1

Mu!tIp!c gcncs affcct scnsItIvIty nf C. e|egans tn thc bactcrIa!

pathngcn M. nematohl|um


Maiia }. Ciavalo-Nolie, Hannah R. NichoIas
1
, Reindeil NijIand
2
, DeIia ORouike,
Deloiah L. Whillinglon, Kaien }. Yook, }onalhan Hodgkin



Cenelics Unil, Depailnenl of iochenisliy, Oxfoid OX1 3QU, Uniled Kingdon

1
Iiesenl addiess: SchooI of MoIecuIai and MicioliaI iosciences, Univeisily of Sydney
2
Iiesenl addiess: Depailnenl of Cenelics, Ciningen ionoIecuIai Sciences and
iolechnoIogy Inslilule

Genetics: Published Articles Ahead of Print, published on August 3, 2005 as 10.1534/genetics.105.045716
2
Running head: Mulanls iesislanl lo ieclaI infeclion

Key voids: C. c|cgans, Micrcoac|criun ncna|cpni|un, ieclaI infeclion, iesislance,
nenalode culicIe

ResponsilIe individuaI:
}onalhan Hodgkin
Cenelics Unil, Depailnenl of iochenisliy
Univeisily of Oxfoid
Soulh Iaiks Road
Oxfoid OX1 3QU
UK
TeI +44 1865 275317
Iax +44 1865 275318
LnaiI jonalhan.hodgkinlioch.ox.ac.uk

3
AB5TRACT
Inleiaclions vilh lacleiia pIay a najoi ioIe in innune iesponses, ecoIogy and
evoIulion of aII aninaIs, lul lhey have leen negIecled unliI iecenlIy in lhe case of C.
c|cgans. We iepoil a genelic invesligalion of lhe inleiaclion of C. c|cgans vilh lhe
nenalode-specific palhogen Micrcoac|criun ncna|cpni|un, vhich coIonizes lhe ieclun
and causes dislinclive laiI sveIIing in ils hosl. 121 nulanls vilh aIleied iesponse lo
infeclion veie isoIaled fion seIeclions oi scieens foi a us (acleiiaIIy Un-SvoIIen)
phenolype, using lolh chenicaI and liansposon nulagenesis. Sone of lhese
coiiespond lo knovn genes, affecling eilhei lacleiiaI adhesion oi coIonizalion (srf-2, srf-
3, srf-5) oi hosl sveIIing iesponse (sur-2, cg|-5). Mosl nulanls define 15 nev genes (ous-
1 lo ous-6, ous-8, ous-10, ous-12 lo ous-18). The najoiily of lhese nulanls exhilil IillIe
oi no ieclaI infeclion vhen chaIIenged vilh lhe palhogen, and aie piolalIy aIleied in
suiface piopeilies such lhal lhe lacleiia can no Iongei infecl voins. A nunlei have
coiiesponding aIleialions in Ieclin slaining and culicIe fiagiIily. Mosl of lhe
uninfeclalIe nulanls giov lellei lhan viId lype in lhe piesence of lhe palhogen, lul
lhe sur-2 nulanl is hypeisensilive, indicaling lhal lhe laiI-sveIIing iesponse is
associaled vilh a specific defense nechanisn againsl lhis palhogen.
4
INTRODUCTION
AII nuIliceIIuIai oiganisns have lo deaI vilh lacleiiaI palhogens of one kind oi
anolhei, and have evoIved efficienl defenses in oidei lo suivive in a voiId doninaled,
al Ieasl nuneiicaIIy, ly polenliaIIy hosliIe piokaiyoles. These defenses can le
physicaI, pioviding nechanicaI laiiieis againsl invasion, oi lioIogicaI, in lhe foin of
anlinicioliaI conpounds and dedicaled phagocylic ceIIs. Veileliales have evoIved
sophislicaled pioleclion in lhe foin of adaplive innunily, lul nosl aninaI and pIanl
species ieIy onIy on innale innune nechanisns, vhich aie incieasingIy iecognized as
inpoilanl and univeisaI. Innale innune defenses can le sludied effecliveIy in nodeI
oiganisns such as DiosophiIa and C. c|cgans (IRADLL and LWANK 2OO4). Ioi
exanpIe, ieseaich on lhe ToII ieceploi in DiosophiIa vas inpoilanl in ieveaIing lhe
appaienlIy conseived ioIe of lhis ieceploi noIecuIe in aclivaling innale innunily.
RecenlIy, C. c|cgans has aIso lecone incieasingIy sludied fion lhe peispeclive of innale
innunily (foi iecenl ievievs, see CRAVATO-NORL and HODCKIN 2OO5, MILLLT
and LWANK 2OO4, SCHULLNURC c| a|. 2OO4, SIIRI c| a|. 2OO5).
C. c|cgans is a species vilh a gIolaI disliilulion, and Iives ly ealing lacleiia, so il
is IikeIy lo encounlei a veiy vide vaiiely of lacleiia in ils diel and daiIy Iife. Iev of
lhese diffeienl lacleiiaI species seen lo have any deIeleiious effecl on lhe voin,
suggesling lhal il has effeclive neans of pioleclion. Recenl voik has idenlified a
vaiiely of diffeienl defense iesponses in lhe voin (DARY c| a|. 1999, KIM c| a|. 2OO2,
2OO4, HUIIMAN c| a|. 2OO4, NICHOLAS and HODCKIN 2OO4). Thus fai, nosl of lhe
lacleiia lhal have leen exanined vilh iespecl lo palhogenic oi loxic effecls on C.
5
c|cgans have leen nicioles knovn lo have danaging effecls on a vaiiely of diffeienl
nelazoan oiganisns, such as 8aci||us |nuringicnsis, Pscudcncnas acrugincsa, Sa|ncnc||a
cn|crica, Scrra|ia narccsccns and S|apnq|cccccus aurcus (COUILLAULT and LWANK
2OO2, MARROQUIN c| a|. 2OOO, AALLAY c| a|. 2OOO, LAROUSSL el aI 2OOO,
MAHA}AN-MIKLOS c| a|. 1999, MALLO c| a|. 2OO2, SIIRI c| a|. 2OO3). Such lioad-
specliun palhogens aie IikeIy lo eIicil ieIaliveIy non-specific defense nechanisns, foi
exanpIe geneiaIized iesponses lo infeclion and sliess. In conliasl, lacleiia vilh a
naiiov hosl iange, vhich have evoIved speciaIized nechanisns lo infecl C. c|cgans,
nay eIicil sepaiale kinds of iesponse, vilh noie dedicaled ioIes in piolecling lhe hosl
fion danage. Moieovei, palhogens laigeled lo a iesliicled nunlei of species nay le
noie fiequenlIy associaled vilh hosl-paiasile co-evoIulion, vilh conconilanl
speciaIizalion and diveisily in nechanisns of allack and defense. SCHULLNURC
and LWANK (2OO4) have piovided evidence lhal co-evoIulion can aIso occui lelveen
C. c|cgans and a lioad-specliun palhogen, Scrra|ia narccsccns. In geneiaI, hovevei, il
seens IikeIy lhal delaiIed exaninalion of inleiaclions vilh speciaIisl palhogens viII
ieveaI diffeienl phenonena fion lhose invoIved in iesponses lo geneiaIisl palhogens.
One palhogen of C. c|cgans vilh an appaienlIy naiiov hosl iange anong
nenalodes, and aIso a veiy unusuaI node of infeclion and palhoIogy, is lhe iecenlIy
discoveied coiynefoin species Micrcoac|criun ncna|cpni|un (HODCKIN c| a|. 2OOO).
This palhogen has leen isoIaled on al Ieasl foui independenl occasions as a chance
conlaninanl of Ialoialoiy cuIluies of nenalodes, as a iesuIl of lhe sliiking
noiphoIogicaI defoinalion lhal il induces in lhe laiI of infecled voins (HODCKIN c|
6
a|. 2OOO, AKIMKINA c| a|. (sulnilled)). The lacleiiun is sIov gioving, so ils piesence
in lhe Iavns of |. cc|i used foi C. c|cgans cuIluie is nol easiIy noliced. NeveilheIess, il is
alIe lo infecl C. c|cgans and eslalIish a coIony in lhe ieclun of lhe aninaI, vilh ceIIs
adheiing lighlIy lo lhe ieclaI and posl-anaI culicIe. In iesponse lo lhe infeclion, lhe
anaI iegion of lhe voin lecones giealIy enIaiged, ciealing a disloiled noiphoIogy
lhal is easiIy scoied ly dissecling nicioscope (lhe Dai, oi Defoined AnaI Region
phenolype, iIIuslialed in Iiguie 1). Woins lecone sonevhal conslipaled as a iesuIl of
lhe infeclion, and giov noie sIovIy, lul lhey do nol geneiaIIy expeiience noie
deIeleiious effecls.
In lhe iniliaI invesligalion of lhis phenonenon, il vas nol olvious vhelhei lhe
laiI-sveIIing iepiesenls pail of a defense ieaclion on lhe pail of lhe voin, oi is a
defoinalion induced ly lhe palhogen foi ils ovn lenefil. Iuilhei anaIysis
(NICHOLAS and HODCKIN 2OO4) has shovn lhal lhe laiI-sveIIing iesuIls fion IocaI
aclivalion of an LRK MAI kinase cascade, and is associaled vilh pioleclion againsl lhe
palhogen. When eIenenls of lhis signaIing cascade aie inhililed, eilhei ly nulalion oi
chenicaIIy, lhe palhogen is alIe lo coIonize lhe ieclun lul no sveIIing ensues.
Mulanls vilh defecls in LRK MAI kinase signaIing aie hypeisensilive lo lhe palhogen,
expeiiencing exliene conslipalion logelhei vilh diaslicaIIy ieduced vialiIily and
feiliIily aflei infeclion. Il lheiefoie appeais lhal lhe voin has evoIved a speciaIized
defense againsl allack ly M. ncna|cpni|un. Diffeienl MAI kinase palhvays have
iecenlIy leen inpIicaled in piolecling C. c|cgans againsl olhei kinds of lacleiiaI
7
infeclion oi loxins (KIM c| a|. 2OO2, 2OO4, HUIIMAN c| a|. 2OO4), lul lhe LRK MAI
kinase palhvay appeais lo le specific foi ieclaI infeclion.
Many queslions ienain lo le ansveied aloul lhis hosl-paiasile inleiaclion, such
as: hov is lhe palhogen alIe lo coIonize lhe voin` Whal nechanisns aie invoIved in
ils adheience lo hosl culicIe` And vilh iespecl lo lhe hosl side of lhe inleiaclion: hov
does lhe hosl delecl lhe piesence of lhe palhogen` Whal ceIIuIai nachineiy is invoIved
in lhe sveIIing ieaclion and olhei aspecls of lhe defense iesponse` Whal olhei faclois
Iinil lhe exlenl and consequences of infeclion`
In oidei lo addiess lhese queslions, and lo expIoie lhe hosl-palhogen inleiaclion
fuilhei, ve sel oul lo isoIale and invesligale C. c|cgans nulanls vilh iesislance oi olhei
aIleialion in iesponse lo infeclion ly M. ncna|cpni|un. We found lhal iesislanl
nulanls can le efficienlIy seIecled, and ve have isoIaled and sludied a Iaige nunlei.
This papei piovides a iepoil on lhe iniliaI chaiacleiizalion of lhese nulanls, vhich
iepiesenl a sulslanliaI nunlei of lolh knovn and pieviousIy unchaiacleiized Ioci. In
iniliaI voik on lhe infeclion (HODCKIN c| a|. 2OOO), il vas olseived lhal nulalions in
lhiee knovn genes affecling suiface anligenicily (srf-2, 3, 5) couId confei iesislance lo
M. ncna|cpni|un, so il vas nol suipiising lhal nev nulalions in aII lhiee of lhese genes
veie iecoveied, lul lhe najoiily of nulanls iepiesenl genes vilh no pieviousIy
iepoiled nulanl phenolype. AnaIysis of lhe liolic inleiaclions lelveen hosl and
palhogen nay ieveaI funclions foi nany hosl genes foi vhich knockoul oi knockdovn
has no olvious consequence, in lhe alsence of chaIIenge ly disease oi olhei sliess.
8
MATERIAL5 AND METHOD5
Cu!turc cnndItInns: Iioceduies foi giovlh and nanipuIalion of C. c|cgans veie
as pulIished (RLNNLR 1974, SULSTON and HODCKIN 1987). Lxpeiinenls veie
caiiied oul al 2O - 22`C unIess olheivise noled. Mulagenesis vilh elhyI nelhane
suIfonale (LMS, O.O5M) vas caiiied oul as desciiled ly RLNNLR (1974).

