38-pant lsd1

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This article was downloaded by: [Frantisek Baluska] On: 11 February 2015, At: 14:31 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Click for updates Plant Signaling & Behavior Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kpsb20 The syntaxin 31-induced gene, LESION SIMULATING DISEASE1 (LSD1), functions in Glycine max defense to the root parasite Heterodera glycines Shankar R Pant a , Aparna Krishnavajhala ab , Brant T McNeece a , Gary W Lawrence b & Vincent P Klink a a Department of Biological Sciences; Mississippi State University; Starkville, MS USA b Department of Biochemistry; Molecular Biology; Entomology and Plant Pathology; Mississippi State University; Starkville, MS USA Accepted author version posted online: 20 Dec 2014.Published online: 05 Feb 2015. To cite this article: Shankar R Pant, Aparna Krishnavajhala, Brant T McNeece, Gary W Lawrence & Vincent P Klink (2015) The syntaxin 31-induced gene, LESION SIMULATING DISEASE1 (LSD1), functions in Glycine max defense to the root parasite Heterodera glycines, Plant Signaling & Behavior, 10:1, e977737, DOI: 10.4161/15592324.2014.977737 To link to this article: http://dx.doi.org/10.4161/15592324.2014.977737 PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions

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Page 1: 38-Pant LSD1

This article was downloaded by: [Frantisek Baluska]On: 11 February 2015, At: 14:31Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,37-41 Mortimer Street, London W1T 3JH, UK

Click for updates

Plant Signaling & BehaviorPublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/kpsb20

The syntaxin 31-induced gene, LESION SIMULATINGDISEASE1 (LSD1), functions in Glycine max defense tothe root parasite Heterodera glycinesShankar R Panta, Aparna Krishnavajhalaab, Brant T McNeecea, Gary W Lawrenceb & Vincent PKlinka

a Department of Biological Sciences; Mississippi State University; Starkville, MS USAb Department of Biochemistry; Molecular Biology; Entomology and Plant Pathology;Mississippi State University; Starkville, MS USAAccepted author version posted online: 20 Dec 2014.Published online: 05 Feb 2015.

To cite this article: Shankar R Pant, Aparna Krishnavajhala, Brant T McNeece, Gary W Lawrence & Vincent P Klink (2015)The syntaxin 31-induced gene, LESION SIMULATING DISEASE1 (LSD1), functions in Glycine max defense to the root parasiteHeterodera glycines, Plant Signaling & Behavior, 10:1, e977737, DOI: 10.4161/15592324.2014.977737

To link to this article: http://dx.doi.org/10.4161/15592324.2014.977737

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) containedin the publications on our platform. However, Taylor & Francis, our agents, and our licensors make norepresentations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of theContent. Any opinions and views expressed in this publication are the opinions and views of the authors, andare not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon andshould be independently verified with primary sources of information. Taylor and Francis shall not be liable forany losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoeveror howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use ofthe Content.

This article may be used for research, teaching, and private study purposes. Any substantial or systematicreproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in anyform to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

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The syntaxin 31-induced gene, LESIONSIMULATING DISEASE1 (LSD1), functions

in Glycine max defense to the rootparasite Heterodera glycines

Shankar R Pant1, Aparna Krishnavajhala1,2, Brant T McNeece1, Gary W Lawrence2, and Vincent P Klink1,*

1Department of Biological Sciences; Mississippi State University; Starkville, MS USA; 2Department of Biochemistry; Molecular Biology;

Entomology and Plant Pathology; Mississippi State University; Starkville, MS USA

Keywords: Golgi, LESION SIMULATING DISEASE1 (LSD1), membrane fusion, pathogen resistance, salicylic acid, signaling,syntaxin 31, vesicle

Abbreviations : EDS1, enhanced disease susceptibility1; PR1, pathogenesis-related 1; NPR1, nonexpressor of PR1; SA, salicylic acid;LSD1, lesion simulating disease1; a-SNAP, alpha soluble N-ethylmaleimide-sensitive factor attachment protein; BIK1, botrytis

induced kinase1; XTH, xyloglucan endotransglycosylase/hydrolase; RNAi, RNA interference; Sed5p, suppressors of the erd2-deletion5; sec, secretion; JA, jasmonic acid; PAD4, phytoalexin deficient 4; SID2, salicylic-acid-induction deficient2; LOL1, LSD1-like;

qPCR, quantitative polymerase chain reaction; INA, 2,6-dichloroisonicotinic acid; SAR, systemic acquired resistance; O2¡, superox-

ide; ROI, reactive oxygen intermediates; CuSOD, copper superoxide dismutase; SHMT, serine hydroxymethyltransferase; GOI, geneof interest; ER, endoplasmic reticulum; MATE, multidrug and toxin extrusion; PCD, programmed cell death.

