4. Systems Biology of Forage Grass
Symbiomes and Microbiomes
N
H
O
O
HO O O
O
HO
H
Chemical Formula: C39 H51 NO7
Exact Mass: 645.36655
N
H
N
H
HN
O
N
O
NHO
H
O
O
Chemical Formula: C29 H35 N5 O5
Exact Mass: 533.26382
N
N
O
N
NH2
H2N
Chemical Formula: C12 H17 N5 O
Exact Mass: 247.14331 Janthitrem I
ergovaline
peramine
[M+H]+
248.15022
[M+H]+
646.37238
[M+H]+
534.27002
N
H
O
HO
O
O
H
O
H O
O H
H
Chemical Formula: C42 H55 NO7
Exact Mass: 685.39785
[M+H]+
686.40369
Lolitrem B
N
H
O
O
HO O O
O
HO
H
Chemical Formula: C39 H51 NO7
Exact Mass: 645.36655
N
H
N
H
HN
O
N
O
NHO
H
O
O
Chemical Formula: C29 H35 N5 O5
Exact Mass: 533.26382
N
N
O
N
NH2
H2N
Chemical Formula: C12 H17 N5 O
Exact Mass: 247.14331 Janthitrem I
ergovaline
peramine
[M+H]+
248.15022
[M+H]+
646.37238
[M+H]+
534.27002
N
H
O
HO
O
O
H
O
H O
O H
H
Chemical Formula: C42 H55 NO7
Exact Mass: 685.39785
[M+H]+
686.40369
Lolitrem B
5. 5
• Asexual filamentous fungi (phylum Ascomycota, family Clavicipitaceae) that
form mutualistic symbioses with temperate grasses (subfamily Pooideae)
• Seed transmissible
• Protect host grasses from biotic (e.g. insects and vertebrate herbivores) and
abiotic (e.g. drought) stresses
• Produce several bioactive secondary metabolites in planta
• Evolved from sexual grass choke Epichloë pathogens
Neotyphodium spp. Endophytes
E. festucae
Loss of
sexual state
N. lolii
(c. 29 + 4 Mb)
N. lolii x E. typhina
Interspecific
hybridisation
N. sp. LpTG-2
(c. 55 + 6 Mb)
6. 6
From Endophyte Discovery to Pangenome
Analysis Exploiting Global Genetic Diversity – Endophytes
from Perennial Ryegrass
Genetically similar endophytes have a similar toxin profile and origin
Endophytes with reduced toxicity effects are genetically divergent
from the main group
Selection of novel candidate endophytes based on:
DNA profiles
Geographic origin
Toxin profiles
Endophytes cluster into groups based
on geographical origin and toxin
production
Ability to predict likely toxin production
based on genotypic profile
Genetic similarity
0.12 0.34 0.56 0.78 1.00
Genetic similarity
0.12 0.34 0.56 0.78 1.00
Middle East
Eastern Europe
Northern Europe
Lolitrem B
Peramine
Middle East
Mediterranean
Western Europe
New World
Lolitrem B
Ergovaline
Peramine
Mediterranean
Western Europe
Eastern Europe
Ergovaline
Peramine
Peramine
LpTG-2
N. lolii
LpTG-3
A broadly-applicable approach for discovery of novel endophytes
Janthitrem
7. 7
In vitro cultures of candidate endophytes
Endophyte genotypes confirmation
Long-term cryopreservation of endophyte cultures
Species No. Isolates Examples
N. lolii 70 ST, NEA2, NEA3, NEA5, NEA6, NEA10, 42 novel endophytes
N. coenophialum 43 E34, E6, 22 novel endophytes
LpTG-2 7 NEA4, NEA11, 3 novel endophytes
LpTG-3 5 NEA12, E1
FaTG-2 4 8907 and 3 novel endophytes
FaTG-3 6 NEA21, NEA23
N. uncinatum 1 E81
Total 136
Discovering Genetically Novel Endophytes
A broad-based, germplasm collection of novel, genetically diverse endophytes7
8. 8
E9
G4
ST
C9
NA6
Lp19
AR1
NEA3
Genetic similarity
0.12 0.34 0.56 0.78 1.00
Genetic similarity
0.12 0.34 0.56 0.78 1.00
Middle East
Eastern Europe
Northern Europe
Lolitrem
Peramine
Middle East
Mediterranean
Western Europe
New World
Lolitrem
Ergovaline
Peramine
Mediterranean
Western Europe
Eastern Europe
Ergovaline
Peramine
Peramine
NEA12, 15310,15311
E1
Ef E2368
N. lolii
LpTG-3
NEA10
15335
15441
NEA2
15714
NEA6
15931
F2
A1
NEA11
NEA4
LpTG-2
Over 80 ryegrass endophyte strains sequenced
16 N. lolii
3 LpTG-2
4 LpTG-3
Reference genome construction - ST
Representatives of global diversity of
perennial ryegrass endophytes
Current commercial endophytes
[e.g. AR1, NEA2, NEA3 and NEA4]
New endophytes in pre-commercial development
[e.g. NEA10, NEA11, NEA12]
Within cluster analysis of genetic diversity
- Endophytes from distinct geographical origins
[e.g. ST (Grasslands Samson) – NA6 (Morocco) and C9 (Spain)]
- Endophytes from the same geographical origin
[e.g. NEA12 (France) – 15310 and 15311]
