Use of a 15 N-tracer Method as a Tool to Indicate the Assimilation of Elicitin-Sterol Complexes by Phytophthorasojae

The mechanism of sterol acquisition and transport in oomycetes of the genus Phytophthora is suspected to involve secreted class-I elicitins, but little information is available to confirm this. The objective of this study was to determine if class-I elicitins of Phytophthorasojaepromote assimilation into this organism when a phytosterol is amended into its growth medium using a qualitative N tracer method. Two P. sojae growth experiments were conducted involving N labeled mycelium or N spent broth containing putative P. sojaeelicitins in the absence or presence of a phytosterol (stigmasterol). Mycelium was harvested after 0, 2, 3 or 6 days of growth and analyzed for N by Elemental Analysis-Isotopic Ratio Mass Spectrometry (EA-IRMS). Results from the two experiments demonstrated that P. sojae assimilated putative elicitins from the spent broth more rapidly when stigmasterol was present in the growth medium. These results provide evidence for the involvement of secreted class-I elicitins in sterol acquisition by P. sojae.


Introduction
Oomycetes in the genus Phytophthora are fungal-like organisms in the kingdom Stramenopiles (Tyler 2006).Phytophthora spp.constitute a large number of destructive pathogens that cause disease on a wide range of herbaceous and woody plants (Erwin and Ribiero 1996), and all are reported to secrete large amounts of low molecular weight proteins (10 kDa) belonging to class-I elicitins (Ponchetet al. 1999).The intrinsic function of class-I elicitins remains largely unknown but is presumed to involve sterol acquisition because in vitro experiments showed they bind sterols and catalyze their transfer across artificial membranes (Mikes et al., 1998) and plasma membrane preparations (Osman et al. 2001b;Vauthrin et al.1999).Phytophthora spp.are sterol auxotrophs because they lack key enzymes such as squaleneepoxydase and the 14α-demethylase that are essential in converting sterol precursors to active sterols (Tyler et al. 2006).Because of the auxotrophic requirement for sterols combined with the in vitro sterol binding activity of class-I elicitins, it has been hypothesized that elicitins act as shuttles that transport sterols from the environment to the pathogen (Ponchet et al. 1999).Yousef et al. (2009) found that nanomolar concentrations of sterols (stigmasterol and cholesterol) caused a down-regulation of class-I elicitin gene expression.This also coincided with a reduction in class-I elicitin protein secretion into the spent broth.These findings suggested class-I elicitins are involved in sterol acquisition by P. sojae.
Elicitins were first characterized on the basis of their ability to induce the hypersensitive response (HR) when infiltrated into tobacco leaves (Ponchet et al. 1999).The HR-eliciting property of elicitins was attractive to plant pathologists because it suggested that elicitins secreted by Phytophthoraspp.may function as determinants of host range for selected plant-Phytophthora interactions (Ponchetet al. 1999).Efforts were made to determine the mechanisms by which elicitins trigger the HR in tobacco.This lead to the discovery that elicitins interact with receptors present in tobacco plasma membrane preparations that exhibit a high affinity for elicitins (Bleinet al. 1991).Interestingly, ligand binding studies determined that the affinity of these receptors in tobacco was higher for elicitins when they were bound to a sterol than when alone, which was also a requisite step to trigger the HR (Osman et al. 2001b).Surprisingly, however, there have been no attempts to identify if a similar mechanism of elicitin-sterol uptake involving receptors exists in Phytophthora, the discovery of which would add to our knowledge of the intrinsic roles of elicitins and the mechanisms of sterol uptake in this important group of plant pathogens.
The use of enriched stable isotopes in tracer experiments is a valuable tool for investigating the fate of elements and biomolecules in biological (Stuurup et al. 2008) and natural ecosystems (e.g.Bohlke et al. 2004;Peterson 1999).Experiments utilizing stable isotopes are very similar to radioisotope tracer experiments, except they have the advantage of being nontoxic and can be used to study active pathways in living organisms (Stuurup et al. 2008).The objective of this study was to utilize a 15N tracer method as a tool for providing evidence for the involvement of 15 N-elicitins in P. sojae sterol acquisition.The hypothesis is that if receptors for elicitins are present on the mycelia of P. sojae, and only become activated to internalize elicitins when they are bound to sterol, then  15 N signature shifts will be observed only when stigmasterol is added to the growth medium of P. sojae.

