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
Hordeum spontaneum
5S(1HS)
mlt 1S(7HS)
Mlf 1L(7HL)
Mlg 4L(4HL)
Mlj 7L(5HL)
Ml(La) Hordeum laevigatum 2L(2HL)
MlHb Hordeum bulbosum 2S(2HS)
mloMutants from Hordeum
vulgare4L(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 proteinsPDF1.2
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 trasduzionedel segnale
LZ: leucine zipper, un sottotipo della struttura Coiled Coil
- queste sequenze ripetute facilitano la interazione proteina-proteina, favorendo la formazione di strutturecoiled-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 dominioLRR del gene Pi-ta e la proteina di avirulenza di M. grisea Avr-Pita (Embo J 2000, 19: 4004-
(4014
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, differisconosolo a livello di dominio LRR; inoltre quando i domini TIR e NBS di L6 e L10 sono stati fusi aldominio 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 alivello del dominio TIR; per questi due alleli la regione di specificità è a livello del dominio TIR (Plant Cell 2000, 12: 1367-1377)
RGH1
RGH2
RGH3 YAC clone from Franka (Mla-6)
BAC clones from Manchuria (No known Mla specificity)
100 Kb 120 Kb140 Kb
FrYAC 120 ID1
80 H14
721 K19
714 K1
175 D16
175D16-T7 721K19-R1.1 MWG2083 MWG 21972X9X
Mla
3600 gametes
Positional cloning of R genes: the Mla locus of barley, 11 R genes homologues clustered together
Comparative mapping of R gene homologues in the monocot species rice, barley, and foxtail millet. A circle diagram was chosen to visualizesyntenic 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 nonsyntenicR 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 .(370-375 :95 ,1998
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 shockanoxia
nutrient stress
.
Barley plants frozen at -10°°°°C
Stress resistance can be developed through exposition to sub-
optimal growing conditions: the acclimation process (hardening)
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
lower water
availability
stomatal closure
lower CO2 level
reduces transpiration and limits the
flow of water from roots to leaves
reduction in the movement of
nitrate and other compounds from
roots to leaves
reduction of
amino acid
synthesis
photoinhibition
ROS production
damages to biological
structures
polysome reduction
altered cell wall extensibility
growth inhibition
lower photosynthetic activity
Physiological traits relevant for response to drought conditions
Timing of phenological phasesEarly / late flowering. Maturity
and growth duration
Wheat and barley advanced
flowering, rice delayed
Rooting depthHigher / lower tapping of soil
water resources
Reduced total mass but increased root/shoot ratio,
growth into wet soil layers
Stomatal resistanceMore / less rapid water consumption
Increase under stress / Response to soil water potential
Cuticular resistance and surface roughness
Higher or lower water loss, modification of boundary layer and reflectance
Partitioning and stem
reserve utilization
Lower / higher remobilisation of
reserves from stems
Compensation of reduced current leaf photosynthesis by
increased remobilization
Osmotic adjustmentAccumulation 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
ERECTA
A putative leucine-rich
repeat receptor-like
kinase is a major
contributor to a locus for
on Arabidopsis
chromosome 2.
ERECTA acts as a regulator of
transpiration efficiency with effects
on stomatal density, epidermal cell
expansion, mesophyll cell
proliferation and cell–cell contact.
Masle et al., 2005
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.
The water use efficiency
Yield under water stress is a function of (i) water extracted from soil (ii) water use efficiency and (iii) harvest index.