Pangenome Analysis of Endophytes
Pangenome analysis across spectrum of genetic, geographic
and taxonomic diversity of endophytes from perennial ryegrass
8
9. 9
Gene present Gene absent Gene partially present
Pangenome Analysis of Endophytes
Sequence Diversity in Alkaloid Production Genes
Identification of core and flexible genomes in Neotyphodium endophytes
9
10. 10
Establishing Symbiota in Isogenic Hosts
Developing Diverse Perennial Ryegrass Isogenic Host Panel
Host cultivar Characteristics
Number of
TCR
genotypesa
TCR genotype
used for
inoculation
Tolosa Distinct forage type 1 Tol 03
Bronsyn
Standard forage type with robust
endophyte performance
3 Bro 08
Impact Late flowering, dense tillering forage type 3 Imp 04
Meridian Early flowering forage type 1 Mer 05
Barsandra Turf type 1 San 02
Bealey Tetraploid forage type 2 Bea 02
Barsintra Tetraploid forage type 4 Sin 04
Barfest
Intergeneric hybrid between Lolium
species parents
3 Fest 02
Materials for symbiome analysis to dissect
endophyte and grass host effects
10
11. 11
Establishing Symbiota in Isogenic Hosts
Inoculating Novel Endophytes into Perennial Ryegrass Isogenic Host Panel
Establishing defined symbiota to study Gp x Ge effects11
12. 12
Endophyte Transcriptome in Symbiota
Perennial ryegrass symbiota; isogenic background; with/without ST endophyte
6 growth conditions: complete media; Low NO3, Low NH4, Low K, Low PO4 and Low Ca
RNAseq libraries; shoots and roots; sequence reads mapped using BLASTn; plant and endophyte transcripts
Endophyte genic sequence reads only observed in tillers of symbiota
Endophyte transcriptome only in symbiotum shoots
genes
0
50000
100000
150000
200000
250000
300000
350000
Full Ca K NH 4 NO 3 PO 4 Full Ca K NH 4 NO 3 PO 4
Free ST Free ST Free ST Free ST Free ST Free ST Free ST Free ST Free ST Free ST Free ST Free ST
Leaves Roots
Reads mapped to endophyte genes with an overlap >40 bp and a percent identity of greater >98genes
0
50000
100000
150000
200000
250000
300000
350000
Full Ca K NH 4 NO3 PO4 Full Ca K NH4 NO3 PO4
Free ST Free ST Free ST Free ST Free ST Free ST Free ST Free ST Free ST Free ST Free ST Free ST
Shoots Roots
Reads mapped to endophyte genes with an overlap >40 bpand a percent identity of greater >98
13. 13
0
10,000,000
20,000,000
30,000,000
40,000,000
50,000,000
60,000,000
LpL_
FR
F
U
LL1_8
0_40
LpL_
FR
C
a1_
80_40
LpL_
FR
K
1_80_40
LpL_
FR
N
H
1_80
_40
LpL_
FR
N
O
1_80_40
LpL_
FR
P
O
1_80
_40
LpL_
S
T
FU
LL
1_80_40
LpL_
S
T
C
a1_80_40
LpL_
S
T
K
1_80_
40
LpL_
S
T
N
H
1
_80_40
LpL_
S
T
N
O
1_80
_40
LpL_
S
T
P
O
1
_80_40
mito
cp
rRNA
gene
Number of reads mapping to plant genes from leaf libraries
0
10,000,000
20,000,000
30,000,000
40,000,000
50,000,000
60,000,000
LpL_
FR
F
U
LL1_8
0_40
LpL_
FR
C
a1_
80_40
LpL_
FR
K
1_80_40
LpL_
FR
N
H
1_80
_40
LpL_
FR
N
O
1_80_40
LpL_
FR
P
O
1_80
_40
LpL_
S
T
FU
LL
1_80_40
LpL_
S
T
C
a1_80_40
LpL_
S
T
K
1_80_
40
LpL_
S
T
N
H
1
_80_40
LpL_
S
T
N
O
1_80
_40
LpL_
S
T
P
O
1
_80_40
mito
cp
rRNA
gene
Number of reads mapping to plant genes from leaf libraries
Plant Transcriptome in Symbiota
0
10,000,000
20,000,000
30,000,000
40,000,000
50,000,000
60,000,000
LpL_
FR
F
U
LL1_8
0_40
LpL_
FR
C
a1_
80_40
LpL_
FR
K
1_80_40
LpL_
FR
N
H
1_80
_40
LpL_
FR
N
O
1_80_40
LpL_
FR
P
O
1_80
_40
LpL_
S
T
FU
LL
1_80_40
LpL_
S
T
C
a1_80_40
LpL_
S
T
K
1_80_
40
LpL_
S
T
N
H
1
_80_40
LpL_
S
T
N
O
1_80
_40
LpL_
S
T
P
O
1
_80_40
mito
cp
rRNA
gene_sum
Number of reads mapping to plant genes from leaf libraries
0
10,000,000
20,000,000
30,000,000
40,000,000
50,000,000
60,000,000
LpL_
FR
F
U
LL1_8
0_40
LpL_
FR
C
a1_
80_40
LpL_
FR
K
1_80_40
LpL_
FR
N
H
1_80
_40
LpL_
FR
N
O
1_80_40
LpL_
FR
P
O
1_80
_40
LpL_
S
T
FU
LL
1_80_40
LpL_
S
T
C
a1_80_40
LpL_
S
T
K
1_80_
40
LpL_
S
T
N
H
1
_80_40
LpL_
S
T
N
O
1_80
_40
LpL_
S
T
P
O
1
_80_40
mito
cp
rRNA
gene_sum
Number of reads mapping to plant genes from leaf libraries
Number of sequence reads mapped to plant sequences in shoot libraries (2.4 to 20.8 million reads per library)
