BACKGROUND
plant parasitic nematodes cause billions of dollars in losses each year worldwide  <cit> .
the root-knot nematode, genus meloidogyne, attacks over  <dig> species of plants  <cit> , while the soybean cyst nematode  has a much narrower host range and is responsible for almost one billion dollars per year in losses in the us  <cit> .
although some soybean genotypes are resistant to specific populations of scn, no soybean genotype is known to be resistant to all scn populations.
several genes conferring partial resistance to heteroderaglycines  have been mapped, and, recently, genes at the rhg <dig> and rhg <dig> loci have been elucidated  <cit> .the defense response of soybean to scn has been examined at the physiological, genetic, and molecular level, and several reports indicate that salicylic acid , jasmonic acid , ethylene , and indole acetic acid  play a role in resistance and susceptibility of plants to nematodes  <cit> .
however, the roles of defense-related hormones and specific components of their synthesis and signaling pathways in providing resistance in plants to nematodes are unknown.
the plant defense response is complex.
plants launch a myriad of local and systemic defense responses to protect themselves from invasion by pests and pathogens.
several hormones are involved in inducing the defense response and regulating defense response networks, including sa, ja, et, and iaa  <cit> .
plants react to pathogen-associated or microbe–associated molecular patterns  which are sensed by plants through leucine-rich repeat  receptors.
pamps signal stomatal closure and stimulate innate immunity in plants.
in general, sa activates the defense response to biotrophic and hemi-biotrophic pathogens, induces systemic acquired resistance , and triggers the expression of sar-associated pathogenesis related genes pr- <dig>  pr- <dig>  and others  <cit> .
the role of specific components of sa regulation, synthesis, and signaling in defending plants against parasitic nematodes is not well understood.
however, sa does play a role in decreasing susceptibility to root-knot nematode  in cow pea  <cit>  and tomato  <cit> , and to the cyst nematode, heterodera schachtii, in arabidopsis <cit> .
likewise, ja also plays a role in resistance of plants to nematodes.
foliar spraying of tomato with ja reduced galling and the final population of rkn ;  <cit> , as did pre-treatment of tomato seeds with ja  <cit> , indicating a role for ja in plant resistance to nematodes.
little is known of the role of specific components of sa regulation and signaling in the interaction of soybean with the soybean cyst nematode , the major pest of soybean in the us.
although soybean genes conferring resistance to scn have been identified, these do not provide resistance to all scn populations.
resistance in soybean to scn is multigenic, and several major loci for resistance have been identified  <cit> .
for example, in soybean cv peking, several genes  have been reported that confer resistance to scn race  <dig>  <cit> , yet none of these genes confers complete resistance to all scn populations.
therefore, we are applying to soybean a portion of the vast knowledge that has been gained from studies on the model plant arabidopsis and its large array of mutants on the role of sa and ja in the plant defense response to identify important components that may be useful in decreasing susceptibility of plants to nematodes, and especially of soybean to scn.
arabidopsis has been used widely as a model system to study plant defense pathways, usually with bacterial and fungal pathogens  <cit> .
much attention has been focused on sa production and signaling pathways using arabidopsis mutants infected with bacterial and fungal pathogens   <cit> .
for example, when a virulent strain of the biotrophic pathogen, pseudomonas syringae, attacks arabidopsis, the avrrps <dig> effector protein of p. syringae secreted by the type iii secretion system is detected by the plant receptor rps <dig>  a toll-interleukin-nucleotide binding-leucine-rich repeat  that mediates the induction of antimicrobial defenses to provide disease resistance.
the nucleo-cytoplasmic protein enhanced disease susceptibility  <dig> , which is a lipase-like protein, connects rps <dig> with downstream defense pathways and regulates the accumulation of sa  <cit> .
eds <dig> is essential for production of the hypersensitive response and mobilization of defense pathways  <cit> .
eds <dig> can dimerize and interact with another lipase-like protein, phytoalexin deficient  <dig>   <cit> .
both eds <dig> and pad <dig> are required for the accumulation of sa and they are involved in ros signaling  <cit> .
pad <dig> protein is required for amplification of weak signals resulting from pathogen infection.
another important component of the defense response is eds <dig>  a multi-drug transporter member of the mate family of transporters  <cit> .
it is postulated that sa can be synthesized through two different pathways in arabidopsis <cit> .
one pathway involves the enzyme isochorismate synthase , which catalyzes the conversion of chorismate to isochorismate.
the enzyme chorismate mutase  catalyzes the competing chemical reaction and converts chorismate to prephenate.
this would divert chorismate to produce other compounds, such as phenylalanine and tyrosine.
the arabidopsis sid2- <dig>  mutation has been mapped to the locus encompassing the ics  gene  <cit> .
upon synthesis, sa can bind directly with npr <dig>  which is encoded by atnpr <dig> , also known as nim <dig> .
npr <dig> is an sa receptor that is a transcriptional regulator of genes involved in the sa-dependent defense response  <cit> , including the sa marker gene pr- <dig> .
npr <dig> interacts with transcription factor tga <dig> family members, including ahbp-1b, and the complex binds to sa-responsive promoter elements of pr- <dig> and other sa-dependent defense genes to regulate expression  <cit> .
sa can also be regulated independently of eds <dig> and pad <dig>  ndr <dig>  is a positive regulator of sa that works independently of eds <dig> and pad <dig>  <cit>  ndr <dig> is an integrin-like protein that can associate with rin <dig>  while rin <dig> can associate with rpm <dig> and rps <dig>  <cit> .
ndr <dig> may play a role in electrolyte release upon infection of arabidopsis by p. syringae, while rin <dig> regulates stomatal apertures in conjunction with h + −atpases of the plasma membrane of arabidopsis during pathogen attack  <cit> .
ja, jaile, and related lipid-derived compounds also act as signals in the plant defense response and are associated with resistance to necrotrophic pathogens  <cit> .
the pathway leading to ja and jaile synthesis and some components related to ja signaling and control are depicted in figure  <dig>  allene oxide synthase  and allene oxide cyclase  are two enzymes important to ja synthesis.
jar <dig> encodes an atp-dependent ja-amido synthase that conjugates isoleucine with ja to form jaile, which plays an essential role in ja signaling.
in this paper, we examine the role of some components of the plant defense response in conferring resistance in soybean to scn.
we show that specific arabidopsis genes, namely atnpr <dig>  attga <dig>  atics <dig>  and atpr <dig> which encode components of sa regulation, biosynthesis, and downstream effectors, can decrease susceptibility of soybean to scn when expressed in transgenic soybean roots.
we also demonstrate that expression of arabidopsis genes encoding aos, aoc, and jar <dig>  which are involved in the synthesis and modification of ja, only modestly decrease susceptibility of soybean roots to scn.
these results indicated that some arabidopsis genes can be directly used in soybean, thus directly applying knowledge of the defense response gained from studies using arabidopsis as a model system to soybean to decrease susceptibility to nematodes.
