北海道大学	 大学院農学院	 修士論文発表会,2014 年 2 月 7 日,10 日	 	 	  Study	 of	 four	 symbiosis	 genes	 required	 for	 root-knot	 nematode	 
infestation	 	  	  -ネコブ線虫感染に必要な４つの共生遺伝子に関する研究-	 	 

応用生物科学専攻	  生命分子化学講座	  植物細胞制御学	  林	  周平	 	 	 1.Background	 &	 Aim	 	 Root-knot	 nematodes	 (RKNs)	 are	 obligate	 plant	 parasites	 that	 collapse	 plant	 root.	 RKNs	 
modify	 plant	 root	 cells	 into	 giant	 cells	 (GCs)	 for	 feeding.	 This	 reprogramming	 step	 is	 
important	 to	 obtain	 nutrient	 because	 RKNs	 take	 nutrient	 from	 the	 GCs.	 However,	 the	 detail	 molecular	 mechanism	 which	 RKNs	 establish	 and	 maintain	 feeding	 area	 is	 unknown.	 
On	 the	 other	 hand,	 there	 are	 microbes	 that	 give	 advantages	 on	 taking	 nutrient	 for	 plant.	 
Rhizobia	 and	 arbuscular	 mycorrhizal	 fungi	 (AMF)	 provide	 host	 plant	 nitrate	 and	 phosphate	 
respectively,	 are	 well-known	 symbiosis	 microbes	 with	 plant.	 Recent	 study	 has	 revealed	 
that	 these	 symbiotic	 interaction	 are	 controlled	 by	 specific	 genes	 called	 as	 symbiosis	 
genes	 in	 plant	 and	 there	 are	 independent	 signalling	 pathway	 for	 symbiosis	 respectively,	 
but	 some	 of	 genes	 are	 necessary	 for	 both	 rhizobia	 and	 AMF	 symbiosis.	 Considering	 RKNs	 
still	 can	 infest	 despite	 RKNs	 are	 plant	 parasites,	 it	 is	 hypothesised	 that	 RKNs	 can	 invade	 and	 infest	 plant	 by	 mimic	 or	 hijack	 this	 symbiosis	 process.	 The	 aim	 of	 this	 
research	 is	 to	 verify	 the	 hypothesis	 and	 investigate	 RKN	 infestation	 mechanism.	 2.Materials	 &	 Methods	 
Lotus	 japonicus	 Gifu	 Wild-type,	 and	 symrk,	 castor,	 ccamk,	 nsp2	 mutants	 which	 are	 defective	 in	 symbiosis	 genes	 and	 RKN	 (Meloidogyne	 hapla)	 were	 used	 for	 infestation	 
assay	 in	 this	 study.	 The	 number	 of	 RKN	 egg	 mass	 8	 week	 post	 infestation	 (wpi)	 are	 counted	 for	 checking	 if	 there	 is	 difference	 of	 completely	 infestation	 phenotype,	 and	 the	 number	 of	 RKNs	 categorised	 in	 several	 growth	 stages	 2	 wpi	 and	 4	 wpi	 were	 counted	 for	 determining	 which	 timing	 RKNs	 infestation	 arrested.	 And	 also	 gall	 
sectioning	 of	 WT,	 ccamk	 and	 nsp2	 at	 2	 wpi	 were	 carried	 out	 for	 observation	 of	 
inside	 gall	 structure.	 	 3.Result	 &	 Discussion	 	 The	 number	 of	 egg	 mass	 at	 8	 wpi	 significantly	 reduced	 in	 symrk,	 castor,	 ccamk,	 and	 
nsp2.	 And	 also	 the	 number	 of	 well-grown	 RKN	 at	 4	 wpi	 was	 reduced	 in	 the	 mutants,	 though	 many	 RKNs	 that	 partially	 grew	 were	 observed	 at	 2	 wpi	 in	 the	 mutants.	 Induction	 of	 GCs	 
was	 observed	 in	 WT,	 ccamk	 and	 nsp2.	 Direct	 connection	 between	 xylem	 and	 GCs	 was	 observed	 
in	 WT,	 however	 indirect	 connection	 was	 observed	 in	 the	 ccamk	 and	 nsp2.	 	 4.Summary	 	 These	 results	 suggest	 that	 infestation	 of	 RKNs	 in	 ccamk	 and	 nsp2	 mutants	 were	 arrested	 
because	 xylem	 connection	 with	 GCs	 was	 indirect	 and	 it	 caused	 malnutrition	 of	 RKNs	 inside	 plant.	 And	 also	 it	 is	 suggested	 that	 these	 symbiosis	 genes	 are	 not	 necessary	 for	 
recognition	 of	 RKNs	 or	 RKNs	 don’t	 mimic	 the	 symbiosis	 pathway,	 but	 have	 important	 roles	 for	 development	 of	 feeding	 sites,	 especially	 xylem	 connection	 with	 GCs	 during	 RKNs	 
infestation.	 Additional	 analysis	 of	 gall	 sectioning	 of	 symrk	 and	 castor	 is	 also	 expected	 to	 have	 difference.