Counts mapping to genes used to identify endophyte-induced or repressed plant genes in symbiota shoots
Of 918 endophyte-regulated plant genes 68 are differentially regulated in shoots
(51 induced and 15 repressed)
Shoot Transcriptome: Endophyte-Regulated Plant Genes
13
14. 14
Plant Transcriptome in Symbiota
Root Transcriptome: Endophyte-Regulated Plant Genes
Number of sequence reads mapped to plant sequences in root libraries (2.4 to 20.8 million reads per library)
Counts mapping to genes used to identify endophyte-induced or repressed plant genes in symbiota roots
0
5,000,000
10,000,000
15,000,000
20,000,000
25,000,000
30,000,000
LpR
_F
R
F
U
LL1_80_4
0
LpR
_F
R
C
a1_80
_40
LpR
_F
R
K
1_
80_40
LpR
_F
R
N
H
1_80_4
0
LpR
_F
R
N
O
1
_80_40
LpR
_F
R
P
O
1_80_4
0
LpR
_S
TF
U
LL1_8
0_40
LpR
_S
TC
a1
_80_40
LpR
_S
TK
1_80_40
LpR
_S
TN
H
1_8
0_40
LpR
_S
TN
O
1_t80_
40
LpR
_S
TP
O
1_8
0_40
mito
cp
rRNA
gene_
Number of reads mapping to plant genes from root libraries
0
5,000,000
10,000,000
15,000,000
20,000,000
25,000,000
30,000,000
LpR
_F
R
F
U
LL1_80_4
0
LpR
_F
R
C
a1_80
_40
LpR
_F
R
K
1_
80_40
LpR
_F
R
N
H
1_80_4
0
LpR
_F
R
N
O
1
_80_40
LpR
_F
R
P
O
1_80_4
0
LpR
_S
TF
U
LL1_8
0_40
LpR
_S
TC
a1
_80_40
LpR
_S
TK
1_80_40
LpR
_S
TN
H
1_8
0_40
LpR
_S
TN
O
1_t80_
40
LpR
_S
TP
O
1_8
0_40
mito
cp
rRNA
gene_
Number of reads mapping to plant genes from root libraries
0
10,000,000
20,000,000
30,000,000
40,000,000
50,000,000
60,000,000
LpL_
FR
F
U
LL1_8
0_40
LpL_
FR
C
a1_
80_40
LpL_
FR
K
1_80_40
LpL_
FR
N
H
1_80
_40
LpL_
FR
N
O
1_80_40
LpL_
FR
P
O
1_80
_40
LpL_
S
T
FU
LL
1_80_40
LpL_
S
T
C
a1_80_40
LpL_
S
T
K
1_80_
40
LpL_
S
T
N
H
1
_80_40
LpL_
S
T
N
O
1_80
_40
LpL_
S
T
P
O
1
_80_40
mito
cp
rRNA
gene_sum
Number of reads mapping to plant genes from leaf libraries
0
10,000,000
20,000,000
30,000,000
40,000,000
50,000,000
60,000,000
LpL_
FR
F
U
LL1_8
0_40
LpL_
FR
C
a1_
80_40
LpL_
FR
K
1_80_40
LpL_
FR
N
H
1_80
_40
LpL_
FR
N
O
1_80_40
LpL_
FR
P
O
1_80
_40
LpL_
S
T
FU
LL
1_80_40
LpL_
S
T
C
a1_80_40
LpL_
S
T
K
1_80_
40
LpL_
S
T
N
H
1
_80_40
LpL_
S
T
N
O
1_80
_40
LpL_
S
T
P
O
1
_80_40
mito
cp
rRNA
gene_sum
Number of reads mapping to plant genes from leaf libraries
Of 918 endophyte-regulated plant genes 728 are differentially regulated in roots
(529 induced and 167 repressed)
14
15. Plant Transcriptome in Symbiota
Cluster 3: root-expressed genes induced
by endophytes, but expressed at lower level
in endophyte-free plants
Largest cluster of endophyte-regulated
plant genes
Annotation of endophyte-regulated plant
genes
Defence response genes
Chitin responsive genes
Innate immunity genes
Patterns of Expression in Endophyte-Regulated Plant Genes
Part of cluster 3 hierachical clusterPart of cluster 3 hierachical cluster
Symbiotum transcriptional
response to endophyte
presence is up-regulation of
defence-related genes in roots15
16. Plant Transcriptome in Symbiota
Hierarchical clustering of C1Hierarchical clustering of C1
Cluster 1: root-expressed genes repressed by
endophytes
Annotation of endophyte-regulated plant genes
Transcription regulators
Max2 F-box LRR gene in
signalling of strigolactones
Patterns of Expression in Endophyte-Regulated Plant Genes
Clusters 10 and 11: shoot-expressed genes
repressed by endophyte
Annotation of 15 endophyte-regulated plant genes
3 MADS-box genes
2 blue light photoreceptors
1 cytokinin oxidase
Carbohydrate metabolism and transporters
16
17. 17
Peramine
N-formylloline
Lolitrem B
Metabolome Analysis of Symbiota
Ergovaline
Metabolic Profiling of Natural Symbiota
Metabolic profiling across spectrum of genetic, geographic
and taxonomic diversity of endophytes from perennial ryegrass
17
18. 18
Barsandra TolosaImpact
Lolitrem B
0.00
0.50
1.00
1.50
2.00
2.50
3.00
NEA10 NEA11 NEA12 E1 STxxx
b
a
Ergovaline
0.00
0.50
1.00
1.50
2.00
2.50
3.00
NEA10 NEA11 NEA12 E1 STxxx
*
Janthitrem
0.00
0.50
1.00
1.50
2.00
2.50
NEA10 NEA11 NEA12 E1 ST
x
a
ab
b
xx
Peramine
0.00