RESULTS
expression of arabidopsis genes in soybean roots
sa and ja are well known regulators of the plant defense response as described through studies of the model plant, arabidopsis.
to determine if some of these arabidopsis genes could be directly used in soybean to translate knowledge from arabidopsis to soybean, we selected and cloned genes encoding numerous components of sa and ja synthesis, regulation, and signaling from the literature describing the defense response of arabidopsis to pathogens .
to broaden the scope of our study, we also selected several arabidopsis genes less well defined in function or that represented other portions of the plant defense response less dependent upon sa and ja.
open reading frames  of thirty-one arabidopsis genes were cloned into the gene expression vector prap <dig>  <cit>  using the primers listed in additional file 1: table s <dig>  the inserted orfs were sequenced to confirm their identity and to ensure their sequence was conserved.
the vector with insert was transformed by agrobacterium rhizogenes k <dig> into cells at the base of the cut stem of soybean seedlings.
approximately 35 days after transformation, untransformed roots were removed from the composite plants, and the transformed roots were inoculated with scn.
the effect of expression of thirty-one genes on the number of scn cysts at  <dig> dai  was determined .
six genes decreased the number of cysts more than 40%, thus conferring partial resistance to scn.
three of these genes, atnpr <dig>  attga <dig>  and atpr- <dig>  decreased the number of cysts more than 60%, while three others, atacbp <dig>  atacd <dig>  and atcm <dig> decreased the number of cysts 40%.
one arabidopsis gene, atdnd <dig>  increased the number of cysts of scn to 175% of the control, thus making the soybean roots more susceptible to scn .
rna was extracted from a subset of transformed roots for genes listed in additional file 2: table s <dig> to check for expression of the arabidopsis gene by pcr.
the amplicons were separated and visualized by gel electrophoresis and staining  to confirm that the orfs were expressed in the composite root.
all roots tested expressed the transcript.
in addition, the abundance of transcript of two genes providing the most protection against scn, atnpr <dig> and attga <dig>  was determined by qrt-pcr using gene specific primers .
transcript number was calculated using the sigmoidal method  <cit> .
the number of transcripts of atnpr <dig> was  <dig>  molecules and for attga <dig> was  <dig>  molecules in transformed roots, while no transcripts of either gene were detectable in the control roots.
in all samples, the expression level was similar for the housekeeping gene encoding ubiquitin- <dig> .
the number of transcripts of three defense-related genes, gmpr <dig>  gmchib <dig>  and gmerf1was also determined using qrt-pcr .
in roots overexpressing atnpr <dig>  there were  <dig> transcripts of gmpr <dig>  while in roots overexpressing attga <dig>  there were  <dig> transcripts.
in control roots, there were only  <dig> transcripts of gmpr <dig>  thus, gmpr <dig> was elevated approximately 4-fold in roots overexpressing attga <dig>  transcripts of gmchib <dig> were also elevated in these roots.
there were  <dig> transcripts of gmchib <dig> in roots overexpressing atnpr <dig> and  <dig> transcripts in roots overexpressing attga <dig>  there were only  <dig> transcripts of gmchib <dig> in control roots.
this represents an increase in expression of gmchib <dig> by approximately 8- and  <dig> -fold in soybean roots overexpressing atnpr <dig> and attga <dig>  respectively.
in contrasts, the number of transcripts of gmerf <dig> decreased in soybean roots overexpressing atnpr <dig>  only  <dig> transcripts of gmerf <dig> were present, whereas control plants contained  <dig> transcripts.
similarly, roots overexpressing attga <dig> contained fewer gmefr <dig> transcripts than in control roots with only  <dig> transcripts present.
thus, gmerf <dig> expression was greatly decreased in both sets of transgenic roots.
gmpr5
,
gmchib1
, and
gmerf <dig>
was determined in roots transformed with
atnpr <dig>
and
attga <dig>
using qrt-pcr.
sa-related genes
activation of the defense response in plants is initiated through several parallel signaling pathways.
in gene-for-gene resistance, host resistance  proteins indirectly recognize pathogen effectors to initiate resistance  <cit> .
the coiled-coiled-nucleotide-binding site-leucine-rich repeat  and the tir-nb-lrr classes of proteins are two major sub-groups of r protein  <cit> .
in this study, we selected the arabidopsis protein non-race-specific disease resistance  <dig>  as a representative cc-nb-lrr r-protein, because of its known role in activating the sa-mediated defense response in arabidopsis.
rps <dig>  encoded by atrps <dig>  was selected as a representative of the tir-nb-lrr class of proteins.
both atndr <dig> and atrps <dig> were overexpressed in transgenic soybean roots to determine their effect on scn growth and maturation as measured by the female index , which expresses the number of mature scn females 35 days after root inoculation as a percent of the control value.
overexpression of atndr <dig> slightly inhibited scn development , while atrps <dig> had a slightly greater inhibitory effect , but the effect was not statistically significant   for either gene.
arabidopsis non-expressor of pathogenesis-related genes  <dig>  is downstream of the r proteins ndr <dig> and rps <dig>  npr <dig> is a key regulator of sar and plays a critical role as a sa signal transducer in arabidopsis <cit> .
when atnpr <dig> was overexpressed, the fi decreased to 33% of the control.
atnpr <dig> had the lowest fi value among the arabidopsis genes tested in this study .
alignment of the  <dig> aa of atnpr <dig> with its closest soybean counterpart, the product of glyma09g <dig> , indicates conservation of only  <dig> aa, although there are also many conservative substitutions .
there are five soybean genes encoding proteins closely related to atnpr <dig>  the protein encoded by glyma09g <dig>  is most closely related to the protein encoded by glyma09g <dig> , and these are closely related to the proteins encoded by glyma15g <dig> , glyma14g <dig> , and glyma02g <dig> .
all five soybean putative npr <dig> proteins contain a btb/poz domain, ankyrin repeats , a npr1/nim1-like defense protein c terminal, and a domain of unknown function , as does atnpr <dig>
expression of attga <dig>  encoding the tga-type basic leucine zipper bzip transcription factor ahbp-1b, in soybean roots decreased the fi of scn to 38% of the control .
there are numerous soybean homologs of attga <dig>  the four most highly conserved are glyma13g <dig> , glyma15g <dig> , glyma20g <dig> , and glyma10g <dig> .
the amino acid sequence of attga <dig> is closely related to glyma20g <dig>  .
the alternative transcript, glyma20g <dig> , is exactly the same, except it lacks the 5′ leader sequence.
the glyma20g <dig>  transcript appears to contain a chloroplast transit sequence as predicted using chlorop <dig> , technical university of denmark, http://www.cbs.dtu.dk/services/chlorop/, while glyma20g <dig>  does not contain this 5′ transit sequence.
we used the alternate sequence glyma20g <dig>  that lacks a 5′ leader sequence that is also missing in attga <dig>  the attga <dig> and gmtga <dig> protein sequences are highly conserved in the bzip domain  <cit> , with only two aa differences in the  <dig> aa domain.
this region is highly conserved with only eight aa substitutions in the  <dig> aa region.
a second, domain, dog <dig>  <cit>  is found toward the carboxy terminus and is involved in the control of seed dormancy  <cit> .
the dog <dig> domain is less conserved between these arabidopsis and soybean proteins.
in arabidopsis, sa interacts with the receptor npr <dig>  which then interacts with the transcription factor tga <dig> to modulate the transcription of some genes, including pr- <dig> and pr- <dig>  thus, we examined the effect of overexpression of atpr- <dig> and atpr- <dig> on the fi of scn.
overexpression of atpr- <dig> did not have a statistically significant effect  on the number of scn cysts.
in contrast, when atpr- <dig> was overexpressed in soybean roots, the fi was decreased to 38% of the control , while overexpression of atpr- <dig> had only a mild effect on the fi, which was 80% of the control.
in soybean there is a large family of over  <dig> gm-pr- <dig> genes with glyma14g <dig> and glyma17g <dig> being the most closely related to the arabidopsis pr- <dig> gene, at1g <dig> .
the sequences of the proteins encoded by the arabidopsis and two most closely related soybean genes are highly conserved.
three well-studied genes involved in sa are atpad <dig>  ateds <dig>  and ateds <dig>  previously, we demonstrated that expression of atpad <dig> greatly decreased the development of female scn to 32% of the control  <cit> .
here, we examined the effect of overexpression of ateds <dig> and ateds <dig>  neither of which significantly affected scn development, with fi values of  <dig> and  <dig>  respectively.
sa can be synthesized through a shorter pathway involving ics, or through a longer pathway through phenylalanine using cm.
therefore, we expressed atics <dig> and atcm- <dig> in soybean roots to determine their effects on scn maturation.
overexpression of atics <dig> in roots had a modest inhibitory effect .
because atics <dig> did not strongly affect the fi, we anticipated that expression of cm would have minimal inhibitory effect on the fi of scn or, perhaps, increase susceptibility, because cm competes with ics for the common substrate chorismate.
however, expression of atcm- <dig> also significantly inhibited scn growth .