Experiments show the membrane fusion genes a soluble NSF attachment protein (a-SNAP) and syntaxin 31 (Gm-SYP38) contribute to the ability of Glycine max to defend itself from infection by the plant parasitic nematodeHeterodera glycines. Accompanying their expression is the transcriptional activation of the defense genes ENHANCEDDISEASE SUSCEPTIBILITY1 (EDS1) and NONEXPRESSOR OF PR1 (NPR1) that function in salicylic acid (SA) signaling. Theseresults implicate the added involvement of the antiapoptotic, environmental response gene LESION SIMULATINGDISEASE1 (LSD1) in defense. Roots engineered to overexpress the G. max defense genes Gm-a-SNAP, SYP38, EDS1,NPR1, BOTRYTIS INDUCED KINASE1 (BIK1) and xyloglucan endotransglycosylase/hydrolase (XTH) in the susceptiblegenotype G. max[Williams 82/PI 518671] have induced Gm-LSD1 (Gm-LSD1–2) transcriptional activity. In reciprocalexperiments, roots engineered to overexpress Gm-LSD1–2 in the susceptible genotype G. max[Williams 82/PI 518671] haveinduced levels of SYP38, EDS1, NPR1, BIK1 and XTH, but not a-SNAP prior to infection. In tests examining the role ofGm-LSD1–2 in defense, its overexpression results in »52 to 68% reduction in nematode parasitism. In contrast, RNAinterference (RNAi) of Gm-LSD1–2 in the resistant genotype G. max[Peking/PI 548402] results in an 3.24–10.42 fold increasedability of H. glycines to parasitize. The results identify that Gm-LSD1–2 functions in the defense response of G. max to H.glycines parasitism. It is proposed that LSD1, as an antiapoptotic protein, may establish an environment whereby theprotected, living plant cell could secrete materials in the vicinity of the parasitizing nematode to disarm it. After thetargeted incapacitation of the nematode the parasitized cell succumbs to its targeted demise as the infected rootregion is becoming fortified.

Introduction

Knowledge of the ability of biological membranes to fuse,resulting in the delivery of vesicle contents to different cellulardestinations, is longstanding.1 Genetic experiments and screensin model organisms have identified the proteins that function inthe process and ordered the events that lead to material deliveryin the form of secretion.2-4 Subsequent work in other systems hasdemonstrated that the core protein machinery involved in

membrane fusion is highly conserved, found in all eukaryotes(Reviewed in 5). The process of membrane fusion requires fidelityand protective measures are taken by the cell to ensure it happensproperly.6

Through recent studies, a link between membrane fusion atthe cell membrane and also the cis face of the Golgi apparatuswith SA signaling has been made in plants.7-9 Genetic work inthe plant genetic model, Arabidopsis thaliana has also identifiedessential roles for proteins involved in membrane fusion.10 The

*Correspondence to: Vincent P Klink; Email: [email protected]: 06/16/2014; Revised: 09/09/2014; Accepted: 09/10/2014http://dx.doi.org/10.4161/15592324.2014.977737

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essential nature of these membrane fusion proteins makes themdifficult to study since their mutants are lethal or cause highlydetrimental developmental anomalies.2,3,10,11 However, it is pos-sible to study these proteins under certain circumstances. Forexample, a genetic screen employed by Mayer et al.10 has deter-mined the role of vesicles in embryo cytokinesis. This approachhas succeeded because the biosynthesis of the phragmoplastwhich relies on vesicles occurs early during embryo development.Subsequent identification of one of the A. thaliana genes involvedin cytokinesis (KNOLLE [At-SYP111]) has determined it to berelated to a Saccharomyces cerevisiae membrane associated proteinknown as suppressors of the erd2-deletion 5 (Sed5p) which isstructurally homologous to syntaxin.12-14 Syntaxin is a proteininvolved in secretion, functioning in the fusion of mem-branes.12,13 Syntaxins perform membrane fusion through theirinteraction with a number of other proteins (Reviewed in 5).One of these proteins is a-SNAP whose relation to plant defensehas been demonstrated.8,12,15,16 Since these discoveries, mem-brane fusion and vesicle transport have been well documented inplants, with many of the related genes having orthologs in yeastand other systems.14,17,18

The roles that these core membrane fusion proteins performin eukaryotes is extensive, ranging from signaling, cell growth,mitosis, the endocytic cycle, exocytosis, hormonal release, neuro-transmission, fertilization, embryogenesis, development, sporula-tion and cell death.2,3,13-15,17-33 A variety of studies showmembrane fusion to be important to the defense process thatplants have toward pathogens as well as different types of defenseresponses.9,11,34-42 While the list of functions that the membranefusion and vesicle transport proteins have is large, it is less clearwhether the proteins also are engaged in other, but relatedfunctions.

Recent experiments in G. max have demonstrated thata-SNAP contributes to the resistance of G. max to the plant para-sitic nematode, Heterodera glycines.8,43 The a-SNAP gene wasfirst identified in S. cerevisiae as Sec17p in a genetic screen fortemperature sensitive secretion (sec) mutants.3 Subsequentresearch has demonstrated Sec17p is required for vesicle transportfrom the endoplasmic reticulum (ER) to the Golgi apparatus asmutants accumulated 50 nm vesicles.4,44 The results presentedby Matsye et al.8 identified the existence of a role for a-SNAPthat went beyond membrane fusion. Matsye et al.8 examined theeffect that the overexpression of an a-SNAP gene had on genesassociated with different types of hormonal signaling that haveknown defense functions. While not comprehensive, these genesincluded an analysis of the SA-regulated cysteine rich secretoryprotein gene, pathogenesis-related 1 (PR1).45 Furthermore, thestudy examined the transcriptional activity of other genes whoseprotein products are secreted. These genes included the ethyleneresponsive b-1,3-glucanase, PR2,46 the ethylene and jasmonicacid (JA) responsive chitinase gene, PR347 and the SA-responsivethaumatin, PR5.48 In those experiments, Matsye et al.8 demon-strated a-SNAP overexpression causes induced expression ofPR1, PR2 and PR5. Thus, the induced expression of componentsof the membrane fusion and vesicular transport machinery(a-SNAP) appears to influence the expression of genes that are

