How close are we to nitrogen-fixing cereals? Myriam Charpentier and Giles Oldroyd (2010) 組員 :...
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Transcript of How close are we to nitrogen-fixing cereals? Myriam Charpentier and Giles Oldroyd (2010) 組員 :...
How close are we to nitrogen-fixing cereals?Myriam Charpentier and
Giles Oldroyd (2010)
組員 : 彭元慶、林柏齡、郭宇翔、陳傑君
Introduction
• Meeting the demand for higher yields
• Decrease the chemical ferterlizer
• Root nodule symbiosis (RNS) is restricted to four related orders within the Eurosid clade of angiosperms
• Establishing nitrogen-fixing symbiosis in cereals will probably require additional genetic engineering for bacterial colonisation and nodule organogenesis.
Introduction
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)
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)
LysM-receptor-like kinase (LysM-RLKs)
• Nod factor receptor• 结合 Nod factor 中 N- 乙酰葡萄糖胺 (N-
acetylglucosamine) 分子结构的 LysM (lysin motif
domain) 功能域。• NFR1 和 NFR5 (L. japonicus) (Medicago
truncatula)• Mt LYK (Medicago truncatula)
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.
Medicago truncatula
Medicago truncatula (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.
Arbuscular mycorrhizal, 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
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
Myc factors
• AM真菌所釋放的信號分子• Chitin oligomers
AM菌根形成過程
(a)土壤中萌發的胞子
(b)植物與真菌雙方信號感知
(c)附著胞的形成,和穿透
(d)菌絲蔓延,叢枝形成
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 Medicago truncatula, 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
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
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
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
symbiotic components(SYMs) pathway gene
• 核孔蛋白如 LjNUP133 、 LjNUP85 和 NENA
• ????
symbiotic components(SYMs) pathway gene
• Others: CYCLOPS
受到 CCAMK 調控促使真菌 or 根瘤菌 繼續感染
Nod factors Mac factors
LjNFR1MtLYKLjNFR1
MtDMI 2SYMRKNORK
MtDM1LjCASTORLjPOLLOX
LjNUP133LjNUP85NENA
Ca2+ 濃度震盪
MtDMI3CCaMK
MtNSP1MtNSP2ERN
CYCLOPs
細胞膜
質體 細胞核模
細胞核內根瘤共生體
AM 共生體
EF-hand motifs
Early recognition
• 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
Bacterial entry
Bacterial entryBacterial entryRoot hair infection Lateral root base Nodal root base
• Additional entry receptors• recognition of bacterial
surface polysaccharides• IT(infection thread)
initiation and develpment • cytoskeleton reorganisation
Bacterial entry
RHI
Genes affected in the IT initiation and progression
• E3 ubiquitin ligase 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
Bacterial entry
RHI
• 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
Bacterial entry
RHI
spatial regulation(lipid-raft localized protein)
• Flotillins
• Plant specific remorin (MtSYMREM1)
Bacterial entry
Animal
Plant
• 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
Bacterial entry
NRB
• Bradyrhizobium ORS278 can invade the rice species Oryza breviligulata
• 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.
Bacterial entry
LRB
• 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
Bacterial entry
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 arbuscular mycorrhiza
Bacterial entry
Nodule organogenesis• The processes of bacterial infection and nodule
organogenesis are genetically separable.
Madsen et al., 2010
• 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 theresultant induction of nodules and nodulation gene expression in the absence of bacterial elicitation.
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
Nodule organogenesis
LHK1
snf2 mutant: gain of functionleucine 266 replaced by a phenylalanine
Nodule organogenesis
• Mutations or silencing the cytokinin receptors CRE1 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 infectionCRE1: use RNAi LHK1 : use 點突變
Nodule organogenesis
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
Nodule organogenesis
• It is likely that there are nodulation specific cytokinin/auxin responses and understanding
these is essential for engineering nodule organogenesis in nonlegumes
Nodule organogenesis
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
• 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
Concluding remarks
• Bacterial entry is still poorly defined
• Crack entry is a more realistic target for transfer to cereals
Concluding remarks
• No need to transfer to cereals every genetic component for nodulation.
• Engineering nitrogen-fixing cereals will involve A gradual enhancement of rhizobial
colonisation of the root Stepwise improvements to the efficiency of
nitrogen fixation
Concluding remarks
Thanks for attention!