0.50
1.00
1.50
2.00
NEA10 NEA11 NEA12 E1 ST
b
a
a
*
*b
a
xxx
NEA10 NEA11 NEA12 E1 ST
LolitremBErgovalineJanthitremPeramine
Metabolome Analysis of Symbiota
Metabolic Profiling of Novel Symbiota
in Isogenic Hosts
Strong Gp x Ge effects on alkaloid toxin profiles
in defined symbiota with novel endophytes18
Endophyte
strain
Putative
toxin profile
Endogenous
toxin profile
Isogenic
(confirmed)
toxin profile
Taxon
NEA10 Unknown -/E/n.d
a
-/E/P/- (Y) N. lolii
NEA11 E+P -/E/n.d
a
-/E/P/- (Y) Lp TG-2
NEA12 Unknown -/-/- -/-/-/J (Y) Lp TG-3
E1 Unknown n.d -/-/-/-
ST L/E/P L/E/P/- (Y) N. lolii
a
Peramine not measured
Lp TG-3
19. 19
Metabolome Analysis of Symbiota
XX X
X
Adapted from Young et al, 2009
10 lolitrem biosynthetic genes
3 gene clusters
2 deletions (LtmE, LtmJ)
Pathway Analysis – Lolitrem Biosynthesis
19
21. 21
Assessing Endophyte Stability and Symbiota Performance
Barsandra
E- ST NEA11 NEA12
Bronsyn
A. Number of inoculations performed
ST NEA10 NEA11 NEA12 Total
Bea02 40 30 70
Bro08 80 75 155
Imp04 90 50 140
San02 80 50 130
Tol03 80 40 120
Total 0 370 0 245 615
B. Number of inoculations tested
ST NEA10 NEA11 NEA12 Total
Bea02 31 21 52
Bro08 59 50 109
Imp04 60 21 81
San02 64 31 95
Tol03 32 27 59
Total 246 150 396
C. Number of successful inoculations
ST NEA10 NEA11 NEA12 Total
Bea02 0 1 1
Bro08 1 0 1
Imp04 1 2 3
San02 0 1 1
Tol03 0 2 2
Total 2 6 8
D. Percent of successful inoculations
ST NEA10 NEA11 NEA12 Total
Bea02 0 1 1.0
Bro08 1.7 0 1.7
Imp04 1.7 9.5 11.2
San02 0 3.2 3.2
Tol03 0 7.4 7.4
Total 3.4 21.2 24.5
Stable association
Unstable association
Stable association
Unstable association
E- ST NEA11 NEA12
Phenome Analysis of Symbiota
21
22. 22
Assessing Endophyte Effect on Symbiota PerformanceShoot Fresh Weight in Response to Nitrate
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
E- NEA10 NEA11 NEA12 ST
Host-Endophyte Association
FreshWeight(g)
0.5 mM NO3-
2.5 mM NO3-
10.0 mM NO3-
Shoot fresh weight
Tiller Number in Response to Nitrate
0
10
20
30
40
50
60
70
80
E- NEA10 NEA11 NEA12 ST
Host-Endophyte Association
TillerNumber
0.5 mM NO3-
2.5 mM NO3-
10.0 mM NO3-
Tiller number
Root fresh weight
Root Fresh Weight in Response to Nitrate
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
E- NEA10 NEA11 NEA12 ST
Host-Endophyte Association
RootFreshWeight(g)
0.5 mM NO3-
2.5 mM NO3-
10.0 mM NO3-
Root Dry Weight in Response to Nitrate
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
E- NEA10 NEA11 NEA12 ST
Host-Endophyte Association
RootDryWeight(g)
0.5 mM NO3-
2.5 mM NO3-
10.0 mM NO3-
Root dry weight
Shoot Fresh Weight in Response to Nitrate
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
E- NEA10 NEA11 NEA12 ST
Host-Endophyte Association
FreshWeight(g)
0.5 mM NO3-
2.5 mM NO3-
10.0 mM NO3-
Shoot fresh weight
Tiller Number in Response to Nitrate
0
10
20
30
40
50
60
70
80
E- NEA10 NEA11 NEA12 ST
Host-Endophyte Association
TillerNumber
0.5 mM NO3-
2.5 mM NO3-
10.0 mM NO3-
Tiller number
Root fresh weight
Root Fresh Weight in Response to Nitrate
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
E- NEA10 NEA11 NEA12 ST
Host-Endophyte Association
RootFreshWeight(g)
0.5 mM NO3-
2.5 mM NO3-
10.0 mM NO3-
Root Dry Weight in Response to Nitrate
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
E- NEA10 NEA11 NEA12 ST
Host-Endophyte Association
RootDryWeight(g)
0.5 mM NO3-
2.5 mM NO3-
10.0 mM NO3-
Root dry weight
0.5 mM NO3
-
2.5 mM NO3
-
10.0 mM NO3
-
Phenome Analysis of Symbiota
22
23. 23
23
N-formylloline
Peramine
• Perennial ryegrass and
• Tall fescue
Establish symbiota
with both:
NEA21
Morocco
NEA23
Tunisia
Novel endophytes for broad deployment discovered and characterised23
Novel Fungal Endophytes for Forage Grasses
Discovering Endophytes with Novel Bioactivity
and Broad Host Specificity
25. 25
Forage Grass Microbiomes
Shoot and Root Microbiomes in Perennial Ryegrass
Hierarchical clustering of bacterial counts classifies root treatments but not shoot treatments.
