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Plant Signaling & Behavior 5:2, 187-189; February 2010; 2010 Landes Bioscience

ARTICLE ADDENDUM ARTICLE ADDENDUM

Key words:Heterodera glycine,Heteroderaschachtii, arabidopsis, 10A06 effector pro-tein, spermidine synthase 2

Submitted: 12/24/09

Accepted: 12/25/09

Previously published online:

www.landesbioscience.com/journals /psb/article/11084

*Correspondence to: Thomas J. Baum;

Email: tbaum@iastate.edu

Addendum to: Hewezi T, Howe PJ, Maier TR,

Hussey RS, Mitchum MG, Davis EL, Baum TJ.

Arabidopsis spermidine synthase is targeted

by an effector protein of the cyst nematode

Heterodera schachtii. Plant Physiol 2009;

152:96884; PMID: 19965964; DOI: 10.1104/

pp.109.150557.

The soybean cyst nematode(Heterodera glycines) and theclosely related sugar beet cyst nematode(Heterodera schachtii) are devastatingpathogens of plant roots that use secreted

effector proteins to engage in sophisti-cated host-parasite interactions. WhileH. schachtiiinfects and reproduces read-ily on the roots ofArabidopsis thaliana,H. glycines rarely is able to infect thismodel plant. The molecular basis fordiffering host ranges remains obscurebut likely involves differences betweennematode effector proteins and the rec-ognition of host factors. Recently wereported that constitutive expression ofthe H. schachtii 10A06 effector protein

gene (Hs-10A06) in Arabidopsis affectedplant morphology and increased suscep-tibility to H. schachtiiand that the 10A06protein functions through its interaction

with Arabidopsis spermidine synthase2 (SPDS2). Therefore, we investigated

whether differences between cyst nema-tode effector protein orthologs in twonematode species have a role in mediat-ing host specificity. Here, we show that,similar to Hs-10A06, ectopic expressionof H. glycines 10A06 (Hg-10A06) in

Arabidopsis affected leaf number and root

length, however, to a much lesser extent.More importantly, no effect of Hg-10A06overexpression on Arabidopsis suscep-tibility to H. schachtii was observed.

While we found that Hg-10A06 canweakly interact with Arabidopsis SPDS2in yeast-two hybrid assays, this abilityto interact with SPDS2 was decreasedapproximately five-fold compared withHs-10A06. Collectively, these data sug-gest that sequence divergence between

cyst nematode effector protein orthologscould contribute in determining cystnematode host range.

Cyst nematodes are sedentary pathogens

of roots of many economically importantcrop plants and induce the formation ofspecialized feeding cells, so-called syn-cytia, that provide the nematodes withnourishment. The infection process ismediated through secretion of an arrayof nematode effector proteins inside planttissues and cells. One of these effectorproteins is 10A06, which was initiallyidentified from a gland cell cDNA libraryfrom H. glycines, the soybean cyst nema-tode.1 The 927 bp full-length H. glycines

Hg-10A06 cDNA (GenBank AccessionAF502391) encoded a predicted proteinof 308 amino acids with an N-terminalsignal peptide of 17 amino acids for secre-tion. Recently, we identified the ortholo-gous 10A06 sequence from the sugar beetcyst nematode H. schachtii (Hs-10A06),which is able to infect the model plantArabidopsis thaliana. The Hs-10A06cDNA(GenBank Accession GQ373256) con-tained an open reading frame of 858 bpencoding a 285-amino acid protein withan N-terminal signal peptide for secre-

tion.2

Sequence alignment of H. glycinesand H. schachtii 10A06 proteins revealeda strong homology between both ortho-logues with 86% identity and 87% simi-larity. The largest difference between thetwo proteins is the lack of a stretch of 23amino acids in Hs-10A06. Additionally, aregion of 15 amino acid residues locatedbetween amino acid 167 and 181 exhib-ited a high degree of divergence betweenboth proteins. Constitutive expression of

Sequence divergences between cyst nematode effector protein orthologsmay contribute to host specificity

Tarek Hewezi and Thomas J. Baum*Department of Plant Pathology; Iowa State University; Ames, IA USA

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Hs-10A06 in Arabidopsis affected plantmorphology and increased susceptibilityto H. schachtii.2 We uncovered in yeasttwo-hybrid assays that the Hs-10A06 pro-

tein interacts with Arabidopsis SPDS2, akey enzyme involved in polyamine biosyn-thesis, to mediate susceptibility. Here, weassessed the effects of ectopic Hg-10A06expression in the non-host Arabidopsison plant morphology and nematode sus-ceptibility. Moreover, we assayed whetherHg-10A06 also is able to interact withSPDS2 from Arabidopsis.