vesicle cargo. To expand on this concept further, related experi-ments have been performed analyzing the effect that the overex-pression of the a-SNAP binding partner, syntaxin 31 has ontranscription.9 In these experiments, the overexpression ofa-SNAP or SYP38 also results in the transcriptional induction ofthe SA signaling genes EDS1 and NPR1.9 In A. thaliana, SA bio-synthesis and signaling occurs through a well-understood path-way including the EDS1 protein binding to the lipasePHYTOALEXIN DEFICIENT 4 (PAD4).49-51 This hetero-dimer functions upstream of SALICYLIC-ACID-INDUCTIONDEFICIENT2 (SID2), a putative chloroplast-localized isochoris-mate synthase, its allelic EDS16, along with the multidrug andtoxin extrusion (MATE) efflux transporter EDS5 to activate SAbiosynthesis.52-54 Downstream, a complex composed of SA, theSA hormone receptor protein NPR1, copper ions and the tran-scription factor TGA2 forms.55,56 The complex binds to a DNApromoter sequence composed of TGACG which results in theinduction of PR1 transcription.55,61 Another gene that relates toSA signaling in A. thaliana is LESION SIMULATING DIS-EASE1 (LSD1).62 In A. thaliana, the LSD1 gene is a negative reg-ulator of programmed cell death (PCD) and its activity isantagonized by a related positive regulator of cell death genecalled LSD1-like (LOL1).63-68 Currently, it is unknown whetherthe G. max LSD1 functions in defense. However, its involvementin establishing a tight boundary between cells targeted and nottargeted for apoptosis makes it an intriguing candidate.

In the analysis presented here, the relationship between the G.max a-SNAP, Gm-SYP38 and SA signaling is examined further,adding to information generated in prior experiments.9 Geneexpression experiments have identified induced levels of Gm-LSD1 (Gm-LSD1–2) in roots engineered to overexpressa-SNAP or SYP38. These results further strengthen a linkbetween vesicle transport and SA signaling. Genetic engineeringexperiments reveal that the overexpression of Gm-LSD1–2results in engineered resistance. In contrast, RNAi of Gm-LSD1–2 in a G. max genotype that is normally resistant toH. gly-cines infection results in roots that permit parasitism at a higherfrequency. It is shown the Gm-LSD1–2 overexpression positivelyinfluences the transcriptional activity of G. max SYP38, EDS1,NPR1 and BIK1. Furthermore, the overexpression of Gm-LSD1–2 also results in the induction of the expression of thehemicellulose-modifying, vesicle-cargo gene XTH43. In contrast,their expression is suppressed in roots expressing an LSD1–2RNAi construct. The experiments presented here identify anantiapoptotic aspect of defense in the G. max-H. glycinespathosystem.

Results

Gm-LSD1 is expressed in roots overexpressing a-SNAP,SYP38 and genes relating to SA signaling

Deep sequencing experiments show that the overexpression ofthe G. max Gm-SYP38, results in the induction of 5 a-SNAPparalogs, including the rhg1 component Glyma18g02590 andGlyma11g35820 (Table 1). This result strengthened prior

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observations of the importance of a-SNAP to the process ofdefense.8 Furthermore, Pant et al.9 has demonstrated that alongwith the involvement of Gm-SYP38 during the defense of G.max to H. glycines, its overexpression also results in induced levelsof the SA signaling gene EDS1. The demonstration that SA sig-naling genes function in the defense of G. max to H. glycines hasled to an analysis showing that Gm-LSD1 (Gm-LSD1–2) isinduced in roots overexpressing Gm-SYP38 (Table 2). Duringparasitism, a well demarcated boundary is established betweenparasitized and nonparasitized cells in the G. max-H. glycinespathosystem (Fig. 1). To understand the nature of Gm-LSD1–2in relation to resistance (Fig. 2), qPCR experiments have beenperformed using cDNA template from genetically engineered G.max roots that acquired the ability to defend itself from H. gly-cines parasitism. Roots genetically engineered to overexpress G.max a-SNAP, SYP38, NPR1, EDS1, BIK1 or XTH43 exhibitinduced levels of LSD1–2 (Table 2). The association of Gm-LSD1–2 expression in roots undergoing defense indicates that itmay be performing an important role in the process. To test thishypothesis, the susceptible G. max[Williams 82/PI 518671] has beenengineered to overexpress Gm-LSD1–2 (Fig. 3). No statisticallysignificant effect is observed in root growth (Fig. S1). In experi-ments presented here, the overexpression of the Gm-LSD1–2results in a significant reduction in parasitism (Fig. 4). To exam-ine the specificity of the overexpression experiments, the expres-sion of an RNAi cassette for Gm-LSD1–2 in the normallyresistant genotype G. max[Peking/PI 548402] was done (Fig. 3). Nostatistically significant effect is observed in root growth (Fig. S1).The expression of an RNAi cassette for Gm-LSD1–2 in the nor-mally resistant genotype G. max[Peking/PtdIns 548402] results in anincreased capability of H. glycines to parasitize the resistant G.max[Peking/PI 548402] (Fig. 5).