Culture Source and Growth Media
The Phytophthorasojae culture (race 1) was obtained from Dr. A. Dorrance (OARDC, Wooster OH, USA) and maintained on non-clarified V8 agar (18% v/v V8 juice, 0.3% CaCO 3 , and 2% agar) at 25 o C in the dark.A defined minimal broth similar to (Wu et al., 2003) was made by dissolving the following in one L of distilled water: 0.2 g K 2 HPO 4 , 0.1 g MgSO 4 , 0.1 g CaCl 2 , 0.1 g L-asparagine, 10 g glucose and 1 mL trace elements.The trace element solution was prepared by dissolving 200 mg FeEDTA, 10 mg CuSO 4 , 10 mg MnCl 2 , 10 mg Na 2 MoO 4 , 10 mg Na 2 B 4 O 7 , and 20 mg ZnSO 4 , and 100 mg thiamine hydrochloride in 100 mL distilled water.All chemicals were obtained from Sigma Aldrich (St. Louis, MO).To obtain 15 N labeled or unlabeled mycelium or broth for Experiments 1 and 2, described below, 0.1g Na 14 NO 3 or Na 15 NO 3 (98+ atom% 15 N), was added to the above growth medium.
The growth medium of P. sojaecultures that received NaNO 3 -15 N (98% atom) was enriched by 37 % atom 15 N.This means that 37 % of all the N atoms present in the growth medium are 15 N.This was calculated as follows: A total of 0.1g L -1 L-asparagine (representing 28.7 mg total N) and 0.1 g L -1 Na 15 NO 3 -98% atom (representing 17 mg total N) were used as the sole N source in the growth medium.N enrichment was then calculated as follows: 15 N enrichment (%) = (17 mg) (0.98; this is % atom enrichment in Na 15 NO 3 )/ 45.7 mg (100).

Preparation of 15 N Labeled and Unlabelled Mycelium and Broth
Four 500 mL Erlenmeyer flasks containing 200 mL each of growth media (as described in section 2.1) that included Na 15 NO3 (98+ atom% 15 N) were inoculated with three mycelial plugs (9 mm diameter).These were incubated in the dark for 4 weeks after which the mycelial plugs were separated from the combined four flasks containing the spent broth using vacuum filtration through a sterile 250 mL Stericup unit having a 0.22 µm PES membrane (Millipore Billerica, Ma).The mycelia was washed with and maintained in sterilized distilled water.The labeled 15 N-labelled mycelium and broth were saved and stored for the subsequent growth incubation treatments.The broth contains the presumed labeled sterol-binding elicitins.
The same growth conditions as described in the section above for preparation of 15 N-labeled mycelium and broth were imposed, except that the NaNO 3 was not enriched in 15 N. Again after 4-weeks of incubation, the combined spent broth from each replicate was vacuum-filtered and separated from the mycelium with the mycelium preserved in sterilized distilled water.The unlabeled mycelium and broth were saved and stored for subsequent growth incubation treatments.The broth contains the presumed unlabelled sterol-binding elicitins.
Published by Canadian Center of Science and Education 121

