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!