Gm-LSD1–2 overexpression induces the expression of genesrelating to membrane fusion and SA signaling

To understand the relationship between Gm-LSD1–2 andresistance, a series of qPCR analyses have been performed usingcDNA synthesized from RNA isolated from roots overexpressingGm-LSD1–2 (Table 3). The gene expression analysis demon-strates that Gm-LSD1–2 overexpression results in inducedmRNA levels of LSD1–2 as well as EDS1–2, NPR1–2, BIK1–6,XTH43 and SYP38. In contrast, Gm-LSD1–2 overexpressionresults in suppressed levels of a-SNAP prior to infection. Thisresult is not surprising since recent experiments have shown thata-SNAP becomes highly induced later during the resistant reac-tion.9 In reciprocal experiments, the expression of an RNAi cas-sette for Gm-LSD1–2 in the normally resistant genotype G.max[Peking/PtdIns 548402] results in suppressed transcriptional activ-ity for LSD1–2 as well as EDS1–2, NPR1–2, BIK1–6, XTH43and SYP38 (Table 3). Expression of a-SNAP was not detectedunder the experimental conditions. The results confirm and pro-vide further context for the existence of a link between the mem-brane fusion gene SYP38 and SA signaling.

Discussion

LSD1 was first discovered in A. thaliana in a forward geneticscreen designed to identify spontaneous lesion simulatingmutants.62 The 5 identified lsd mutants have been divided into 2

Table 2. qPCR of G. max roots overexpressing defense-related genes. qPCRwas performed using primers designed specifically against LSD1–2 Theexperiments used the ribosomal S21 gene8 as a control to standardize theexperiments

qPCR using LSD1 primers

Transgenic line 0 dpiEDS1-OE 47.679NPR1-OE 82.061SYP38-OE 335.571a-SNAP-OE 228.011BIK1-OE 89.195XTH43-OE 190.915

Table 1. Deep sequencing of mRNA isolated from uninfected Gm-SYP38 overexpressing roots reveals altered transcriptional activity of the rhg1 resistancegene, a-SNAP (Glyma18g02590) and paralogs of a-SNAP

a-SNAP log2(fold_change) test_stat P_value q_value significant

Glyma18g02590 0.396298 1.49148 0.0204 0.03961 yesGlyma11g35820 0.39959 1.51642 0.0192 0.0375849 yesGlyma14g05920 0.936435 2.72327 5.00E-05 0.000175486 yesGlyma02g42820 2.64661 1.74839 0.00365 0.00866875 yesGlyma09g41590 1.31903 3.53869 5.00E-05 0.000175486 yes

Figure 1. A 3 dpi image of H. glycines successfully parasitizing a root of G.max[Williams 82/PI 518671]. Black arrow, nematode; red arrows, boundary ofthe nurse cell (syncytium). Bar D 100 mm.

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classes. One class forms spontaneous necrotic lesions that aredeterminate in nature.62 In this class, the expansion of necrosisinto adjacent tissue is limited.62 Furthermore, lesion formation isnot influenced by pathogens or chemicals such as SA and the nonSA-inducing 2,6-dichloroisonicotinic acid (INA) that induce theonset of systemic acquired resistance (SAR).62,70 The second classof lsd mutants, defined by LSD1, is described as a feedback orpropagation mutant.62 The lsd1 mutant forms spontaneouslesions under long day growth conditions.62 In contrast, lesionformation is suppressed under short days.62 These characteristicsindicate that light influences the process at some level. The lsd1mutant is characterized by indeterminate lesions that eventuallyconsume the whole leaf or plant.62 Another characteristic of lsd1mutants is that plants grown under permissive short day condi-tions develop lesions that eventually consume the whole plantwhen switched to long day.62 Furthermore, the lsd1 mutant

initiates lesion formation by fungal or bacte-rial pathogens and inducers of SAR, includ-ing SA and INA.62 Related experimentsusing lsd1 mutants demonstrate that super-oxide (O2¡) accumulates in the cells adja-cent to the cells undergoing cell death.63

This result demonstrates that O2¡ is both

necessary and sufficient to initiate lesion for-mation and promote its spreading into adja-cent cells.63 This result also identifies a linkbetween photorespiration and lesiondevelopment.

It is clear from these studies that lsd1mutants are impaired in their ability toestablish a boundary beyond which theneighboring cells are not consumed in thewave of cell death. Sequence analysis ofLSD1 demonstrates it to be a novel zinc fin-ger, GATA-type transcription factor.64 Inthis regard, the data presented here providesan example of a GATA-type transcriptionfactor involved in G. max defense against H.glycines. From observations made in A. thali-ana it has been hypothesized that LSD1 isresponsible either to negatively regulate apro-death pathway or activate a repressor ofcell death.64 As a regulator, LSD1 wouldfunction very early in the process. In A.thaliana, LSD1 has since been shown tofunction in relation to genes composing theSA signaling pathway, including EDS1,PAD4 and NPR1 as well as the signalingmolecule SA.65,71,72 Notably, LSD1 as anantiapoptotic gene, functions in the cellsadjacent to the infected cell that is undergo-ing cell death.65,71,72 Experiments haveshown that runaway cell death was depen-dent on SA and NPR1 in lsd1 mutants.72 Incontrast, LSD1 has been shown to nega-tively regulate SA and NPR1-independent

basal disease resistance.72 From these studies, it has been pro-posed that SA and NPR1 function in runaway cell death in thelsd1 mutant through their participation in a signal amplificationloop that promotes apoptosis.71,72 It has been shown that animportant component of runaway cell death is the generation ofreactive oxygen intermediates (ROI) such as O2

¡.63,65,71,72 Addi-tional studies further link the lsd1 mutant to impaired photores-piration, leading to the accumulation of excess excitation energyand subsequent cell death.73 In contrast, cell death is preventedin the lsd1mutants by impeding conditions that lead to photores-piration.73 These results explain the link between the lsd1 andphoto-oxidative damage. Thus, it has been proposed that theLSD1 protein functions like a rheostat whereby above a ROIthreshold, the cell would undergo cell death.63,64,65,71,72,73 Incontrast, below a certain threshold, the cell would survive. Fromthis work, a signal potentiation loop has been coined to describe

