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

The effects of soil amendment with rapeseed meal from Brassica napus cv. 'Dwarf Essex' (high glucosinolate concentrations) and 'Stonewall' (low glucosinolate concentrations) on the biological control activity of Trichoderma harzianum towards Sclerotinia sclerotiorum and Aphanomyces euteiches were evaluated. Trichoderma harzianum added to soil reduced myceliogenic germination of S. sclerotiorum by 94%, but did not affect carpogenic germination. In contrast, 100% reduction in carpogenic germination was observed in soil amended with Dwarf Essex meal, along with a 33% reduction in myceliogenic germination. With Stonewall meal as soil amendment, carpogenic germination was reduced by 44% and myceliogenic germination was not affected. Both Dwarf Essex and Stonewall meals inhibited colonization of sclerotia in soil by T. harzianum, from 100% to 0% and 8%, respectively, so that biocontrol activity of T. harzianum was reduced in the presence of either meal. Aphanomyces euteiches root rot of pea was significantly reduced by T. harzianum alone (100%), by amendment with Dwarf Essex meal alone (77%), and by T. harzianum in combination with Dwarf Essex meal (100%). Amendment with Stonewall meal alone did not control root rot, and combination of Stonewall meal with T. harzianum reduced the biocontrol efficacy of T. harzianum.

Combined effects of Brassica napus seed meal and Trichoderma harzianum on two soilborne plant pathogens.

Seven filamentous fungal species were isolated from individual eggs of Globodera pallida cysts collected from infested fields in Shelley Idaho, USA and identified as Chaetomium globosum, Fusarium oxysporum, Fusarium solani, Fusarium tricinctum, Microdochium bolleyi, Purpureocillium lilacinum, and Plectosphaerella cucumerina. Their ability to reduce infection by G. pallida in planta were assessed in simple, reproducible micro-rhizosphere chambers (micro-ROCs). All fungi reduced G. pallida infection in potato, but greatest reduction was observed with C. globosum at an average reduction of 76%. Further non-destructive methods were developed to rapidly assess biological control potential of putative fungal strains by staining the infectious second stage juveniles of G. pallida with the live fluorescent stain PKH26. In comparisons between the standard, invasive acid fuchsin method and use of the live stain PKH26, no significant difference in infection level of G. pallida was observed whether roots were stained with PKH26 or acid fuchsin. For both methods, a similar reduction (77% for acid fuchsin, and 78% for PKH26 stain) in invasion of infectious stage of G. pallida was observed when potato plants were inoculated with C. globosum compared to non-inoculated potato.

Prospecting fungal parasites of the potato cyst nematode Globodera pallida using a rapid screening technique.

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 .

The eradication program for the potato cyst nematode (PCN), , in the Northwest of the United States revolves around the use of soil fumigation. Alternative, integrated strategies are needed to continue to battle this invasive nematode. Laboratory, greenhouse, and field experiments were conducted with  and another cyst nematode found in the United States, , to evaluate the efficacy of a new formulated  seed meal extract, as well as a traditional  seed meal, as alternate eradication strategies. This is the first report on the efficacy of  seed meal extract against plant-parasitic nematodes. Rates of  seed meal greater than 2.2 t/ha and 4.5 t/ha for  and , respectively, were required for egg hatch suppression, as determined by a potato root diffusate (PRD) bioassay. Reproduction of  on potato after exposure to  seed meal at a rate of 2.2 t/ha was also significantly reduced. In the field, 8.9 t/ha  seed meal almost eliminated egg hatch of . Rates needed for  spp. suppression were greatly reduced when using the  seed meal extract. When compared side-by-side, half as much  seed meal extract, 1.1 t/ha, was required to suppress  egg hatch to the same extent as  seed meal. Exposure of  to  seed meal extract at 4.5 t/ha reduced egg hatch by 90% compared with a nonamended control. The ability to reduce the amount of material being applied to soil by using an extract has the potential for integration into a  eradication program.

Control of  spp. Using  Seed Meal and Seed Meal Extract.

, 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, .

The Complex Object Parametric Analyzer and Sorter (COPAS) is a large particle flow cytometer designed for analyzing, sorting, and dispensing objects of varying sizes. We explored the potential of using this instrument to analyze and sort various developmental stages and egg viability of Globodera pallida. Cysts were successfully examined and sorted from debris by optimizing side-scatter and red-fluorescence parameters on the COPAS. We were able to separate eggs and second-stage juveniles from samples of mixed population using extinction and time of flight. Separation of live and dead eggs was examined following staining eggs with SYTOX Green and application of time of flight and green peak height. Data were compared with a commonly used viability assay by which eggs were stained with Meldola's Blue and examined by a microscope. COPAS proved to be effective in assessing viability by detecting two separate gates: live eggs having green fluorescence peaks <190 and dead eggs with the peaks >190. The application of COPAS in combination with SYTOX Green detected a greater number of live eggs than the Meldola's assay, suggesting that SYTOX Green provided an overestimate of live eggs. COPAS noticeably increased the accuracy and reduced the time required for screening and analyzing nematode populations.

An Improved Technique for Sorting Developmental Stages and Assessing Egg Viability of Globodera pallida using High-Throughput Complex Object Parametric Analyzer and Sorter.

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.

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.

The potato cyst nematode, , is a globally regulated potato pest. Detected in the U.S. in the state of Idaho in 2006, the infestation, as of February 2019, is limited to 1,326 hectares.  is an obligate sedentary endoparasite that reduces yields up to 80% and is spread through movement of soil, tubers or equipment. The objectives of this study were to describe the spatiotemporal pattern of  in infested fields and to model its dispersal patterns in Idaho. We used geostatistical tools and simulation models for mapping and description of the relationships between  incidence and spatial configurations. We found that the nematode is spatially clustered, prevalent around field edges, and its dispersal pattern followed the direction of cultivation. The absence of potato in an infested field significantly reduced the number of cysts sampled each year subsequent to the initial delimitation sampling in 2007. Phytosanitary measures prohibiting potato contributed in stopping nematode reproduction, and the use of chemical fumigants and biofumigant cover crops contributed to a significant reduction in egg viability. We observed a process of a non-linear decline in the prevalence of cysts as the distance separation from the primary infestation focus increased. A power-law model was used to fit  dispersal capabilities. This study contributed in describing  infestation for Idaho and provided information on the spatial pattern and landscape ecology of  in Idaho to facilitate common understandings on the challenges and opportunities for controlling this pest in Idaho.

Louise-Marie Dandurand

Department of Entomology,
Plant Pathology and Nematology,
Department of Entomology, Plant Pathology and Nematology

University of Idaho

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

Louise-Marie Dandurand

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Entomology,Plant Pathology and Nematology,Department of Entomology, Plant Pathology and Nematology

University of Idaho

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

Department of Entomology,
Plant Pathology and Nematology,
Department of Entomology, Plant Pathology and Nematology