How close are we to nitrogen-fixing cereals?

1. How close are we to nitrogen-fixing cereals? MyriamCharpentier and Giles Oldroyd (2010) 組員: 彭元慶、林柏齡、郭宇翔、陳傑君

2. Introduction • Meeting the demand for higher yields • Decrease the chemical ferterlizer

3. Introduction • Root nodule symbiosis (RNS) is restricted to four related orders within the Eurosidclade of angiosperms • Establishing nitrogen-fixing symbiosis in cereals will probably require additional genetic engineering for bacterial colonisation and nodule organogenesis.

4. Early recognition • plant root flavonoids activate the bacterial production and secretion of lipochito-oligosaccharide Nod factors • Nod factors are perceived by LysM-receptor-like kinase (LysM-RLKs)

5. Nod factors • 類黃酮化合物　→　根瘤菌細胞內膜的NodD　→lipochitooligosaccharides(Nod factors) • Nod gene nodA至 nodX 1.共同結瘤基因，nodABDIJ 2.寄主專一性基因nodEFL, nodMNT, nodO 3. 結瘤的調節基因，nodD基因 • 豆科植物根上的凝集素也能夠識別根瘤菌的Nod facetors，表面的酸性胞外多糖(EPS)和脂多糖(LPS)

8. LysM-receptor-like kinase (LysM-RLKs) • Nodfactorreceptor • 结合Nod factor中N-乙酰葡萄糖胺(N-acetyl glucosamine)分子结构的 LysM(lysin motif domain)功能域。 • NFR1和NFR5(L. japonicus) (Medicagotruncatula) • Mt LYK (Medicagotruncatula)

9. Lotus japonicus Lotus japonicus is a wild legume that belongs to family Fabaceae. Members of this family are very diverse, constituting about 20,000 species. They are of significant agricultural and biological importance as many of the legume species are rich sources of protein and oil and can also fix atmospheric nitrogen.

10. Medicagotruncatula Medicagotruncatula (Barrel Medic or Barrel Medick or Barrel Clover) is a small legume native to the Mediterranean region that is used in genomic research. It is a low-growing, clover-like plant 10–60 cm tall with trifoliate leaves. Each leaflet is rounded, 1–2 cm long, often with a dark spot in the center. The flowers are yellow, produced singly or in a small inflorescence of 2-5 together; the fruit is a small spiny pod.

11. Arbuscularmycorrhizal, AMAM共生體 • 真菌Glomeromycota • 嚴格寄生 • 80% 以上的植物 (除了十字花科、石竹科、藜科、蓼科和莎草科) • 多個可能共同參與AM與RNS訊息傳導基因 • AM 比根瘤菌共生起源還早

12. AM共生體形成 • “Branching factors, BFs”促進AM真菌孢子萌發，菌絲生長與分支 • 類黃酮物質促進AM真菌孢子萌發，菌絲生長與分支 • 從Lotus japonicus發現Strigolactone (5-deoxy-strigol) • 植物BFs 　→　孢子分支向寄主延伸

13. Myc factors • AM真菌所釋放的信號分子 • Chitin oligomers

14. ＡＭ菌根形成過程 土壤中萌發的胞子 植物與真菌雙方信號感知 附著胞的形成，和穿透 菌絲蔓延，叢枝形成

15. AM　與　RNS相同的共生途徑!? • 根瘤菌與　AM真菌享有一些相同的共生途徑 • Nod factors 　→　 NFR1　和　NFR5 (receptor) → SYMRK (Symbiosis receptor-like kinase)類似基因 →下游基因 　在 Medicagotruncatula, 三個突變體基因dmi1、dmi2和dmi3 　在 Lotus japonicus中，九個基因　LjSYMRK、LjCASTOR、LjPOLLUX、LjSYM3、LjSYM6、LjSYM15、LjSYM24、LjNUP133 和LjNUP85

16. symbiotic components(SYMs) pathway gene • (一). 含有Leucine rich repeat的受體激酶 (Kinase) LjSYMRK、MsNORK (Nodule receptor –like kinase) 和 MtDMI2 (does not make infection) • 蛋白質結構中有一個細胞外的受體，透過特殊訊號結合後將會磷酸化下游基因產生訊息傳遞

17. symbiotic components(SYMs) pathway gene • (二).　陽離子通道蛋白，如　LjCASTOR、LjPOLLUX 和MtDMI1 Ｋ+離子進出平衡

18. symbiotic components(SYMs) pathway gene • (三)Calcium/calmodulin-dependent protein kinase, (CCaMK) 如如MtCCaMK/MtDMI3 Ca2+ & CaM　→　CCaMK 蛋白酶自體激化 →　下游調控表現

19. symbiotic components(SYMs) pathway gene • 核孔蛋白如　LjNUP133 　、　LjNUP85　和　NENA • ？？？？

20. symbiotic components(SYMs) pathway gene • Others: CYCLOPS 受到CCAMK調控 促使真菌 or 根瘤菌 繼續感染

21. Mac factors Nod factors LjNFR1 MtLYK LjNFR1 MtDMI 2 SYMRK NORK 細胞膜 MtDM1 LjCASTOR LjPOLLOX LjNUP133 LjNUP85 NENA 質體 細胞核模 Ca2+ 濃度震盪 CYCLOPs MtNSP1 MtNSP2 ERN 細胞核內 MtDMI3 CCaMK 根瘤共生體 AM共生體