GcnctIc markcrs: The foIIoving slandaid nulalions veie used in lhis sludy,
and aie Iisled in HODCKIN (1997) and on Woinase (hllp://vvv.voinlase.oig/),
unIess olheivise indicaled. iisloI N2 vas used as viId lype (WT).
|G|. dpq-5(c61), unc-13(c51), srf-2(qj262), spc-9(nc52ls), sur-2(|u9)
|G||. o|i-1(c769)
|G|||. cg|-5(n486), unc-32(c189), nu|-7(p|204)
|G|V. dpq-9(c12), cg|-4(n478), dpq-13(184), unc-5(c53), unc-24(c138), nin-8(c1489), dpq-
20(c1282), nin-6(c1423), srf-3(qj10), unc-30(c191), dpq-4(c1166)
|GV. dpq-11(c224), unc-42(c270), nin-5(c1490), |cn-3(c2175), fcg-2(q71), unc-51(c369)
|GX. unc-2(c55), |cn-2(c678), unc-97(su110), dpq-8(c130), srf-5(c|115), unc-7(c5)
The nuIlipIy naiked sliain DA438 (o|i-4(c937) |, rc|-6(c187) ||, daf-2(c1368ls) tao-7(c1562)
|||, unc-31(c928) |V, dpq-11(c224) V, |cn-2(c678) X), consliucled ly L. AVLRY, vas used
foi sone napping and consliuclion.

Assays: Ioi convenience, ve adopled a slandaid pioceduie foi piepaiing
infeclive Iavns: |. cc|i sliain OI5O and M. ncna|cpni|un sliain CX1O2 veie lolh
9
giovn lo slalionaiy phase in L liolh and nixed al a voIuneliic ialio of 9:1. Infeclion
aIso occuis al nuch Iovei piopoilions of lhe palhogen, lul a 1O nixluie vas used lo
ensuie lhal infeclion vas iapid and ieIialIe. The nixluie vas used lo seed Iavns on
slandaid NCM agai nedia (RLNNLR 1974). These Iavns aie iefeiied lo as ML
(nixed lacleiiaI Iavn). The ialio of |. cc|i lo M. ncna|cpni|un in lhe nixed Iavns is
unIikeIy lo le conslanl vilh line, foi al Ieasl lvo ieasons: iniliaI doulIing line is nuch
Iovei foi M. ncna|cpni|un lhan foi |. cc|i, lul M. ncna|cpni|un ceIIs aie alIe lo suivive
lellei in slalionaiy phase, and conlinue lo divide in lhe nixed Iavns. ConsequenlIy,
lheii piopoilion giaduaIIy iises. Hovevei, no gieal vaiialiIily vas olseived in lhe
iesponse of voins lo Iavns of diffeienl ages. Iuie Iavns of M. ncna|cpni|un veie nol
used in nosl of lhis voik, lecause aflei a fev days of inculalion lhey lecone
exlieneIy slicky and laieIy edilIe even ly iesislanl voins.

5taInIng adhcrcnt bactcrIa wIth 5YTO 13: As in NICHOLAS and HODCKIN (2OO4)

LcctIn staInIng: 3O-6O aduIl voins and Iaivae veie picked and vashed lvice foi five
ninules in 1 nI of IS-T (phosphale luffeied saIine, O.5 Tiilon X-1OO). Woins veie
inculaled in 3O I of 5O g/nI IITC- oi TRITC-conjugaled Ieclin foi 45 ninules al 25`C,
lhen vashed lhiee lines foi 1O ninules in 1 nI of IS-T. Slained voins veie
anaeslhelized in 3O I O.5 phenoxypiopanoI, excess anaeslhelic vas lhen ienoved and
1O I of IS-cilifIuoi (Agai Scienlific) added lo ieduce fading, lefoie nounling on
agaiose pads and vieving vilh appiopiiale fiIleis. Six fIuoiescenl Ieclins (suppIied ly
10
Signa) vilh diffeienl specificilies veie used: WCA (vheal gein aggIulinin), SA (soy
lean aggIulinin), TIA (Tc|ragcnc|cous purpurcas aggIulinin), INA (peanul Ieclin), CON
(concanavaIin A), LLA (|qccpcrsiccn cscu|cn|un, lonalo Ieclin).

TcstIng b!cach scnsItIvIty: Ioi each lesl, a 1O I diop of fieshIy piepaied aIkaIine
hypochIoiile soIulion (1 N NaOH, 4O NaOCI soIulion (>12 avaiIalIe chIoiine)) vas
pIaced on NCM agai, and 15 aduIl heinaphiodiles innedialeIy liansfeiied logelhei lo
lhe diop, using a pIalinun viie voin pick. The line laken lefoie aII voins slopped
lhiashing, and lhe line laken foi lhe fiisl voins lo legin lo lieak up, veie lolh noled.
Ioi WT voins, lhese lines veie usuaIIy 2O - 3O and 9O - 12O seconds, iespecliveIy, foi
lhe nosl sensilive voins, Iess lhan 5 and Iess lhan 2O seconds, iespecliveIy. Scoies
veie assigned accoidingIy (+ = WT, ++++ = nosl sensilive).

Ana!ysIs nf mutants: AII LMS-induced nulanls veie oulciossed al Ieasl once againsl
WT, and in nosl cases a nin (high incidence of naIes) deiivalive sliain vas lhen
consliucled, foi convenience in fuilhei sliain consliuclion and napping, using eilhei
nin-8 |V oi nin-5 V. DifficuIly in consliucling eilhei doulIe nulanl vas an indicalion
of Iinkage lo eilhei LCIV oi LCV, vhich convenienlIy happens lo le vheie nosl of lhe
nev genes idenlified in lhis sludy aie Iocaled.
Mulanls isoIaled fion lhe nu|-7 sliain NL917 (KLTTINC c| a|. 1999) veie
oulciossed noie exlensiveIy, in oidei lo ienove al Ieasl sone of lhe exlianeous
liansposon inseilions lhal inevilalIy accunuIale in lhis sliain. The nu|-7 Iocus vas
11
expIicilIy ienoved, ly consliucling doulIe nulanls vilh unc-32 (lighlIy Iinked lo nu|-
7), and lhen ienoving lhe unc-32 naikei ly fuilhei ciosses vilh WT. This aIso seived
as a lesl foi Iinkage lo LCIII.