Figure 2. The Golgi apparatus serves a central role in resistance as a defense engine, processingproteins for their eventual transport. The overexpression of a-SNAP resulted in engineered resis-tance.8 Furthermore, a-SNAP overexpression results in the induction of Gm-SYP38 transcription.9

In reciprocal experiments, Gm-SYP38 overexpression results in the transcriptional activation ofa-SNAP and its paralogs (Table 1). The overexpression of Gm-SYP38 results in the transcriptionalactivation of EDS1 which functions upstream of SA biosynthesis (dashed lines). The overexpres-sion of Gm-SYP38 also results in the transcriptional activation of the SA receptor, NPR1, the DNAbinding b-ZIP transcription factor TGA2 and the GATA-like transcription factor LSD1. The bindingof SA to NPR1 results in its translocation to the nucleus. NPR1 and TGA2 are directly involved inthe transcriptional activation of PR1 and PR5. For presentation purposes, on the right side of theGolgi apparatus are shown vesicles undergoing anterograde transport while those on the left areundergoing retrograde transport. Vesicles are shown released from the trans-Golgi network, mov-ing toward the endosome. Ultimately, secretory vesicles fuse with the plasma membrane todeliver receptor components and secrete contents into the apoplast. Some of these secreted con-tents, like Gm-XTH43, play important roles in defense.9 In contrast, vesicles emerge from theplasma membrane and fuse with the endosome, recycling contents. Not shown, Gm-SYP38 anda-SNAP overexpression results in induced expression of the cytoplasmic receptor-like kinase BIK1that is important for defense 9

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how in the absence of LSD1protein, the accumulationof signaling componentsleads to runawayapoptosis.72

These experimentsfocused in on the aboveground portions of A. thali-ana. Subsequently, a num-ber of experimentsexamining LSD1 have stud-ied specific aspects of rootbiology. Under certainadaptive environmental cir-cumstances (i.e. water satu-rated conditions and lowoxygen [hypoxia]), rootcells become targeted forapoptosis through a processcalled lysigeny. As a conse-quence of this process, theroots develop aerenchymawhich increases the abilityof roots to maintain higher O2 levels. Experiments in A. thalianahave shown that lysigeny is under the control of LSD1.74 Underconditions of hypoxia, LSD1, EDS1 and PAD4 functionupstream of H2O2 production and ethylene signaling events thatlead to lysigeny.74 Under normal conditions in A. thaliana,LSD1 functions as a negative regulator of the apoptosis-promot-ing EDS1 and PAD4. In contrast, under hypoxia, LSD1 is nega-tively regulated, permitting EDS1 and PAD4 to promote celldeath in A. thaliana.74 To understand how H2O2 productioncould be regulated in the roots, earlier experiments performed onaerial portions of A. thaliana demonstrated that LSD1 controlsH2O2 production through SA-regulated transcription ofCuSOD.65 This is an important finding since plants can producethe highly toxic O2¡ during plant defense by the activities ofNADPH oxidase.75 Recent findings performed in A. thalianahave shown a direct link between NADPH oxidase and BIK1.76

In those experiments, BIK1 directly phosphorylates NADPHoxidase to produce O2¡ and activate defense pathways. Plantsthen detoxify O2

¡ to H2O2 through major antioxidant enzymeslike CuSOD. Thus, certain aspects of LSD1 function in A. thali-ana are similar between the shoot and root. Furthermore, recentfindings in A. thaliana have also revealed LSD1 has many func-tions with regard to basic aspects of plant growth, developmentand its ability to function under different environmental condi-tions and stresses.77 These observations place some context intothe observation that Gm-BIK1 functions in defense in the G.max-H. glycines pathosystem.9

LSD1 transcription is induced in G. max rootsoverexpressing the membrane fusion gene a-SNAP

Two major H. glycines resistance loci have been identifiedfrom screening ecological collections of G. max.78,79 These loci,the recessive rhg1 and the dominant Rhg4, have been mapped

and cloned through traditional means and aided further by tran-scriptomics and candidate gene approaches.43,78,79,80-83 Geneticcrosses of rhg1 and Rhg4-containing genotypes leads to progenywith further-enhanced, nearly full resistance. The additive effectthat these loci have, regarding H. glycines resistance, indicate thatthe genes function in different genetic pathways that converge onthe same outcome (resistance). The rhg1 locus, depending on theresistant genotype examined, is composed of multiple tandem

Figure 3. Representative control and transgenic LSD1–2 overexpressing and LSD1–2 RNAi G. max plants. (A) Controlsusceptible G. max[Williams 82/PtdIns 518671] plant. (B) Genetically engineered G. max[Williams 82/PI 518671] overexpressingGm-LSD1–2. (C) Control resistant G. max[Peking/PtdIns 548402] plant. (D) A resistant G. max[Peking/PI 548402] plant geneti-cally engineered to express an LSD1–2 RNAi construct. Scale provided on left of each image.

