Transcript*Profiling*of*Nematode*and*Aphid*Infectome*in*Soybean*
Priyanka'Surana,'Iowa'State'University'
'
Soybean' aphids' and' cyst' nematodes' are' economic' pests' of' soybean' in' the' north'
central' region' of' America' and' they' both' originated' from' China' and' Japan.' They' alone'
account' for' 20' –' 50%' yield' reduction' in' a' soybean' crop.' Understanding' the' relationship'
between' these' pathogens' can' help' improve' plant' yield' within' agroecosystems.' Although'
studies'have'shown'effect'of'nematode'and'aphid'on'soybeans,'only'limited'information'is'
known'how'coIinfection'with'these'pathogens'affect'gene'expression'in'both'the'host'and'
pathogen.'
'
Intellectual*Merit''
To' study' interaction' between' a' below' ground' herbivore' and' an' above' ground'
insect,' research' will' be' conducted' on' soybean' cyst' nematode' (Heterodera( glycines'
Ichinohe),' soybean' aphid' (Aphis( glycines' Matsumura)' and' soybean' (Glycine( max)' as' a'
model' system.' Heeren' and' colleagues' (2012)' have' previously' studied' this' model' system.'
The'proposed'experiments'will'have'radical'impact'in'the'research'area'by'identifying'host'
and'pathogen'genes'involved'in'the'three'–'way'interaction.''
The' main' hypothesis' is' that' H.(glycines(and' A.(glycines(change' gene' expression' on'
their' host' (Glycine( max)' when' co–infected' affecting' plant' yield.' Soybean' lines' will' be'
exposed' to' four' different' treatments:' no' pest' (control),' cyst' nematode' infection,' aphid'
infection'and'coIinfection'by'both'pathogens.'Each'treatment'will'be'repeated'five'times'in'
a'split'plot'design.'Soybean'lines'and'pathogen'treatments'will'be'the'whole'and'split'plot'
factor' respectively.' RNA' samples' from' roots' and' leaves' will' be' collected' and' sequenced.'
Physical'attributes'of'the'plant'and'pathogens'will'be'measured'as'well.'
This'study'will'determine'which'genes'are'regulated'during'infection'by'one'or'both'
pathogens.' Genome' sequence' of' soybean,' aphid' and' cyst' nematode' makes' accurate'
transcriptome'assembly'for'the'plant'–'pathogen'infectomes'possible.'Further'annotation,'
category' enrichment,' sequence' and' structural' analysis' will' be' performed' on' transcripts'
identified' by' gene' expression' analysis.' Statistical' analysis' of' the' physical' variables' of' the'
host'and'its'herbivores'will'identify'the'attributes'affected'by'co–infection.''
'
Broader*Impact'
Pest'infestation'results'in'significant'economic'loss'in'soybean'plants'(Glycine(max).'
The' project' will' improve' our' understanding' of' two' major' soybean' pathogens' (H.(glycines(
and'A.(glycines),'and'how'their'infection'and'coIinfection'affects'plants'at'both'the'physical'
and'transcriptomic'level.'This'information'can'be'used'to'create'transgenic'soybean'plant'
lines'with'pathogen'resistance'and'better'yield.'
The' proposed' project' will' contribute' to' training' a' graduate' student' in' plant'
pathology,'molecular'biology'and'bioinformatics.'Postdoctoral'researcher'trained'through'
this'project'will'be'well'prepared'for'a'research'career.'Additionally,'the'main'investigator'
will' be' involved' in' ongoing' outreach' efforts' to' educate' the' community' about' plant'
pathology'and'bioinformatics.'
'
'
Surana,'Project'Summary'–'1'
Transcript*Profiling*of*Nematode*and*Aphid*Infectome*in*Soybean*
Priyanka'Surana,'Iowa'State'University'
*
Introduction'
Plants' face' challenges' from' both' above' and' below' ground' pests' that' may' be'
interacting' by' inducing' changes' in' their' shared' host' (Scheu' 2001).' Effects' of' root'
herbivores'on'shoot'herbivores'range'from'positive'(Gange'and'Brown'1989;'Masters'and'
colleagues'1993;'Poveda'and'colleagues'2005)'to'neutral'(Moran'and'Whitham'1990;'Salt'
and' colleagues' 1996)' to' negative' (van' der' Putten' and' colleagues' 2001;' Bezemer' and'
colleagues' 2003;' van' Dam' and' colleagues' 2005).' Belowground' herbivores' enhance'
aboveground'herbivores'probably'via'water'stress'causing'altered'concentrations'of'plant'
metabolites.'Positive'effects'have'been'shown'mostly'in'phloemIfeeding'aphids'(Gange'and'
Brown' 1989;' Poveda' and' colleagues' 2005)' and' leaf' miners' (Masters' and' Brown' 1992).'
Belowground' herbivores' suppress' aboveground' herbivores' probably' via' systemic'
induction'of'plant'defenses'(van'der'Putten'and'colleagues'2001).'The'disparity'in'results'
indicate'that'above'and'below'ground'pathogen'interactions'are'not'only'organism'specific'
but'are'also'limited'by'indirect'effects'(Wardle'and'colleagues'2004).''