Ectopic Expression of Hg-10A06in Arabidopis Alters Plant

Morphology but not Susceptibilityto H. schachtii

To investigate whether Hg-10A06 hasbiological activity in Arabidopsis and canmodulate susceptibility to H. schachtii,we used the CaMV 35S promoter toexpress Hg-10A06in Arabidopsis. Twentyfour independent transgenic T1 lineswere generated and three homozygousT3 lines (lines 4-2, 16-12 and 30-9)were selected for phenotypic analyses.Similar to Hs-10A06, ectopic expression

of Hg-10A06 in Arabidopsis affected rootlength and leaf number. A slight increasein root length of about 10% relative to thenon-transgenic control was detected at 10days after planting (Fig. 1A). Similarly,the total leaf number in the transgeniclines was increased and ranged between22.55 1.37 and 26.0 1.39, whereas inwild-type plants the average leaf numberwas 16.6 0.58 at the onset of flowering(Fig. 1B). As a comparison, expression

Figure 1.Characterization of H. glycines

10A06. Ectopic expression of Hg-10A06in

-

cantly increased root lengths (A) and higher

numbers of leaves than wild-type plants

(B). Transgenic plants expressing Hg-10A06

H. schachtii

susceptibility (C). Homozygous T3 lines ex-

pressingHg-10A06

(lines 4-2, 16-12 and 30-9)

and 2-week-old seedlings were inoculated

with approximately 250 surface-sterilized

J2 H. schachtiinematodes. Three weeks

after inoculation, the number of J4 female

nematodes per root system was counted and

used to quantify plant susceptibility. Data are

presented as the mean SE.

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different effector mechanisms in the twopathosystems.

H. glycines and H. schachtii are twoclosely related cyst nematode species thatshow very similar infection behavior onArabidopsis roots up to the formation offeeding sites.8 While H. schachtii infects

and reproduces efficiently on Arabidopsisroots, H. glycines does so only very rarely.Our findings that Hg-10A06 only slightlyaffects root length and leaf numbers andnot nematode susceptibility when expressedin Arabidopsis along with the observedweak Hg-10A06/SPDS2 interaction couldpartially explain the limited ability of H.glycinesto parasitize Arabidopsis.

Acknowledgements

This is a journal paper of the IowaAgriculture and Home Economics Station,

Ames, IA, supported by Hatch Act andState of Iowa funds. This work was fundedby USDA National Research InitiativeCompetitive Grants Program Award 2005-35604-15434 and by grants from theIowa Soybean Association and the UnitedSoybean Board.

References

1. Gao B, Allen R, Maier TR, Davis EL, Baum TJ,Hussey RS. The parasitome of the phytonematodeHeterodera glycines. Mol Plant-Microbe Interact 2003;16:720-6.

2. Hewezi T, Howe PJ, Maier TR, Hussey RS, Mitchum

MG, Davis EL, et al. Arabidopsis spermidine synthaseis targeted by an effector protein of the cyst nematodeHeterodera schachtii. Plant Physiol 2010; 152:968-84.

3. Stevens C, Bennett MA, Athana ssopoulos E, TsiamisG, Taylor JD, Mansfield JW. Sequence variationsin alleles of the avirulence gene avrPphE.R2 fromPseudomonas syringaepv.phaseolicolalead to loss of rec-ognition of the AvrPphE protein within bean cells anda gain in cultivar-specific virulence. Mol Microbiol1998; 29:165-77.

4. Dodds PN, Lawrence GJ, Catanzariti AM, Teh T,Wang CIA , Aylif fe MA, e t al. Direct protein interac-tion underlies gene-for-gene specificity and coevolu-tion of the flax resistance genes and flax rust avirulencegenes. Proc Natl Acad Sci USA 2006; 103:8888-93.

5. Joosten MHAJ, Cozijnsen TJ, DeWit PJGM. Hostresistance to a fungal tomato pathogen lost by a singlebase-pair change in an avirulence gene. Nature 1994;

29:384-6.

6. Joosten MHAJ, Vogelsang R, Cozijnsen TJ,Verberne MC, DeWit PJGM. The biotrophic fungusCladosporium fulvumcircumvents Cf-4-mediated resis-tance by producing unstable AVR4 elicitors. Plant Cell1997; 29:367-79.

