Genetics of biotic and abiotic stress
resistance: basic concepts
luigi cattivelli
Disease resistance
Different types of resistance
Identification of sources of disease resistance
The regions of species diversification/evolution also contain most of the sources of
resistance due to the long co-evolution between plants and pathogens
Sources of resistance can be found in cultivated accessions as well as
in landraces, wild accessions and wild related species
Resistance to powdery mildew in Hordeum
Gene
Source
chromosome
Mla
5S(1HS)‫‏‬
‫‏‬
mlt
1S(7HS)‫‏‬
‫‏‬
Mlf
Hordeum spontaneum
1L(7HL)‫‏‬
‫‏‬
Mlg
4L(4HL)‫‏‬
‫‏‬
Mlj
7L(5HL)‫‏‬
‫‏‬
Ml(La)‫‏‬
‫‏‬
Hordeum laevigatum
2L(2HL)‫‏‬
‫‏‬
MlHb
Hordeum bulbosum
2S(2HS)‫‏‬
‫‏‬
mlo
Mutants from Hordeum
vulgare
4L(4HL)‫‏‬
‫‏‬
The Efficiency of resistance is based on two parameters:
1) the degree of resistance 2) The duration/stability of resistance
1) Degree of resistance
Resistance with qualitative genetic bases: they confer a full (or
almost full) resistance against specific races of the pathoges. They
behave as mendelian, often dominant, traits
Resistance with quantitative genetic bases: they usually are
independent on the specific race of the pathogen but
show a non complete level of resistance
From evolutionary point of view this condition limits the evolution of
new virulent strains of the pathogen.
Co-evolution between plant and pathogen
In agricultural systems the diffusions of resistant
cultivars might lead to the evolution of new pathogen
strains/races with new virulences.
To slow down this process three different strategies
might be considered:
1) Pyramiding: 2 o more R genes in the same
genotype
2) Utilization of different cultivars carrying different
R genes in the same region‫‏‬
3) Utilization of multi-lines cultivars
R-gene polycultures are proposed to give more durable resistance (1) Any pathogen race that can overcome
only one R gene will give rise to a much slower epidemic. (2) Any such pathogen race that undergoes an
additional mutation to overcome another R gene is likely to be less fit than a race that can overcome only
one R gene, because avr genes are likely to encode pathogenicity factors. (3) High inoculum of avirulent
races is likely to promote systemic acquired resistance, reducing the susceptibility of the plants. (Curr.
Opin. Plant Biol. 2001, vol. 4, no 4 pp. 281-287)‫‏‬
Plant pathogen interaction
The R-Avr combinations leading to
incompatibility are epistatic toward the
combination leading to compatibility.
Signalling networks triggered by R genes
(Curr Opin Biotech 2003, 14: 177-193)‫‏‬
PR proteins
PR proteins
PDF1.2
The origin of Avr products
The product of Avr genes
may be involved in the
virulence process in host
without the corresponding
R.
A virulence protein can
became Avr once it has
been recognized by a R
gene.
R genes conserved domains
LRR: leucine rich repeats (consensus XX(L)X(L)XXXX);
- sono ripetizioni in serie di circa 24 aminoacidi contenenti Leu o altri residui idrofobici ad
intervalli regolari
- per la specificità sono importanti gli AA idrofilici esposti
- in proteine di Drosophila, uomo e lievito LRR mediano interazioni proteina-proteina
NBS: nucleotide binding sites = Ploop
- dominio per legame di ATP o GTP
- questo legame può alterare la interazione tra gene R e altre proteine della via di trasduzione
del segnale
LZ: leucine zipper, un sottotipo della struttura Coiled Coil
- queste sequenze ripetute facilitano la interazione proteina-proteina, favorendo la formazione
di strutture
coiled-coil
TIR:
- similarità al dominio citoplasmatico della proteina Toll di Drosophila e ai recettori
dell’interleukina 1 dei mammiferi
- questi domini, in seguito a legame con un ligando, causano attivazione di fattori di
trascrizione; è quindi verosimile che anche nei geni R svolgano questa funzione.
The LRR domains (often) confers the race specificity
1) L’allele resistente del gene Pi-ta in riso (resistenza a Magnaporthe grisea) differisce
dall’allele suscettibile per un solo aminoacido presente nel dominio LRR (Ala invece di Ser)
(Plant Cell 2000, 12: 2033-2045) Inoltre è stata dimostrata una interazione diretta tra il dominio
LRR del gene Pi-ta e la proteina di avirulenza di M. grisea Avr-Pita (Embo J 2000, 19: 40044014)‫‏‬
2) in molti sistemi pianta patogeno variazioni nella sequenza LRR o variazioni nel numero di
ripetizioni di copie LRR sono responsabili di diverse capacità di riconoscimento, come al locus
Cf4/Cf9 in pomodoro.