Figure 4. The female index for transgenic G. max plants genetically engi-neered to overexpress Gm-LSD1–2 and infected with H. glycines. Repli-cate 1 (R1) control plants had 28.39 cysts per gram (12 plants); LSD1–2-R1-overexpressing plants (LSD1–2-R1: oe) had 13.66 cysts per gram (12plants). The FI D 47.92; P-value D 0.0216541 which is statistically signifi-cant (P < 0.05). R2 control plants (replicate 2) had 30.40 cysts per gram(16 plants); LSD1–2-R2-overexpressing plants (LSD1–2-R2: oe) had 9.85cysts per gram (12 plants). The FI D 32.4; P-value D 0.000059234 which isstatistically significant (P < 0.05). R3 control plants had 32.98 cysts pergram (20 plants); LSD1–2-R3 overexpressing plants (LSD1–2-R3: oe) had14.07 cysts per gram (18 plants). The FI D 42.662; P-value D 3.36219e-06which is statistically significant (P < 0.05).

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repeated copies of 3 or 4 genes. These genes include an aminoacid transporter, a-SNAP, a wound inducible protein and insome genotypes, a gene known as placenta-specific gene 8 protein(PLAC8).43,81,82,84 Among these genes, the overexpression ofa-SNAP has been shown to yield a resistant reaction when over-expressed on its own. As part of the secretory pathway, a-SNAPwould function in many essential cellular processes.3 The otherresistance gene, Rhg4, gene is a SHMT which plays a role in pho-torespiration. In overexpression studies, SHMT suppresses theability of H. glycines to parasitize G. max.69,83

The overexpression of a-SNAP leads to an increase in expres-sion of its binding partner, syntaxin 31 (Gm-SYP38). Syntaxin31 functions at the cis face of the Golgi apparatus to facilitate thefusion of transport vesicles transported from the endoplasmicreticulum.3,4,44,85-88 In G. max, the overexpression of a-SNAPand Gm-SYP38 results in induced levels of the SA signalinggenes EDS1, NPR1 and PR1.9 While the observation of an influ-ence of vesicle transport on SA signaling is not a new concept,7

the results of Pant et al.9 indicates that SA signaling may beimportant to the process of defense in the G. max-H. glycinespathosystem. To test this hypothesis, the overexpression of Gm-EDS1 and NPR1 has been shown to lead to resistance.9 Inrelated experiments, the overexpression of EDS1 and NPR1 inG. max leads to induced levels of SHMT prior to infection.9 Fur-thermore, the overexpression of G. max syntaxin 31 leads toslightly induced levels of EDS1 and SHMT during infection.9

While these experiments were not comprehensive, they indicatethat genes composing the rhg1 locus can influence the expressionof Rhg4.

The observation in G. max that EDS1 and NPR1 function inresistance to H. glycines indicated other genes relating to themmay also function in the process. An obvious candidate is Gm-LSD1. In qPCR experiments examining G. max roots overex-pressing a-SNAP, it is shown that Gm-LSD1–2 transcription isinduced. Complimentary experiments presented here show thatGm-LSD1–2 is also induced in roots engineered to overexpressGm-SYP38. Furthermore, Gm-LSD1–2 transcription is alsoinduced in roots overexpressing BIK1, EDS1, NPR1 or XTH.The strong association of Gm-LSD1–2 with engineered forms ofresistance led to the idea that it may perform a direct role in theprocess. Since A. thaliana LSD1 is known to play roles in estab-lishing and maintaining a tight boundary around the cells and tis-sues involved in pathogen infection, it is possible that theexpression of Gm-LSD1–2 could be performing an importantrole in regulating the expansion and/or initial survival of parasit-ized cells. The H. glycines-parasitized root cells undergo a slowprocess taking days to conclude that ultimately leads to resis-tance.89 During this time, the parasitized root cell would havetime to synthesize and secrete molecules in the vicinity of thenematode to neutralize its activities while fortifying the parasit-ized area. One such enzyme is Gm-XTH43. Notably, XTH con-tains a signal peptide and is transported through the vesicletransport machinery to the apoplast where it modifies hemicellu-lose.9,90 Furthermore, the parasitized cell may produce O2¡whose subsequent metabolism to H2O2 has been shown in A.thaliana to be under regulation by LSD1.63,65,70-74 In the analysispresented here, the overexpression of Gm-LSD1–2 in G. max[Wil-

liams 82/PI 518671] roots that are otherwise susceptible to H. glycinesparasitism, resulted in »52 to 68% reduction in nematode para-sitism. Roots overexpressing Gm-LSD1–2, when tested for theexpression of markers of resistance (i.e., XTH43, SYP38, NPR1,EDS1 and BIK1) show that each is induced in its expressionprior to H. glycines infection. In examining molecular markers ofdifferent signaling processes, highly induced levels of PR2 wereobserved in Gm-LSD1–2 overexpressing roots prior to theirinfection by H. glycines. The induction of PR2 transcription

Figure 5. G. max plants genetically engineered for RNAi of Gm-LSD1–2and infected with H. glycines have an increased capability, shown as foldchange, for parasitism. Replicate 1 (R1) control plants (resistant G. max[-Peking/PtdIns 548402]) had 1.98 cysts per gram (10 plants). LSD1–2-RNAi-R1(LSD1–2-R1: RNAi) in resistant G. max[Peking/PI 548402]) had 6.41 cysts pergram (11 plants). The results were statistically significant (p D0.00255251). Replicate 2 (R2) control plants (resistant G. max[Peking/PtdIns548402]) had 0.79 cysts per gram (12 plants). LSD1–2-RNAi-R2 (LSD1–2-R2:RNAi) in resistant G. max[Peking/PI 548402]) had 8.63 cysts per gram (5plants). The results were statistically significant (p D 0.0117053). Repli-cate 3 (R3) control plants (resistant G. max[Peking/PtdIns 548402]) had 2.51cysts per gram (10 plants). LSD1–2-RNAi-R3 (LSD1–2-R3: RNAi) in resis-tant G. max[Peking/PI 548402]) had 11.7 cysts per gram (7 plants). The resultswere statistically significant (p D 0.0120138).