MappIng data fnr ncw gcncs: Linkage of aII nulalions vas exanined using lhe
nuIlipIy naiked sliain DA438, peinilling unanliguous assignnenl lo a Iinkage gioup
of aII lul a fev ous nulalions. NolalIy, ous-14(c2779) couId nol le easiIy Iocaled ly
lhis nelhod, and vas lheiefoie napped using SNI naikeis, vhich denonslialed
Iinkage lo lhe iighl end of LCV. Thiee olhei nulalions (c2706, c2701, c2725) gave
iniliaIIy nisIeading napping iesuIls, foi vhich ieason lhey veie al fiisl assigned lo
dislincl conpIenenlalion gioups (ous-7, ous-9, ous-11), lul fuilhei chaiacleiizalion
shoved lhal c2701 vas a ous-5 aIIeIe, and lhe olhei lvo veie aIIeIes of knovn genes
(sur-2(c2706) (NICHOLAS and HODCKIN 2OO4), and cg|-5(c2725) (H.R.N. and }. H.,
nanusciipl in piepaialion)). MininaI dala lhal define lhe appioxinale nap posilions
of lhe 15 nev genes idenlified in lhis sludy aie given leIov. Wheie noie lhan one
aIIeIe vas ollained foi a given gene, nosl napping iesuIls veie ollained using onIy
lhe iefeience aIIeIe foi each gene, in a fev cases confiinaloiy dala veie ollained vilh
olhei aIIeIes. These, pIus noie exlensive addilionaI nap dala, viII le deposiled in lhe
pulIic dalalases ACeD and Woinase. Map posilions aie shovn in Iiguie 2.
ous-1. Dpy non-Unc and Unc non-Dpy ieconlinanls picked fion lhe piogeny of dpq-
11 + unc-42/+ ous-1(c2678) + gave lhe oidei dpq-11 4/6 ous-1 2/6 unc-42.
12
ous-2. LgI non-Dpy ieconlinanls picked fion lhe piogeny of dpq-9 + cg|-4/ + ous-
2(c2687) + gave lhe oidei dpq-9 1O/13 ous-2 3/13 cg|-4.
ous-3. Dpy non-Unc and Unc non-Dpy ieconlinanls picked fion lhe piogeny of dpq-5
unc-13 + / + + ous-3 gave lhe oidei dpq-5 9/9 unc-13 O/9 ous-3.
ous-4. Dpy non-Unc and Unc non-Dpy ieconlinanls picked fion lhe piogeny of dpq-13
ous-4 unc-5 / + + + gave lhe oidei dpq-13 37/57 ous-4 2O/57 unc-5
ous-5. Unc non-Dpy and Dpy non-Unc ieconlinanls picked fion lhe piogeny of unc-2
+ dpq-8 / + ous-5 + gave lhe oidei unc-2 11/98 ous-5 87/98 dpq-8.
ous-6. Dpy non-Unc and Unc non-Dpy ieconlinanls picked fion lhe piogeny of dpq-11
+ unc-42 / + ous-6 + gave lhe oidei dpq-11 7/16 ous-6 9/16 unc-42.
ous-8. Lon non-Unc ieconlinanls picked fion lhe piogeny of |cn-2 + unc-97 / + ous-8 +
gave lhe oidei |cn-2 8/18 ous-8 1O/18 unc-97.
ous-10. Unc non-Dpy and Dpy non-Unc ieconlinanls picked fion lhe piogeny of unc-
24 + dpq-20 / + ous-10 + gave lhe oidei unc-24 7/8 ous-10 1/8 dpq-20.
ous-12. Unc non-Dpy ieconlinanls picked fion lhe piogeny of unc-30 + dpq-4 / + ous-12
+ gave lhe oidei unc-30 1/8 ous-12 7/8 dpq-4.
ous-13. Dpy non-Unc and Unc non-Dpy ieconlinanls picked fion lhe piogeny of dpq-
11 + unc-42 / + ous-13 + gave lhe oidei dpq-11 2/13 ous-9 11/13 unc-42.
ous-14. us non-Unc ieconlinanls picked fion lhe piogeny of ous-14 unc-51 + / + + fcg-
2 gave lhe oidei ous-14 7/7 fcg-2 O/O unc-51.
ous-15. Unc non-Dpy ieconlinanls picked fion lhe piogeny of dpq-20 nin-6 + unc-30 /
+ + ous-15 + gave lhe oidei dpq-20 3/1O nin-6 1/1O ous-15 6/1O unc-30.
13
ous-16. us non-Dpy ieconlinanls picked fion lhe piogeny of dpq-5 + ous-16 / + spc-9 +
gave lhe oidei dpq-5 1O/13 spc-9 3/13 ous-16.
ous-17. Lon non-Unc ieconlinanls picked fion lhe piogeny of |cn-2 + unc-7 / + ous-17 +
gave lhe oidei |cn-2 11/12 ous-17 1/12 unc-7.
ous-18. Lon non-Unc ieconlinanls picked fion lhe piogeny of unc-42 + |cn-3 / + ous-18
+ gave lhe oidei unc-42 28/34 ous-18 6/34 |cn-3.
14
RE5ULT5
Isn!atInn nf EM5-Induccd mutants rcsIstant tn InfcctInn by M. nematohl|um: WT
voins of lhe slandaid N2 (iisloI) iace of C. c|cgans can le infecled ly feeding on a
nixed lacleiiaI Iavn conlaining |. cc|i OI5O and liaces of M. ncna|cpni|un. They
iespond ly deveIoping a chaiacleiislic svoIIen laiI, easiIy delecled undei lhe dissecling
nicioscope, vhich ve iefei lo as lhe Dai (Defoined AnaI Region) phenolype (Iiguie
1). Dai aninaIs exhilil a sIovei giovlh iale and ieduced fecundily (HODCKIN c| a|.
2OOO, HILICH c| a|. 2OO4). In a suivey of pieviousIy chaiacleiized C. c|cgans nulanls,
il vas found lhal sone of lhe srf nulanls, oiiginaIIy isoIaled on lhe lasis of aIleied
suiface anligenicily, veie iesislanl lo infeclion. SpecificaIIy, nulanls of srf-2, srf-3 and
srf-5 did nol deveIop svoIIen laiIs and exhililed IillIe oi no giovlh inpaiinenl in lhe
piesence of M. ncna|cpni|un (HODCKIN c| a|. 2OOO). We lheiefoie soughl addilionaI
nulanls of lhis lype, ly seIecling foi noinaI giovlh on nixed lacleiiaI Iavns, and
scieening foi alsence of lhe Dai phenolype.
M. ncna|cpni|un does nol noinaIIy kiII ils hosl, lul lhe giovlh inpaiinenl il
causes is sufficienl lo peinil eslalIishnenl of seni-seIeclive condilions foi lhe isoIalion
of iesislanl nulanls. The foIIoving piolocoI vas used foi lhe isoIalion of nosl of lhe
chenicaIIy-induced nulanls iepoiled heie: popuIalions of C. c|cgans heinaphiodiles
veie nulagenized vilh LMS and giovn foi lvo geneialions on |. cc|i. The popuIalion
vas lhen liealed ly lIeaching vilh aIkaIine hypochIoiile and lhe iesuIling eggs veie
aIIoved lo halch on a nixed Iavn of |. cc|i and M. ncna|cpni|un. Undei such
condilions, halched Iaivae noinaIIy aII lecone infecled ly L2/L3 slage, and deveIop a
15
Dai (svoIIen laiI) phenolype. RapidIy gioving voins veie easiIy olseived on lhese
Iavns aflei lvo days, and nosl of lhen did nol have svoIIen laiIs. We iefei lo lhis as
lhe us (acleiiaIIy Un-SvoIIen) phenolype, since noinaI laiI analony is lhe nosl
easiIy olseived aspecl of lhe iesislance phenolype. Candidale iesislanl nulanls veie
picked individuaIIy lo nixed Iavn pIales, lo veiify lheii phenolype. Iuilhei genelic
chaiacleiizalion, sunnaiized leIov, ieveaIed lhal nosl of lhese nulanls defined nev
genes, lo vhich ve have given lhe nane ous.
A lolaI of 37 independenl iesislanl nulanls veie isoIaled fion luIk scieens of
lhis naluie. In addilion, six nulanls veie isoIaled fion cIonaI scieens, in vhich
individuaI voins fion an I
2
popuIalion aflei nulagenesis veie seIfed foi one day on
|. cc|i and lhen liansfeiied lo nixed Iavn pIales. These scieens veie caiiied oul vilh
lhe inlenlion of finding nulanls vilh enhanced suscepliliIily lo M. ncna|cpni|un (D.
W. and }. H.), lul lhe scieens aIso yieIded sone appaienlIy iesislanl nulanls. CIonaI
scieens of lhis lype offei lhe oppoilunily of idenlifying nulanls vhich have non-
svoIIen laiIs in lhe piesence of lhe palhogen, lul aie olheivise conpionised in giovlh
oi iesislance lo olhei aspecls of palhogenic allack. In conliasl, lhe luIk scieen is liased
lovaids nulanls vilh sliong iesislance and fev pIeioliopic defecls. Hovevei, none of
lhe six nulanls isoIaled fion lhe piIol cIonaI scieens appeaied iniliaIIy lo le veiy
inpaiied in giovlh oi novenenl.
Resislanl nulanls veie idenlified al high fiequency in lhese seaiches, suggesling
lhal nuIlipIe diffeienl genes can le nulaled lo yieId a us phenolype. On aveiage,
each nulagenized heinaphiodile yieIded al Ieasl one iesislanl nulanl in hei I
2

16
piogeny. In lhe luIk scieens, oflen nany ous nulanls veie iecoveied fion lhe piogeny
of a given I
O
, lul onIy one vas ielained foi each conpIenenlalion gioup, lo ensuie
independence. The six independenl nulanls found in cIonaI scieens veie deiived fion
639 picked I
2
voins. Consislenl vilh lhese high fiequencies, il vas found lhal lhe
43 nulanls defined 17 diffeienl Ioci (TalIe 1).

Isn!atInn nf mut-7-Induccd rcsIstant mutants: In addilion lo lhese seaiches foi LMS-
induced nulanls, scieens foi iesislanl nulanls veie caiiied oul using lhe nulaloi
sliain nu|-7. This sliain is de-ieguIaled foi geinIine liansposilion of seveiaI diffeienl
C. c|cgans liansposons, so nulanls isoIaled on lhis lackgiound aie IikeIy lo iesuIl fion
liansposon inseilions in lhe ieIevanl genes (KLTTINC c| a|. 1999). Tiansposon-lagging
of genes offeis a polenliaIIy iapid ioule foi lheii noIecuIai idenlificalion, as opposed lo
convenlionaI posilionaI cIoning (COLLINS c| a|., 1987). Mulalion fiequencies in nu|-7
popuIalions aie Iovei lhan aflei LMS nulagenesis, and isoIaled nulanls fiequenlIy do
nol lieed liue. Theiefoie, a diffeienl piolocoI vas used foi lhe isoIalion of iesislanl
nulanls fion a nu|-7 sliain. IndividuaI heinaphiodiles veie picked fion a nu|-7
popuIalion lo sepaiale pIales spiead vilh nixed Iavns, and lheii piogeny exanined foi
Dai phenolype. AInosl aII segiegaled onIy Dai aninaIs in lhe fiisl lvo geneialions,
lheiely eslalIishing independenl sensilive Iines. These popuIalions veie lhen
piopagaled foi successive geneialions, passaging lo fiesh pIales vhen lhe suppIy of
lacleiiaI food vas exhausled. IIales veie scieened peiiodicaIIy foi lhe appeaiance of
17
nany us aninaIs. IopuIalions giovn in lhis vay aie undei suslained seIeclion foi
iesislanl nulanls, lecause any us aninaI viII have a giovlh advanlage ovei ils
silIings, and ils piogeny viII lend lo lake ovei lhe popuIalion. Suslained piopagalion
of an unnulagenized WT popuIalion on nixed Iavns iesuIled in lhe isoIalion of one
sponlaneous us nulanl, denonslialing lhe effecliveness of Iong-lein seIeclion foi
iesislance (lhis vas found lo le an aIIeIe of ous-1). Mosl nu|-7 popuIalions evenluaIIy
yieIded a us nulanl, vilh lhe exceplion of a fev popuIalions lhal died oul in Iale
passages, piesunalIy as a iesuIl of accunuIaled exlianeous nulalions. The slailing
popuIalions veie aII unifoinIy sensilive, so lhe diffeienl nulanls iepiesenl
independenl evenls.
In lhe couise of lhese scieens, il lecane appaienl lhal sone Ioci appeai lo le hol-
spols (piefeienliaIIy nulalIe Ioci) foi nu|-7-induced nulalion lo a us phenolype, in
pailicuIai ous-1 and ous-10 (iespecliveIy 1O and 16 aIIeIes veie isoIaled). Theiefoie,
aII slalIe nulanls isoIaled fion lhe nu|-7 scieen veie fiisl conpIenenlalion-lesled
againsl lhese lvo genes, lefoie fuilhei anaIysis. Lighl addilionaI ous-1 sliains and 15
addilionaI ous-10 sliains veie idenlified lul nol given aIIeIe nunleis oi ielained foi
fuilhei anaIysis, as il seened unIikeIy lhal lhey vouId piovide fuilhei infoinalion.
The facl lhal lvo-lhiids of lhe Iines iecoveied fion lhe nu|-7 scieen caiiied
aIIeIes of eilhei ous-1 oi ous-10 suggesls lhal lhese Ioci nay le piefeiied laigels foi
liansposilion. An aIleinalive expIanalion is lhal lhese nulalions iepiesenl fiequenl
inpiecise excisions fion siIenl liansposons aIieady piesenl in lhe lvo genes in lhe
slailing popuIalion of lhe nulaloi sliain. Hovevei, lhis expIanalion can le excIuded
18
in lhe case of ous-1, lecause sulsequenl noIecuIai anaIysis of lhis gene has shovn lhal
lhe 1O ielained nu|-7 aIIeIes aII caiiy diffeienl inseilion evenls, eilhei of Tc1 oi of Tc3
(M. }. C.-N and }. H., in piepaialion). The nu|-7 Iine used in lhese expeiinenls nay
aIso exhilil sone piefeience foi liansposilion inlo sone chionosonaI iegions, lecause
nosl (61/74) of lhe nulalions iecoveied veie on LCIV oi LCV.
A lolaI of 74 slalIe us sliains veie iecoveied fion lhe nulaloi scieen (TalIe 1).
In addilion, a nunlei of Iines exhililing a veak oi vaiialIe us phenolype veie
iecoveied, lul lhese pioved lo le noie difficuIl lo anaIyse oi nap genelicaIIy, and viII
nol le fuilhei discussed heie.