23. EF-hand motifs

25. Early recognition

26. Bacterial entry • Legumes have developed two major rhizobial invasion processes 1.root-hair dependent (75%) : Lotus.Japonicus(百脈根屬) Medicago. Truncatula(截形苜蓿) 2.root-hair independent infection (25%) • some legumes can potentially operate both mechanisms

27. Bacterial entry Bacterial entry Root hair infection Lateral root base Nodal root base

28. Bacterial entry • Additionalentry receptors • recognition of bacterial surface polysaccharides • IT(infection thread) initiation and develpment • cytoskeleton reorganisation RHI

29. Bacterial entry Genes affected in the IT initiation and progression • E3 ubiquitinligase is necessary for IT initiation • SCAR/ WAVE complex PIR1 and NAP1 are essential for mediating actin rearrangement • VAPYRIN(dual) and RPG containing putative protein–protein interaction domains • Ex : vapyrin loss-of-function mutant RHI

30. Bacterial entry • Silencing SYMRK : nodules containing ITs, but limited or no bacterial release • transformation of the Nod factor receptors : infection threads formed, but bacterial release was not efficient or sustained • implies additional ligand–receptor complexes are necessary for sustained IT development and bacterial release RHI

31. Bacterial entry spatial regulation (lipid-raft localized protein) • Flotillins • Plant specific remorin (MtSYMREM1) Animal Plant

32. Bacterial entry • Nod factor dependent • In Sesbania rostrata CCaMK is necessary, while SYMRK is not required • new signalling pathway to activate CCaMK • Ethylene, GA and reactive oxygen species play essential positive roles during crack entry NRB

33. Bacterial entry • Bradyrhizobium ORS278 can invade the rice species Oryzabreviligulata • Bradyrhizobium proliferate in the fissures caused by protruding lateral roots and reach four to five cell layers deep. • Bradyrhizobium ORS278 also elicits root- and stem-nodules on some Aeschynomene(合萌) species in a NF-independent process. LRB

34. Bacterial entry • nodule-specific cysteine-rich peptides, processed in their active form by a plant peptidase highly expressed in nodules, are required to promote the development of bacteria

35. Bacterial entry nod: nodulation; has: root-hair swelling; hab: root-hair branching; hac: root-hair curling; Iti: IT initiation; Ith: IT growth in the root-hair cell; thi: thick IT; bulb: IT ending with bulbous protrusion; Ite: IT growth in epidermal cells; Itc: IT growth in cortical cells; Npi: bacteria release in nodule primordium; Type I: small bump, nitrogen fixation ineffective; Type II: rare pale pink nodule; n.d: not determined; myc-:defective in arbuscularmycorrhiza

36. Bacterial entry

37. Nodule organogenesis • The processes of bacterialinfection and nodule organogenesis are geneticallyseparable. Madsen et al., 2010

38. Nodule organogenesis • gain-of-function mutations in CCaMK lead to the development of spontaneous nodule formation in the absence of rhizobia Tirichine L et al., 2006 CCaMK EMS 點突變 snf1 (spontaneous nodule formation)mutant of Lotus japonicus, a single amino-acid replacement Autoactivation of the nodulation signalling pathway in the plant, with the resultant induction of nodules and nodulation gene expression in the absence of bacterial elicitation.

39. Nodule organogenesis • gain-of-function mutations in CCaMK lead to the development of spontaneous nodule formation in the absence of rhizobia Gleason C et al., 2006 Tirichine L et al., 2006

40. Nodule organogenesis LHK1 snf2 mutant: gain of function leucine 266 replaced by a phenylalanine

41. Nodule organogenesis • Mutations or silencing the cytokinin receptorsCRE1 in M. truncatula (Gonzalaz et al., 2006 )and LHK1 in L. japonicus (Murray et al.,2007) show impairments in nodule organogenesis without impacting on bacterial infection CRE1: use RNAi LHK1 : use 點突變

42. Nodule organogenesis

43. Nodule organogenesis • Cytokinin is likely to act in concert with auxin to initiate nodulation, with evidence implying that suppression of auxin levels promotes nodulation. • Cytokinin and auxin regulate several plant development programs such as lateral root formation and root meristem maintenance

44. Nodule organogenesis • It is likely that there are nodulation specific cytokinin/auxin responses and understanding these is essential for engineering nodule organogenesis in nonlegumes

45. Concluding remarks Rhizobial bacteria already colonise the rhizosphere of cereals There is sufficient knowledge of engineering the SYM pathway to allow cereal recognition of rhizobial bacteria

46. Concluding remarks Such further engineering processes are likely to be nodule organogenesis and bacterial entry Not knowing the specific responses in changes in auxin and cytokinins that lead to nodulation

47. Concluding remarks • Bacterial entry is still poorly defined • Crack entry is a more realistic target for transfer to cereals

48. Concluding remarks • No need to transfer to cereals every genetic component for nodulation. • Engineering nitrogen-fixing cereals will involve A gradual enhancement of rhizobialcolonisation of the root Stepwise improvements to the efficiency of nitrogen fixation

49. Thanks for attention!