Nematodes' dominate' the' root' herbivore' population,' and' impair' translocation' of'
water' and' nutrients' in' many' plant' species' (Stanton' 1988;' Ingham' and' Detling' 1990).'
Phytophagous'insects'dominate'the'shoot'herbivore'population'and'damage'plants'in'both'
natural'and'controlled'habitats'(Marquis'1992).'Even'though'both'nematodes'and'insects'
infest'almost'all'terrestrial'environments,'interactions'between'these'plant'pathogens'are'
not' yet' understood.' However,' recent' studies' have' shown' that' nematode' –' plant' –' insect'
interactions'do'occur'(Wardle'and'colleagues'2004;'van'Dam'and'colleagues'2005;'De'Deyn'
and' colleagues' 2007;' Wurst' and' van' der' Putten' 2007).' This' experiment' proposes' to'
investigate' the' economically' relevant,' soybean' cyst' nematode' –' soybean' I' soybean' aphid'
interaction.'
Soybean' cyst' nematodes' hibernate' as' eggs' within' cysts' in' the' soil.' Second' stage'
juveniles' parasitize' soybean' roots' in' the' spring.' For' the' rest' of' their' lives,' female'
nematodes'feed'on'parasitized'roots'(Riggs'and'Schuster'1998).'Numerous'generations'in'
one' soybean' season' cause' large' densities' of' eggs' by' fall' (Jacobsen' and' colleagues' 1983;'
Abawi'and'Jacobsen'1984).'Soybean'cyst'nematodes'reduce'seed'production'efficiency'by'
inhibiting'root'growth'and'nodulation'(Alston'and'colleagues'1991;'Porter'and'colleagues'
2001).' They' also' impede' water' and' nutrient' uptake' (Smith' and' colleagues' 2001).'
Chlorosis,' delayed' phenological' development,' failure' of' the' crop' canopy' to' close' and'
stunting' are' seen' on' plants' infested' with' soybean' cyst' nematodes' (Alston' and' Schmitt'
1987).' Yield' loss' often' occurs' without' visual' symptoms,' leading' producers' to'
underestimate'the'economic'loss'caused'by'this'pest'(Wang'and'colleagues'2003).'
Soybean' aphids' hibernate' as' eggs' on' buckthorn' (Rhamnus' spp.).' Soybean' aphids'
undergo' two' generations' on' buckthorn' in' spring,' before' spreading' to' soybean' (Ragsdale'
and' colleagues' 2004).' ' After' 15' generations' via' viviparous' parthenogenesis' during' the'
growing' season,' soybean' aphids' return' to' buckthorn' to' produce' eggs' via' sexual'
reproduction' (Wang' and' colleagues' 1962).' Soybean' aphids' primarily' locate' soybeans'
through' olfactory' chemical' signaling.' Detrimental' effects' on' soybeans' are' variable' and'
'
Surana,'Project'Description'–'1'
influenced'by'factors'such'as'aphid'density,'temperature,'plant'density'and'stage'(Dai'and'
Fan'1991).''
Soybean'cyst'nematodes'and'soybean'aphids'infect'the'root'and'foliage'respectively'
of' the' same' host,' soybean.' Recent' studies' suggest' that' soybean' aphids' prefer' host' plants'
not'infested'by'soybean'cyst'nematode'to'infested'plants'(Hong'and'colleagues'2010).'One'
of' the' objectives' of' this' research' is' to' evaluate' the' effect' of' an' interaction' between' these'
pathogens'on'the'yield'of'resistant'and'susceptible'soybean'lines.'
The' availability' of' reference' genome' for' soybean' (Glycine( max),' soybean' cyst'
nematode' (Heterodera( glycines)' and' soybean' aphid' (Aphis( glycines)' will' enable' studying'
the'molecular'interactions'between'the'host'and'its'pathogens.'The'expression'profiles'of'
the'host'plant'and'associated'pathogens'in'a'spatially'and'temporally'equivalent'biological'
sample' will' promote' understanding' of' the' molecular' mechanisms' of' host' defense' and'
pathogen' infection' strategies' simultaneously' under' single' and' double' infection' in'
susceptible' and' resistant' lines.' No' study' was' found' to' have' analyzed' the' genomeIwide'
expression' profile' of' this' threeIway' interaction' (soybean' cyst' nematode' –' soybean' –'
soybean' aphid).' Thus,' it' is' necessary' to' obtain' the' responsive' expression' profiles' of' the'
infected'leaves'and'roots'to'understand'the'true'molecular'interactions'between'soybean'
and'its'pathogens.'