GcnctIc charactcrIzatInn: Mulanls veie ciossed al Ieasl once againsl WT (noie
exlensiveIy in lhe case of nu|-7 aIIeIes) and lesled foi chionosonaI Iinkage. AInosl aII
nulanls veie found lo lehave as sinpIe MendeIian iecessives. The onIy significanl
exceplion vas lhe sur-2 nulalion c2706, vhich exhililed a naiked naleinaI iescue
effecl: c2706/+ piogeny of c2706 honozygous nolheis ciossed vilh WT naIes exhililed
a veak us phenolype.
Mulalions veie assigned lo diffeienl Ioci ly conpIenenlalion lesling vilh genes
napped lo lhe sane chionosone. Lxlensive conpIenenlalion lesling lelveen aIIeIes
of lhe sane gene and of diffeienl genes look pIace duiing lhe assignnenl of lhe 118
nulalions lo 2O Ioci, lul aInosl no cases of inliagenic conpIenenlalion, oi conveiseIy
non-aIIeIic non-conpIenenlalion, veie olseived. One case of anonaIous non-
19
conpIenenlalion vas olseived, in lhal aninaIs of genolype ous-3(c2696)/+, ous-
17(c2695)/+ aie sonelines us, aIlhough lhe lvo Ioci conceined aie unIinked.
Ioi nosl of lhe nine genes vilh nuIlipIe independenl nulalions, aII aIIeIes
appeaied supeificiaIIy siniIai in piopeilies. Lxceplions veie a lenpeialuie-sensilive
aIIeIe of srf-3 (desciiled in noie delaiI ly HILICH c| a|. 2OO4) and sone aIIeIes of ous-
5, vhich exhililed dislinclive diffeiences in culicIe fiagiIily, as desciiled leIov, one
ous-5 aIIeIe vas aIso veakIy lenpeialuie-sensilive foi us phenolype.
Mosl Ioci (12/2O) idenlified in lhis voik aie defined ly a singIe nulalion, and
lhe nunlei of nulalions does nol foIIov a Ioisson disliilulion, even aIIoving foi hol-
spols (piefeienliaIIy nulalIe Ioci). Il foIIovs fion lhis lhal lhese scieens aie fai fion
saluialed, so nuneious fuilhei genes affecling sensilivily lo M. ncna|cpni|un avail
discoveiy.

Thc spcctrum nf EM5 mutants and mut-7 mutants Is substantIa!!y dIffcrcnt: olh
LMS and nu|-7 scieens yieIded nuIlipIe nulalions in seven genes (srf-2, srf-3, ous-1,
ous-2, ous-4, ous-6, ous-10) lul nosl genes veie idenlified onIy in one scieen (TalIe 1).
This indicales lhal lhe specliun of nulanls is significanlIy diffeienl lelveen lhe lvo
scieens, even lhough neilhei has ieached saluialion. Such diffeience is scaiceIy
suipiising, given lhal liansposon inseilion is IikeIy lo pioduce eilhei no phenolype
(inseilion inlo an inlion) oi an exliene Ioss-of-funclion phenolype. Weak vialIe
nulalions in essenliaI genes aie unIikeIy lo le iecoveied aflei liansposon nulagenesis
and aie noie IikeIy lo le iecoveied aflei chenicaI nulagenesis. Cenes such as ous-5
20
(eighl LMS aIIeIes, no nu|-7 aIIeIes) nay faII inlo lhis cIass. The gene ous-8, defined ly
a singIe LMS aIIeIe, is denonslialIy of lhis lype, lecause a non-conpIenenlalion scieen
foi fuilhei aIIeIes yieIded nuIlipIe addilionaI nulalions, sone of vhich aie IelhaI as
honozygoles (}. H., unpulIished iesuIls).

PhcnntypIc charactcrIzatInn: grnwth In thc abscncc nf pathngcn: Refeience nulanls
foi each of lhe 2O Ioci veie giovn on noinaI |. cc|i Iavns and conpaied vilh WT foi
diffeiences in giovlh iale, vialiIily and feiliIily, as veII as olhei phenolypic
chaiacleiislics such as noiphoIogy and Ioconolion, vilh iesuIls sunnaiized in TalIe
2. None shoved olvious defecls in giovlh iale oi feiliIily, apail fion cg|-5, vhich
shoved a sliong egg-Iaying defecl and uncooidinaled novenenl, Iike nosl olhei cg|-5
nulanls. The sur-2 nulanl exhililed an egg-Iaying defecl and sone vuIvaI induclion
alnoinaIilies (Hin, oi hypeiinduced, phenolype) al Iov lenpeialuies, as iepoiled
eIsevheie (NICHOLAS and HODCKIN 2OO4). One nulanl, ous-18, exhililed sIov
giovlh and sonevhal ieduced lody size. SIighl and vaiialIe ieduclion in size vas
aIso noliced in sone of lhe olhei nulanls (ous-4, ous-13, ous-16).
A nunlei of nulanls shoved a dislinclive novenenl defecl, iefeiied lo as lhe
'skiddy phenolype, chaiacleiized ly noinaI sinusoidaI novenenl of lhe lody lul
appaienlIy pooi liaclion on lhe agai suiface. ConsequenlIy, aninaIs nake ieIaliveIy
IillIe foivaid piogiess foi each Ioconoloiy vave lhal passes dovn lhe lody. The
skiddy phenolype is conspicuous in ous-16, ous-17 and ous-18 nulanls, and aIso
deleclalIe in srf-3, ous-5 and ous-8. Il is coiieIaled vilh culicIe alnoinaIily, as
21
desciiled fuilhei leIov. CulicuIai aIae, vhich aie leIieved lo le inpoilanl in
pioviding liaclion, appeai giossIy noinaI in lhese nulanls.
A noie dislinclive veak coiIei uncooidinaled phenolype vas seen in ous-3
aninaIs. This phenolype co-segiegaled vilh lhe us phenolype duiing exlensive
ciosses, so is IikeIy lo le caused ly lhe sane nulalion, lul il is conceivalIe lhal il aiises
fion a dislincl lighlIy Iinked nulalion. Conpaiison of nuIlipIe aIIeIes vouId iesoIve
lhis queslion, lul onIy one ous-3 aIIeIe has leen idenlified lhus fai.
One nulanl, ous-16, exhililed a phenolype of vuIvaI iupluie al oi soon aflei lhe
Iasl IaivaI noIl (Rup), in aloul 4O of individuaIs. A siniIai phenolype vas seen al
Iovei peneliance (<1O) in sone of lhe olhei nulanls (ous-13, ous-18).

Ma!c phcnntypcs: MaIe phenolypes veie exanined ly consliucling nin-5 oi nin-8
sliains foi each of lhe 2O nulanls. The cg|-5 nulanl exhililed seveieIy alnoinaI naIe
laiI deveIopnenl and vas conpIeleIy defeclive in naIe naling, consislenl vilh ils
genolype. Olhei nulanls did nol shov olvious aIleialions in naIe analony, and aII
veie capalIe of naling, aIlhough in sone cases Iess efficienlIy lhan WT. The ous-18
nulanl naIes had veiy Iov naling efficiency, vhich can le expIained ly lheii
ieIaliveIy snaII size and pooi novenenl.

Grnwth In thc prcscncc nf pathngcn: Sliains veie giovn lolh on nixluies of |. cc|i
and M. ncna|cpni|un (ML pIales), and on puie Iavns of M. ncna|cpni|un, al 2O`C and
25`C. As expecled fion lhe nelhod of isoIalion, aII nulanls exhililed a conpIele oi
22
aInosl conpIele alsence of laiI-sveIIing on nixed Iavns, in conliasl lo lhe fuIIy
penelianl Dai phenolype of WT voins, and nosl nulanls giev nuch noie vigoiousIy
lhan WT on such Iavns, vilh lhe exceplion of sur-2. OccasionaI individuaIs of sone
sliains, such as ous-12, exhililed a sIighl laiI sveIIing (veak Dai phenolype), as
indicaled in TalIe 2 and iIIuslialed in Iiguie 1 (paneIs L and I).
Ciovlh on puie Iavns vas sIovei lhan on nixed Iavns, in aII cases. Mosl
nulanls giev noie iapidIy lhan WT on puie Iavns of lhe palhogen, even nulanls vilh
significanl pIeioliopic alnoinaIilies such as cg|-5, ous-16 and ous-18. A conspicuous
exceplion, again, vas lhe sur-2 nulanl, vhich exhilils veiy pooi giovlh on puie M.
ncna|cpni|un. In sone, lul nol aII, expeiinenls ous-14 voins giev pooiIy ieIalive lo
WT. Moie nulanls exhililed a Ieaky phenolype (vaiialIe veak laiI sveIIing) on puie
Iavns lhan on nixed Iavns, nolalIy ous-5, ous-8, ous-12, ous-13, ous-15.
WT naIe voins aie noie seveieIy affecled lhan WT heinaphiodiles ly M.
ncna|cpni|un (M.}.C-N. and }.H., in piepaialion). The iesponse of ous naIes lo
infeclion has nol yel leen syslenalicaIIy exanined, lul vheie lesled, lolh sexes appeai
lo le siniIaiIy iesislanl.

