor, is fine-tuned on both the transcriptional and the post-translational level. The New phytologist\| PubMed ID: 23278405	2013
The ubiquitin ligase SEVEN IN ABSENTIA (SINA) ubiquitinates a defense-related NAC transcription factor and is involved in defense signaling. The New phytologist\| PubMed ID: 26879496	2016
Assessment of RNA Extracted from Roots. Journal of nematology\| PubMed ID: 28512373	2017
Microaspiration of root cells at early stages of infection by . Plant methods\| PubMed ID: 28855955	2017
Assessment of an Organ-Specific Transcriptome of the Nematode Trap-Crop, . G3 (Bethesda, Md.)\| PubMed ID: 29769290	2018
Resistance of Potato Breeding Clones and Cultivars to Three Species of Potato Cyst Nematode. Plant disease\| PubMed ID: 30156963	2018
Identification and Molecular Characterization of Recombinant Potato Virus Y (PVY) in Potato from South Korea, PVY Strain. Plant disease\| PubMed ID: 30412456	2019
The potato cyst nematode effector RHA1B is a ubiquitin ligase and uses two distinct mechanisms to suppress plant immune signaling. PLoS pathogens\| PubMed ID: 30978251	2019
Current Status of Potato Cyst Nematodes in North America. Annual review of phytopathology\| PubMed ID: 31100997	2019
Transcriptome analysis of Globodera pallida from the susceptible host Solanum tuberosum or the resistant plant Solanum sisymbriifolium. Scientific reports\| PubMed ID: 31519937	2019
Lso-HPE1, an effector of 'Candidatus Liberibacter solanacearum' can repress plant immune response. Phytopathology\| PubMed ID: 31697198	2019

Joseph C. Kuhl

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