win <dig>  encoded by hopw1-1-interacting  <dig> , is involved in regulating sa and disease resistance  <cit> , though the mechanism is unclear  <cit> .
overexpression of atwin <dig> decreased the fi of scn to 47% of the control.
arabidopsis acbp <dig> is an acyl-coenzyme a -binding protein  <cit> .
transgenic arabidopsis overexpressing atacbp <dig> displayed constitutive expression of the pathogenesis-related genes pr- <dig> , pr- <dig> , and pr- <dig> , and resistance to p. syringae dc <dig> was dependent upon the npr <dig> mediated signaling pathway  <cit> .
overexpression of atacbp <dig> in soybean roots resulted in a decrease of the fi of scn to 53% of the control.
overexpression of atcpr <dig>  in soybean roots had little effect on the female index of scn.
cpr <dig> mutants constitutively express pr genes at a high level  <cit> ; display defects in cell division, endoreduplication, and cell wall production  <cit> ; and are of reduced stature and exhibit the formation of spontaneous lesions  <cit> .
ja-related genes
ja and related compounds are important in defense responses, especially the response to necrotrophic pathogens  <cit> .
ja and jaile are synthesized through a series of enzymatic steps , including the enzymes allene oxide synthase ; ec  <dig> .
<dig> ); allene oxide cyclase ; and jasmonic acid-amido synthetase .
jar <dig> conjugates ja with isoleucine to form ja-ile, which is considered to be one of the active forms of ja  <cit> .
overexpression of the arabidopsis genes ataos, ataoc, and atjar <dig> did not influence the fi of scn in a statistically significant manner , 76% , and 69%  of the control, respectively; table 1).
these data do not suggest overexpression of  <cit>  these genes will improve resistance in soybean to scn.
other arabidopsis genes
overexpression of atrin <dig> genes in soybean roots had little effect on the female index of scn .
rin <dig> is a negative regulator of innate immunity in plants  <cit> .
it regulates stomatal closure.
it appears to be peripheral to the defense response of soybean roots to nematode attack, as it did not significantly alter the fi of scn.
in arabidopsis, the chloroplast protein accelerated cell death  <dig>  modulates the amount of cell death that occurs in arabidopsis leaves infected with p. syringae <cit> .
when the atacd <dig> gene was overexpressed in soybean roots, the fi of scn was reduced to 55% of the control .
cysteine endopeptidases containing a c-terminal endoplasmic reticulum retention signal, kdel, are involved plant cell death  <cit> .
overexpression of the cysteine endopeptidase encoded by atcep <dig> reduced the fi of scn to 66% of the control which was not statistically significant .
arabidopsis genes that increased susceptibility when overexpressed
the atdnd <dig> gene at5g <dig>  encodes the cyclic nucleotide-gated cation channel protein defense no death  <dig> , and is involved in production of the hypersensitive response  <cit> .
the arabidopsis dnd <dig> mutant produces elevated amounts of sa.
overproduction of atdnd <dig> in soybean roots did not provide resistance to scn; rather, it enhanced susceptibility.
the fi of transgenic soybean roots containing atdnd <dig> was 175% of the control, the largest increase in susceptibility of the genes tested here .
the protein sequence of dnd <dig> is highly conserved between arabidopsis and soybean  as indicated in figure  <dig>  it contains a cyclic nucleotide-binding domain as indicated by a significant  pfam-a match.
overexpression of two other arabidopsis genes did not alter susceptibility of soybean to scn at a statistically significant level.
the first gene, constitutive disease resistance  <dig> , encodes an aspartic protease  <cit> .
when atcdr <dig> was overexpressed in soybean roots, the fi was 142% of the control  .
the second gene atmc <dig> ) encodes the positive regulator of cell death during the hypersensitive response and is a conserved paralog of lsd <dig>  <cit> .
lsd <dig> is a negative regulator of plant programmed cell death.
overexpression of atmc <dig> in soybean roots yielded a fi of 135%  .
discussion
resistance to scn is a multigenic trait and several genetic loci have been mapped  <cit> .
recently, the identity of genes residing at the rhg <dig> and rhg <dig> loci have been reported  <cit>  which confer some resistance to scn.
however, none of these loci alone provides full resistance to any one scn population.
for example, in a cross between soybean cv essex and forrest, rhg <dig> and rhg <dig> accounted for about 65% of the variation in resistance found in the resultant inbred population to scn  <cit> .
other soybean genes have been identified that confer partial resistance to scn when overexpressed in roots  <cit> .
an option to developing resistance to nematodes is to use defense-related genes that have been described in the literature.
much of the literature describing work with the defense response of arabidopsis is concerned with elucidating defense response signaling, regulation, and pathways important to bacterial and fungal pathogens that attack the leaf of the plant.
although this research may be applicable to resistance of plants to nematodes and to agronomic crops such as soybean, little published work has yet translated the knowledge gained from these important studies in arabidopsis to soybean and other important crops.
direct translation of research in arabidopsis, includes transforming arabidopsis genes directly into crop plants to determine if they have a positive or negative effect in that crop on disease resistance.
here we have shown that some arabidopsis genes, when overexpressed in soybean roots, are compatible and confer resistance to scn.
sa plays an important role in the plant defense response to pathogens.
sa regulates sar, local disease resistance, host cell death, and expression of genes involved in the defense response  <cit> .
in tomato, sa is important to resistance to three rkn species  <cit> .
transgenic tomato expressing nahg, encoding salicylate hydrolase which degrades sa, was less resistant to rkn.
however, resistance to rkn induced in tomato through the application of cell suspensions of the biocontrol bacterium pseudomonas aeruginosa is independent of the accumulation of sa  <cit> .
thus, it may be that sa plays a role in providing resistance to rkn in tomato, but there may be other sa-independent mechanisms that also confer resistance.
uehara et al.
<cit>  showed that inhibition of the sa signaling pathway in tomato harboring the hero a gene increased susceptibility to globodera rostochiensis.
a protective effect against gall eelworm was seen in tomatoes when seeds were soaked in sa  <cit> .
these and other reports show a strong link between sa and nematode resistance.
examination of arabidopsis mutants has played a key role in our understanding of the defense response and is the subject of many reviews  <cit> .
it is postulated that sa can be synthesized through two different biochemical pathways  <cit> .
in the first pathway, chorismate is converted to isochorismate via the action of ics; then, sa is produced from isochorismate by isochorismate pyruvate lyase.
examination of ics <dig> mutants, sid2- <dig> and sid2- <dig>  of arabidopsis indicate that loss of ics <dig> activity dramatically decreases sa levels  <cit> .
most sa synthesized and relevant to plant defense in arabidopsis appears to be made through this pathway.
the sid <dig> mutant does not accumulate sa upon inoculation with p. syringae, and pr- <dig> expression is reduced greatly.
however, pr- <dig> and pr- <dig> are expressed  <cit> .
the second possible pathway diverts chorismate via cm to phenylalanine, which is converted to cinnamic acid by phenylalanine ammonia lyase  and progresses through a series of reactions to form sa.
previously, we demonstrated that overexpression in transgenic soybean roots of two different soybean genes encoding pal did not greatly affect scn maturation, with fi values of  <dig> and 111%  <cit> .
however, here we show that overexpression of cm and ics, representatives of the two different pathways, decrease the fi to 57% and 67% of the control, respectively.
however, overexpression of pal, cm, or ics alone does not confer resistance to the level provided by overexpression of several other sa-related genes individually.
genes decreasing susceptibility of soybean to scn
if the sa-related defense response is a major factor in soybean resistance to scn, then components regulated by sa may confer resistance to scn when overexpressed.
our results show that several arabidopsis genes involved in sa regulation, synthesis, and signaling conferred resistance to scn when overexpressed in soybean roots.
the arabidopsis genes atnpr <dig>  attga <dig>  atpr- <dig>  and several others related to sa strongly decreased the fi of scn in transgenic soybean roots .
npr <dig> is a master regulator of the sa-related defense response, and it is a receptor for sa  <cit> .
npr <dig> binds sa and interacts with tga transcription factors, such as the transcription factor ahbp-1b/tga <dig>  perhaps through its ankyrin domain  <cit> .
npr <dig> and tga transcription factors work downstream of sa and are important to the expression of the genes encoding pr- <dig>  pr- <dig>  and others  <cit> .
expression of these genes is completely abolished in arabidopsis plants carrying the npr <dig> mutation  <cit> .
recently, pant et al.