Tcsts fnr cn!nnIzatInn by thc pathngcn: Woins exhililing a us nulanl phenolype
nighl le un-svoIIen lecause of a faiIuie in lhe iniliaI adheience of lacleiia lo lhe
suiface of lhe voin, oi lecause of an alnoinaIily in lhe sulsequenl iesponse lo
infeclion. In oidei lo dislinguish lelveen lhese possiliIilies, WT and nulanl voins
veie giovn on nixed lacleiiaI Iavns, vashed in oidei lo ienove non-adheiing |. cc|i
23
and lo conpIele digeslion of ingesled lacleiia, and slained liiefIy vilh lhe fIuoiescenl
vilaI dye SYTO 13. This pioceduie aIIovs visuaIizalion of adheienl lacleiia in lhe
Iiving voins. WT voins infecled vilh M. ncna|cpni|un exhilil sliong ieclaI and posl-
anaI fIuoiescence, iefIecling exlensive coIonizalion of lhe ieclun ly lhe palhogen,
vheieas uninfecled voins exhilil no fIuoiescence (Iiguie 3).
Repiesenlalive aIIeIes of 2O diffeienl nulanls veie exanined ly lhis pioceduie,
vilh iesuIls sunnaiized in TalIe 3 and Iiguie 3. The nulanls faII ioughIy inlo foui
gioups: lhiee nulanls (cg|-5, sur-2, ous-14) shoved sliong ieclaI fIuoiescence,
conpaialIe lo WT, vhich suggesls lhal lhese nulanls aie inpaiied in iesponse lo lhe
palhogen, ialhei lhan lhe iniliaI infeclion. Tvo nulanls (ous-12, ous-15) exhililed
sulslanliaI ieclaI infeclion, lul Iess lhan WT, suggesling lhal lolh infeclion and
iesponse aie ieduced. Six nulanls shoved veiy Iov oi vaiialIe ieclaI fIuoiescence (srf-
3, ous-2, ous-3, ous-5, ous-8, ous-13), suggesling lhal sone lacleiiaI adheience can occui,
lul nol enough lo eslalIish a fuII infeclion oi eIicil a sliong iesponse. The ienaining
nine nulanls exhililed essenliaIIy no ieclaI fIuoiescence, indicaling lhal no infeclion
had occuiied. The nosl IikeIy inleipielalion of lhe Iasl lvo cIasses is lhal lhese
nulanls have aIleialions in ieclaI culicIe piopeilies lhal pievenl oi inpaii lhe iniliaI
adheience ly infecling lacleiia. AddilionaI and noie geneiaI aIleialions in lhe culicIe
piopeilies of sone of lhese nulanls aie aIso suggesled ly lhe olseivalion lhal seveiaI
nulanls (srf-3, srf-5, ous-3, ous-12, ous-13, ous-14, ous-16, ous-18) exhilil occasionaI
palches of lacleiiaI adheience eIsevheie on lhe oulside of lhe voins, vhich aie nevei
seen in WT voins, an exanpIe is shovn in Iiguie 3I (ous-16). One nulanl, ous-12,
24
sonelines exhililed an unusuaI pallein of sliong eclopic lacleiiaI allachnenl, lul onIy
in aduIl naIes.

LcctIn staInIng: The dala in lhe pievious seclion suggesl lhal lhe najoiily of
nulanls iepoiled heie aie iesislanl lo M. ncna|cpni|un lecause lhe lacleiia aie unalIe
lo allach lo lhe ieclaI oi anaI culicIe, and lheiefoie cannol iniliale an infeclion. This
faiIuie lo adheie couId le due lo aIleied suiface piopeilies of lhe voin culicIe, eilhei
IocaIIy oi ovei lhe vhoIe lody. Such aIleialions have aIieady leen denonslialed in
lhe case of lhe lhiee srf nulanls (LINK c| a|. 1992), vhich shov changes in lhe linding
of lolh anlilodies and IaleIed Ieclins lo lhe suiface of lhe voin. We lheiefoie
exanined Ieclin-linding piopeilies of lhe nulanls isoIaled in lhis voik, lesling one oi
lvo aIIeIes foi each of lhe lvenly Ioci idenlified as affecling suscepliliIily lo M.
ncna|cpni|un. Six Ieclins veie lesled, as desciiled in noie delaiI in MATLRIALS AND
MLTHODS.
As expecled fion pievious olseivalions (LINK c| a|. 1992), srf-2 and srf-5 aduIl
nulanls exhililed sliongei linding of WCA and SA, as conpaied lo WT. None of
lhe olhei nulanls shaied exaclIy lhis phenolype, and none exhililed consislenl sliong
slaining vilh any of lhe olhei foui Ieclins. Hovevei, dislinclive palleins of slaining
veie olseived vilh a nunlei of lhe ienaining nulanls. SeveiaI nulanls exhililed
incieased lul vaiialIe and palchy slaining, nosl nolicealIy vilh SA lul sonelines
aIso vilh olhei Ieclins. This phenolype is chaiacleiislic of srf-3, lul a siniIai and oflen
sliongei phenolype vas seen vilh ous-8, ous-16, ous-17 and ous-18. One of lvo lesled
25
aIIeIes of ous-5 shoved lhis phenolype (c2701) lul lhe olhei did nol (c2688). This
pallein of Ieclin slaining is coiieIaled vilh culicIe fiagiIily, as desciiled leIov. The
fiagiIily vas sonelines aIso diieclIy appaienl in lhese slaining expeiinenls, lecause
lhe oulei Iayei of lhe aduIl culicIe lended lo disinlegiale duiing lhe piocess of
nounling lhe voins foi nicioscopy, and vas sonelines sIoughed off (Iiguie 4C).
Thiee nulanls (ous-13, ous-14, ous-15) had olhei palleins of alnoinaI Ieclin
slaining. The ous-13 nulanl exhililed incieased lul vaiialIe slaining ly nosl of lhe six
Ieclins lesled (aII lul WCA), vilh slaining lending lo le sliongesl aIong lhe aIae (IaleiaI
iidges lhal iun aIong lhe sides of lhe aninaI), as iIIuslialed in Iiguie 4 (paneIs I,}). Il is
possilIe lhal lhis effecl iesuIls fion non-specific liapping of Ieclins on lhe culicIe
suiface, ialhei lhan a noie specific inleiaclion, lul il sliII indicales lhal lhe suiface of
ous-13 voins is significanlIy diffeienl fion WT. SiniIaiIy, sone ous-14 nulanl
aninaIs exhililed incieased and palchy slaining vilh SA, ILA and LLA Ieclins, lul
lhe peneliance of lhis phenolype vas Iov. IinaIIy, ous-15 nulanls couId le IaleIed
vilh SA in lhe head, aIlhough lhe iesl of lhe lody vas unslained (Iiguie 4L).
Laivae of lhese sliains veie aIso exanined aflei slaining vilh lhe sane sel of six
Ieclins, and sone fuilhei diffeiences fion WT slaining veie olseived, nosl nolalIy
incieased slaining of ous-12 Iaivae ly WCA. AIso, lolh WCA and SA lind veakIy lo
lhe culicIe of WT Iaivae, and sone nulanls (ous-1, ous-4, ous-15) shoved a possilIe
ieduclion of linding of lhese lvo Ieclins in Iaivae. Olseivalions aie sunnaiized in
TalIe 4.
26
AIleied suiface piopeilies in sone of lhese nulanls nay aIso expIain lhe
alnoinaI skiddy novenenl phenolype lhal lhey exhilil, desciiled alove. Skiddiness
is seen lo a giealei oi Iessei degiee in nany of lhe nulanls vilh Ieclin slaining
phenolypes.

CutIc!c fragI!Ity: SeveiaI olhei phenolypes, in addilion lo lhe Ieclin-slaining dala
desciiled alove, indicaled lhal sone of lhese nulanls aie defeclive in culicIe inlegiily.
Il has leen iepoiled lhal dauei Iaivae of srf-3 nulanls aie Iess iesislanl lo deleigenl
liealnenl. We found lhal sone of lhe ous nulanls exhililed nolalIy pooi suivivaI on
slaived oi desiccaled pIales. The vasl najoiily of C. c|cgans nulanls ienain vialIe foi
veeks on slandaid NCM agai pIales, Iong aflei lacleiiaI food has leen exhausled, and
even vhen lhe pIale has diied dovn lo haIf oi Iess of ils oiiginaI voIune. Woins
usuaIIy iecovei iapidIy aflei 'chunk liansfei lo a fiesh pIale vilh a lacleiiaI Iavn
(SULSTON and RLNNLR, 1988). In conliasl, sone of lhe ous and srf nulanls died oul
undei desiccalion condilions.
The ieduced suivivaI of lhese sliains vas coiieIaled vilh incieased sensilivily lo
aIkaIine hypochIoiile liealnenl. The slandaid lIeach sleiiIizalion pioceduie foi
cIeaning conlaninaled voin slocks invoIves picking a fev giavid heinaphiodiles lo a
diop of aIkaIine hypochIoiile (see MATLRIALS AND MLTHODS). Woins iapidIy
slop noving and sulsequenlIy lieak up and dissoIve, Ieaving lehind onIy deliis and
suiface-sleiiIized eggs. Ieach-sensilive voins exhilil a naikedIy veakei iesislance
lo lhis liealnenl: on exposuie lo aIkaIine hypochIoiile lhey slop noving aInosl
27
inslanlIy, and slail lo disinlegiale nuch faslei lhan WT voins. The line in seconds lo
lieak-up piovides a convenienl neasuie of lIeach-sensilivily, and aIIovs lhe nulanls
lo le giouped inlo foui cIasses (TalIe 5). Aloul haIf of lhe nulanls shoved IillIe oi no
diffeience fion WT, lul lhe ienaindei veie dislinclIy noie sensilive, eilhei niIdIy
(ous-2, ous-13), sliongIy (srf-3, ous-8) oi veiy sliongIy (ous-5, ous-16, ous-17, ous-18).
Wheie lesled, diffeienl aIIeIes of a given gene usuaIIy exhililed siniIai
sensilivily, lul aIIeIes of ous-5 veie sliikingIy heleiogeneous: foui aIIeIes veie
hypeisensilive (c2685, c2701, c2704, c2801) vheieas foui veie onIy sIighlIy noie
sensilive lhan WT (c2686, c2688, c2699, c2794). AII eighl shoved siniIai iesislance lo M.
ncna|cpni|un infeclion, hovevei.
Diug sensilivily of sone nulanls vas aIso exanined, lo expIoie lhe possiliIily of
noie iapid peinealion of diugs inlo lhe aninaI. Consislenl vilh lhis, ous-16 and ous-
18 nulanls veie found lo le unalIe lo giov on pIales conlaining concenlialions of
eneline oi coIchicine (O.5 - 1 nM) lhal did nol pievenl giovlh of WT voins.
Hovevei, no diiecl neasuienenls of diug uplake oi culicIe peinealiIily have yel leen
nade.
Iuilhei evidence of culicIe fiagiIily vas piovided ly naking doulIe nulanls
vilh o|i-1(c769). This and olhei o|i nulanls exhilil fIuid fiIIed lIisleis on lhe suiface of
lhe culicIe al lhe aduIl slage, vhich has a doulIe-Iayeied culicIe and can lheiefoie
lecone lIisleied ly fIuid accunuIalion lelveen lhe culicIe Iayeis. DoulIe nulanls
such as ous-16, o|i-1 veie found lo deveIop onIy snaII lIisleis, piesunalIy lecause
Iaigei lIisleis iupluie as a iesuIl of veakness in lhe oulei culicIe Iayei. This phenolype
28
coiieIales vilh lhe disinlegialion and sIoughing of lhe culicIe seen in sone nulanls
aflei Ieclin slaining (ous-8, ous-16, ous-17, ous-18).
The vuIvaI iupluie (Rup) phenolype noled in sone of lhe culicIe nulanls,
nolalIy ous-16, nay aIso iesuIl fion culicIe fiagiIily, lul lhis phenolype seens lo occui
al high fiequency onIy in ous-16, and is nol olviousIy coiieIaled vilh lhe ieIalive
fiagiIily of diffeienl nulanls.