Parallel' sequencing' techniques' have' made' large' amounts' of' genomic' and'
transcriptomic' sequence' data' available' at' low' costs' (Wang' and' colleagues' 2009).' Until'
recently' gene' expression' has' been' studied' mainly' using' microarrays' for' organisms' with'
high'quality'gene'annotation'data'available.'RNAISeq'has'higher'technical'reproducibility,'a'
wider' dynamic' range' and' provides' better' absolute' expression' estimates' compared' to'
microarrays' (Marioni' and' colleagues' 2008;' Fu' and' colleagues' 2009).' Also,' unannotated'
transcripts' cannot' be' analyzed' using' microarrays' as' probes' are' constructed' using'
annotation' data.' Another' NGS' technology,' HTISuperSAGE' is' much' more' cost' effective'
compared'to'RNAISeq'and'has'similar'sensitivity.'However,'it'does'require'gene'structure'
information'to'convert'tag'counts'to'expression'values'and'does'not'provide'complete'gene'
structures.' RNAISeq,' on' the' other' hand,' measures' expression' values' for' all' transcribed'
regions' without' any' previous' information.' Several' programs' can' construct' transcript'
structures' and' provide' expression' levels' based' on' reference' genome' and' RNAISeq' reads'
(Langmead' and' colleagues' 2009;' Trapnell' and' colleagues' 2009;' Trapnell' and' colleagues'
2010).'Using'RNAISeq,'this'research'will'elucidate'the'gene'expression'profiles'for'soybean'
and' its' pathogens' simultaneously' under' different' conditions' (no' pest,' single' and' dual'
infection'in'both'resistant'and'susceptible'soybean'lines).'It'will'also'aid'in'identifying'host'
genes'and'pathogen'effectors'that'interact'with'each'other.'
'
This'project'combines'the'expertise'of'the'principal'investigator'in'the'assembly'and'
quantification' of' RNAISeq' data' for' hostIpathogen' infectomes,' followed' by' statistical'
analysis' to' identify' differentially' expressed' and' interacting' genes,' and' biological'
interpretation' of' the' analysis.'This' research' will' provide' a' comprehensive' analysis' of' the'
interaction' between' soybean' and' its' pathogens' (soybean' cyst' nematode' and' soybean'
aphid).' The' results' of' this' research' could' aid' in' the' design' of' new' pest' management'
techniques'and'improved'control'over'the'two'most'economically'significant'pathogens'of'
soybean.''
'
'
Surana,'Project'Description'–'2'
Hypothesis*and*Objectives'
Soybeans'(Glycine'max)'in'midwestern'United'States'suffer'most'economic'loss'due'
to' soybean' aphids' (Aphis( glycines' Matsumura)' and' soybean' cyst' nematodes' (Heterodera(
glycines'Ichinohe).'Soybean'cyst'nematodes'caused'more'soybean'crop'losses'than'all'other'
soybean' pests' combined,' before' soybean' aphids' were' introduced' in' North' America'
(Wrather' and' colleagues' 2001a,b).' Exposure' to' high' soybean' aphid' densities' can' cause'
yield'losses'of'50'–'70%'(Wang'and'colleagues'1962;'Dai'and'Fan'1991;'Wu'and'colleagues'
2004).'Soybean'crops'undergo'a'yield'loss'of'about'7%'for'every'10,000'cumulative'aphid'
days'(Ragsdale'and'colleagues'2007).''Their'life'cycles'overlap'on'soybeans'with'H.(glycines'
growing'below'ground'and'A.(glycines'growing'on'foliage.'However,'effects'of'coIinfection'
by'these'pests'on'soybean'yield'are'currently'unknown.'
'
Aim*1:*Identify* physical* attributes* affected* by* soybean* –* cyst* nematode* –* aphid*
interaction*as*compared*with*single*or*no*infection*
Hypotheses:' 1.1' Reduced' yield' and' number' of' mature' seeds' per' pod' in' soybeans'
compared'to'single'or'no'infection'
'
1.2' Reduced' aphid' density' and' cumulative' aphid' days' as' compared' to'
only'aphid'infection'but'increased'compared'to'no'infection'
'
1.3' Nematode' egg' density' and' number' of' root' cysts' increased' as'
compared'to'only'cyst'nematode'infection'
'
Aim*2:*Identify*and*characterize*host*genes*and*pathogen*effectors*
Hypotheses:' 2.1' Effectors' responsible' for' successful' infection' are' differentially'
expressed'
'
2.2'' Effectors'from'cyst'nematode'and'aphids'co–operate'to'increase'plant'
susceptibility'
2.3' Interacting' host' genes' and' pathogen' effectors' have' correlated'
expression'patterns'
'
'
Rationale*and*Significance'
This' project' will' investigate' the' interaction' between' soybean,' soybean' cyst'
nematode' and' soybean' aphid.' This' will' be' achieved' at' a' phenotypic' and' transcriptomic'
level' by' the' two' main' objectives.' First,' evaluate' the' effects' of' cyst' nematode' and' aphid'
interaction' on' plant' yield' in' resistant' and' susceptible' lines.' Second,' elucidate' the' gene'
expression' profiles' for' soybean' and' its' pathogens' simultaneously' under' different'
conditions' (no' pest,' single' and' dual' infection' in' both' resistant' and' susceptible' soybean'
lines).'