Meta-transcriptomics reveals differences in bacterial species
predominance in shoot and root microbiomes
Root microbiome profiles ‘descriptive’ of treatment (e.g. nutritional status)
25
26. 26
Forage Grass Microbiomes
Shoot and Root Microbiomes in Perennial RyegrassL_F_FULL1
L_F_FULL3
L_F_FULL5
L_F_Ca2
L_F_Ca4
L_F_K1
L_F_K3
L_F_K5
L_F_NH2
L_F_NH4
L_F_NO1
L_F_NO3
L_F_NO5
L_F_P02*
L_F_P04*
L_S_FULL1
L_S_FULL3
L_S_FULL5
L_S_Ca2
L_S_Ca4
L_S_K1
L_S_K3
L_S_K5
L_S_NH2
L_S_NH4
L_S_NO1
L_S_NO3
L_S_NO5
L_S_P02*
L_S_P04*
R_F_FULL1
R_F_FULL3
R_F_FULL5
R_F_Ca2
R_F_Ca4
R_F_K1
R_F_K3
R_F_K5
R_F_NH2
R_F_NH4
R_F_NO1
R_F_NOL3
R_F_NO5
R_F_PO2
R_F_PO4
R_S_FULL1
R_S_FULL3
R_S_FULL5
R_S_Ca2
R_S_Ca4
R_S_K1
R_S_K3
R_S_K5
R_S_NH2
R_S_NH4
R_S_NO1
R_S_NO3
R_S_NO5
R_S_PO2
R_S_PO4
Azospirillum sp
0
50
100
150
200
250
Azospirillum sp
Azospirillum sp
Azospirillum amazonense
Azospirillum sp
Azospirillum sp
Azospirillum amazonense
Azospirillum brasilense
Azospirillum brasilense
Azospirillum lipoferum
Azospirillum sp
Azospirillum sp
Azospirillum brasilense
Analysis of bacterial microbiome in symbiota reveals range of bacterial
species known to be N fixers and phytostimulators of grasses
Azospirillum species induced in number (under low N)
Associative nitrogen fixation
Synthesis of phytohormones
26
28. Lessons and Prospects?
1. Breeding and Selection of Host Grass Only
Current Paradigm
2. Few Selective Recombinations in Long Breeding Cycle
4. Evaluation of Symbiota (i.e. Grass-Endophyte Associations)
3. Inoculation of Single Unselected Endophytes
5. Seed Generational Advance Limiting Heterosis
6. No Hybrid Varieties Limiting Value Capture
28
29. Lessons and Prospects?
1. Ab Initio Breeding and Selection of Symbiota
New Paradigm?
2. More Selective Recombinations in Shorter Breeding Cycle
4. More Accurate Evaluation of Symbiota
3. Exploit Broader Endophyte Diversity and Endophyte Effects
5. Exploit Heterosis and High-Impact Traits
6. Hybrid Varieties Enhancing Value Capture
29
30. Capture Ab Initio Plant Genotype X Endophyte Genotype Effects
Capture and Exploit Broader Endophyte Genotype Effects
on Symbiota Performance
Extend Concept of Synthetic Varieties to Both Partners of
the Symbiotum i.e. Grass Host and Endophyte
→ Deploy multiple endophyte and grass genotypes in populations
selected for optimal symbiota compatibility and performance
→ Breed and select ab initio symbiota for optimal symbiota compatibility
and performance rather than breed and select grass host only followed
by endophyte inoculation and symbiota evaluation
→ Exploit significant endophyte genotype effects on symbiota performance
well beyond pest resistance (and reduced animal toxicosis)
What Does This Mean?
30
31. Maximise Heterosis in Farmers’ Seed
Deliver F1 Hybrid Symbiota Varieties for Maximal On-Farm Impact
Reduce Generation Interval and Increase Selection Intensity
of Symbiota
→ Tailor genomic selection interventions in breeding cycle building on
simulated breeding schemes and sward-relevant phenotypes
→ Implement integrative, F1 hybrid symbiota breeding schemes building on
self-incompatibility allele typing
→ Produce F1 hybrid seed of symbiota deploying multiple endophytes and
high-impact traits
What Does This Mean?
31
32. Overcoming Bottle-Necks
• New tools for efficient, robust, low-cost, large-scale
generation of grass-endophyte symbiota
Method applicable to inoculation of 10s of endophytes in
100s of grass genotypes
Method applicable to inoculation of novel and designer
endophytes with de novo generated genetic variation
[i.e. induced mutagenesis (ionizing radiation, colchicine), genome editing, transgenesis]
Enabling tool for next-generation ab initio molecular breeding, selection
and evaluation of grass-endophyte symbiota [rather than breeding and selection of
grass host followed by endophyte inoculation and symbiota evaluation only]
High-Throughput, Large-Scale Endophyte Inoculation
32
33. Day 1 Day 3 Days 4-5 Days 6-7 Days 8-10Day 1 Day 3 Days 4-5 Days 6-7 Days 8-10
Production of Artificial Seeds
Large-Scale Generation of Symbiota
Coating with single or multiple Ca-alginate matrix layers of ryegrass mature seed-derived embryos
Assessing germination frequency of artificial seeds
33
34. Inoculation of isolated
seed-derived embryos
with endophyte mycelia
followed by Ca-alginate
coating into artificial
seeds or double-coating
of isolated seed-derived
embryos with endophyte-
containing inner Ca-
alginate matrix
Coating seed-derived embryos with multiple endophytes into viable symbiota artificial seeds
a b c
First coating Second coatingEndophyte outgrowth Germinating symbiota
Large-Scale Inoculation of Endophytes into Artificial Seeds
Large-Scale Generation of Symbiota
Generating > 1,000 viable symbiota artificial seeds per FTE and day
Established symbiota plants with live endophytes in <50% artificial seeds.