29
DI5CU55ION
This papei iepoils lhe iesuIls of an iniliaI suivey foi nulanls aIleied in iesponse
lo infeclion ly M. ncna|cpni|un. We have shovn lhal il is easy lo find such nulanls,
and lhal a Iaige nunlei of genes aie invoIved.
The najoiily of nulanls veie isoIaled ly seIeclion lolh foi iesislance lo lhe
deliIilaling effecls of lhe palhogen, and foi alsence of lhe dislinclive svoIIen laiI
phenolype caused ly infeclion. Many of lhese nulanls appeai lo le iesislanl lecause
lhe lacleiiun is unalIe lo adheie lo lhe culicIe and iniliale an infeclion. Ioi
nenalodes, lhe culicIe iepiesenls lhe fiisl and nosl geneiaI Iine of defense againsl
lacleiiaI allack, and ils suiface piopeilies aie ceilain lo le ciilicaI in deleinining
vhelhei nany diffeienl lacleiia aie alIe lo acl as palhogens oi olheivise inleiacl vilh
a hosl species.
A fev nulanls lhal aie alIe lo peinil lhe eslalIishnenl of infeclion, lul Iack lhe
sveIIing iesponse, veie ollained. Hovevei, il appeais IikeIy lhal nosl such nulanls
aie aIso hypeisensilive lo lhe palhogen, as iIIuslialed ly lhe sur-2 nulanl (NICHOLAS
and HODCKIN 2OO4, lhis papei), and lheiefoie nol diieclIy seIeclalIe. The cg|-5 nulanl
is anonaIous in lhis iegaid, in lhal ils giovlh is ieIaliveIy uninpaiied ly lhe palhogen
despile ieclaI infeclion, lul lhis nay le a consequence of lhe nuIlipIe aIleialions caused
ly defecls in lhis gene, vhich encodes a Hox piolein. Iuilhei nulanls defeclive in
iesponse aie leing soughl in cIonaI scieens such as lhe one lhal yieIded lhe sur-2
nulanl, ialhei lhan in diiecl seIeclions.
30
Diiecl seIeclions foi iesislanl nulanls do nol appeai lo le saluialed, as noled in
lhe lexl, even lhough fifleen nev Ioci have leen idenlified lhus fai. Diffeienl seIeclion
piolocoIs and diffeienl nulagens viII piolalIy yieId yel noie cIasses of nulanls,
indeed, expeiinenls using Mos liansposon nulagenesis (LSSLRLAU c| a|. 2OO1) have
aIieady defined fuilhei ous genes (K.}.Y. and }.H., unpulIished iesuIls). Il is aIso IikeIy
lhal a Iaige cIass of veakIy iesislanl nulanls exisl, vhich exhilil a ieduced oi vaiialIe
iesponse lo M. ncna|cpni|un, dislinguishalIe lolh fion lhe slandaid Dai phenolype
exhililed ly WT voins, and fion lhe iolusl us phenolype of lhe nulanls desciiled
heie. Sone nulanls of lhis lype veie iecoveied fion lhe nu|-7 scieen, lul lhey have
nol yel leen fuilhei invesligaled. Such veak nulanls aie necessaiiIy haidei lo voik
vilh, lul nay le infoinalive aloul olhei aspecls of lhe lioIogy of lhis hosl-palhogen
inleiaclion.
The nulanls sludied so fai can le lenlaliveIy pIaced in diffeienl gioups, on lhe
lasis of lhe diffeienl piopeilies sel oul in TalIes 3 - 5. Ioi exanpIe, nulalions of srf-3,
ous-5, ous-8, ous-16, ous-17, ous-18 aII iesuIl in ialhei siniIai phenolypes, vilh no ieclaI
coIonizalion, incieased Ieclin slaining, culicIe fiagiIily and 'skiddy novenenl.
Hovevei, il vouId le pienaluie lo cIassify lhese six genes logelhei, in lhe alsence of
fuilhei infoinalion aloul lheii piopeilies. Ioi nosl of lhen, lhe nuII phenolype is as
yel unceilain, and il is aIieady cIeai lhal nulalions of ous-8, ous-16 and ous-18 can have
nuch noie seveie consequences lhan nuII nulalions of srf-3. Iiopei cIassificalion
nusl avail cIoning and noIecuIai anaIysis of lhe ieIevanl Ioci.
31
The najoiily of nulanls exhilil IillIe oi no accunuIalion of lacleiia in lhe
ieclun vhen exposed lo M. ncna|cpni|un, and aIso exhilil addilionaI phenolypes lhal
suggesl lhal culicIe piopeilies have leen aIleied. The sinpIesl expIanalion foi
iesislance in lhese nulanls is lhal lhe culicIe suiface has leen aIleied in such a vay lhal
M. ncna|cpni|un can no Iongei adheie lo lhe culicuIai Iining of lhe ieclun, and is
lheiefoie unalIe lo iniliale an infeclion. Consislenl vilh lhis idea, noIecuIai
chaiacleiizalion of srf-3 has shovn lhal il encodes a nucIeolide sugai lianspoilei and
appeais lo le defeclive in suiface gIycosyIalion (HILICH c| a|. 2OO4, CIIOLLO c| a|.,
2OO4).
Hovevei, sone of lhe nulanls do nol have olviousIy aIleied culicIe. This nay
le lecause oui assays foi suiface aIleialion viII nol delecl alnoinaIilies such as sullIe
changes in gIycosyIalion oi olhei suiface piopeilies. Il is aIso possilIe lhal lhe culicIe
is aIleied onIy in lhe iegion of infeclion, as opposed lo lhe vhoIe lody suiface.
NeveilheIess, olhei expIanalions foi lhe faiIuie lo eslalIish a ieclaI coIony can le
consideied: foi exanpIe, il nay le lhal lhe palhogen needs lo delecl signaIs fion lhe
hosl in oidei lo aclivale lhe infeclion piocess, and lhal lhese nulanls aie defeclive in
pioducing such signaIs.
Il is significanl lhal lhe najoiily of lhe nulanls ve have exanined no Iongei
peinil ieclaI coIonizalion ly lhe palhogen and do nol exhilil a Dai phenolype even
vhen giovn on a puie Iavn of M. ncna|cpni|un. This suggesls lhal lhe signaI oi
signaIs lhal liiggei lhe sveIIing iesponse acl onIy ovei a shoil iange, oi aie onIy
enilled ly lacleiia oi danaged lissues aflei successfuI infeclion. Ioinalion of a ieclaI
32
coIony of a sufficienl size (a quoiun) couId le an essenliaI slep in eIiciling lhe iesponse,
lecause sone of lhe nulanls did exhilil significanl adheience of lacleiia lo lhe ieclun,
lul nuch Iess lhan seen in WT oi sur-2 nulanls.
Tesling foi a us phenolype is a convenienl, sinpIe and iolusl assay, vhich can
le used effecliveIy in lhe anaIysis and cIoning of genes lhal affecl lhis iesponse. Ioi
exanpIe, M. ncna|cpni|un and lhe nev srf-3 aIIeIes isoIaled in lhis voik veie essenliaI
in lhe noIecuIai cIoning and chaiacleiizalion of srf-3 (HILICH c| a|. 2OO4). These
scieens have aIso sulslanliaIIy incieased lhe nunlei of aIIeIes of srf-2 and srf-5, and lhe
us phenolype is IikeIy lo le equaIIy usefuI in lhe fuilhei invesligalion of lhese genes.
The facl lhal nosl of lhe nulanls iepoiled heie affecl adheience ly M.
ncna|cpni|un and aie eilhei piolalIy oi denonslialIy aIleied in suiface piopeilies
neans lhal lhey nay aIso le significanlIy aIleied in iesponse lo olhei palhogens lhal
allack nenalodes lhiough an iniliaI suiface allachnenl slep. Il has aIieady leen
iepoiled lhal srf-2, srf-3 and srf-5 nulanls aie incieased in suscepliliIily lo lhe
nenalopalhogenic fungus Dudding|cnia f|agrans (MLNDOZA DL CIVLS c| a|. 1999).
ConveiseIy, lhe sane lhiee nulanls aie iesislanl lo foinalion of a suiface liofiIn on
lhe head ly Ycrsinia spp. (DARY c| a|. 2OO2, TAN and DARY 2OO4). Lxaninalion of
sone of lhe olhei nulanls iepoiled heie has shovn lhal a sulsel of lhe ous nulanls aie
iesislanl lo Ycrsinia liofiIn allachnenl (CRLC DARY, peisonaI connunicalion).
Olhei suiface palhogens of C. c|cgans nay aIso shov changes in inleiaclion vilh ous
nulanls.
33
Moieovei, lhe isoIalion of lhese nulanls sulslanliaIIy incieases lhe nunlei of
genes knovn lo affecl lhe suiface of C. c|cgans. Iievious scieens have uliIized suiface
anligenicily and/oi Ieclin slaining lo idenlify such genes, Ieading lo lhe definilion of
nine srf genes, onIy one of vhich has leen cIoned lo dale (LINK c| a|. 1992, IOLITZ c| a|.
1987). Al Ieasl nine of lhe fifleen ous genes iepoiled heie appeai lo affecl lhe culicIe in
one vay oi anolhei, and lheii fuilhei invesligalion shouId piovide nev infoinalion on
lhe deveIopnenl and piopeilies of nenalode culicIe, vhich is one of lhe defining
fealuies of lhe phyIun and a najoi ieason foi ils gieal evoIulionaiy success. A
nunlei of lhe ous nulanls aie cIeaiIy aIleied in nechanicaI and sliucluiaI piopeilies of
lhe culicIe, exhililing alnoinaI 'skiddy novenenl, fiagiIily and incieased sensilivily
lo chenicaIs and diugs. They piovide a nev neans of Iooking al lhe inleiface lelveen
nenalodes and lheii suiiounding enviionnenl.
The najoiily of lhe nulanls do nol exhilil any olvious najoi aIleialions in
phenolype, olhei lhan in iesponse lo M. ncna|cpni|un. Many of lhen neveilheIess
piolalIy iesuIl fion conpIele Ioss-of-funclion in lhe gene conceined, and lheiefoie
lhey aie nenleis of lhe Iaige sel of C. c|cgans genes foi vhich knockoul has no olvious
effecl. olh cIassicaI and ieveise genelic appioaches (such as RNAi) indicale lhal ovei
7O of C. c|cgans genes faII inlo lhis sel. As discussed eIsevheie (HODCKIN 2OO1), il
nay le lhal lhe funclions of nany of lhese genes peilain lo liolic inleiaclions such as
lhal vilh M. ncna|cpni|un, and lhal in oidei lo undeisland lhen, il viII le necessaiy lo
have a lellei undeislanding of lhe naluiaI ecoIogy of C. c|cgans, and lo idenlify noie
dedicaled palhogens Iike M. ncna|cpni|un.
34