<cit>  demonstrated that a gm ortholog, glyma09g <dig>  of arabidopsis npr <dig> reduced scn cysts to approximately 30% of the control, in agreement with our data for overexpression of atnpr .
there are reports in numerous plants indicating that overexpression of npr <dig> results in defense against fungal and bacterial pathogens.
overexpression of atnpr <dig> in arabidopsis conferred resistance to p. syringae and peronospora parasitica <cit> .
overexpression of atnpr <dig> in rice conferred resistance to the rice bacterial blight pathogen xanthomonas oryzae <cit> .
overexpression of atnpr <dig> in wheat conferred resistance to fusarium head blight, caused by fusarium graminearum.
the apple mpnpr <dig> gene confers resistance to two fungal pathogens of apple, venturia inaequalis and gymnosporangium juniper-virginianae <cit>  complemented arabidopsis npr1- <dig> mutants with soybean homologs gmnpr1- <dig> and npr1- <dig>  and pr- <dig> was induced in the transformed plants after infection with p. syringae and after treatment with the sar inducer,  <dig> -dichloroisonicotinic acid.
npr <dig> interacts with the transcription factor tga <dig> to modulate expression of some plant defense genes, such as pr- <dig> and pr <dig>  <cit> .
when we overexpressed attga <dig>  the fi of scn was decreased to  <dig>  showing that tga <dig> can also confer resistance to scn.
this is further supported by our data showing the reduction of the fi to 38% of the control due to pr- <dig> overexpression.
pr- <dig> is a thaumatin-like protein involved in the defense response  <cit> , perhaps creating transmembrane pores to disrupt the membranes of pathogens  <cit> .
some pr- <dig> proteins exhibit anti-fungal activity  <cit> .
when prunus domestica pr- <dig> was overexpressed in transgenic arabidopsis, the plants displayed more resistance to the fungal pathogen alternaria brassicola <cit> .
pad <dig> is found upstream of sa production .
pad <dig> is a lipase like protein  <cit>  that can form molecular complexes with eds <dig> to modulate sa defense signaling  <cit> .
ectopic expression of pad <dig> reduces feeding time of green peach aphids on transgenic arabidopsis plants.
the aphid spends more time actively feeding on pad <dig> mutants  <cit> .
previously, we showed that atpad <dig> conferred resistance to scn and rkn  <cit> .
eds <dig> is also upstream of sa, and it can interact with pad <dig>  <cit> .
recently, pant et al.
<cit>  demonstrated that gmeds <dig>  had a great effect on scn and reduced scn cysts by approximately 80%.
this is in contrast to our data indicating that overexpression of ateds <dig> did not decrease the fi of scn.
gmeds <dig> is composed of  <dig> aa and ateds <dig> is composed of  <dig> aa .
the amino acid sequences of gmeds <dig> and ateds <dig> have  <dig> aa in common.
furthermore, they have another  <dig> aa that are closely related substitutions.
apparently, this conservation is not enough for ateds <dig> to provide resistance to scn as did gmeds <dig>
the eds <dig> gene, also found upstream of sa, encodes a membrane protein with homology to multidrug and toxin extrusion  transporters  <cit> .
the eds <dig> mutant accumulates very little sa and exhibits a reduction in pr- <dig> transcripts when infected with nematodes  <cit> .
we show that overexpression of ateds <dig> reduced the number of mature female cysts only modestly, to 75% of the control.
acbp <dig> is one of six acyl-coenzyme a -binding proteins in arabidopsis <cit> .
acbp binds to acyl-coa esters and protects acyl-coas from degradation  <cit> .
bovine acbp overexpression in yeast leads to an increase in the acyl-coa pool size  <cit> .
overexpression of atacbp <dig> increased freezing tolerance in arabidopsis  <cit> .
these plants also showed a decrease in phosphatidyl choline and an increase in phosphatidic acid.
infection of arabidopsis by either botrytis cinerea or p. syringae pv tomato dc <dig> induces the expression of atacbp <dig>  as does treatment with the fungal elicitor arachidonic acid  <cit> .
the authors also showed that resistance to p. syringe was conferred by acbp <dig> overexpression in an npr1-dependent manner and that pr- <dig>  pr- <dig>  and pr- <dig> were constitutively expressed.
when we overexpressed atacbp in transgenic soybean roots, the number of scn cysts decreased to 53% of the control at  <dig> dai.
the acd <dig> gene in arabidopsis encodes red chlorophyll reductase  <cit> , which catalyzes the degradation of the porphyrin portion of chlorophyll  <cit> .
acd <dig> modulates cell death in arabidopsis infected with p. syringae.
it is localized to the chloroplast.
upon infection by p. syringae, localization of the protein changes, and it is localized in chloroplasts, mitochondria, and to a lesser degree the cytosol  <cit> .
the accumulation of chlorophyll breakdown products may trigger cell death  <cit> .
when we overexpressed the atacd <dig> gene in soybean roots, the fi of scn was reduced to 55% of the control.
jasmonic acid
ja and ethylene are also important in the plant defense response, especially the response to necrotrophic pathogens  <cit> .
ja and ethylene interact antagonistically with sa.
mechanical damage and wounding activates ja synthesis in arabidopsis, potato, tomato, and other plants  <cit> .
the role of ja in defense against nematodes is being examined by several laboratories.
there are recent reports that ja is involved in defense in rice plants against nematodes  <cit> .
exogenous application of methyl-ja on rice shoots reduced galls by 63% per plant.
in contrast, bhattarai et al.
<cit>  showed that ja is not required for resistance in tomato to rkn.
they used nearly isogenic tomato cultivars resistant and susceptible to rkn to study gene expression using microarrays.
the tomato jai <dig> mutant, altered in ja signaling, reduced the susceptibility of tomato to rkn.
furthermore, they showed that auxin-related genes were differentially expressed in compatible and incompatible interactions with rkn.
neither foliar spray nor soil-drenching of tomato plants with sa, ja, or methyl-ja affected galling of roots by rkn  <cit> .
we show that overexpression of three genes involved in ja/jaile production, ataos, ataoc, and atjar1reduced the fi of scn to 66%  , 75%  and 69%  of the control.
although, the data are at the borderline of significance, the trend suggests that ja/ jaile may provide some degree of resistance to scn in soybean.
further work is needed in this area, as the effects of sa and ja in roots has not been explored.
nor has sa-ja antagonism been documented in root systems.
perhaps, sa and ja interactions are not completely antagonistic at all times in all tissues.
or perhaps, exogenous application of plant hormones can provide resistance to nematodes, but the level of the plant hormone necessary to achieve resistance is not normally achieved during nematode attack.
genes increasing susceptibility of soybean to scn
overexpression of atdnd <dig> resulted in the greatest increase in susceptibility of soybean roots to scn of all genes tested here.
dnd <dig> is a known negative regulator of plant immunity  <cit> .
its promoter is the target of the transcriptional co-repressor, topless-related  <dig> , which may function through repression of negative regulators to activate r protein-mediated immunity responses  <cit> .
when atdnd <dig> was overexpressed in soybean roots, it decreased resistance to scn as reflected by the female index of 175% as compared to the control.
arabidopsis defense genes not impacting scn susceptibility
overexpression of atcdr <dig>  which encodes an apoplastic aspartic protease, resulted in a non-significant increase in susceptibility to scn.
this result contrasts with those of previous studies on plant resistance to bacterial and fungal pathogens.
overexpression of cdr <dig> in t-dna activation tagging studies yielded dwarf arabidopsis plants and increased resistance to p. syringae <cit> .
antisense cdr <dig> arabidopsis plants were compromised in resistance to p. syringae.
a rice aspartic protease, encoded by oscdr <dig>  was identified by presad et al.