3) gli alleli L6 e L11 in lino, che conferiscono resistenza a razze diverse di ruggine, differiscono
solo a livello di dominio LRR; inoltre quando i domini TIR e NBS di L6 e L10 sono stati fusi al
dominio LRR di L2, la specificità conferita era quella di L2 (Plant Cell 1999, 11: 495-506)‫‏‬
4) malgrado ciò, gli alleli L6 e L7 hanno identiche LRR e la loro sequenza differisce solo a
livello del dominio TIR; per questi due alleli la regione di specificità è a livello del dominio TIR
(Plant Cell 2000, 12: 1367-1377)‫‏‬
Positional cloning of R genes: the Mla locus of barley, 11 R
genes homologues clustered together
Mla
175D16-T7
9X
100 Kb
721K19-R1.1
MWG2083
140 Kb
120 Kb
MWG 2197
2X
3600 gametes
175 D16
721 K19
FrYAC 120 ID1
714 K1
80 H14
RGH1
RGH2
RGH3
Comparative mapping of R gene
homologues in the monocot species
rice, barley, and foxtail millet. A circle
diagram was chosen to visualize
syntenic relationships that align the
genomes of barley (green), rice
(red), and foxtail millet (blue). Map
locations of NBS-LRR genes that
could be mapped in at least two of
the three tested species are given.
Syntenic map positions are marked
by bold red spokes and nonsyntenic
R gene homologue loci are boxed in
black. Clusters containing at least
two highly divergent NBS-LRR genes
in rice and foxtail millet (RHC-A to
RHC-D) are highlighted in the
periphery. Barley chromosomes are
numbered 1H to 7H, rice
chromosomes 1 to 12, and foxtail
millet chromosomes I to IX (PNAS
1998, 95: 370-375)‫‏‬.
BAC clones from Manchuria (No known Mla specificity)‫‏‬
‫‏‬
YAC clone from Franka (Mla-6)‫‏‬
‫‏‬
Abiotic stress tolerance
ABIOTIC STRESSES: situations where environmental stimuli that
normally influence plant development, growth, and productivity, exceed
thresholds (species-specific), damaging the plant
drought
cold (chilling and freezing)‫‏‬
salt
heavy metals
heat shock
anoxia
nutrient stress
Stress resistance can be developed through exposition to suboptimal growing conditions: the acclimation process (hardening)
Barley plants frozen at -10°
°C
.
Plant response to drought stress
(generally conserved across species)‫‏‬
• Development:
– Growth reduction
– Alterations in flowering times
– Increase in the root/shoot ratio
• Morophological adaptation
– Stomatal closure
– wilty
– Abscission
• Physiological changes
– Decrease in transpiration
– Reduction of water potential
reduces transpiration and limits the
flow of water from roots to leaves
stomatal closure
reduction in the movement of
nitrate and other compounds from
roots to leaves
lower CO2 level
lower water
reduction of
amino acid
synthesis
lower photosynthetic activity
availability
photoinhibition
ROS production
growth inhibition
polysome reduction
altered cell wall extensibility
damages to biological
structures
Physiological traits relevant for response to drought conditions
Timing of phenological phases
Rooting depth
Stomatal resistance
Cuticular resistance and
surface roughness
Partitioning and stem
reserve utilization
Osmotic adjustment
Early / late flowering. Maturity
and growth duration
Higher / lower tapping of soil
water resources
More / less rapid water
consumption
Wheat and barley advanced
flowering, rice delayed
Reduced total mass but increased root/shoot ratio,
growth into wet soil layers
Increase under stress / Response to soil water
potential
Higher or lower water loss, modification of boundary layer and
reflectance
Lower / higher remobilisation of
reserves from stems
Compensation of reduced current leaf photosynthesis by
increased remobilization
Accumulation of solutes: ions,
sugars, poly-sugars, amino
acids, glycinebetaine
Slow response to water potential. OA higher in
sorghum, wheat, indica rice than in maize, cowpea
The water use efficiency
The carbon isotope discrimination can be used as a surrogate for water use
efficiency to select lines with high water use efficiency in drought-prone
environments. During photosynthesis plants discriminate against the heavy isotope
of carbon (13C) and, as a result, in several C3 species, ∆ is positively correlated with
the ratio of internal leaf CO2 concentration to ambient CO2 concentration (Ci/Ca) and
negatively associated with transpiration efficiency. Thus, a high Ci/Ca leads to a
higher ∆ and a lower transpiration efficiency.