This' research' will' provide' a' comprehensive' analysis' of' the' interaction' between'
soybean'and'its'pathogens'(soybean'cyst'nematode'and'soybean'aphid).'The'results'of'this'
research'could'aid'in'the'design'of'new'pest'management'techniques'and'improved'control'
over' the' two' most' economically' significant' pathogens' of' soybean.' Nematode' and' aphid'
resistant' soybean' line' might' protect' against' two' of' the' most' economically' important'
soybean' pathogens.' It' could' change' how' soybean' producers' make' pest' management'
decisions'by'reducing'insecticide'application.'Most'importantly,'results'from'this'work'will'
improve'our'understanding'of'how'these'significant'soybean'pests'interact.''
Surana,'Project'Description'–'3'
Experimental*Approach'
'
Experimental'Design'
Research'will'be'conducted'at'a'site'that'has'not'been'used'to'grow'soybeans'in'the'
recent' past' to' avoid' infestation' with' preIexisting' soybean' cyst' nematode' population.' The'
area'will'be'extensively'tilled'to'loosen'soil'before'sowing'seeds.'The'experimental'design'
will'be'a'splitIplot'with'five'replicates.'Soybean'lines'and'pathogen'treatments'as'the'whole'
and' split' plot' factor' respectively.' Two' soybean' lines' that' will' be' used' are' SD01I76R'
(ASNS—aphidIsusceptible,' nematodeIsusceptible)' and' LD05I16529' (ARNR—aphidI
resistant,'nematodeIresistant).'Each'soybean'line'will'be'tested'under'four'pest'treatments'
–'no'pest'(control),'soybean'aphids'only,'soybean'cyst'nematodes'only'and'both'pests.''
Replicates'will'be'separated'with'strips'of'grass'and'divided'into'8'plots.'Seeds'will'
be' sowed' in' plots' by' hand' in' a' furrow' and' inoculated' with' Bradyrhizobium(japonicum' to'
ensure' nodulation.' Plots' will' be' watered' as' needed,' and' stand' counts' and' growth' stages'
will'be'recorded'weekly.'Soybean'cyst'nematodes'will'be'introduced'in'plots'before'closing'
the'furrows'by'hand.'An'exclusion'cage'will'be'put'around'each'plot'to'prevent'predators'
from'feeding'on'soybean'aphids.'Insecticide'will'also'applied'to'each'cage'to'kill'any'preI
existing' predators.' Lawn' edging' to' prevent' movement' between' plot' of' soybean' cyst'
nematodes'will'also'enclose'each'cage.''
'
Aim*1:*Identify* physical* attributes* affected* by* soybean* –* nematode* –* aphid*
interaction*as*compared*with*single*or*no*infection*
*
Hypothesis(1.1:(Reduced(yield(and(number(of(mature(seeds(per(pod(in(soybeans(compared(to(
single(or(no(infection'
Plants'from'each'plot'will'be'harvested'by'hand'and'stored'for'processing.'Samples'
will'be'threshed'mechanically.'Soybeans'will'be'separated'using'a'small'plot'harvester'and'
stored'for'weighing.'A'moisture'meter'will'be'used'to'measure'moisture'samples'and'the'
average'soybean'moisture'for'each'plot'will'be'calculated.'
Pest'infestation'is'expected'to'significantly'reduce'plant'yield'and'seeds'per'pod.'CoI
infestation'is'expected'to'reduce'plant'yield'and'seeds'per'pod'more'than'infestation'with'
only'one'pathogen'or'no'pathogen.'Soybean'lines'are'also'expected'to'affect'plant'yield'and'
seeds'per'pod.'Irrespective'of'pest'infestation,'aphid'resistant'and'nematode'resistant'line'
(LD05I16529)' is' expected' to' have' higher' yield' and' seeds' per' pod' compared' to' aphid'
susceptible'and'nematode'susceptible'line'(SD01I76R).''Interaction'between'soybean'line'
and'pest'infestation'is'also'to'be'expected.''
'
Hypothesis(1.2:(Reduced(aphid(density(and(cumulative(aphid(days(as(compared(to(only(aphid(
infection(but(increased(compared(to(no(infection'
Apterous' aphid' nymphs' will' be' used' to' infest' designated' plots' when' plants' reach'
the'beginning'pod'stage.'Aphids'will'be'infested'in'the'early'growth'stage'to'correlate'with'
natural'populations.'Aphids'will'be'placed'on'ten'plants'to'uniformly'infest'each'plot'with'
the' pest.' Until' senescence' occurs,' soybean' aphids' will' be' counted' weekly' on' three'
randomly'selected'plants'to'ensure'all'plants'in'a'cage'are'observed.'Cumulative'aphid'days'
will' be' calculated' using' aphid' densities' at' the' end' of' the' soybean' season.' It' represents' a'
measure'of'infection'intensity'and'duration'of'aphid'pressure.'An'aphid'day'is'defined'as'
Surana,'Project'Description'–'4'
one'aphid'feeding'on'one'plant'for'a'period'of'24'hours.'Aphid'days'are'calculated'by'using'
the'formula,'AD'='([XD'+'XDI1]/[2])*t,'where'AD'is'the'number'of'aphid'days'between'the'
current' and' previous' sampling' date,' XD' is' the' mean' number' of' aphids' per' plant' on' the'
current' sampling' date,' XDI1' is' the' mean' number' of' aphids' per' plant' on' the' previous'
sampling'date'and't'is'the'number'of'days'between'sampling'(Ruppel'1983).'