34
35. Predicting Endophyte Stability in Stored Seed and Selecting
Stable Associations Using Accelerated Ageing
A method for Accelerated Ageing [i.e. 80% -100% RH for 4-7 days]
of seed (natural and artificial) with resident endophytes developed
The method allows to predict endophyte stability in stored
seed [range of endophytes assessed in single and different host genetic backgrounds]
The method allows to rank novel endophytes according to predicted
stability/viability in stored seed
[range of endophytes assessed in single host genetic background]
The method allows to select and rank symbiota according to their
stability
Overcoming Bottle-Necks
35
36. 36
NEA12
0
20
40
60
80
100
120
Alto Bealey Bronsyn Trojan
Control
80% 4d
80% 7d
100% 4d
100% 7d
NEA10
0
20
40
60
80
100
120
Alto Bealey Bronsyn Trojan
Control
80% 4d
80% 7d
100% 4d
100% 7d
NEA11
0
20
40
60
80
100
120
Alto Bealey Bronsyn Trojan
Control
80% 4d
80% 7d
100% 4d
100% 7d
E1
E1
0
20
40
60
80
100
120
Al to Beal ey Br onsyn T r oj an Endo
E1
0
20
40
60
80
100
120
A l t o B eal ey B r onsyn T r oj an E ndo
Cont r ol
80%4d
80%7d
100%4d
100%7d
Accelerated ageing [i.e. 100%RH for 4d or 7d] allows ranking endophytes for
compatibility and selecting for endophyte genotype-host genotype stability
Using Accelerated Ageing to Select for Symbiota Viability and Stability
Assessed endophyte viability and stability of symbiota after accelerated ageing treatment of seed
Selection for Symbiota Stability
36
37. 37
Rapid Early Assay of Endophyte Viability in Symbiota
37
Overcoming Bottle-Necks
A fast, reliable and low-cost method for determining endophyte
viability in perennial ryegrass seeds, seedlings and established
symbiota
Assay Requirements:
1. Rapid determination: 3-5 day old epicotyls
2. Robust and reliable
3. Sensitive for use in single seed to seed batches
4. Specific to Neotyphodium endophytes
(i.e. does not detect other fungi)
5. Detects live endophyte only
Seed with endophyte
38. 3838
Assaying Endophyte Viability
Metabolomics-Based Assay
Assays developed based on:
• Genotyping
• Early gene expression
• Production of indicator metabolites
Seed
germination
Harvest
epicotyls
Dark
Light
Day 1 Day 4 Day 5 Day 6
Metabolite
extraction
Direct
Injection MS
Set-up
Data
analysis
With Endophyte
Without Endophyte
Detection of E- seed
Rapid (≤6 days), low cost (<$1/sample) assay – 5X cheaper and 5X faster
39. 3939
Increasing Accuracy and Reducing Cost of Phenotyping
Low-cost, high-throughput, accurate methods for large-scale
phenotyping of individual plants for herbage quality traits
Robust, reliable methods enabled by automated workflows
Overcoming Bottle-Necks
Low-cost, high-throughput, accurate methods for large-scale,
multisite phenotyping of key traits at sward level
Field-based phenomics (from individual plant to farmer’s paddock)
Laboratory-based molecular phenomics
Generating low-cost, high-throughput, accurate, relevant
phenotypes for genomics-assisted molecular breeding
40. 40
Rainout Shelters
Precise water-stress treatments
Automated Assessments
Vegetative biomass
Quality traits – CP/WSC/ME/Minerals
Persistence traits – Biomass
over time
Stress related traits
Active Optical Sensors
Canopy greenness &
photosynthetic capacity
Normalised difference
vegetative index output
Forage quality
Field-Based Phenomics
40
41. 41
Non-Destructive Forage Yield Estimation Using Normalized
Difference Vegetation Index
Field-Based Phenomics
GreenSeeker Aphex hexacopter
41
43. 43
Molecular Phenomics of Symbiota
0
10000000
20000000
30000000
40000000
50000000
60000000
70000000
80000000
1 7 13 19 25 31 37 43 49 55 61 67 73 79 85 91 97 103 109 115 121 127 133 139 145 151 157 163 169 175
lolitrem B
ergovaline
peramine
0
10000000
20000000
30000000
40000000
50000000
60000000
70000000
80000000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
lolitrem B
ergovaline
peramine
Top 20 peramine producing symbiota
Molecular Phenotyping to Enable Symbiota Selection
Selection of symbiota within breeding population with favourable toxin profiles
• high peramine, low ergovaline, no lolitrem B
Molecular breeding of symbiota capturing ab initio Gp x Ge effects
43
44. 4444
Refined breeding schemes
Phenotyping tools at acceptable cost
Genotyping tools at acceptable costs
Computational tools to handle data
and empowering breeders
Genomic Selection Selection candidates
Genotypes
Selected parents
Estimated
breeding
values
Prediction equation
Genomic Breeding Value =
w1x1+w2x2+w3x3……..