ACKNOWLEDGEMENT5
This voik vas suppoiled ly lhe MedicaI Reseaich CounciI UK, ly an NalionaI
Inslilules of HeaIlh feIIovship lo K.}.Y., and ly a ConnonveaIlh SchoIaiship lo H.R.N.
Sone of lhe sliains uliIized in lhis sludy veie ollained fion lhe Caenoihaldilis
Cenelics Cenlei, vhich is funded ly lhe NalionaI Inslilules of HeaIlh (NIH) NalionaI
Cenlei foi Reseaich Resouices (NCRR). We lhank Cieg Daily foi connunicaling
unpulIished dala and Iieddie Iailiidge foi connenls on lhe nanusciipl.
35
TABLE 1
Isn!atInn nf mutants
Cene LMS -induced aIIeIes nu|-7-induced aIIeIes TolaIs
srf-2 c2679, c2703, c2793, c2799, c2718, c2729, c2730, c2734, 5 , 11
c2833 c2766, c2767, c2768, c2771,
c2777, c2778, c2787
srf-3 c2680, c2689, c2797 c2789 3, 1
srf-5 c2697 - 1, O

sur-2 e2706 - 1, O
cg|-5 - e2725 O, 1

ous-1 e2678, c2681, c2682, c2683, c2713, c2720, c2721, c2738, 6, 18
c2684, c2692 c2743, c2746, c2750, c2751,
c2757, c2758
ous-2 c2676, c2677, e2687, c2705 c2776, c2780, c2781 4, 3
ous-3 e2696 - 1, O
ous-4 e2693, c2700, c2803 c2752, c2788 3, 2
ous-5 c2685, c2686, c2688, c2699, - 8, O
e2701, c2704, c2794, c2801
ous-6 c2690, e2691 c2728, c2756, c2759, c2772, 2, 5
c2790
36
TABLE 1 cnntd
Isn!atInn nf mutants
Cene LMS -induced aIIeIes nu|-7-induced aIIeIes TolaIs
ous-8 e2698 1, O
ous-10 c2694, e2702 c2714, c2715, c2716, c2717, 2, 31
c2719, c2722, c2723, c2724,
c2731, c2732, c2733, c2736,
c2737, c2742, c2745, c2753
ous-12 - e2740 O, 1
ous-13 e2710 - 1, O
ous-14 - e2779 O, 1
ous-15 e2709 - 1, O
ous-16 e2802 - 1, O
ous-17 c2695, e2800 - 2, O
ous-18 e2795 - 1, O
-----
43, 74
______________________________________________________________________________

AIIeIes in loId veie chosen as iepiesenlalive foi each Iocus, and used in fuilhei
chaiacleiizalion.
Nol aII isoIaled aIIeIes of ous-1 and ous-10 veie ielained and assigned aIIeIe nunleis.
37
TABLE 2
Mutant charactcrIstIcs and grnwth In thc abscncc and prcscncc nf pathngcn

Sliain Iuie |. cc|i Mixed Iavn Iuie M. ncn
N2 WT Dai Dai

srf-2(qj262) Sif us us
srf-3(qj10) Sif, sI Skd us us
srf-5(c|115) Sif, sI Cio us us

sur-2(c2706) LgI-cs, Hin-cs us (pooi giovlh) us (pooi giovlh)
cg|-5(c2725) LgI, Unc us us

ous-1(c2678) WT us us
ous-2(c2687) WT us us
ous-3(c2606) SI Unc us us
ous-4(c2693) SI Sna us us
ous-5(c2701) SI Skd us us/sI Dai
ous-6(c2728) WT us us
ous-8(c2698) SI Skd us/v sI Dai us/sI Dai
ous-10(c2702) WT us us
ous-12(c2740) WT us/sI Dai us/sI Dai
38
TABLE 2 cnntd.
Mutant charactcrIstIcs and grnwth In thc abscncc and prcscncc nf pathngcn

Sliain Iuie |. cc|i Mixed Iavn Iuie M. ncn
ous-13(c2716) Raie Rup us/v sI Dai us/sI Dai
ous-14(c2779) WT us us/sI Dai
ous-15(c2709) WT us us/sI Dai
ous-16(c2802) Skd, Rup us us
ous-17(c2800) Skd us us
ous-18(c2795) Sna, Skd, Cio, Rup us/sI Dai us/sI Dai
______________________________________________________________________________
Allievialions: Skd, skiddy, Hin, vuIvaI hypeiinduced, Rup, iupluied, Cio, sIov
giovlh, sI, sIighl, v sI, veiy sIighl. Ioi olhei allievialions, see lexl oi MATLRIALS
AND MLTHODS.




39
TABLE 3
Tcsts fnr rccta! cn!nnIzatInn by vIta! dyc staInIng fnr bactcrIa

Sliain ReclaI Olhei Connenl
slaining slaining____________________
N2 +++ O

srf-2(qj262) O O
srf-3(qj10) + ++ Sone geneiaI suiface slaining
srf-5(c|115) O + Sone aIae slaining

sur-2(c2706) +++ O
cg|-5(c2725) +++ O

ous-1(c2678) O O
ous-2(c2687) + O
ous-3(c2606) + +
ous-4(c2693) O O
ous-5(c2701) + O Raie ieclaI slaining
ous-6(c2728) O O OccasionaI veak slaining
ous-8(c2698) + O
ous-10(c2702) O O
40
TABLE 3 cnntd.
Tcsts fnr rccta! cn!nnIzatInn by vIta! dyc staInIng fnr bactcrIa

Sliain ReclaI Olhei Connenl
slaining slaining____________________
ous-12(c2740) ++ + AlnoinaI naIe slaining
ous-13(c2716) + + VaiialIe slaining
ous-14(c2779) +++ +
ous-15(c2709) ++ O
ous-16(c2802) O + Ialches on culicIe
ous-17(c2800) O O
ous-18(c2795) O + Iie- and posl-anaI palches
______________________________________________________________________________
Legend: O = no lacleiiaI slaining, + = deleclalIe slaining, ++ = sone slaining, +++ =
sliong slaining.
41
TABLE 4
LcctIn staInIng

Sliain WCA SA TIA ILA CON LLA
N2 - - - - - -

srf-2(qj262) ++ ++ - - - -
srf-3(qj10) V V V V - V
srf-5(c|115) ++ ++ - - - -

cg|-5(c2725) - - - - - -
sur-2(c2706) - - - - - -

ous-1(c2678) - - - - - -
ous-2(c2687) - - - - - NL
ous-3(c2606) - - - - - -
ous-4(c2693) - - - - - -
ous-5(c2688) - - - - - -
ous-5(c2701) V - - - - V
ous-6(c2728) - - - - - -
ous-8(c2698) V V - V V V
ous-10(c2702) - - - - - -
42
TABLE 4 cnntd.
LcctIn staInIng

Sliain WCA SA TIA ILA CON LLA
ous-12(c2740) - - - - V -
ous-13(c2716) - A A A A A
ous-14(c2779) - V - - - V
ous-15(c2709) H H - H - NL
ous-16(c2802) V V - V V V
ous-17(c2800) - V - V V NL
ous-18(c2795) - V - V V -
______________________________________________________________________________
LLCLND
The six Ieclins used aie desciiled in MATLRIALS AND MLTHODS.
-: no slaining. ++: sliong slaining.
V: vaiialIe, palchy slaining. A: vaiialIe, nainIy aIae slaining. H: vaiialIe, nose/head
slaining. NL, nol exanined.
43
TABLE 5
CutIc!c fragI!Ity

Sliain Ieach sensilivily Connenl

N2 +

srf-2(qj262) +
srf-3(qj10) +++ Iooi Iong-lein suivivaI
srf-5(c|115) +

cg|-5(c2725) +
sur-2(c2706) +

ous-1(c2678) +
ous-2(c2687) ++
ous-3(c2606) +
ous-4(c2693) ++
ous-5(c2688) ++
ous-5(c2701) ++++ Iooi Iong-lein suivivaI
ous-6(c2728) +
ous-8(c2698) +++
44
TABLE 5 cnntd.
CutIc!c fragI!Ity

Sliain Ieach sensilivily Connenl
ous-10(c2702) +
ous-12(c2740) +
ous-13(c2716) ++
ous-14(c2779) +
ous-15(c2709) +
ous-16(c2802) ++++ Iooi Iong-lein suivivaI
ous-17(c2800) ++++ Iooi Iong-lein suivivaI
ous-18(c2795) ++++ Iooi Iong-lein suivivaI
______________________________________________________________________________
LLCLND
Ioi scoiing, see MATLRIALS AND MLTHODS


45

FIGURE LEGEND5
FIGURE 1
InfcctInn nf WT and rcsIstant wnrms.
AII paneIs shov lhe laiI iegion of aduIl heinaphiodiles. (A) Uninfecled WT. ()
Infecled WT vilh veak Dai phenolype. Adheienl lacleiia aie visilIe (aiiov). (C)
Infecled WT vilh sliong Dai phenolype. (D) Infecled ous-1, us phenolype. (L)
Infecled ous-12, us phenolype. (I) Infecled ous-12 vilh veak Dai phenolype, seen in a
ninoiily of aninaIs.

FIGURE 2
GcnctIc map.
Linkage gioups (aulosones I - V, and X chionosone) aie shovn in slandaid veilicaI
foinal, vilh scaIe lai indicaling 5 cenliMoigans. Zeio cooidinale foi each Iinkage
gioup is shovn as a lai lo lhe Iefl of each veilicaI Iine. Appioxinale posilions of genes
sludied in lhis papei (five pieviousIy napped, 15 nev) aie as shovn.

FIGURE 3
Tcsts fnr cn!nnIzatInn: 5YTO 13 staInIng.
IaneIs A-L shov lhe laiI iegion of aduIl heinaphiodiles aflei infeclion, exlensive
vashing lo ienove |. cc|i ceIIs, and liief slaining vilh SYTO 13 lo ieveaI infecling
lacleiia (gieen fIuoiescence). (A) WT: exlensive ieclaI coIonizalion and sliong Dai
46
phenolype. () ous-13. liace infeclion and veiy veak Dai iesponse. (C) ous-4. no
coIonizalion and no Dai iesponse. (D) ous-14: sliong coIonizalion and no Dai iesponse.
(L) sur-2. sliong coIonizalion and no Dai iesponse. (I) Head iegion of aduIl ous-16
heinaphiodile, shoving palchy adheience of lacleiia lo lhe culicIe suiface.