<cit> .
when they overexpressed oscdr <dig> in arabidopsis, the plants were more resistant to the necrotrophic fungal pathogen alternaria brassicicola.
when the gene was overexpressed in rice plants, the plants were more resistant to xanthomonas oryzae, the rice blast fungus, and to magnaporthe oryzae, which causes bacterial blight.
CONCLUSIONS
expression of several arabidopsis genes provided protection to soybean against scn.
in fact, several genes provided increases in resistance comparable to or better than that provided by rhg <dig> and rhg <dig>  two naturally occurring resistance gene loci in soybean.
however, not all arabidopsis genes provided resistance.
in fact, overexpression of ateds <dig> did not increase resistance, although a gmeds <dig> ortholog was recently reported as providing resistance.
these data indicate that some arabidopsis genes can be used directly in soybean to confer resistance, especially genes associated with sa regulation, signaling, and synthesis, but not all arabidopsis orthologs will provide the same results as the orthologous soybean gene.
these and similar studies may provide useful insights into protein conservation and function, and several of these arabidopsis genes may prove useful in engineering plants with broad resistance to nematodes.

 plant parasitic nematodes cause billions of dollars in losses each year worldwide. the root-knot nematode, genus meloidogyne, attacks over species of plants, while the soybean cyst nematode has a much narrower host range and is responsible for almost one billion dollars per year in losses in the us. although some soybean genotypes are resistant to specific populations of scn, no soybean genotype is known to be resistant to all scn populations. several genes conferring partial resistance to heteroderaglycines have been mapped, and, recently, genes at the rhg and rhg loci have been elucidated.the defense response of soybean to scn has been examined at the physiological, genetic, and molecular level, and several reports indicate that salicylic acid, jasmonic acid, ethylene, and indole acetic acid play a role in resistance and susceptibility of plants to nematodes. however, the roles of defense-related hormones and specific components of their synthesis and signaling pathways in providing resistance in plants to nematodes are unknown. the plant defense response is complex. plants launch a myriad of local and systemic defense responses to protect themselves from invasion by pests and pathogens. several hormones are involved in inducing the defense response and regulating defense response networks, including sa, ja, et, and iaa. plants react to pathogen-associated or microbe–associated molecular patterns which are sensed by plants through leucine-rich repeat receptors. pamps signal stomatal closure and stimulate innate immunity in plants. in general, sa activates the defense response to biotrophic and hemi-biotrophic pathogens, induces systemic acquired resistance, and triggers the expression of sar-associated pathogenesis related genes pr- pr- and others. the role of specific components of sa regulation, synthesis, and signaling in defending plants against parasitic nematodes is not well understood. however, sa does play a role in decreasing susceptibility to root-knot nematode in cow pea and tomato, and to the cyst nematode, heterodera schachtii, in arabidopsis. likewise, ja also plays a role in resistance of plants to nematodes. foliar spraying of tomato with ja reduced galling and the final population of rkn;, as did pre-treatment of tomato seeds with ja, indicating a role for ja in plant resistance to nematodes. little is known of the role of specific components of sa regulation and signaling in the interaction of soybean with the soybean cyst nematode, the major pest of soybean in the us. although soybean genes conferring resistance to scn have been identified, these do not provide resistance to all scn populations. resistance in soybean to scn is multigenic, and several major loci for resistance have been identified. for example, in soybean cv peking, several genes have been reported that confer resistance to scn race, yet none of these genes confers complete resistance to all scn populations. therefore, we are applying to soybean a portion of the vast knowledge that has been gained from studies on the model plant arabidopsis and its large array of mutants on the role of sa and ja in the plant defense response to identify important components that may be useful in decreasing susceptibility of plants to nematodes, and especially of soybean to scn. arabidopsis has been used widely as a model system to study plant defense pathways, usually with bacterial and fungal pathogens. much attention has been focused on sa production and signaling pathways using arabidopsis mutants infected with bacterial and fungal pathogens. for example, when a virulent strain of the biotrophic pathogen, pseudomonas syringae, attacks arabidopsis, the avrrps effector protein of p. syringae secreted by the type iii secretion system is detected by the plant receptor rps a toll-interleukin-nucleotide binding-leucine-rich repeat that mediates the induction of antimicrobial defenses to provide disease resistance. the nucleo-cytoplasmic protein enhanced disease susceptibility, which is a lipase-like protein, connects rps with downstream defense pathways and regulates the accumulation of sa. eds is essential for production of the hypersensitive response and mobilization of defense pathways. eds can dimerize and interact with another lipase-like protein, phytoalexin deficient. both eds and pad are required for the accumulation of sa and they are involved in ros signaling. pad protein is required for amplification of weak signals resulting from pathogen infection. another important component of the defense response is eds a multi-drug transporter member of the mate family of transporters. it is postulated that sa can be synthesized through two different pathways in arabidopsis. one pathway involves the enzyme isochorismate synthase, which catalyzes the conversion of chorismate to isochorismate. the enzyme chorismate mutase catalyzes the competing chemical reaction and converts chorismate to prephenate. this would divert chorismate to produce other compounds, such as phenylalanine and tyrosine. the arabidopsis sid2- mutation has been mapped to the locus encompassing the ics gene. upon synthesis, sa can bind directly with npr which is encoded by atnpr, also known as nim. npr is an sa receptor that is a transcriptional regulator of genes involved in the sa-dependent defense response, including the sa marker gene pr-. npr interacts with transcription factor tga family members, including ahbp-1b, and the complex binds to sa-responsive promoter elements of pr- and other sa-dependent defense genes to regulate expression. sa can also be regulated independently of eds and pad ndr is a positive regulator of sa that works independently of eds and pad ndr is an integrin-like protein that can associate with rin while rin can associate with rpm and rps. ndr may play a role in electrolyte release upon infection of arabidopsis by p. syringae, while rin regulates stomatal apertures in conjunction with h + −atpases of the plasma membrane of arabidopsis during pathogen attack. ja, jaile, and related lipid-derived compounds also act as signals in the plant defense response and are associated with resistance to necrotrophic pathogens. the pathway leading to ja and jaile synthesis and some components related to ja signaling and control are depicted in figure  allene oxide synthase and allene oxide cyclase are two enzymes important to ja synthesis. jar encodes an atp-dependent ja-amido synthase that conjugates isoleucine with ja to form jaile, which plays an essential role in ja signaling. in this paper, we examine the role of some components of the plant defense response in conferring resistance in soybean to scn. we show that specific arabidopsis genes, namely atnpr attga atics and atpr which encode components of sa regulation, biosynthesis, and downstream effectors, can decrease susceptibility of soybean to scn when expressed in transgenic soybean roots. we also demonstrate that expression of arabidopsis genes encoding aos, aoc, and jar which are involved in the synthesis and modification of ja, only modestly decrease susceptibility of soybean roots to scn. these results indicated that some arabidopsis genes can be directly used in soybean, thus directly applying knowledge of the defense response gained from studies using arabidopsis as a model system to soybean to decrease susceptibility to nematodes.  