7. Elling AA, Davis EL, Hussey RS, Baum TJ. Activeuptake of cyst nematode parasitism proteins into theplant cell nucleus. Int J Parasitol 2007; 37:1269-79.

8. Puthoff DP, Nettleton D, Rodermel SR, Baum TJ.Arabidopsis gene expre ssion changes during cystnematode parasitism revealed by statistical analysesof microarray expression profiles. Plant J 2003;33:911-21.

does not have the same ability as Hs-10A06to facilitate a compatible interactionbetween H. schachtiiand Arabidopsis. I.e.,Hg-10A06 does not work as a functioningeffector in the non-host Arabidopsis. Thebasis for host range determination amongHeterodera species is unknown, but our

data now suggest a likely involvement ofsequence differences between orthologouseffector proteins. In Pseudomonas syringaepv. phaseolicola, DNA sequencing of avr-PphEalleles has demonstrated that aminoacid substitutions or an insertion of 104 bpare responsible for the difference betweenvirulence and avirulence and lead to a gainin bean cultivar-specific virulence.3 Morerecently, it has been reported that aminoacid variation between flax rust fungusAvrL567 proteins alters host recognition.4

The finding that Hg-10A06 can only

weakly interact with Arabidopsis SPDS2suggests that this reduced protein-proteininteraction may be responsible for a lackof altered susceptibility in the Hg-10A06plants. Careful examination of the10A06 sequence that shows strong vari-ability between the two nematode speciesrevealed that the Hs-10A06 cysteine resi-dues in position 193 and 196 are replacedby valine in Hg-10A06. This changemay prevent the formation of disulphidebonds in the Hg-10A06 protein, which

may affect a protein structure necessaryto bind to Arabidopsis SPDS2. In thefungal pathogen Cladosporium fulvum,sequence analysis of avr4alleles demon-strated single base changes leading to theloss of cysteine residues5and subsequentfailure to act as an effective elicitor of theplants resistance response.6Furthermore,as mentioned above, there is a 23-aminoacid stretch missing in the H. schachtii10A06 orthologue and in this region,Hg-10A06 is predicted to have twoSV40-like nuclear localization signals

(94

PVPKGKK100

and96

PKGKKVE102

).7

Reporter gene fusions of Hg-10A06sequences containing the predicted NLSsshowed strong reporter gene activity inthe nucleus with some minor accumu-lation in the cytoplasm. This suggeststhat Hg-10A06 may act as both a cyto-plasmic and nuclear effector in soybeanroots whereas Hs-10A06 may functiononly in the Arabidopsis cytoplasm. Moreresearch is needed to fully understand the

of Hs-10A06 in Arabidopsis had muchmore substantial effects on these param-eters.2These transgenic lines were used ina nematode susceptibility assay (Fig. 1C).Surprisingly, no statistically significanteffects of transgene expression on nema-tode susceptibility were observed. These

results show that the sequence differencesbetween H. schachtiiand H. glycines10A06are sufficiently large to a lter biological activ-ity in Arabidopsis. Taken one step further,these results suggest that effector proteindivergence may be one mechanism of hostrange determination.

with Arabidopsis SPDS2 in Yeast

Two-Hybrid Assay

To determine whether 10A06 from H.

glycines also interacts with ArabidopsisSPDS2, the full-length Hg-10A06 (minusthe signal peptide coding sequence) wasinserted into the bait vector and trans-formed into yeast cells along with the preyvector containing Arabidopsis SPDS2. Thestrength of binding between Hg-10A06and SPDS2 was estimated by measuringo-nitrophenyl--D-galactoside concentra-tions as a function of the -galactosidaseactivity produced by 10 yeast colonies.These -galactosidase quantitative assays

revealed that Hg-10A06 interacts withSPDS2 but its ability to interact withSPDS2 decreased by about five-fold whencompared with Hs-10A06.

Conclusion and Perspective

Hg-10A06 binds Arabidopsis SPDS2, albeitweaker than the Hs-10A06 orthologue,which most likely results in similar to butweaker than the alterations of polyaminesignaling found in Hs-10A06 overexpress-ing Arabidopsis lines.2 Consequently, the

observed morphological changes in rootlength and leaf numbers that are less pro-nounced than observable in Hs-10A06lines comes as no surprise. However,unlike Hs-10A06, constitutive expressionof Hg-10A06 in Arabidopsis produced nosignificant differences in nematode suscep-tibility between the transgenic lines andwild-type plants despite the high sequenceidentity between both orthologs (86%). Inother words, the H. glycines10A06 protein