Effects of cyst components and low temperature exposure of Heterodera glycines eggs on juvenile hatching in vitro

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Proteases from Heterodera glycines and Meloidogyne incognita juveniles were inhibited by heat-stable content from H. glycines cysts (hHglCE), and by a polyphenol (EGCG) similar to a compound previously identified in Globodera cysts. General protease activities detected using the nematode peptide KSAYMRFa were inhibited by EGCG (IC50 1.19 mM, H. glycines', 0.34 mM, M. incognita) but not by hHglCE. However, hHglCE and EGCG each inhibited proteasome-associated chymotrypsin-like (CT-L) activity. EGCG IC50 values were 0.47 mM (H. glycines) and 0.15 mM (M. incognita). hHglCE IC50 values were 0.16 and 0.005 mM hHglCEeq for H. glycines and M. incognita, respectively. Across all substrate-inhibitor combinations, M. incognita proteases were inhibited more robustly than those from H. glycines, particularly by hHglCE. In addition to CT-L protease, post-glutamate peptide hydrolysing (PGPH) and trypsin-like (T-L) proteasome proteases were detected in M. incognita, and each of these was also strongly inhibited by hHglCE. hHglCE inhibited CT-L, PGPH and T-L proteases within catalytic subunits from yeast (Saccharomyces cerevisiae) and human proteasomes. Proteasome inhibitors MG-132 and aclacinomycin A each inhibited M. incognita CT-L and PGPH activities by more than 80% at 20-100 gM , and hHglCE inhibited the same proteases by 70-80% at 0.04 hHglCEeq /zl_ l . hHglCE completely inhibited M. incognita T-L activity, but CT-L activity in native content from H. glycines cysts was not inhibited. Evidence that H. glycines cysts contain inhibitors of all proteases associated with the proteasome establishes the cyst as an important new target to explore for potential nematode control compounds. In addition, characterisation of protease activities from a core cellular metabolic component using M. incognita is novel for plant-parasitic nematodes.

Section: Discussionmentioning

confidence: 91%

mentioning

confidence: 99%

Protease inhibition by Heterodera glycines cyst content: evidence for effects on the Meloidogyne incognita proteasome

Masler¹

2015

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“…Although all eggs {i.e., not eggshells) examined on Day 14 were intact, and none showed any signs of degradation, it was not possible with the experimental design used here to determine if the embryos were still alive. Experiments designed to monitor and evaluate individually identified eggs (Masler & Rogers, 2011) need to be used to address this question. Nonetheless, the present results demonstrate that H. glycines responds to BITC with behavioural and reproductive changes involving multiple developmental stages.…”

Section: Discussionmentioning

confidence: 99%

“…DEVELOPMENT All materials (eggs, eggshells and J2) collected from each sieve on D14 from the above experiments were examined at 40x and lOOx using an inverted microscope. Embryos (intact eggs) were assigned to one of four development stages using an arbitrary classification scale (Masler & Rogers, 2011). Briefly, eggs that were opaque or dark with distinct uniform spherical structures were considered as eariy embryos and classified as multicellular (MC).…”

Section: Hatch Assaysmentioning

confidence: 99%

Benzyl isothiocyanate affects development, hatching and reproduction of the soybean cyst nematode Heterodera glycines

Masler²,

Rogers³

et al. 2014

Benzyl isothiocyanate (BITC), prepared in 1% methanol and applied at micro-molar doses, decreased Heterodera glycines infective second-stage juvenile (J2) movement, hatching and reproduction on soybean. Glycine max. Direct exposure of J2 to 30 ßM BITC caused an immediate decrease (17%) in J2 movement relative to 1% methanol controls, and within 3 h exposure eliminated >99% of movement. Continuous exposure of H. glycines eggs to 30 ßU or 60 /xM BITC significantly decreased percentage hatch of J2 measured at 7 days (30 ßU, 15.8 ± 2.4%; 60 ßU, 7.9 ± 2.2%) relative to controls (44.9 ± 4.1%). Control percentage hatch increased between days 7 and 14 (87.5 ± 2.3%), whereas there was no significant increase in hatch after Day 7 in either treatment group. The effect of BITC on hatch was rapid and persistent. Following 4 h exposure of eggs to either 30 ßM or 60 ßM BITC and transfer to water, percentage hatch at Day 14 was 51.3 ± 6.3% and 15.6 ± 1.4%, respectively. Each value was significantly less than the control percentage hatch (96.5 ± 0.2%). The same treatment method also resulted in decreased reproduction. The mean number of cysts per plant harvested 5 weeks after inoculation with control eggs (173.0 ± 12.1) was significantly greater than the numbers from plants inoculated with either 30 ^M (78.9 ± 8.2) or 60 ßM (38.3 ± 4.5) BITC treated eggs. Reduction of hatch and reproduction were strongly correlated, with r^ > 0.60 for both 30 ßM or 60 ßM BITC treatments. The effects of BITC were primarily on the early embryo. The percent of all embryos in the multicellular stage at Day 14 in control eggs was 4.9 ± 0.7%, but increased to 35.1 ± 4.3% (30 ßM) and 56.3 ± 4.5% (60 ßM) in BITC treated eggs. BITC has multiple effects on H. glycines, affecting both infective juvenile movement and embryonic development, and decreasing reproduction.

“…requiring host plant signals to trigger hatching may remain quiescent until such signals are available, at which time hatch is initiated (Perry, 2002;Palomares-Rius et al, 2013;Zasada etal., 2013). In a lab oratory population of H. glycines where hatching in wa ter is normally spontaneous, exposure to low temperature repressed hatch until normal temperature was restored (Masler et al, 2008a;Masler & Rogers, 2011). Diapause is a more stringent dormancy (Jones, P.W.…”

Section: External Survival and Hatchingmentioning

confidence: 99%

Targeting internal processes of plant-parasitic nematodes in the pursuit of novel agents for their control

Masler

2014

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The success of plant-parasitic nematodes as competitors with humans for crops is evidenced by the parasites' significant and continuous economic drain on global agriculture. Scientific efforts dedicated to the control of plant-parasitic nematodes employ strategies from the environmental to molecular levels. Understanding the interaction of the nematode with its environment, and the molecules involved, offers great promise for novel control agent development. Perhaps more significantly, such knowledge facilitates the generation of ever more detailed and sophisticated information on nematode biology and new molecular targets. Among the most economically important groups of plant-parasitic nematodes are those comprising the cyst-forming species and the root-knot nematodes. Presented here is a brief overview of research into the biology of these parasites relative to their life cycles. Recent advances in elucidating the molecular biology and biochemistry of nematode-plant interactions during the internal parasitic stages of the life cycle have been driven by advances in genomics and transcriptomics. The remarkable discoveries regarding parasitism, and the application of genetic resources in these findings, provide a template for advanced investigation of external, survival stages biology. While survival biology research lags somewhat behind that of parasitism with regard to the molecular genetics of signalling and response, its extensive catalogue promises explosive rates of discovery as progress in genomics and transcriptomics allows a molecular genetic examination of embryogenesis, dormancy and hatching. Our group is interested in behaviour, development and hatching of cyst and root-knot nematodes, and the effects of the environment on the mechanisms of these activities. Phytochemical and temperature effects are discussed, and evidence is presented that the cyst may provide useful molecules for exploring nematode physiology.