expression of arabidopsis genes in soybean roots sa and ja are well known regulators of the plant defense response as described through studies of the model plant, arabidopsis. to determine if some of these arabidopsis genes could be directly used in soybean to translate knowledge from arabidopsis to soybean, we selected and cloned genes encoding numerous components of sa and ja synthesis, regulation, and signaling from the literature describing the defense response of arabidopsis to pathogens. to broaden the scope of our study, we also selected several arabidopsis genes less well defined in function or that represented other portions of the plant defense response less dependent upon sa and ja. open reading frames of thirty-one arabidopsis genes were cloned into the gene expression vector prap using the primers listed in additional file 1: table s the inserted orfs were sequenced to confirm their identity and to ensure their sequence was conserved. the vector with insert was transformed by agrobacterium rhizogenes k into cells at the base of the cut stem of soybean seedlings. approximately 35 days after transformation, untransformed roots were removed from the composite plants, and the transformed roots were inoculated with scn. the effect of expression of thirty-one genes on the number of scn cysts at dai was determined. six genes decreased the number of cysts more than 40%, thus conferring partial resistance to scn. three of these genes, atnpr attga and atpr- decreased the number of cysts more than 60%, while three others, atacbp atacd and atcm decreased the number of cysts 40%. one arabidopsis gene, atdnd increased the number of cysts of scn to 175% of the control, thus making the soybean roots more susceptible to scn. rna was extracted from a subset of transformed roots for genes listed in additional file 2: table s to check for expression of the arabidopsis gene by pcr. the amplicons were separated and visualized by gel electrophoresis and staining to confirm that the orfs were expressed in the composite root. all roots tested expressed the transcript. in addition, the abundance of transcript of two genes providing the most protection against scn, atnpr and attga was determined by qrt-pcr using gene specific primers. transcript number was calculated using the sigmoidal method. the number of transcripts of atnpr was molecules and for attga was molecules in transformed roots, while no transcripts of either gene were detectable in the control roots. in all samples, the expression level was similar for the housekeeping gene encoding ubiquitin-. the number of transcripts of three defense-related genes, gmpr gmchib and gmerf1was also determined using qrt-pcr. in roots overexpressing atnpr there were transcripts of gmpr while in roots overexpressing attga there were transcripts. in control roots, there were only transcripts of gmpr thus, gmpr was elevated approximately 4-fold in roots overexpressing attga transcripts of gmchib were also elevated in these roots. there were transcripts of gmchib in roots overexpressing atnpr and transcripts in roots overexpressing attga there were only transcripts of gmchib in control roots. this represents an increase in expression of gmchib by approximately 8- and -fold in soybean roots overexpressing atnpr and attga respectively. in contrasts, the number of transcripts of gmerf decreased in soybean roots overexpressing atnpr only transcripts of gmerf were present, whereas control plants contained transcripts. similarly, roots overexpressing attga contained fewer gmefr transcripts than in control roots with only transcripts present. thus, gmerf expression was greatly decreased in both sets of transgenic roots. gmpr5, gmchib1, and gmerf was determined in roots transformed with atnpr and attga using qrt-pcr. sa-related genes activation of the defense response in plants is initiated through several parallel signaling pathways. in gene-for-gene resistance, host resistance proteins indirectly recognize pathogen effectors to initiate resistance. the coiled-coiled-nucleotide-binding site-leucine-rich repeat and the tir-nb-lrr classes of proteins are two major sub-groups of r protein. in this study, we selected the arabidopsis protein non-race-specific disease resistance as a representative cc-nb-lrr r-protein, because of its known role in activating the sa-mediated defense response in arabidopsis. rps encoded by atrps was selected as a representative of the tir-nb-lrr class of proteins. both atndr and atrps were overexpressed in transgenic soybean roots to determine their effect on scn growth and maturation as measured by the female index, which expresses the number of mature scn females 35 days after root inoculation as a percent of the control value. overexpression of atndr slightly inhibited scn development, while atrps had a slightly greater inhibitory effect, but the effect was not statistically significant for either gene. arabidopsis non-expressor of pathogenesis-related genes is downstream of the r proteins ndr and rps npr is a key regulator of sar and plays a critical role as a sa signal transducer in arabidopsis. when atnpr was overexpressed, the fi decreased to 33% of the control. atnpr had the lowest fi value among the arabidopsis genes tested in this study. alignment of the aa of atnpr with its closest soybean counterpart, the product of glyma09g, indicates conservation of only aa, although there are also many conservative substitutions. there are five soybean genes encoding proteins closely related to atnpr the protein encoded by glyma09g is most closely related to the protein encoded by glyma09g, and these are closely related to the proteins encoded by glyma15g, glyma14g, and glyma02g. all five soybean putative npr proteins contain a btb/poz domain, ankyrin repeats, a npr1/nim1-like defense protein c terminal, and a domain of unknown function, as does atnpr expression of attga encoding the tga-type basic leucine zipper bzip transcription factor ahbp-1b, in soybean roots decreased the fi of scn to 38% of the control. there are numerous soybean homologs of attga the four most highly conserved are glyma13g, glyma15g, glyma20g, and glyma10g. the amino acid sequence of attga is closely related to glyma20g. the alternative transcript, glyma20g, is exactly the same, except it lacks the 5′ leader sequence. the glyma20g transcript appears to contain a chloroplast transit sequence as predicted using chlorop, technical university of denmark, http://www.cbs.dtu.dk/services/chlorop/, while glyma20g does not contain this 5′ transit sequence. we used the alternate sequence glyma20g that lacks a 5′ leader sequence that is also missing in attga the attga and gmtga protein sequences are highly conserved in the bzip domain, with only two aa differences in the aa domain. this region is highly conserved with only eight aa substitutions in the aa region. a second, domain, dog is found toward the carboxy terminus and is involved in the control of seed dormancy. the dog domain is less conserved between these arabidopsis and soybean proteins. in arabidopsis, sa interacts with the receptor npr which then interacts with the transcription factor tga to modulate the transcription of some genes, including pr- and pr- thus, we examined the effect of overexpression of atpr- and atpr- on the fi of scn. overexpression of atpr- did not have a statistically significant effect on the number of scn cysts. in contrast, when atpr- was overexpressed in soybean roots, the fi was decreased to 38% of the control, while overexpression of atpr- had only a mild effect on the fi, which was 80% of the control. in soybean there is a large family of over gm-pr- genes with glyma14g and glyma17g being the most closely related to the arabidopsis pr- gene, at1g. the sequences of the proteins encoded by the arabidopsis and two most closely related soybean genes are highly conserved. three well-studied genes involved in sa are atpad ateds and ateds previously, we demonstrated that expression of atpad greatly decreased the development of female scn to 32% of the control. here, we examined the effect of overexpression of ateds and ateds neither of which significantly affected scn development, with fi values of and respectively. sa can be synthesized through a shorter pathway involving ics, or through a longer pathway through phenylalanine using cm. therefore, we expressed atics and atcm- in soybean roots to determine their effects on scn maturation. overexpression of atics in roots had a modest inhibitory effect. because atics did not strongly affect the fi, we anticipated that expression of cm would have minimal inhibitory effect on the fi of scn or, perhaps, increase susceptibility, because cm competes with ics for the common substrate chorismate. however, expression of atcm- also significantly inhibited scn growth. win encoded by hopw1-1-interacting, is involved in regulating sa and disease resistance, though the mechanism is unclear. overexpression of atwin decreased the fi of scn to 47% of the control. arabidopsis acbp is an acyl-coenzyme a -binding protein. transgenic arabidopsis overexpressing atacbp displayed constitutive expression of the pathogenesis-related genes pr-, pr-, and pr-, and resistance to p. syringae dc was dependent upon the npr mediated signaling pathway. overexpression of atacbp in soybean roots resulted in a decrease of the fi of scn to 53% of the control. overexpression of atcpr in soybean roots had little effect on the female index of scn. cpr mutants constitutively express pr genes at a high level; display defects in cell division, endoreduplication, and cell wall production; and are of reduced stature and exhibit the formation of spontaneous lesions. ja-related genes ja and related compounds are important in defense responses, especially the response to necrotrophic pathogens. ja and jaile are synthesized through a series of enzymatic steps, including the enzymes allene oxide synthase; ec. ); allene oxide cyclase; and jasmonic acid-amido synthetase. jar conjugates ja with isoleucine to form ja-ile, which is considered to be one of the active forms of ja. overexpression of the arabidopsis genes ataos, ataoc, and atjar did not influence the fi of scn in a statistically significant manner, 76%, and 69% of the control, respectively; table 1). these data do not suggest overexpression of these genes will improve resistance in soybean to scn. other arabidopsis genes overexpression of atrin genes in soybean roots had little effect on the female index of scn. rin is a negative regulator of innate immunity in plants. it regulates