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

Pyridine-2,6-bis(thiocarboxylic acid) (pdtc) is a small secreted metabolite that has a high affinity for transition metals, increases iron uptake efficiency by 20% in Pseudomonas stutzeri, has the ability to reduce both soluble and mineral forms of iron, and has antimicrobial activity towards several species of bacteria. Six GenBank sequences code for proteins similar in structure to MoeZ, a P. stutzeri protein necessary for the synthesis of pdtc.

Structural, functional, and evolutionary analysis of moeZ, a gene encoding an enzyme required for the synthesis of the Pseudomonas metabolite, pyridine-2,6-bis(thiocarboxylic acid).

Eukaryotes use a common set of genes to perform two mechanistically similar autophagic processes. Bulk autophagy harvests proteins nonselectively and reuses their constitutents when nutrients are scarce. In contrast, different forms of selective autophagy target protein aggregates or damaged organelles that threaten to interfere with growth. Yeast uses one form of selective autophagy, called cytoplasm-to-vacuole targeting (Cvt), to engulf two vacuolar enzymes in Cvt vesicles ("CVT-somes") within which they are transported to vacuoles for maturation. While both are dispensable normally, bulk and selective autophagy help sustain life under stressful conditions. Consistent with this view, knocking out several genes participating in Cvt and specialized autophagic pathways heightened the sensitivity of Saccharomyces cerevisiae to inhibitory levels of Zn(2+). The loss of other autophagic genes, and genes responsible for apoptotic cell death, had no such effect. Unexpectedly, the loss of members of a third set of autophagy genes heightened cellular resistance to zinc as if they encoded proteins that actively contributed to zinc-induced cell death. Further studies showed that both sensitive and resistant strains accumulated similar amounts of H2O2 during zinc treatments, but that more sensitive strains showed signs of necrosis sooner. Although zinc lethality depended on autophagic proteins, studies with several reporter genes failed to reveal increased autophagic activity. In fact, microscopy analysis indicated that Zn(2+) partially inhibited fusion of Cvt vesicles with vacuoles. Further studies into how the loss of autophagic processes suppressed necrosis in yeast might reveal whether a similar process could occur in plants and animals.

Identification of autophagy genes participating in zinc-induced necrotic cell death in Saccharomyces cerevisiae.

Autophagy is essential for prolonging yeast survival during nutrient deprivation; however, this report shows that some autophagy proteins may also be accelerating population death in those conditions. While leucine starvation caused YCA1-mediated apoptosis characterized by increased annexin V staining, nitrogen deprivation triggered necrotic death characterized by increased propidium iodide uptake. Although a Δatg8 strain died faster than its parental strain during nitrogen starvation, this mutant died slower than its parent during leucine starvation. Conversely, a Δatg11 strain died slower than its parent during nitrogen starvation, but faster during leucine starvation. Curiously, although GFP-Atg8 complemented the Δatg8 mutation, this protein made ATG8 cells more sensitive to nitrogen starvation, and less sensitive to leucine starvation. These results were difficult to explain if autophagy only extended life but could be an indication that a second form of autophagy could concurrently facilitate either apoptotic or necrotic cell death.

Autophagy proteins play cytoprotective and cytocidal roles in leucine starvation-induced cell death in Saccharomyces cerevisiae.

Yersinia pestis grown with physiologic glucose increased cell autoaggregation and deposition of extracellular material, including membrane vesicles. Membranes were characterized, and glucose had significant effects on protein, lipid, and carbohydrate profiles. These effects were independent of temperature and the biofilm-related locus pgm and were not observed in Yersinia pseudotuberculosis. 

Physiological levels of glucose induce membrane vesicle secretion and affect the lipid and protein composition of Yersinia pestis cell surfaces.

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 .

Yr10 is an important gene to control wheat stripe rust, and the search for Yr10 needs to be continued. Wheat stripe rust or yellow rust is a devastating fungal disease caused by Puccinia striiformis f. sp. tritici (Pst). Host disease resistance offers a primary source for controlling wheat stripe rust. The stripe rust resistance gene Yr10 confers the race-specific resistance to most tested Pst races in China including CYR29. Early studies proposed that Yr10 was a nucleotide-binding site, leucine-rich repeat gene archived as GenBank accession AF149112 (hereafter designated the Yr10 candidate gene or Yr10  ). In this study, we revealed that 15 Chinese wheat cultivars positive for Yr10  are susceptible to CYR29. We then expressed the Yr10  cDNA in the common wheat 'Bobwhite'. The Yr10  -cDNA positive transgenic plants were also susceptible to CYR29. Thus, it is highly unlikely that Yr10  corresponds to the Yr10 resistance gene. Using the Yr10 donor 'Moro' and the Pst-susceptible wheat 'Huixianhong', we generated two F populations that displayed a single Mendelian segregation on the Yr10 gene, and used them to remap the Yr10 gene. Six markers were placed in the Yr10 region, with the Yr10  gene now mapping about 1.2-cM proximal to the Yr10 locus and the Xsdauw79 marker is completely linked to the Yr10 locus. Apparently, the Yr10 gene has not yet been identified. Fine mapping and positional cloning of Yr10 is important for gene pyramiding for stripe rust resistance in wheat.

Remapping of the stripe rust resistance gene Yr10 in common wheat.