Table 3. qPCR of G. max roots either overexpressing LSD1-2 or geneticallyengineered with a RNAi construct targeting LSD1–2 The experiments usedthe ribosomal S21 gene as a control to standardize the experiments.*expression not detected

qPCR LSD1–2 OE LSD1–2 RNAi

Gene tested 0 dpi 0 dpiLSD1 293.784 ¡1.851EDS1 40.129 ¡2.094NPR1 145.11 ¡2.346SYP38 581.545 ¡1.889a-SNAP ¡3.104 N/A*BIK1 161.048 ¡1.359XTH43 37.536 ¡1.223PR1 4.276 3.192PR2 159.282 ¡3.222PR3 3.206 1.388PR5 ¡2.005 1.2

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indicates ethylene may also be a component of in Gm-LSD1–2-mediated resistance. The contribution of PR2 to resistance hasbeen demonstrated, linking ethylene to the process.69 In contrast,RNAi of Gm-LSD1–2 in the resistant genotype G. max[Peking/PI548402] demonstrates specificity. In these experiments, the nor-mally resistant G. max[Peking/PI 548402] roots engineered with theGm-LSD1–2 RNAi cassette lacked the induction of LSD1–2expression and exhibited an increase in parasitism capability.These results provide direct evidence that Gm-LSD1–2 plays animportant role in the ability of G. max to prevent parasitism byH. glycines, contrasting with recent heterologous expression stud-ies.91 In examining this discrepancy between the heterologousexpression of A. thaliana LSD1 and Gm-LSD1–2 further, theconceptually translated At-LSD1 gene studied in Matthewset al.91 is 66.5% identical to the tested G. max LSD1–2 protein(Glyma08g13630) presented here. Thus, part of the differenceobserved between the capability of At-LSD1 and Gm-LSD1–2proteins to function in G. max may arise from gene sequence var-iation. To reinforce our observation that Gm-LSD1–2 func-tioned in resistance, we present through a double-blind analysisexperimental and biological replicates in both the Gm-LSD1–2overexpression and RNAi experiments.

Spatial and temporal aspects regarding LSD1The demonstration that Gm-LSD1–2 is important to the

defense process clarifies the paradox that parasitized G. max rootcells tolerate the establishment and maintenance of the attackedcell early during H. glycines parasitism prior to the commitmentof the parasitized cell for demise. The association of LSD1 withthe antiapoptotic activities of photorespiration in A. thalianalinks its function to G. max Rhg4-mediated defense.63,65,70-73

The demonstration that induced levels of Gm-LSD1–2 transcrip-tion in roots overexpressing the rhg1 gene a-SNAP and SYP38links LSD1 to the process of vesicle transport at some level. Atthis point, many details remain concerning the genetic programresponsible for the establishment and maintenance parasitizedcell and surrounding root cells. From these observations, it isplausible that Gm-LSD1–2 functions initially in both the parasit-ized cell and surrounding cells to prevent cell death and establisha boundary. The demonstration that Gm-BIK1 is important toresistance implicates NADPH oxidase performing a role in theprocess.9,76 NADPH oxidase would provide the O2

¡ that couldantagonize H. glycines. During this time, as the cell is protectedfrom apoptosis, the vesicle transport machinery including therhg1 gene a-SNAP would function to deliver antimicrobials, cellwall modifying enzymes and other substances to the site of para-sitism. However, the process of resistance is not limited to thisframework.

Methods

Gene cloningThe candidate gene overexpression study presented here has

been done according to our published procedures using thepRAP15 and pRAP17 vectors.8,9 The primers used to clone Gm-

LSD1–2 (Glyma08g13630) are provided (Table S1). The natureof the hairy root system is that each transgenic root system func-tions as an independent transformant line.9,92 Amplicons, repre-senting the gene of interest (GOI) generated by PCR have beengel purified in 1.0% agarose using the Qiagen� gel purificationkit, ligated into the directional pENTR/D-TOPO� vector andtransformed into chemically competent E. coli strain One ShotTOP10. Chemical selection has been done on LB-kanamycin(50 mg/ml) according to protocol (Invitrogen�). Amplicons havebeen confirmed by sequencing and comparing the sequence to itsoriginal Genbank accession. The G. max amplicon has been shut-tled into the pRAP15 or pRAP17 destination vector using LRclonase (Invitrogen�). The engineered pRAP15 or pRAP17 vec-tor have been transformed into chemically competent A. rhizo-genes strain K599 (K599)93 using the freeze-thaw method94 onLB-tetracycline (5 mg/ml).