stomatal closure. it appears to be peripheral to the defense response of soybean roots to nematode attack, as it did not significantly alter the fi of scn. in arabidopsis, the chloroplast protein accelerated cell death modulates the amount of cell death that occurs in arabidopsis leaves infected with p. syringae. when the atacd gene was overexpressed in soybean roots, the fi of scn was reduced to 55% of the control. cysteine endopeptidases containing a c-terminal endoplasmic reticulum retention signal, kdel, are involved plant cell death. overexpression of the cysteine endopeptidase encoded by atcep reduced the fi of scn to 66% of the control which was not statistically significant. arabidopsis genes that increased susceptibility when overexpressed the atdnd gene at5g encodes the cyclic nucleotide-gated cation channel protein defense no death, and is involved in production of the hypersensitive response. the arabidopsis dnd mutant produces elevated amounts of sa. overproduction of atdnd in soybean roots did not provide resistance to scn; rather, it enhanced susceptibility. the fi of transgenic soybean roots containing atdnd was 175% of the control, the largest increase in susceptibility of the genes tested here. the protein sequence of dnd is highly conserved between arabidopsis and soybean as indicated in figure  it contains a cyclic nucleotide-binding domain as indicated by a significant pfam-a match. overexpression of two other arabidopsis genes did not alter susceptibility of soybean to scn at a statistically significant level. the first gene, constitutive disease resistance, encodes an aspartic protease. when atcdr was overexpressed in soybean roots, the fi was 142% of the control. the second gene atmc ) encodes the positive regulator of cell death during the hypersensitive response and is a conserved paralog of lsd. lsd is a negative regulator of plant programmed cell death. overexpression of atmc in soybean roots yielded a fi of 135%. discussion resistance to scn is a multigenic trait and several genetic loci have been mapped. recently, the identity of genes residing at the rhg and rhg loci have been reported which confer some resistance to scn. however, none of these loci alone provides full resistance to any one scn population. for example, in a cross between soybean cv essex and forrest, rhg and rhg accounted for about 65% of the variation in resistance found in the resultant inbred population to scn. other soybean genes have been identified that confer partial resistance to scn when overexpressed in roots. an option to developing resistance to nematodes is to use defense-related genes that have been described in the literature. much of the literature describing work with the defense response of arabidopsis is concerned with elucidating defense response signaling, regulation, and pathways important to bacterial and fungal pathogens that attack the leaf of the plant. although this research may be applicable to resistance of plants to nematodes and to agronomic crops such as soybean, little published work has yet translated the knowledge gained from these important studies in arabidopsis to soybean and other important crops. direct translation of research in arabidopsis, includes transforming arabidopsis genes directly into crop plants to determine if they have a positive or negative effect in that crop on disease resistance. here we have shown that some arabidopsis genes, when overexpressed in soybean roots, are compatible and confer resistance to scn. sa plays an important role in the plant defense response to pathogens. sa regulates sar, local disease resistance, host cell death, and expression of genes involved in the defense response. in tomato, sa is important to resistance to three rkn species. transgenic tomato expressing nahg, encoding salicylate hydrolase which degrades sa, was less resistant to rkn. however, resistance to rkn induced in tomato through the application of cell suspensions of the biocontrol bacterium pseudomonas aeruginosa is independent of the accumulation of sa. thus, it may be that sa plays a role in providing resistance to rkn in tomato, but there may be other sa-independent mechanisms that also confer resistance. uehara et al. showed that inhibition of the sa signaling pathway in tomato harboring the hero a gene increased susceptibility to globodera rostochiensis. a protective effect against gall eelworm was seen in tomatoes when seeds were soaked in sa. these and other reports show a strong link between sa and nematode resistance. examination of arabidopsis mutants has played a key role in our understanding of the defense response and is the subject of many reviews. it is postulated that sa can be synthesized through two different biochemical pathways. in the first pathway, chorismate is converted to isochorismate via the action of ics; then, sa is produced from isochorismate by isochorismate pyruvate lyase. examination of ics mutants, sid2- and sid2- of arabidopsis indicate that loss of ics activity dramatically decreases sa levels. most sa synthesized and relevant to plant defense in arabidopsis appears to be made through this pathway. the sid mutant does not accumulate sa upon inoculation with p. syringae, and pr- expression is reduced greatly. however, pr- and pr- are expressed. the second possible pathway diverts chorismate via cm to phenylalanine, which is converted to cinnamic acid by phenylalanine ammonia lyase and progresses through a series of reactions to form sa. previously, we demonstrated that overexpression in transgenic soybean roots of two different soybean genes encoding pal did not greatly affect scn maturation, with fi values of and 111%. however, here we show that overexpression of cm and ics, representatives of the two different pathways, decrease the fi to 57% and 67% of the control, respectively. however, overexpression of pal, cm, or ics alone does not confer resistance to the level provided by overexpression of several other sa-related genes individually. genes decreasing susceptibility of soybean to scn if the sa-related defense response is a major factor in soybean resistance to scn, then components regulated by sa may confer resistance to scn when overexpressed. our results show that several arabidopsis genes involved in sa regulation, synthesis, and signaling conferred resistance to scn when overexpressed in soybean roots. the arabidopsis genes atnpr attga atpr- and several others related to sa strongly decreased the fi of scn in transgenic soybean roots. npr is a master regulator of the sa-related defense response, and it is a receptor for sa. npr binds sa and interacts with tga transcription factors, such as the transcription factor ahbp-1b/tga perhaps through its ankyrin domain. npr and tga transcription factors work downstream of sa and are important to the expression of the genes encoding pr- pr- and others. expression of these genes is completely abolished in arabidopsis plants carrying the npr mutation. recently, pant et al. demonstrated that a gm ortholog, glyma09g of arabidopsis npr reduced scn cysts to approximately 30% of the control, in agreement with our data for overexpression of atnpr. there are reports in numerous plants indicating that overexpression of npr results in defense against fungal and bacterial pathogens. overexpression of atnpr in arabidopsis conferred resistance to p. syringae and peronospora parasitica. overexpression of atnpr in rice conferred resistance to the rice bacterial blight pathogen xanthomonas oryzae. overexpression of atnpr in wheat conferred resistance to fusarium head blight, caused by fusarium graminearum. the apple mpnpr gene confers resistance to two fungal pathogens of apple, venturia inaequalis and gymnosporangium juniper-virginianae complemented arabidopsis npr1- mutants with soybean homologs gmnpr1- and npr1- and pr- was induced in the transformed plants after infection with p. syringae and after treatment with the sar inducer, -dichloroisonicotinic acid. npr interacts with the transcription factor tga to modulate expression of some plant defense genes, such as pr- and pr. when we overexpressed attga the fi of scn was decreased to showing that tga can also confer resistance to scn. this is further supported by our data showing the reduction of the fi to 38% of the control due to pr- overexpression. pr- is a thaumatin-like protein involved in the defense response, perhaps creating transmembrane pores to disrupt the membranes of pathogens. some pr- proteins exhibit anti-fungal activity. when prunus domestica pr- was overexpressed in transgenic arabidopsis, the plants displayed more resistance to the fungal pathogen alternaria brassicola. pad is found upstream of sa production. pad is a lipase like protein that can form molecular complexes with eds to modulate sa defense signaling. ectopic expression of pad reduces feeding time of green peach aphids on transgenic arabidopsis plants. the aphid spends more time actively feeding on pad mutants. previously, we showed that atpad conferred resistance to scn and rkn. eds is also upstream of sa, and it can interact with pad. recently, pant et al. demonstrated that gmeds had a great effect on scn and reduced scn cysts by approximately 80%. this is in contrast to our data indicating that overexpression of ateds did not decrease the fi of scn. gmeds is composed of aa and ateds is composed of aa. the amino acid sequences of gmeds and ateds have aa in common. furthermore, they have another aa that are closely related substitutions. apparently, this conservation is not enough for ateds to provide resistance to scn as did gmeds the