Salicylic acid (SA) plays an important role in signal transduction and disease resistance. In Arabidopsis, SA can be made by either of two biosynthetic branches, one involving isochorismate synthase (ICS) and the other involving phenylalanine ammonia-lyase (PAL). However, the biosynthetic pathway and the importance of SA remain largely unknown in Triticeae. Here, we cloned one ICS and seven PAL genes from barley, and studied their functions by their overexpression and suppression in that plant. Suppression of the ICS gene significantly delayed plant growth, whereas PAL genes, both overexpressed and suppressed, had no significant effect on plant growth. Similarly, suppression of ICS compromised plant resistance to Fusarium graminearum, whereas similar suppression of PAL genes had no significant effect. We then focused on transgenic plants with ICS. In a leaf-based test with F. graminearum, transgenic plants with an up-regulated ICS were comparable with wild-type control plants. By contrast, transgenic plants with a suppressed ICS lost the ability to accumulate SA during pathogen infection and were also more susceptible to Fusarium than the wild-type controls. This suggests that ICS plays a unique role in SA biosynthesis in barley, which, in turn, confers a basal resistance to F. graminearum by modulating the accumulation of H O , O2- and reactive oxygen-associated enzymatic activities. Although SA mediates systemic acquired resistance (SAR) in dicots, there was no comparable SAR response to F. graminearum in barley. This study expands our knowledge about SA biosynthesis in barley and proves that SA confers basal resistance to fungal pathogens.

Isochorismate-based salicylic acid biosynthesis confers basal resistance to Fusarium graminearum in barley.

, 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, .

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.

Plant male sterility is a valuable trait in breeding and hybrid seed production. The barley male-sterility gene msg26 was mapped to a 0.02-cM region that anchors to a 506-kb low-quality assembly between two cleaved amplified polymorphic sequence (CAPS) markers, SP1M14 and SP1M49. The barley gene HORVU4Hr1G074840, which encodes a putative cytochrome P450 CYP704B protein, appears to be a strong candidate for the MSG26 trait. Barley (Hordeum vulgare L.) is an important cereal crop worldwide. Traditional breeding in barley is time-consuming and labor-intensive. The use of male-sterile genotypes may significantly improve the efficacy of hybrid breeding and seed production. The barley accession 'GSHO745' is a spontaneous male-sterile mutant from the barley variety, 'Unitan'. The male sterility in 'GSHO745' is controlled by the recessive gene, msg26 (originally named as ms-u). We revealed that the barley plants homozygous for msg26 proceeded normally through Meiosis II until the tetrad stage, but became fully defective in the late uninucleate microspores and developed pollen-less anthers. Using seven barley F populations, we mapped MSG26 to a 0.02-cM region that anchored to a 506-kb low-quality assembly between two cleaved amplified polymorphic sequence markers, SP1M14 and SP1M49. The HORVU4Hr1G074840 gene that encodes a putative cytochrome P450 protein (CYP704B) was identified as the most plausible candidate for MSG26. First, HORVU4Hr1G074840 is located in a collinear region of the rice CYP704B2 and the maize CYP704B1. Both of these genes are essential for male gamete production. Second, the male-sterile allele of HORVU4Hr1G074840 in GSHO745 contained a 4-bp deletion in the last exon. The resulting frame shift causes a Gly436Gln substitution, scrambles the sequence of the remainder of the protein, and forms a new termination site at the 70th triplet of the shifted reading frame. We thus called the variant protein CYP704B:p.G436Qfs*70. Third, the barley HORVU4Hr1G074840 gene was specifically expressed in anthers. Altogether, HORVU4Hr1G074840 represents a strong candidate for MSG26 in barley.

The anther-specific CYP704B is potentially responsible for MSG26 male sterility in barley.

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.

Article	Year
Structural, functional, and evolutionary analysis of moeZ, a gene encoding an enzyme required for the synthesis of the Pseudomonas metabolite, pyridine-2,6-bis(thiocarboxylic acid). BMC evolutionary biology\| PubMed ID: 11972321	2002
Identification of autophagy genes participating in zinc-induced necrotic cell death in Saccharomyces cerevisiae. Autophagy\| PubMed ID: 21317551	2011
Autophagy proteins play cytoprotective and cytocidal roles in leucine starvation-induced cell death in Saccharomyces cerevisiae. Autophagy\| PubMed ID: 22361650	2012
Physiological levels of glucose induce membrane vesicle secretion and affect the lipid and protein composition of Yersinia pestis cell surfaces. Applied and environmental microbiology\| PubMed ID: 23686263	2013
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
Remapping of the stripe rust resistance gene Yr10 in common wheat. TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik\| PubMed ID: 29476226	2018
Isochorismate-based salicylic acid biosynthesis confers basal resistance to Fusarium graminearum in barley. Molecular plant pathology\| PubMed ID: 29517854	2018
Assessment of an Organ-Specific Transcriptome of the Nematode Trap-Crop, . G3 (Bethesda, Md.)\| PubMed ID: 29769290	2018
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
The anther-specific CYP704B is potentially responsible for MSG26 male sterility in barley. TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik\| PubMed ID: 31209536	2019
Transcriptome analysis of Globodera pallida from the susceptible host Solanum tuberosum or the resistant plant Solanum sisymbriifolium. Scientific reports\| PubMed ID: 31519937	2019

Allan Caplan

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