15 N Tracer Incubation Growth Experiments
Two incubation experiments were conducted that each had four treatments and three replications.The starting materials ( 15 N-labelled and unlabelled mycelium and spent broth) for the two experiments were obtained from the incubations described in section 2.2.These two experiments were designed to have treatments that isolate the availability of N (either unlabeled or 15 N-labeled N sources) in the presence or absence of a phytosterol during the growth of P. sojae.The hypothesis was that if elicitins form complexes with sterols and are taken up by P. sojae, then this will be reflected in a change in 15 N content in mycelium.In the first experiment in which the starting material is unlabelled mycelium of P. sojae growing in the presence of 15 N-labelled spent broth containing labeled elicitins, the 15N content will increase as mycelium growth proceeds.In the reverse incubation experiment, in which the starting material is 15 N-enriched -mycelium of P. sojae growing in spent broth containing unlabelledelicitins, a decrease in 15 N content in the mycelium will occur.
Fifty ml of spent broth containing 15 N -putative elicitins originating from the 4-week 15 N-labeled incubation cultures, were transferred into 75 mL Erlenmeyer flasks and inoculated with a 9mm plug of unlabeled mycelium of P. sojae that was recovered from the 4-week unlabelled incubation cultures (see section 2.2).Then the following treatments were established in the flasks (Table 1):.These treatments were (1) labeled spent broth control (no amendment); (2) labeled spent broth amended a plant sterol which was 1 µM stigmasterol (final concentration); (3) labeled spent broth amended with 10 g L -1 glucose and 0.1 g L -1 unlabelled NaNO 3 ; and (4) labeled spent broth amended with 10 g L -1 glucose, 0.1 g L -1 unlabelled NaNO 3 and 1 µM stigmasterol.Flasks were incubated in the dark at 25 o C and mycelia sampled at days 0, 2, 3 and 6.
Fifty mL of spent broth containing unlabelledputative elicitins originating from the 4-week incubation cultures were transferred into 75 mL Erlenmeyer flasks and inoculated with a 9mm plug of 15 N-labeled mycelium of P. sojae that was recovered from the 4-week labeled incubation cultures (see section 2.2).Then four different treatments were established in the flasks (Table 1).These treatments were (1) unlabelled spent broth control (no amendment); (2) unlabelled spent broth amended with 1 µM of the plant sterol stigmasterol (final concentration); (3) unlabelled spent broth amended with 10 g L -1 glucose and 0.1 g L -1 Na 15 NO 3 ; and (4) unlabelled spent broth amended with 10 g L -1 glucose, 0.1 g L -1 Na 15 NO 3 and 1 µM stigmasterol.Flasks were incubated in the dark at 25 o C and mycelia sampled at days 0, 2, 3 and 6.

Mycelia Harvest
At each sampling day, mycelia were harvested from the treatments using a sterilized glass pipette in a non-destructive fashion by suctioning out a portion of the mycelia mass, and repeated washing with sterilized distilled water over a nylon filter.The mycelia were then recovered from the filter using toothpicks and placed on the surface of glass plates and air-dried overnight.The dried mycelia on the glass plates were ground using a mortar and a round glass rod.Approximately 150-250 µg were weighed into tin (Sn) capsules (Costech, Valencia, CA) in preparation for combustion and determination of the 15 N to 14 N isotopic ratio.

Stable Isotope Measurements
The 15 N to 14 N isotopic ratio values of the tissue samples were determined using an elemental analyzer (EA) (Carlo Erba CHN EA 1108, now Thermo Fisher Scientific, Waltham, MA) coupled to an isotope ratio mass spectrometer (IRMS) (FinniganConflo III Interface and a ThemoFinnigan Delta V Advantage mass spectrometer, Bremen, Germany).Samples weighed in Sn capsules were combusted at 1600 ºC in the elemental analyzer under a stream of oxygen.A standard N 2 gas of known 15N/14N ratio was introduced into the IRMS with every sample run.Acetanilide (purchased from Arndt Schimmelmann, Indiana University, Bloomington, IN,) was calibrated against IAEA-N-1 and IAEA-N-2 and was used as the instrumental reference material after every 20 samples.Isotope ratios are expressed as δ 15 N values per mille [‰] relative to an established N 2 reference gas, and were calculated using software integrated in the EA-IRMS instrument via the following equation: Where R = ratio of the heavy ( 15 N) to light ( 14 N) isotope in the sample and reference determined by mass spectrometry.

Base-line δ 15 N values on Day 0
The growth medium of P. sojae cultures from experiments that received NaNO 3 -15 N was enriched by 37% atom 15 N.The δ 15 N values of non-enriched mycelia typically ranged from 4 to 6 ‰ on day 0 of the experiment (Fig. 1).In comparison, δ 15 N values of the 37% 15 N enrichment of P. sojae ranged from 2160 to 3122 ‰ on day 0 of the experiment (Fig. 2).