Soybean' lines' may' have' a' considerable' effect' on' cumulative' aphid' days.' Aphid'
susceptible'line'(SD01I76R)'is'expected'to'have'significantly'greater'cumulative'aphid'days'
compared' to' aphid' resistant' line' (LD05I16529).' Soybean' cyst' nematode' infestation' may'
have' an' effect' on' cumulative' aphid' days.' Both' lines' are' expected' to' have' reduced'
cumulative' aphid' days' when' soybean' cyst' nematode' is' also' present.' This' may' be' caused'
due' to' systemic' induction' of' plant' defenses' by' soybean' cyst' nematode' before' soybean'
aphid'is'introduced.'Soybean'aphids'are,'however,'expected'to'survive'in'both'lines'under'
all'treatments.'
'
Hypothesis(1.3:(Nematode(egg(density(and(number(of(root(cysts(increase(as(compared(to(only(
cyst(nematode(infection'
Soybean'cyst'nematodes'will'be'obtained'from'a'soybean'field'in'Iowa.'Soil'from'the'
field' will' be' removed' and' dried' for' two' days.' Cysts' will' be' collected' using' a' large' pore'
sieve,'and'separated'from'soil'particles'and'other'debris'using'sedimentation.'Cysts'will'be'
then' rinsed' into' a' small' pore' sieve' and' ground' with' a' rubber' stopper' attached' to' a' drill'
press' bit' (Faghihi' and' Ferris' 2000).' Eggs' will' be' collected' from' the' small' pore' sieve' and'
further'separated'from'debris'using'sucrose'centrifugation'method'(Niblack'and'colleagues'
1993).' The' cleaned' eggs' will' be' removed,' rinsed' and' stored' using' an' aquarium' aerator.'
Number'of'eggs'will'be'increased'by'infesting'seedlings'of'nematode'susceptible'cultivar,'
removing'females'30'days'later'and'repeating'the'process'as'needed.''
Soybean'cyst'nematode'eggs'will'be'aerated'for'two'days'in'lab,'then'evenly'divided'
into' several' test' tubes' and' suspended' in' tap'water.' Each' test' tube' will' be' emptied' into' a'
watering'can'to'distribute'eggs'evenly'over'the'open'furrow'containing'soybean'seeds.'Soil'
samples'will'be'collected'using'a'soil'probe'for'each'plot.'For'each'plot,'the'cores'will'be'
mixed' and' two' subIsamples' will' be' processed.' Cysts' from' each' subIsample' will' be'
separated'from'the'soil.'Eggs'from'the'cysts'will'be'collected,'rinsed,'stained'and'counted'
(as' described' above).' Egg' densities' are' calculated' per' 100' cubic' centimeter' of' soil.' The'
average'number'of'cysts'per'plot'is'also'calculated.''
Soybean' lines' may' have' a' considerable' effect' on' soybean' cyst' nematode' egg'
densities'and'number'of'cysts.'Nematode'susceptible'line'(SD01I76R)'is'expected'to'have'
significantly' higher' egg' densities' and' greater' number' of' cysts' compared' to' nematode'
resistant' line' (LD05I16529).' Soybean' aphid' infestation' is' expected' to' increase' egg'
densities'and'number'of'cysts.''
'
Statistical(Analysis(and(Timeline'
Plant'yield,'number'of'seeds'per'pod,'soybean'aphid'density,'cumulative'aphid'days,'
soybean'cyst'nematode'egg'density'and'number'of'cysts'per'plot'will'be'log'transformed'
and' analyzed' using' SAS' (current' version).' Only' relevant' plots' (example,' cyst' nematode'
infested'plots'will'be'exclude'from'aphid'analysis,'Hypothesis'1.2)'will'be'included'in'the'
analysis.'PROC'MIXED'will'be'used'to'calculate'variance'for'random'effects'and'significance'
level'for'fixed'effects.'LSMEANS'option'of'PROC'MIXED'will'be'used'to'perform'all'pairwise'
Surana,'Project'Description'–'5'
comparisons'for'main'and'interaction'effects.'False'discover'rates'will'be'calculated'using'
the'histogramIbased'method.'
Increasing'soybean'cyst'nematodes'and'soybean'aphid'will'take'about'3'–'6'months.'
This' will' be' followed' by' growing' the' plant,' infection' with' nematodes' and' aphids,' and'
recording' the' physical' parameters,' which' will' occur' during' the' soybean' growing' season'
from'spring'to'before'fall.'The'data'will'then'be'analyzed,'interpreted'and'published,'which'
will'take'about'a'year'more.'This'aim'will'take'approximately'3'years'to'accomplish.'
'
Aim*2:*Identify*and*characterize*host*genes*and*pathogen*effectors*
Two'replicates'will'be'randomly'selected'for'each'combination'of'soybean'line'and'
pest' infestation.' Approximately' 4' weeks' after' aphid' infestation,' plants' will' be' sampled'
from'the'selected'plots.'Roots'and'leaves'with'no'pest,'aphid'only,'nematode'only'and'both'
pests’'conditions'are'collected.''