Reference population
Genotypes
Phenotypes
Increasing Rate of Genetic Gain via Genomic Selection
Overcoming Bottle-Necks
45. 4545
Rates of Selective Breeding, Genetic Gain and Improvement
M1 B M2
(A) (B)
(C)
(D)
(E)
Genomic selection
Update
prediction
equation
Multi-site environment trials
F1 Production
Seed production
(F2 Production)
Selection under grazing and/or
visual assessment
Varietal construction
Seed production
Multi-environment
plot trials
1 varietal release
Base population
establishment
c. 1,000 – 10,000
Individuals
c. 100,000 Individuals
Reduction in
individuals by a
factor of 10
Selective
Recombination
Non-Selective
Recombination
Selective
Recombination
c. 1-10 Varieties
Multi-environment
plot trials
Less than 100
varieties
Non-Selective
Recombination
Less than 100 varieties
F1 Production
Seed production
(F2 Production)
Selection under grazing and/or
visual assessment
Varietal construction
Seed production
Multi-environment
plot trials
1 varietal release
Base population
establishment
c. 1,000 – 10,000
Individuals
c. 100,000 Individuals
Reduction in
individuals by a
factor of 10
Selective
Recombination
Non-Selective
Recombination
Selective
Recombination
c. 1-10 Varieties
Multi-environment
plot trials
Less than 100
varieties
Non-Selective
Recombination
Less than 100 varieties
F1 Production
Seed production
(F2 Production)
Selection under grazing and/or
visual assessment
Varietal construction
Seed production
Multi-environment
plot trials
1 varietal release
Base population
establishment
c. 1,000 – 10,000
Individuals
c. 100,000 Individuals
Reduction in
individuals by a
factor of 10
Selective
Recombination
Non-Selective
Recombination
Selective
Recombination
c. 1-10 Varieties
Multi-environment
plot trials
Less than 100
varieties
Non-Selective
Recombination
Less than 100 varieties
2 selective recombination steps
– 10 years
2 selective recombination steps – 3 years
Genomic Selection
Computational simulation of commercial ryegrass breeding program to
optimise application of genomic selection
Genomic-estimated breeding values for key traits in ryegrass breeding
46. 4646
Exploiting Heterosis via Novel Hybrid Breeding Scheme
Candidate genes for Self-Incompatibility loci (S and Z) discovered and
functionally characterised
An F1 hybrid breeding scheme designed and being piloted
Overcoming Bottle-Necks
Fertilisation
S1Z1
S1Z2 S2Z2 S1Z3
S3Z1
S3Z3
S1S2Z1Z2
S1S2Z1Z2
S1Z1
S1Z2
S2Z1
S2Z2
Pistil
Pollen
(haploid)
(diploid)
Pistil
Anther
A method for SI allele prediction developed
48. 48
Advances in Forage Systems Biology
Summary
Genome, Transcriptome, Proteome, Metabolome and Phenome
Forage Symbiomes and Microbiomes – Exploiting Supplementary
Genomes
Lessons from Systems Biology of Forage Symbiomes
Integrative, Genomics-Assisted Hybrid Breeding of Symbiota
Prospects for Trebling Genetic Gain
49. 49
Acknowledgements
P. Badenhorst, N. Cogan, H. Daetwyler, S. Davidson,
P. Ekanayake, S. Felitti, J. Forster, K. Fulgueras, K. Guthridge,
M. Hand, B. Hayes, M. Hayden, I. Hettiarachchi, D. Isenegger,
J. Kaur, G. Latipbayeva, T. Le, Z. Lin, Z. Liu, C. Ludeman,
E. Ludlow, R. Mann, L. Pembleton, M. Rabinovich,
M. Ramsperger, P. Rigault, S. Rochfort, T. Sawbridge, K. Shields,
L. Schultz, H. Shinozuka, K. Smith, G.Tao, P. Tian, P.X. Tian,
J. Tibbits, Y. Ran, E. van Zijll de Jong, J. Wang, T. Webster
C. Inch, S. van der Heijden, M. Willocks
50.
51. Cluster 1 Cluster 3Cluster 2 Cluster 4
Cluster 5 Cluster 7 Cluster 8Cluster 6
Cluster 9 Cluster 10 Cluster 11 Heat map depiction of average cluster
expression.
Columns are
Cluster Number, Cluster popn., Cluster
Diversity .
Cluster 1 Cluster 3Cluster 2 Cluster 4
Cluster 5 Cluster 7 Cluster 8Cluster 6
Cluster 9 Cluster 10 Cluster 11
Cluster 1 Cluster 3Cluster 2 Cluster 4
Cluster 5 Cluster 7 Cluster 8Cluster 6
Cluster 9 Cluster 10 Cluster 11 Heat map depiction of average cluster
expression.
Columns are
Cluster Number, Cluster popn., Cluster
Diversity .
Heat map depiction of average cluster
expression.
Columns are
Cluster Number, Cluster popn., Cluster
Diversity .
Samples in order
Leaf Free Root Free Leaf ST Root ST
Replete Ca K NH4 NO3 PO4 Replete Ca K NH4 NO3 PO4 Replete Ca K NH4 NO3 PO4 Replete Ca K NH4 NO3 PO4
Plant Transcriptome in Symbiota
11 clusters generated by Self Organizing Trees Algorithm analysis of endophyte-regulated plant genes
Cluster 1: root-expressed genes repressed by endophytes
Clusters 3 and 4: root-expressed genes induced by endophytes
Patterns of Expression in Endophyte-Regulated Plant Genes
51
52. Plant Transcriptome in Symbiota
Cluster 2: root-expressed genes
repressed by endophytes as well as
root-expressed genes induced by
endophytes
Differential gene expression
driven by NH4 responsiveness
Patterns of Expression in Endophyte-Regulated Plant Genes
Hierarchical clustering of genes in cluster 2Hierarchical clustering of genes in cluster 2
Endophyte-regulated
plant genes differentially
regulated in roots
depending on nutritional
symbiota status
52
53. 53
1. Alkaloid (LEPJ) profiles of symbiota
(i.e. E+) versus E- isogenic host plants
2. Alkaloid profiles of symbiota with diverse
endophyte panel in a single isogenic host
3. Alkaloid (LEPJ) profiles of symbiota with
endophytes from different taxonomic
groups across same host panel
Metabolic Profiling of Novel Symbiota in Isogenic Hosts
Detailed characterisation of known
alkaloids and their precursors
Metabolome Analysis of Symbiota
Analysis of Gp x Ge effects on symbiota stability and toxin profile53
54. We MUST We NEED
2XProductivity
Growth 3XGenetic
Gain
56. 56
0
0.1
0.2
0.3
0.4
0.5
0.6
control
1
2
3
*4
5
6
7
8
9
10
11
12
13
14
15
16
*17
18
NEA12 colonies treated (no.)