ERECTA
A putative leucine-rich
repeat receptor-like
kinase is a major
contributor to a locus for
on Arabidopsis
chro moso me 2.
ERECTA acts as a regulator of
transpiration efficiency with effects
on stomatal density, epiderma l cell
e xpansion, mesophyll cell
proliferation and cell–cell contact.
Yield under water stress is a function
of (i) water extracted from soil (ii)
water use efficiency and (iii) harvest
index.
Masle et al., 2005
Cell response to drought stress
(generally conserved across species)‫‏‬
• Loss of turgor,
• Increase of intracellular solutes
• Changes in cell volume
• Denaturation of proteins
• Disruption of membrane integrity
• Changes of gene expression
• Changes of metabolism
REGULATORY PROTEINS
FUNCTIONAL PROTEINS
Membrane proteins
Transcription factors
( Water channel protein,
transporters
(MYC, MYB,bZIP,
EREBP/AP2)‫‏‬
‫‏‬
Proteinases
Protein Kinases
(cytoplam, chloroplast)‫‏‬
‫‏‬
(MAPK, MPKKK,
CDPK,S6K)‫‏‬
‫‏‬
Protection
of macromolecules
Drought
Stress
(Chaperons, LEA proteins
Protein phosphatases
(PTP)‫‏‬
‫‏‬
Osmoprotectant
synthases
(Proline, Gly betane,
sugar)‫‏‬
‫‏‬
Detoxification
enzymes
(GST,sEH, SOD
PI turover
(Phospholipase C,
PIP5K, DGK, PAP)‫‏‬
‫‏‬
Drought stress-inducible genes and their posibles functions in stress
tolerance and response.
• Resistance to abiotic stress is always a quantitative traits
that can be resolved in few or many QTLs.
• Often the expression of the QTLs for tolerance to abiotic
stress is dependent on the environments (QTL x E
interaction).
QTLs for frost tolerance in barley
QTLs for drought tolerance in barley
QTLs da Teulat et al. (2001, 2002 e 2003) e da Diab et al. (2004).
• OA (Osmotic Adjustment) e OP (Osmotic Potential)‫‏‬
‫‏‬
• RWC (relative water content)‫‏‬
‫‏‬
• WSC (Water-soluble carbohydrate)‫‏‬
‫‏‬
Genetic analysis of cold tolerance
Exposure to stress promotes the accumulation of stressregulated mRNAs
3°
°C
20°C 1h 5h 12h 1d
3d 5d 8d 10d 15d
cor14b
tmc-ap3
blt14
cor18
af93
CN
N
CS
TS
P
CS
/C
N
CS N5
A
/
CN TSP
N 5A
CS
TS
P
CS
/C
N
CS N5
A
CN/TSP
N 5A
CS
TS
P
CS
/C
N
CS N5
A
/
CN TSP
N 5A
CS
TS
P
CS
/C
N
CS N5
A
/T
SP
5A
The chromosome 5A controls the expression of cor14b
18°
°/13°
°C
2°
°C
18°
°/13°
°C + ABA 18°
°/13°
°C + PEG
The molecular bases of QTL for stress tolerance: the frost
tolerance QTL of winter cereals
Cor14b expression polymorphism in T.monococcum
DV92
5°
° 10°
°
25°
°
15°
°
G3116
20°
° 25°
°
5°
°
10°
°
15°
°
20°
°
cor14b
blt14
Cor14b expression polymorphism in T.monococcum
segregating population
Cor14b expression-QTL co-segregates with frost resistance QTL
and Cbf locus
9
LOD score
Frost resistance QTL
10cM
8
7
6
5
Cor14b expression QTL
4
3
2
1
Chr 5Am
Xpsr426
Xwg644
Xcdo465
Xpsr2021
XDhn2.1
Xmwg2062
Xpsr120
Xbcd9
XEsi14
Xgwm639
XCbf3
Xbcd508
Xwg530
Xcdo57
Xbcd351
Xbcd926
Xgwm186
cor14b expression and frost resistance I
5A T. monococcum
Rcg1
Xbcd508
Fr2
X-cbf3
Xpsr2021-dhn2
Vrn1
COR proteins accumulation in barley DH population (N x T)
under field conditions
N T
COR14b
N T
TMC-AP3
cor14b expression and frost resistance II
5A T. monococcum
5 H. vulgare
cbf8
Rcg1
Xbcd508
Rcg1
Fr2
X-cbf3
cbf3-4