Multiple'samples'will'be'collected'from'each'plot'and'their'RNA'pooled'together'to'
create'one'sample'per'designated'plot.'Total'RNA'will'be'extracted'using'TRIzol®'reagent'
(Life' Technologies,' Rockville,' MD,' USA)' for' all' samples.' Oligo(dT)' selection' using' Dynal®'
magnetic' beads' (Invitrogen)' will' be' used' to' isolate' total' mRNA.' DoubleIstranded' cDNA'
molecules' will' be' generated' using' random' hexadeoxynucleotide' primers,' then' barcoded'
and'sequenced'using'Illumina'mRNAISeq'single'end'protocol'on'a'HiSeq'2500/1500'with'
100'cycles.'
FASTX' Toolkit' will' be' used' to' remove' adapters,' trim' low' quality' bases' from' each'
end' and' discard' reads' if' they' are' shorter' than' 60' nucleotides' or' of' low' overall' quality.'
Tophat' (along' with' Bowtie2)' will' be' used' to' align' all' reads' against' their' respective'
genomes' and' build' the' transcriptome.' Trinity' (transcriptome' assembly' program)' will' be'
used'if'the'alignment'with'Tophat'is'unsuccessful'due'to'memory'or'other'reasons.'Reads'
that' align' to' multiple' positions' will' be' discarded.' Cufflinks' will' be' used' to' predict' novel'
transcripts.' RSEM' will' be' used' to' generate' raw' and' normalized' read' counts,' and' fold'
change'values.'
Same' plants' used' for' Aim' 1,' can' be' used' to' collect' samples' for' this' aim.' Sample'
collection' will' be' followed' by' RNA' extraction,' library' preparation' and' sequencing' of' the'
transcriptome,' which' can' take' over' a' year' to' a' year' and' a' half.' The' data' will' then' be'
analyzed,'interpreted'and'published,'which'will'take'another'year'or'year'and'a'half.'This'
aim'will'take'approximately'4'years'to'accomplish.'
'
Hypothesis(2.1:(Effectors(responsible(for(successful(infection(are(differentially(expressed'
Raw'read'counts'for'soybean'cyst'nematode'under'nematode'only,'both'pest'and'no'
pest' conditions' will' be' analyzed' using' DESeq.' QuasiSeq' will' be' used' as' an' alternate'
differential'expression'testing'method'to'DESeq.''Raw'read'counts'for'soybean'aphid'under'
aphid' only,' both' pest' and' no' pest' conditions' will' be' analyzed' similarly.' Differentially'
expressed' genes' will' be' annotated' using' the' available' genome' annotation.' Homology'
searches'against'various'protein'databases'(SwissIProt'enteries'in'UniProt;'validated'and'
reviewed'protein'entries'in'RefSeq)'will'be'performed'using'BLASTx'for'the'unannotated'
transcripts' to' infer' function.' To' identify' putative' secretory' proteins,' SignalP' and' TargetP'
will' be' used' to' predict' signal' peptides' and' cellular' localizations' for' each' transcript'
respectively.' Depending' on' the' results,' these' transcripts' can' be' further' examined' using'
category'enrichment,'sequence'and'structural'domain'analysis.'
Surana,'Project'Description'–'6'
'
Result'interpretation'will'depend'on'the'transcripts'identified.'However,'pathogen'
genes'associated'with'defense'inhibition,'nutrient'uptake'and'transcription'are'expected'to'
be' upregulated' in' infected' plants' versus' nonIinfected' plants.' Aphids' are' known' to' cause'
both' salicylic' and' jasmonic' acid' expression' to' increase' but' reduced' recognition,' may' be'
genes' responsible' for' this' process' will' be' recognized' using' this' method.' The' results' will'
also'aid'in'further'hypotheses'generation.'
'
Hypothesis( 2.2:( Effectors( from( cyst( nematode( and( aphids( co–operate( to( increase( plant(
susceptibility(
'
An' effector' is' generally' secreted' from' a' pathogen' to' alter' the' host' organism' and'
enable' infection,' e.g.' by' suppressing' the' host's' immune' system' capabilities.' Effectors'
identified'in'soybean'cyst'nematodes'and'soybean'aphids'could'be'interacting'to'weaken'
plant'defenses.'This'coIoperative'behavior'could'be'seen'if'effectors'from'both'pathogens'
interact' with' the' same' host' protein' or' inhibit' the' same' pathway' using' two' different'
mechanisms.''
In' order' to' achieve' this' aim,' gene' networks' will' be' constructed' for' effectors' and'
host' genes' using' mutual' information,' distance' matrices' and' annotation' information' (as'
obtained'above).'Transcripts'that'have'similar'expression'patterns'and'are'involved'in'the'
same' biological' process' will' cluster' together.' Effectors' that' cluster' together' are' probably'
interacting'with'host'proteins'from'the'same'pathway/process.'Pairs'of'effector'and'host'
protein'involved'in'increasing'plant'susceptibility'can'be'further'studied'using'a'pull'down'
assay'to'identify'true'interactions.'If'pull'down'assay'does'not'work,'yeast'two'hybrid'assay'
will' be' used.' The' results' will' be' further' validated' using' BiFC' (Bimolecular' Fluorescent'
Complementation)'assay.'