eds gene, also found upstream of sa, encodes a membrane protein with homology to multidrug and toxin extrusion transporters. the eds mutant accumulates very little sa and exhibits a reduction in pr- transcripts when infected with nematodes. we show that overexpression of ateds reduced the number of mature female cysts only modestly, to 75% of the control. acbp is one of six acyl-coenzyme a -binding proteins in arabidopsis. acbp binds to acyl-coa esters and protects acyl-coas from degradation. bovine acbp overexpression in yeast leads to an increase in the acyl-coa pool size. overexpression of atacbp increased freezing tolerance in arabidopsis. these plants also showed a decrease in phosphatidyl choline and an increase in phosphatidic acid. infection of arabidopsis by either botrytis cinerea or p. syringae pv tomato dc induces the expression of atacbp as does treatment with the fungal elicitor arachidonic acid. the authors also showed that resistance to p. syringe was conferred by acbp overexpression in an npr1-dependent manner and that pr- pr- and pr- were constitutively expressed. when we overexpressed atacbp in transgenic soybean roots, the number of scn cysts decreased to 53% of the control at dai. the acd gene in arabidopsis encodes red chlorophyll reductase, which catalyzes the degradation of the porphyrin portion of chlorophyll. acd modulates cell death in arabidopsis infected with p. syringae. it is localized to the chloroplast. upon infection by p. syringae, localization of the protein changes, and it is localized in chloroplasts, mitochondria, and to a lesser degree the cytosol. the accumulation of chlorophyll breakdown products may trigger cell death. when we overexpressed the atacd gene in soybean roots, the fi of scn was reduced to 55% of the control. jasmonic acid ja and ethylene are also important in the plant defense response, especially the response to necrotrophic pathogens. ja and ethylene interact antagonistically with sa. mechanical damage and wounding activates ja synthesis in arabidopsis, potato, tomato, and other plants. the role of ja in defense against nematodes is being examined by several laboratories. there are recent reports that ja is involved in defense in rice plants against nematodes. exogenous application of methyl-ja on rice shoots reduced galls by 63% per plant. in contrast, bhattarai et al. showed that ja is not required for resistance in tomato to rkn. they used nearly isogenic tomato cultivars resistant and susceptible to rkn to study gene expression using microarrays. the tomato jai mutant, altered in ja signaling, reduced the susceptibility of tomato to rkn. furthermore, they showed that auxin-related genes were differentially expressed in compatible and incompatible interactions with rkn. neither foliar spray nor soil-drenching of tomato plants with sa, ja, or methyl-ja affected galling of roots by rkn. we show that overexpression of three genes involved in ja/jaile production, ataos, ataoc, and atjar1reduced the fi of scn to 66%, 75% and 69% of the control. although, the data are at the borderline of significance, the trend suggests that ja/ jaile may provide some degree of resistance to scn in soybean. further work is needed in this area, as the effects of sa and ja in roots has not been explored. nor has sa-ja antagonism been documented in root systems. perhaps, sa and ja interactions are not completely antagonistic at all times in all tissues. or perhaps, exogenous application of plant hormones can provide resistance to nematodes, but the level of the plant hormone necessary to achieve resistance is not normally achieved during nematode attack. genes increasing susceptibility of soybean to scn overexpression of atdnd resulted in the greatest increase in susceptibility of soybean roots to scn of all genes tested here. dnd is a known negative regulator of plant immunity. its promoter is the target of the transcriptional co-repressor, topless-related, which may function through repression of negative regulators to activate r protein-mediated immunity responses. when atdnd was overexpressed in soybean roots, it decreased resistance to scn as reflected by the female index of 175% as compared to the control. arabidopsis defense genes not impacting scn susceptibility overexpression of atcdr which encodes an apoplastic aspartic protease, resulted in a non-significant increase in susceptibility to scn. this result contrasts with those of previous studies on plant resistance to bacterial and fungal pathogens. overexpression of cdr in t-dna activation tagging studies yielded dwarf arabidopsis plants and increased resistance to p. syringae. antisense cdr arabidopsis plants were compromised in resistance to p. syringae. a rice aspartic protease, encoded by oscdr was identified by presad et al.. when they overexpressed oscdr in arabidopsis, the plants were more resistant to the necrotrophic fungal pathogen alternaria brassicicola. when the gene was overexpressed in rice plants, the plants were more resistant to xanthomonas oryzae, the rice blast fungus, and to magnaporthe oryzae, which causes bacterial blight.  expression of several arabidopsis genes provided protection to soybean against scn. in fact, several genes provided increases in resistance comparable to or better than that provided by rhg and rhg two naturally occurring resistance gene loci in soybean. however, not all arabidopsis genes provided resistance. in fact, overexpression of ateds did not increase resistance, although a gmeds ortholog was recently reported as providing resistance. these data indicate that some arabidopsis genes can be used directly in soybean to confer resistance, especially genes associated with sa regulation, signaling, and synthesis, but not all arabidopsis orthologs will provide the same results as the orthologous soybean gene. these and similar studies may provide useful insights into protein conservation and function, and several of these arabidopsis genes may prove useful in engineering plants with broad resistance to nematodes. 
the ability of thirty-one arabidopsis genes encoding important components of sa and ja synthesis and signaling in conferring resistance to soybean cyst nematode are investigated. we demonstrate that overexpression of three of thirty-one arabidoposis genes in transgenic soybean roots of composite plants decreased the number of cysts formed by scn to less than 50% of those found on control roots, namely atnpr attga, and atpr-. arabidopsiscomposite plantsgene overexpressionjasmonic acidresistancesalicylic acidsoybeansoybean cyst nematodetransgenic roots extensive studies using the model system arabidopsis thaliana to elucidate plant defense signaling and pathway networks indicate that salicylic acid is the key hormone triggering the plant defense response against biotrophic and hemi-biotrophic pathogens, while jasmonic acid and derivatives are critical to the defense response against necrotrophic pathogens. when the genes, atnpr atga and atpr- encoding specific components involved in sa regulation, synthesis, and signaling, are overexpressed in soybean roots, resistance to scn is enhanced. knowledge of the pathogen defense system gained from studies of the model system, arabidopsis, can be directly translated to soybean through direct overexpression of arabidopsis genes. three additional arabidopsis genes decreased the number of scn cysts by 40% or more: atacbp, atacd, and atcm-. overexpression of atdnd greatly increased susceptibility as indicated by a large increase in the number of scn cysts. this demonstrates functional compatibility of some arabidopsis genes with soybean and identifies genes that may be used to engineer resistance to nematodes.
 extensive studies using the model system arabidopsis thaliana to elucidate plant defense signaling and pathway networks indicate that salicylic acid is the key hormone triggering the plant defense response against biotrophic and hemi-biotrophic pathogens, while jasmonic acid and derivatives are critical to the defense response against necrotrophic pathogens. several reports demonstrate that sa limits nematode reproduction.  here we translate knowledge gained from studies using arabidopsis to soybean. the ability of thirty-one arabidopsis genes encoding important components of sa and ja synthesis and signaling in conferring resistance to soybean cyst nematode are investigated. we demonstrate that overexpression of three of thirty-one arabidoposis genes in transgenic soybean roots of composite plants decreased the number of cysts formed by scn to less than 50% of those found on control roots, namely atnpr attga, and atpr-. three additional arabidopsis genes decreased the number of scn cysts by 40% or more: atacbp, atacd, and atcm-. other genes having less or no effect included ateds, atndr, ateds, and atpr-, as compared to control. overexpression of atdnd greatly increased susceptibility as indicated by a large increase in the number of scn cysts.  knowledge of the pathogen defense system gained from studies of the model system, arabidopsis, can be directly translated to soybean through direct overexpression of arabidopsis genes. when the genes, atnpr atga and atpr- encoding specific components involved in sa regulation, synthesis, and signaling, are overexpressed in soybean roots, resistance to scn is enhanced. this demonstrates functional compatibility of some arabidopsis genes with soybean and identifies genes that may be used to engineer resistance to nematodes. arabidopsiscomposite plantsgene overexpressionjasmonic acidresistancesalicylic acidsoybeansoybean cyst nematodetransgenic roots 