FIGURE 4
LcctIn staInIng.
(A) and (): WT voins aflei slaining vilh SA and WCA, iespecliveIy, Iong exposuie.
No suiface slaining is seen, onIy fainl lackgiound aulofIuoiescence fion lhe gul. (C)
SA-slained srf-2, vuIvaI iegion: sliong suiface slaining , especiaIIy of vuIva and aIae.
(D) WCA-slained srf-5. sliong slaining. (L) SA-slained ous-15. specific slaining of nose.
(I) WCA-slained ous-8. palchy suiface slaining. (C) SA-slained ous-16, shoving
disinlegialion of oulei culicIe. (H) WCA-slained ous-16. sliong palchy suiface slaining.
(I) TIA-slained ous-13. aIae slaining. (}) ConA-slained ous-13. aIae slaining. In paneIs
C, I and }, aIae aie indicaled vilh an aiiov.
47
LITERATURE CITED

AALLAY, A., I. YORCLY and I.M. AUSULL, 2OOO Sa|ncnc||a |qpninuriun
pioIifeiales and eslalIishes a peisislenl infeclion in lhe inlesline of Cacncrnaodi|is
c|cgans. Cuii ioI. 10: 1539-1542.

LSSLRLAU, }.L., A. WRICHT, D.C. WILLIAMS, K. SCHUSKL, M.W. DAVIS and
L.M. }ORCLNSLN LM, 2OO1 MoliIizalion of a DiosophiIa liansposon in lhe
Cacncrnaodi|is c|cgans gein Iine. Naluie 413: 7O-74.

RLNNLR, S., 1974 The genelics of Cacncrnaodi|is c|cgans. Cenelics 77: 71 - 94.

CIIOLLO, }.I., A.M. AWAD, C.L. COSTLLLO and C.. HIRSCHLRC, 2OO4 srf-3, a
nulanl of Cacncrnaodi|is c|cgans, iesislanl lo lacleiiaI infeclion and lo liofiIn linding, is
deficienl in gIycoconjugales. } ioI Chen. 279: 52893-529O3

COLLINS, }., . SAARI and I. ANDLRSON, 1987 Aclivalion of a liansposalIe eIenenl
in lhe gein Iine lul nol lhe sona of Cacncrnaodi|is c|cgans. Naluie 382: 726 -728.

COUILLAULT, C. and }.}. LWANK (2OO2) Diveise lacleiia aie palhogens of
Cacncrnaodi|is c|cgans. Infecl Innun. 70: 47O5-47O7.

48
DARY, C., C.L. COSMA, }.H. THOMAS and C. MANOIL, 1999 LelhaI paiaIysis of
Cacncrnaodi|is c|cgans ly Pscudcncnas acrugincsa. Iioc NalI Acad Sci U S A. 96: 152O2-
152O7.

DARY, C., }. W. HSU, N. CHORI and S. IALKOW, 2OO2 Cacncrnaodi|is c|cgans: pIague
lacleiia liofiIn lIocks food inlake. Naluie 417: 243-244.

CRAVATO-NORL, M. }. and }. HODCKIN, 2OO5 Cacncrnaodi|is c|cgans as a nodeI foi
innale innunily lo palhogens. CeIIuIai MiciolioI. 7: 741-751.

HODCKIN, }., 2OO1 Whal does a voin vanl vilh 2O,OOO genes` Cenone ioI. 2: 2OO8.1
- 2OO8.4

HODCKIN, }., 1997 Appendix 1: Cenelics, pp. 881-1O47 in C. c|cgans ||, ediled ly DL.
RIDDLL, T. LUMLNTHAL, .}. MLYLR and }.R. IRILSS. CoId Spiing Hailoi
Laloialoiy, CoId Spiing Hailoi, NY.

HODCKIN, }., I. L. KUWAARA and . CORNLLIUSSLN, 2OOO A noveI lacleiiaI
palhogen, Micrcoac|criun ncna|cpni|un, induces noiphoIogicaI change in lhe nenalode
C. c|cgans. Cuii ioI. 10: 1615-1618.

49
HILICH, }., I. LRNINSONL, C. COLLL, M. }. CRAVATO-NORL, . }. LIY, C.
DARY, S. M. IOLITZ, }. HODCKIN, C. . HIRSCHLRC, and R. AUMLISTLR, 2OO4
Loss of srf-3 encoded nucIeolide sugai lianspoilei aclivily in Cacncrnaodi|is c|cgans aIleis
suiface anligenicily and pievenls lacleiiaI adheience. } ioI Chen. 279: 3O44O-3O448.

HUIIMAN, D.L., L. ARAMI, R. SASIK, }. CORLIL, I. C. VAN DLR COOT and R.V.
AROIAN, 2OO4 Milogen-aclivaled piolein kinase palhvays defend againsl lacleiiaI
poie-foining loxins. Iioc NalI Acad Sci U S A. 101: 1O995-11OOO.

KLTTINC, R.I., T. H. HAVLRKAMI, H.C. VAN LULNLN AND R.H.A. ILASTLRK,
1999. Mul-7 of C. eIegans, iequiied foi liansposon siIencing and RNA inleifeience, is a
honoIog of Weinei syndione heIicase and RNaseD. CeII 99: 133-141.

KIM, D.H., R. ILINAUM, C. ALLOINC, I.L. LMLRSON, D.A. CARSIN, H. INOUL,
M. TANAKA-HINO, N. HISAMOTO, K. MATSUMOTO, M.W. TAN and I.M.
AUSULL, 2OO2. A conseived p38 MAI kinase palhvay in Cacncrnaodi|is c|cgans innale
innunily. Science 297: 623-626.

KIM, D.H., N.T. LILRATI, T. MIZUNO, H. INOUL, N. HISAMOTO, K. MATSUMOTO
and I.M. AUSULL, 2OO4 Inlegialion of Cacncrnaodi|is c|cgans MAIK palhvays
nedialing innunily and sliess iesislance ly MLK-1 MAIK kinase and VHI-1 MAIK
phosphalase. Iioc NalI Acad Sci U S A. 101: 1O99O-1O994.
50

LAROUSSL, A., S. CHAUVLT, C. COUILLAULT, C.L. KURZ and }.}. LWANK, 2OOO
Cacncrnaodi|is c|cgans is a nodeI hosl foi Sa|ncnc||a |qpninuriun. Cuii ioI. 10: 1543-
1545.

LINK, C.D., M.A. SILVLRMAN, M. RLLN, K.L. WATT and S. A. DAMLS, 1992
Chaiacleiizalion of Cacncrnaodi|is c|cgans Ieclin-linding nulanls. Gcnc|ics 131: 867-881

MAHA}AN-MIKLOS, S., M.W. TAN, L.C. RAHML and I.M. AUSULL, 1999
MoIecuIai nechanisns of lacleiiaI viiuIence eIucidaled using a Pscudcncnas acrugincsa-
Cacncrnaodi|is c|cgans palhogenesis nodeI. CeII 96: 47-56.

MALLO, C.V., C.L. KURZ, C. COUILLAULT, N. IU}OL, S. CRAN}LAUD, Y.
KOHARA, and }.}. LWANK, 2OO2 InducilIe anlilacleiiaI defense syslen in C. c|cgans.
Cuii ioI. 12: 12O9-1214.

MARROQUIN L.D., D. LLYASSNIA, }. S. CRIIIITTS, }. S ILITLLSON and R. V.
AROIAN, 2OOO. 8aci||us |nuringicnsis (l) loxin suscepliliIily and isoIalion of
iesislance nulanls in lhe nenalode Cacncrnaodi|is c|cgans. Cenelics 155: 1693-1699.

MILLLT, A.C. and }.}. LWANK, 2OO4 Innunily in Cacncrnaodi|is c|cgans. Cuii Opin
InnunoI. 16: 4-9.
51

MLNDOZA DL CIVLS, I.M., K.C. DAVILS, S.}. CLARK and }.M. LHNKL, }.M., 1999
Iiedaloiy lehavioui of liapping fungi againsl srf nulanls of Cacncrnaodi|is c|cgans and
diffeienl pIanl and aninaI paiasilic nenalodes. Parasi|c|cgq 119: 95-1O4.

NICHOLAS, H. R. and }. HODCKIN, 2OO4 The LRK MAI kinase cascade nediales laiI-
sveIIing and a pioleclive iesponse lo ieclaI infeclion in C. c|cgans. Cuiienl ioIogy 14:
1256 - 1261.

IOLITZ, S.M., K.}. CHIN and D.L. HLRMAN, 1987 Cenelic anaIysis of aduIl-specific
suiface anligenic diffeiences lelveen vaiielies of lhe nenalode Cacncrnaodi|is c|cgans.
Cenelics 117: 467-476.

IRADLL, L. and }.}. LWANK, 2OO4 Cenelic nodeIs in palhogenesis. Annu. Rev.
Cenel. 38: 347-363.

SCHULLNURC, H. and }.}. LWANK, 2OO4 Diveisily and specificily in lhe inleiaclion
lelveen Cacncrnaodi|is c|cgans and lhe palhogen Scrra|ia narccsccns. MC LvoI ioI. 4:
49.

SCHULLNURC, H., C.L. KURZ and }.}. LWANK, 2OO4 LvoIulion of lhe innale
innune syslen: lhe voin peispeclive. InnunoI Rev. 198: 36-58.
52

SIIRI, C.D., }. LCUN, I.M. AUSULL, and S.. CALDLRWOOD, 2OO3. Cacncrnaodi|is
c|cgans as a nodeI hosl foi S|apnq|cccccus aurcus palhogenesis. Infecl. Innun. 71: 22O8-
2217.

SIIRI, C.D., }. LCUN and I.M. AUSULL, 2OO5 The voin has luined - nicioliaI
viiuIence nodeIed in Cacncrnaodi|is c|cgans. Tiends MiciolioI. 13: 119-127.

SULSTON, }. L., and }. HODCKIN, 1988 Melhods, pp. 587-6O6 in Tnc Ncna|cdc
Cacncrnaodi|is c|cgans, ediled ly W. . WOOD. CoId Spiing Hailoi Laloialoiy, CoId
Spiing Hailoi, NY.

TAN, L., and C. DARY, 2OO4 A novalIe suiface: foinalion of Ycrsinia sp. liofiIns on
noliIe Cacncrnaodi|is c|cgans. } acleiioI. 186: 5O87-5O92.

Figure 1: Infection of wild-type and resistant worms
WT (uninfected)
A
WT
C
bus-12
E
WT
B
bus-1
D
bus-12
F
Figure 2: Genetic map
II
V X
bus-13
bus-6
bus-1
bus-14
bus-5
bus-8
srf-5
bus-17
I
bus-3
srf-2
sur-2
bus-16
III
egl-5
IV
bus-2
bus-4
bus-10
srf-3
bus-15
bus-12
bus-18
5 cM
Figure 3: Tests for colonization: SYTO 13
staining
bus-13
B
bus-4
C
bus-14
D
sur-2
E
WT
A
bus-16
F
Figure 4: lectin staining
WT - SBA
A
WT - WGA
B
srf-2 - SBA
C
srf-5 - WGA
D
bus-15 - SBA
E
bus-8 - WGA
F
bus-16 - SBA
G
bus-16 - WGA
H
bus-13 - TPA
I
bus-13 - ConA
J