Shifts in δ 15 N over time for unlabeled mycelia transferred into 15 N spent broth
In Experiment 1, the δ 15 N values of mycelia increased over-time when unlabeled mycelia were transferred into enriched 1 spent broth containing 15 N-enriched elicitins (Fig. 1).The same was true for mycelia recovered from the same spent broth that had been amended with 1 µM stigmasterol (Fig. 1), but on day 6 the δ 15 N values were approximately 2-fold greater than the δ 15 N value of mycelia recovered from spent broth that did not have stigmasterol (Fig. 1 top).Thusthe presence of stigmasterol caused a greater incorporation of the 15 N-label into P. sojae than if no stigmasterol was present.
The addition of glucose and 14 NO 3 increased the δ 15 N values of mycelia by day 6 (Fig. 1 bottom).These δ 15 N values on day 6 were approximately 45-fold higher for mycelia recovered from spent broth containing stigmasterol than the δ 15 N values of mycelia recovered from spent broth not containing stigmasterol (Fig. 1 bottom).

Shifts in δ 15 N over time for 15 N-labeled mycelia transferred into unlabelled spent broth
In Experiment 2, the δ 15 N values of harvested mycelia decreased over time when enriched 15 N mycelia were transferred into unlabelled spent broth suggesting the uptake and assimilation of unlabelled material (Fig. 2).The δ 15 N values of mycelia in spent broth that contained stigmasterol were approximately 2-fold and 30-fold lower on days 2 and 3, respectively, when compared to the δ 15 N values of mycelia recovered from non-stigmasterol supplemented broth (Fig. 2 top).Thus, the presence of stigmasterol enhanced the incorporation of the unlabelled material from the spent broth by P. sojae.However, the δ 15 N values on day 3 began to increase and by day 6 had reached δ 15 N values similar to those on day 0.
Up to day 3, the addition of glucose and 15 NO 3 to the spent broth resulted in a δ 15 N profile (Fig. 2 bottom) similar to the one observed for mycelia recovered from spent broth that had no glucose or nitrate (Fig. 2

top).
After day 3, however, the δ 15 N values of mycelia recovered from spent broth containing stigmasterol continued to decline over time when glucose and 15 N labeled nitrate were present (Fig. 2 bottom) but increased when they were not present (Fig. 2