Xpsr2021-dhn2
Vrn1
psr911
Fr2
psr637
Xpsr2021-dhn2
Fr1
Vrn1
Two loci control frost resistance in triticeae
The expression of cor14b is genetically link to
frost resistance and to a cbf-like locus
Expression of cor14b is controlled at transcriptional level
TATA
box
- 643
-1
GUS
GUS
GUS
cor14-GUS 20°C
ubi-GUS 20°C
cor14-GUS 3°C
ubi-GUS 3°C
Mutational analysis of the cor14b promoter region
-274-247: identification of a potential cis-element
-274 5’ AGCTTACCCAAAGGTACGTGAGGTCGG3’ -247
DRE
The expression level of Cbf is associated to frost resistance in wheat
Control (20°
°C)
2 hours +2°
°C
Different expression level of Cbf is
associated with a QTL for frost resistance
in wheat
QTL for frost resistance
Cbf
ribosomal
In the triticeae, Cbfs is a large family of 20-40 members, most of them
clustered under the LT tolerance QTL Fr-2 (Chr 5)
Badawi et al., 2007
Most cbfs are clustered under the LT tolerance QTL Fr-2
Knox et al., 2008
Francia et al., 2007
Cbfs are differentially expressed in
resistant vs susceptible plants
Cbf c onserve domain
XpsrB85
XpsrB89
Xpsr911 (Rcg1)
Xpsr637
Xpsr2021 (Rcg2)
Fr-A1
Xcdo504
Xwg644
Xpsr426
Vrn-A1
Xpsr805
Q
Xpsr370
Xpsr164
b-amy-A1
C
C
C
C
C
C
C
C
C
C
T
T
T
C T C T T T
C T C T T T
C C T T T T
C C T T T T
C - - - - T
C T - C C T
C T C C C T
C T C C C T
C T C C C T
C T C C C T
T T C T C C
T - - - - C
T T C T C C
T T C T C C
C T C T C T
C
C
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
Cbf15
Cbf16
Cbf14
Selected Cbfs have been used for association mapping in a
population of about 130 european barley cultiuvars
The ratio of variable to maximal fluorescence (Fv/Fm) in
dark-adapted state measures the maximum quantum yield
for PSII photochemistry and represents a diagnostic
probe for measuring low temperature stress-induced
injury of photosynthesis
Knox et al., 2008
HvCbf6
HvCbf1
4
HvCbf3
HvCbf9
Linkage disequilibrium at CBF loci in
130 European barley cultivars
The LD threshold beyond two sites to declare them in
disequilibrium was fixed at r²=0.20 on the basis of the
evaluation of LD among unlinked molecular markers
(Breseghello, 2006)
Gene
H vCbf14
H vCbf14
Vrn-H 1
H vCbf6
H vCbf14
H vCbf14
SNP
SN P 10 (C/T )
A llele
C
F-value
27.11
P-value
<0.0001
S N P 7 (C/T )
C
21.13
<0.0001
S N P1 (G /A )
G
6.89
0.0016
SN P 13 (G /C)
C
5.64
0.0047
SN P 10 (C/T )
T
1.59
0.2106
S N P 7(C/T )
T
1.41
0.2375
Association analysis between genetic variants in genes involved in frost
tolerance and frost tolerance. Only genetic variants with stronger effects on
freezing tolerance trait are reported.
Conclusions
The Cbf gene cluster is the molecular base of the Fr-2 locus of
triticeae and Cbf14 is (one of) the best candidate
Most of the natural variation for frost tolerance is encoded by
genetic variations at the Cbf locus
Acknowledgements
CRA – Genomic Research Centre, Fiorenzuola d’Arda
¢Cristina Crosatti
¢Caterina Marè
¢Anna Mastrangelo
¢Betty Mazzucotelli
¢Chiara Campoli
¢Fulvia Rizza
Collaborations
¢Nicola
Pecchioni (Univ. Reggio E. - Italy)
¢Gabor Galiba & Attila Vagujfalvi (Martonvasar - Hungary)
¢Jouge Dubkovsky (UC - Davis - Ca)
¢Pietro Piffanelli & Agostino Fricano (PTP – Lodi)