Sizeofmycelia(cm,LogN)
*
**
Analysis of growth rate in culture
after 8 weeks
Initial Screen: Analysis of variance identified two
colonies significantly different to the control
NEA12v17 grows significantly faster (p<0.01**)
NEA12v4 grows significantly slower (p<0.05*)
Validation Screen: Student’s t-tests identified
two colonies significantly different to the control
NEA12v17 grows significantly faster (p<0.01**)
NEA12v15 grows significantly slower (p<0.01**)
Analysis of growth rate in culture
over 5 weeks
In Vitro Growth of NEA12 Variant Strains
Phenome Analysis of Variant Endophytes
Altered phenotypes (e.g. growth rates) observed in variant endophytes56
0
1
2
3
4
5
6
7
8
9
10
0 1 2 3 4 5
Week
Growth(mm)
NEA12
NEA12v4
NEA12v5
NEA12v6
NEA12v13
NEA12v14
NEA12v15
NEA12v17
57. 57
Gene present Gene absent Gene partially present
Pangenome Analysis of Endophytes
Sequence Diversity in Alkaloid Production Genes
Identification of core and flexible genomes in Neotyphodium endophytes
57
58. 58
easH easA
Intergenic deletions in eas gene cluster in AR1 endophyte
AR1
(Ergo-)
ST
(Ergo+)
CONTIG_29770
lpsBeasG easF easE
CONTIG_29770
Pangenome Analysis of Endophytes
Sequence Diversity in Alkaloid Production Genes
Intergenic deletions and SNP causing truncated lpsA also lead to inability to produce ergovaline
58
60. F1 Hybrid Breeding Designs
Endophyte Trait Diversity
Some Key Considerations
Endophyte Deployment
Self-Incompatibility
Value Modelling and Impact Delivery
Accurate, Low-Cost Genotypes and Phenotypes
60
61. Selection for Symbiota Stability
61
Germination of seeds after AA
treatment and storage
Growth of germinated seedlings
in soil for eight weeks
Assessment of endophyte status by
ELISA
Accelerated ageing treatment
• Optimised conditions identified (e.g. 80% humidity, 4-7 days)
• Variation identified between endophytes and grass cultivar combinations
Predicting Endophyte Stability in Stored Seed and Selecting
Stable Associations Using Accelerated Ageing
62. 62
Experimental Work Flow for Colchicine Mutagenesis
n
nucleus
n and 2n?
Colchicine treatment
(0-0.2% w/v)
3 weeks, 22oC, 150rpm, dark
Protoplast
preparation
4 weeks, 22oC, dark
Colony
subculture
Analyse for change in nuclei
size via flow cytometry
Stained cells
Colony
regeneration
A
C D
B
BA
C
D
Protoplast
preparation
(single colonies)
4 weeks, 22oC, dark
SYBR Green I
staining of nuclei
Generation of Novel N. lolii Genotypes
De Novo Generation of Variant Endophytes
62
63. 63
Experimental Work Flow for X-Ray Mutagenesis
Detection of target gene mutants using high through-put multiplex PCR
analysis for target gene presence and absence and by genome survey
sequencing
Single colonies
isolated
Mutant
detection
Protoplast
preparation
Potato dextrose broth
for 4-14 days
Recovery period (10-14 days)
Repeated radiation
Exposure to ionising radiation
caesium source
(10-30 Gy)- )
Recovery: 4 -6 weeks, 22oC, dark- o
BA BAA
-
-
15 days, 22oC, dark
Generation of Novel N. lolii Genotypes
De Novo Generation of Variant Endophytes
X-ray mutagenesis for generation of variant endophytes
63
64. 64
De Novo Generation of Variant Endophytes
Generation of Fluorescently Marked Endophytes
ST:sgfpE1:DsRed NEA12:sgfp
e
e
e
e
e
e
Reporter Endophytes to Develop Endophyte Hybridisation
Methodologies and Study Host Colonisation
Agrobacterium-mediated transformation of N. lolii
and LpTG-3 endophytes with fluorescent reporter genes64
65. 65
De Novo Generation of Variant Endophytes
65
Proof-of-Concept for Enhancing Bio-protective Properties
Agrobacterium-mediated transformation of janthitrem-producing
endophyte for perA expression and peramine production
248.15022
N
H
O
O
H O
O
O
O
H O
H
C h e m i c a l F o r m u l a : C 3 9 H 5 1 N O 7
E x a c t M a s s : 6 4 5 . 3 6 6 5 5
N
N
O
N
N H 2
H 2 N
C h e m i c a l F o r m u l a : C1 2 H1 7 N 5 O
E x a c t M a s s : 2 4 7 . 1 4 3 3 1
Janthitrem I
peramine
[M+H]
[M+H] 646.37238
pEND0025
20395 bp
25bp RB
25bp LB
attB1
attB2
SpecR/ StrepR
pBR322 origin
PVSI origin
PVSI STA region
hph
PerA gene
p gpd
P trpC
T trpC
T trpC
Peramine Biosynthesis perA Gene
Expression Vector
Generation of Transgenic LpTG-3 Endophytes for Peramine Production
N. lolii ST LpTG-2 NEA11 LpTG-3 NEA12
P P J
201bp
PerA gene
414bp
Selectable marker gene
66. 66
HTP method as NIR reference Environmental and meteorological
data gathering of each trial site
Harvesting of plant material
No Sample Preparation
e.g. oven drying and
grinding
In-Field Biomass
In-Field Forage Quality
Forage Mobile Pheno-Lab Digital Image Library
Field-Based Phenomics
66