Soybean'cyst'nematodes'are'expected'to'show'improved'growth'in'plants'infested'
with' soybean' aphids.' This' may' be' due' to' coIoperation' between' the' two' pathogens' in'
invading'the'host'defenses'or'activating'decoy'defenses.''
'
Hypothesis( 2.3:( Interacting( host( genes( and( pathogen( effectors( have( correlated( expression(
patterns(
'
Mutual' information' and' correlation' data' on' gene' expression' can' help' detect'
patterns' and' identity' genes' that' follow' similar' expression' patterns.' Complimentary'
expression'patterns'can'help'us'identify'genes'that'may'be'interacting'with'each'other.'In'
this' objective,' hostIpathogen' interactions' at' the' protein' level' will' be' studied' by' finding'
complimentary' expression' patterns' between' soybean' and' its' either' pest.' Once' a' list' of'
candidate'genes'are'identified,'they'will'be'further'analyzed'using'cellular'localization'and'
biological' process' information.' If' a' host' and' pathogen' transcript' is' involved' in' similar'
biological' process,' is' localized' in' the' same' cellular' component' and' has' complimentary'
expression' profile,' they' will' be' selected' as' top' candidates' for' studying' hostIpathogen'
interactions.'These'top'candidates'will'be'analyzed'using'a'pull'down'assay'to'identify'true'
interactions.' If' pull' down' assay' does' not' work,' yeast' two' hybrid' assay' will' be' used.'
Successful'genes'will'further'be'studied'using'VIGS'(Virus'Induced'Gene'Silencing).''
'
Genes' identified' will' give' us' clue' into' which' host' genes' are' essential' for' pathogen'
viability.'These'genes'are'expected'to'be'involved'in'defense'pathways'(example,'jasmonic'
Surana,'Project'Description'–'7'
acid'pathway),'nutrient'and'water'uptake,'transcription'and'translation.'The'results'from'
this'experiment'will'also'assist'in'further'hypothesis'generation.'
'
'
Future*Directions'
This' research' will' provide' a' comprehensive' analysis' of' the' interaction' between'
soybean'and'its'pathogens'(soybean'cyst'nematode'and'soybean'aphid).'The'results'of'this'
research'could'aid'in'the'design'of'new'pest'management'techniques'and'improved'control'
over'the'two'most'economically'significant'pathogens'of'soybean.'Transgenic'lines'for'the'
genes'identified'in'the'last'hypothesis'of'Aim'2,'could'be'tested'for'commercial'purposes.'
Further' research' could' be' followed' into' how' soybean' producers' can' make' pest'
management' decisions' differently' and' reduce' insecticide' application' using' results' from'
this' study' as' it' will' improve' our' understanding' of' how' these' significant' soybean' pests'
interact.''
Transcriptome' data' gives' immense' amount' of' information,' paired' with' already'
public' genomic' data,' could' be' analyzed' and' reIanalyzed' to' answer' a' wide' range' of'
questions,' such' as' SNP' (Single' Nucleotide' Polymorphisms)' and' CNV' (Copy' Number'
Variation)' information.' Money' constraints' prevented' studying' if' the' order' in' which'
pathogen' is' introduced' has' an' effect' on' plant' yield' (aphid' infection' before' nematode'
infestation'is'not'studied'in'this'work).'Also,'multiple'time'points'(0,'12'and'24'hours'after'
infestation,'along'with'1,'2,'3'and'4'weeks'after'infestation)'would'shed'greater'light'into'
how'these'pests'interact'with'each'other'and'their'common'host.''
'
'
Timeline'
'
Maintain' soybean' aphids' and' increase' Combine' SwissProt' and' RefSeq' protein'
soybean' nematode' population;' Setup' and' databases;' Index' genomic' and' annotation'
test'VIGS'system'
information;'Install'all'programs'to'be'used''
Prepare'field'and'grow'plants'as'per'experimental'design'
Collect' physical' parameters' throughout' the' Collect' samples' 4' weeks' after' aphid'
growing'stage''
infestation'for'sequencing''
Analyze'and'interpret'data'
RNA'extraction'and'library'preparation'
Analyze'and'interpret'data'
VIGS'experiments'
Publish'results'
'
'
*
*
Surana,'Project'Description'–'8'
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59. Scheu,'S.'(2001).'"Plants'and'generalist'predators'as'links'between'the'belowIground'
and'aboveIground'system."'Basic'and'Applied'Ecology'2(1):'3–13.'
'
60. Soler,'R.,'W.'V.'d.'Putten,'J.'Harvey,'L.'Vet,'M.'Dicke'and'T.'Bezemer'(2012).'"Root'
Herbivore'Effects'on'Aboveground'Multitrophic'Interactions:'Patterns,'Processes'and'
Mechanisms."'J'Chem.'Ecol.'38(6):'755I767.'