Discussion
In this study, elicitins were labeled with 15 N by adding Na 15 NO 3 to the growth medium of P. sojae.The N-labelled elicitins that are synthesized are then secreted into the spent culture broth.There is the possibility that P. sojae produces and secretes other proteins than just class-I elicitins.However, we previously determined that the most abundant protein in spent filtrates of the organism were class-I elicitins using 2D-PAGE followed by LC-MS analysis (Yousef et al. 2009).This was also confirmed in this study by visually inspecting spent filtrates on PAGE (data not shown), in which class-I elicitins were the only proteins detected in spent filtrates after silver staining.Therefore, the spent broth was assumed to contain predominantly 15 N-labelled elicitins (Experiment 1, Fig. 1) or unlabelledelicitins (Experiment 2, Fig. 2).
Shifts in  15 N values could also result if P. sojae assimilates nitrate present in the spent broth that had been carried over from the initial 4-week incubation.To determine if this was significant, unlabelled NaNO 3 was added to the spent broth in Experiment 1(Figure 1 bottom) and 15 N-labelled NaNO3 was added to the spent broth in Experiment 2 (Figure 2 bottom).Since P. sojae is a heterotrophic organism that derives energy from the oxidation of organic carbon (Tyler 2006), glucose was also amended into the spent broth to avoid possible cannibalism of spent elicitins by P. sojae as a carbon source.
Data from these treatments indicate that in the absence of stigmasterol, some of the  15 N changes in the mycelium of Experiment 1 (Figure 1) could be due to the assimilation of 'left-over' 15 N-nitrate from the spent broth for nutritional purposes.For example, on day 6 the average  15 N value of mycelia from control treatment (non-stigmasterol amended) was approximately 900 (Figure 1 top), whereas it was 200 for the same treatment that had been amended withunlabelled (i.e. 14 NaNO 3 ) (Figure 1 bottom).However, this phenomenon was only observed in Experiment 1 and not in the reverse experiment (Experiment 2, Fig. 2).This suggests that there was a low amount of residualunlabelled NO 3 remaining in the spent broth in experiment 1 (Fig. 1 top) which was comparable to the residual amount oflabeled NO 3 remaining in the spent broth in the reverse experiment (Fig. 2 top).
The addition of stigmasterol to the spent broth caused up to a 51-fold difference in measured  15 N values of mycelia when compared to controls not receiving stigmasterol (Figures 1 and 2).This is a clear indication that stigmasterol is causing a rapid assimilation by P. sojaeof elicitins from the spent broth.Furthermore, the addition of nutrients (glucose and nitrate) to the spent broth caused a much larger increase (Fig. 1 bottom) or decrease (Fig. 2 bottom) in  15 N values over time when compared to when these nutrients were not present (Fig. 1 top and 2 top).This could possibly be due to the energy requirement of biological assimilation/sequestration, and therefore the depleted level of nutrients in the spent broth was insufficient to support bioaccumulation of elicitin-sterol complexes from the surrounding environment.
The conclusion from this study is consistent with other observations from the literature.First, it is known that class-I elicitin genes in P. sojae become down-regulated over time when nanomolar concentrations of stigmasterol are present in the growth medium (Yousef et al. 2009), which also coincided with reduced detection of elicitins in the spent broth.Second, previous studies have shown that elicitins exhibit sterol carrier activity in vitro (Mikes et al. 1998;Vauthrin et al. 1999).Structurally, elicitins are composed of an α-helix fold stabilized by three disulfide bonds, which provides a hydrophobic cavity able to bind sterols in a 1:1 sterol:elicitin stoichiometry (Osman et al. 2001b).
There are two other physiological mechanisms for how P. sojae might take up an elicitin-sterol complex.One possibility is that the elicitin-sterol complex binds to receptors present on P. sojae membranes that have a higher affinity for an elicitin-sterol complex than when the elicitin binds alone.The higher affinity of receptors for an elicitin-sterol complex would insure that only elicitins carrying a sterol are sequestered by P sojae.While there are no direct studies in Phytophthora to support this hypothesis, putative elicitin receptors have been identified in membrane preparations of tobacco which are the sites responsible for activation of hypersensitivity in tobacco (Bourque et al. 1999;Bourque et al. 1998;Ponchet et al. 1999;Svozilova et al. 2009).These receptor sites also exhibited stronger binding characteristics for an elicitin-sterol complex than when the elicitin was alone (Bourque et al. 1998;Ponchet et al. 1999).It is possible that orthologs of elicitin receptors are present in Phytopthora, which would act as recognition sites in the organism that preferentially bind and capture from the environment an elicitin-sterol complex over an elicitin alone.This would explain why, in this experiment, the  15 N values changed more rapidly when stigmasterol was present than when it was not.The gene sequences of the elicitin receptors present in tobacco membranes have not yet been delineated, the sequences of which may be used to search for orthologs in the genome of P. sojae that is available on the Joint Genome Institute web site (http://genome.jgi-psf.org).
The second hypothetical mechanism is that the binding of an elicitin-sterol complex to elicitin receptors leads to receptor-mediated endocystosis of the elicitin-sterol complex.This could be because biological membranes are composed of an amphipathic bilayer (i.e. a hydrophilic exterior and a sandwiched hyrodphobic layer), and the kinetics of moving hydrophic molecules, such as sterols, across biological membranes would be very slow without the assistance of transport proteins (Prinz 2007).Organisms in the kingdom, protozoa, are similar to Phytophthorain that they universally lack the ability to synthesize cholesterol and must acquire it from the environment (Lige et al. 2009).For example, the protozoan organism Toxoplasma gondii, which is an opportunisticparasite under immunosuppressive conditions, has been reported to sequester cholesterol from its host presumably via receptor-mediated endocytosis of cholesterol using a sterol carrier protein (Lige et al. 2009).
If sterol-uptake by Phytophthorainvolves the endocytosis of an elicitin-sterol complex, then the internalization of 15 N-labelled elicitin would also cause a shift in the natural abundance of 15 N in P. sojae mycelia as was observed in Figure 1.Similar experiments utilizing pure elicitin protein will be necessary to validate this proposed mechanism.However, results in this study support the conclusion that secreted elicitins mediate sterol acquisition by Phytophthora spp. to support their growth and reproduction.

Figure 1 .
Figure1.Change of 15 N/ 14 N ratios in non-enriched P. sojae mycelia growing in spent broth containing putative 15 N-labelled elicitins.Mycelia from 15 N-labeled spent broth without amendment (top) and mycelia from the same spent broth that was amended with 0.1 g L -1 Na 14 NO 3 (i.e.unlabelled nitrate) plus 10 g L -1 glucose (bottom)