The infection of G. max by H. glycinesGenetic transformation overexpression experiments have been

performed according to Pant et al.9 in the functionally hypomor-phic rhg1¡/¡ genetic background of G. max[Williams 82/PtdIns

518671], lacking a defense response to H. glycines parasitism. Incontrast, RNAi studies have been performed in the rhg1C/C

genetic background of G. max[Peking/PI 548402] according to Pantet al.9 Female H. glycines[NL1-Rhg/HG-type 7/race 3] have been puri-fied by sucrose flotation.95,96 Each root has been inoculated withone ml of nematodes at a concentration of 2,000 second stagejuveniles (J2s)/ml per root system (per plant), infected for 30 dand confirmed by acid fuchsin staining.97 At the end of theexperiment, the cysts (fully matured females) have been collectedover nested 20 and 100-mesh sieves.9 Furthermore, the soil hasbeen washed several times and the rinse water sieved to assure col-lection of all cysts.9 The accepted assay to accurately reflect if acondition exerts an influence on H. glycines development is thefemale index (FI).98 The FI has been calculated in a double blindanalysis as FI D (Nx/Ns) X 100, where Nx is the average numberof females on the test cultivar and Ns is the average number offemales on the standard susceptible cultivar.98 Nx is thepRAP15-transformed line that had the engineered GOI. Ns isthe pRAP15 control in their G. max[Williams 82/PtdIns 518671]. Theeffect of the overexpressed gene on parasitism has been tested sta-tistically using the Mann–Whitney–Wilcoxon (MWW) Rank-Sum Test, P < 0.05.9

RNA-seqExon sequencing (RNA seq) has been performed according to

our original published work with modifications.81 RNA has beenextracted from G. max roots using the UltraClean� Plant RNAIsolation Kit (Mo Bio Laboratories�, Inc.; Carlsbad, CA) andtreated with DNase I to remove genomic DNA.8,9 RNA-seqanalyses have been performed using the Illumina� HighSeq2500� platform (Eurofins MWG Operon; Huntsville, Alabama).The RNAseq procedures that identified transcript (tag) countsand chromosomal coordinates of the G. max genome84 alongwith the associated gene ontology (GO) annotations99 are out-lined here, subsequently. The qualities of raw reads have been

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checked using program FASTQC. The updated genomesequence and annotation of G. max84 have been obtained fromPhytozome v9.0 (dated: Nov 27, 2011). The abundance of tran-scripts across all samples has been measured and compared100

and default setting of the programs used unless specified. Briefly,the raw reads for each sample have been mapped on G. maxgenome using TopHat v2.0.6.101 Then, Cufflinks v2.0.2102 pro-gram have been used to assemble the mapped reads into tran-scripts. The FPKM values were calculated for all genes in allsamples and their differential transcript expression (log base 2)computed using program Cuffdiff.102

Quantitative real-time PCR (qPCR)The qPCR experiments examining LSD-1–2 overexpression

have been performed according to Pant et al.9 The same rootmRNA used in Pant et al.9 has been used here for the qPCR analy-ses of roots overexpressing G. max SYP38, a-SNAP, EDS1–2,NPR1–2, XTH43, BIK1–6. Primers used in qPCR gene expres-sion experiments are provided (Table S2). The experiments, pre-sented as log base 2, use the ribosomal protein gene S21 as acontrol.8,9 Gene expression has been tested in relation to severaldifferent classes of pathogenesis related (PR) genes, and defensegenes (Table S1). The qPCR experiments have used Taqman� 6-carboxyfluorescein (6-FAM) probes and Black Hole Quencher(BHQ1) (MWGOperon; Birmingham, AL). The qPCR differen-tial expression tests have been performed according to Livak andSchmittgen.103 The qPCR reaction conditions have been preparedaccording to Pant et al.9 and includes a 20 ml Taqman GeneExpression Master Mix (Applied Biosystems; Foster City, CA),0.9 ml of mM forward primer, 0.9 ml of 100 mM reverse primer,2 ml of 2.5 mM 6-FAM (MWG Operon�) probe and 9.0 ml oftemplate DNA. The qPCR reactions have been executed on anABI 7300 (Applied Biosystems�). The qPCR conditions includea preincubation of 50� C for 2 min, followed by 95�C for10 min. This step has been followed by alternating 95�C for 15sec followed by 60�C for 1 min for 40 cycles.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank George Hopper, Reuben Moore and WesBurger (MAFES) whose support has made the research possible.Greenhouse space to support the research has been provided bythe Department of Biochemistry, Molecular Biology, Entomol-ogy and Plant Pathology (BMEP) and the Department of Plantand Soil Sciences (PSS) at Mississippi State University. Theauthors thank Dr. Giselle Thibaudeau and Amanda Lawrence,Institute for Imaging and Analytical Technologies, MississippiState University for imaging expertise and technical suggestionsduring the course of the research. Postdoctoral research supporthas been provided to Aparna Krishnavajhala through a competi-tive Special Research Initiative grant awarded by MAFES. YixiuPinnix (BMEP) has provided technical support. The efforts ofKeshav Sharma, Jian Jiang, Prakash Nirula and Jillian Harris areacknowledged. Much of the research was supported by under-graduate students including Ashley Dowdy, Nishi Sunthwal,John Clune, Hannah Burson, Chase Robinson, Meghan Cal-houn and Austin Martindale and Annedrea McMillan (DBS)and by James McKibben, Cody Roman and Micah Schneider(BMEP). The Shackouls Honors College is acknowledged. VPKacknowledges Dr. Ben Matthews and Dr. Perry Cregan at theUSDA-ARS (Beltsville, MD) for support throughout the process.

Funding

The research has been supported by the start-up package pro-vided by Mississippi State University and the Department of Bio-logical Sciences (DBS). The authors thank the MississippiSoybean Promotion Board for support. The research is supportedjointly between the College of Arts and Sciences (DBS) and theMississippi Agricultural and Forestry Experimental Station(MAFES). The authors acknowledge the Office of the GraduateSchool at Mississippi State University for providing competitiveSummer Research Program for Undergraduate Students researchaward to Tineka Burkhead.

Supplemental Material

Supplemental data for this article can be accessed on thepublisher’s website.

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