'
61. Vandegehuchte,'M.'L.,'E.'De'La'Peña'and'D.'Bonte'(2010).'"Interactions'between'root'
and'shoot'herbivores'of'Ammophila'arenaria'in'the'laboratory'do'not'translate'into'
correlated'abundances'in'the'field."'Oikos'119(6):'1011I1019.'
'
*
*
Surana,'References'–'5'
Budget*Justification*
A' graduate' student' and' postIdoctoral' research' will' be' needed' to' complete' this'
project.' Graduate' student' and' postIdoctoral' researcher' salaries' are' as' per' the' university'
guidelines.' Travel' costs' are' included' for' one' or' two' laboratory' member(s)' to' travel' to' a'
conference'or'workshop'every'year'to'discuss'and'disseminate'knowledge'gained'from'the'
experiments'conducted.'Publication'costs'are'for'3'or'4'publications'in'total'for'the'project'
(as'anticipated).'Materials'are'the'average'cost'for'supplies'every'year'for'each'researcher.'
The'cost'for'sequencing'is'as'quoted'by'Illumina.''
Surana,'Budget'and'Justification'–'1'
Project Budget Worksheet - Iowa State University of Science and Technology
Form updated 7-27-12
Eff. 7-1-12
Program Sponsor
Title
PI
Period of Performance
Deadline
1/1/2013-12/31/2017
Year 1
A
Key Personnel
1 Graduate Student
9
B
Other Personnel
1 Post Doc
Salary
Monthly
$1,500
$0
Monthly
$3,000
Calendar
Months
12.00
0.00
Calendar
Months
12.00
Academic
Months
8.00
0.00
Summer
Months
4.00
0.00
Number of
persons
1.00
Year 2
Year 3
Year 4
Year 5
Total
$36,000
$37,080
$38,192
$39,338
$40,518
$191,129
$36,000
$0
$37,080
$0
$38,192
$0
$39,338
$0
$40,518
$0
$191,129
$0
$36,000
$37,080
$38,192
$39,338
$40,518
$191,129
$36,000
$37,080
$38,192
$39,338
$40,518
$191,129
$72,000
$74,160
$76,385
$78,676
$81,037
$382,258
$18,900
$19,467
$20,051
$20,653
$21,272
$100,343
$10,980
$7,920
$11,309
$8,158
$11,649
$8,402
$11,998
$8,654
$12,358
$8,914
$58,294
$42,048
$90,900
$93,627
$96,436
$99,329
$102,309
$482,600
$0
$0
$0
$0
$0
$0
Travel
$0
$2,000
$2,000
$2,000
$0
1. Domestic Travel
2. Foreign Travel
$0
$2,000
$0
$2,000
$0
$2,000
$0
$0
$0
$6,000
$6,000
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$12,155
$28,246
$17,340
$7,438
$0
$10,000
$0
$0
$0
$0
$0
$0
$0
$2,155
$0
$0
$0
$10,000
$0
$0
$0
$0
$0
$0
$0
$2,246
$0
$16,000
$0
$10,000
$5,000
$0
$0
$0
$0
$0
$0
$2,340
$0
$0
$0
$0
$5,000
$0
$0
$0
$0
$0
$0
$2,438
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$65,178
$30,000
$10,000
$0
$0
$0
$0
$0
$0
$9,178
$0
$16,000
$0
Subtotal: Total Direct Costs (TDC)
$103,055
$123,873
$115,776
$108,767
$102,309
$553,779
Subtotal: Modified Total Direct Costs
$100,900
$121,627
$113,436
$106,329
$102,309
$544,600
$48,432
$58,381
$54,449
$51,038
$49,108
$261,408
$48,432
$58,381
$54,449
$51,038
$49,108
$151,487
$182,253
$170,225
$159,805
$151,417
$191,128.89
$0.00
Subtotal: Salaries and Wages
C
Fringe Benefits
Graduate Student
Post Doc
Rate
30.5%
22.0%
Subtotal: Salaries, Wages, and Benefits
D
Equipment (List Item and $ amount for each item > $5k)
$191,128.89
1
2
E
F
Participant Support Cost
See notes below
1. Stipend
2. Travel
3. Subsistence
4. Other
G
Other Direct Costs
1
2
3
4
5
6
7
8
9
10
Materials and Supplies
Publication cost
Computing support
Instrumentation facility
Subcontractor1 - Subject to IDC (first $25,000)
See notes below
NOT subject to IDC (Amount over $25,000)
Subcontractor2 - Subject to IDC (first $25,000)
See notes below
NOT subject to IDC (Amount over $25,000)
Tuition - Non-Engineering
(Click on "Tuition" sheet)
Tuition - Engineering
(Click on "Tuition" sheet)
Illumina Sequencing
Other
$0
$0
$0
$0
$0
$553,778.87
[ MTDC = TDC - Tuition - Equipment - Participant Support Cost ]
H
Indirect Costs
IDC on MTDC
Rate
48.0%
[ IDC = MTDC * Indirect Rate ]
I
Total Project Costs
[ Total = TDC + IDC ]
$815,187
$815,187.08