• 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
Cell response to drought stress (generally conserved across species)
Transcription factors(MYC, MYB,bZIP,
EREBP/AP2)
Protein Kinases(MAPK, MPKKK,
CDPK,S6K)
Protein phosphatases(PTP)
PI turover(Phospholipase C,
PIP5K, DGK, PAP)
Membrane proteins( Water channel protein,
transporters
Proteinases(cytoplam, chloroplast)
Protection
of macromolecules(Chaperons, LEA proteins
Osmoprotectant
synthases(Proline, Gly betane,
sugar)Detoxification
enzymes(GST,sEH, SOD
Drought
Stress
REGULATORY PROTEINSFUNCTIONAL PROTEINS
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 stress-regulated mRNAs
3°°°°C
cor14b
tmc-ap3
blt14
cor18
af93
20°C 1h 5h 12h 1d 3d 5d 8d 10d 15d
The chromosome 5A controls the expression of cor14b
18°°°°/13°°°°C 2°°°°C 18°°°°/13°°°°C + ABA 18°°°°/13°°°°C + PEG
CN
NCS
TSP
CS/C
NN
5AC
S/T
SP5A
CN
NCS
TSP
CS/C
NN
5AC
S/T
SP5A
CN
NCS
TSP
CS/C
NN
5AC
S/T
SP5A
CN
NCS
TSP
CS/C
NN
5AC
S/T
SP5A
Cor14b expression polymorphism in T.monococcum
blt14
DV92 G31165°°°° 10°°°° 15°°°° 20°°°° 25°°°° 5°°°° 10°°°° 15°°°° 20°°°°25°°°°
cor14b
The molecular bases of QTL for stress tolerance: the frost tolerance QTL of winter cereals
Cor14b expression polymorphism in T.monococcum segregating population
Cor14b expression-QTL co-segregates with frost resistance QTL and Cbf locus
LOD score
1
2
3
4
5
6
7
8
910cM
Frost resistance QTL
Cor14b expression QTL
Xp
sr4
26
Xw
g644
Xcd
o465
Xp
sr2
021
XD
hn
2.1
Xm
wg
2062
Xp
sr1
20
Xb
cd
9
XE
si1
4
Xg
wm
639
XC
bf3
Xb
cd
508
Xw
g530
Xb
cd
351
Xcd
o57
Xb
cd
926
Xg
wm
186
Chr 5Am
cor14b expression and frost resistance I
Rcg1 Fr2Xbcd508
Xpsr2021-dhn2
5A T. monococcum
Vrn1
X-cbf3
COR proteins accumulation in barley DH population (N x T)
under field conditions
COR14b
N T
TMC-AP3
N T
cor14b expression and frost resistance II
Rcg1
Fr1
Fr2
cbf3-4
cbf8
psr637
Xpsr2021-dhn2
5 H. vulgare
Vrn1
psr911Rcg1 Fr2Xbcd508
Xpsr2021-dhn2
5A T. monococcum
Vrn1
X-cbf3
Two loci control frost resistance in triticeaeThe expression of cor14b is genetically link to
frost resistance and to a cbf-like locus
Expression of cor14b is controlled at transcriptional level
GUS- 643TATAbox
-1
GUS
GUSubi-GUS 20°C ubi-GUS 3°Ccor14-GUS 20°C cor14-GUS 3°C
� -274 5’ AGCTTACCCAAAGGTACGTGAGGTCGG3’ -247
Mutational analysis of the cor14b promoter region-274-247: identification of a potential cis-element
DRE
The expression level of Cbf is associated to frost resistance in wheat
2 hours +2°°°°C
Control (20°°°°C)
Different expression level of Cbf is associated with a QTL for frost resistancein 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
Francia et al., 2007Knox et al., 2008
XpsrB85 C C T C T T T C T C
XpsrB89 C C T C T T T C T C
Xpsr911 (Rcg1) C C C T T T T T T C
Xpsr637 C C C T T T T T T C
Xpsr2021 (Rcg2) - C - - - - T - T C
Fr-A1 C C T - C C T T T C
Xcdo504 C C T C C C T T T C
Xwg644 C C T C C C T T T C
Xpsr426 C C T C C C T T T C
Vrn-A1 C C T C C C T T T C
Xpsr805 C T T C T C C T T C
Q - T - - - - C - T C
Xpsr370 T T T C T C C T T C
Xpsr164 T T T C T C C T T C
b-amy-A1 T C T C T C T T T C
XpsrB85 C C T C T T T C T C
XpsrB89 C C T C T T T C T C
Xpsr911 (Rcg1) C C C T T T T T T C
Xpsr637 C C C T T T T T T C
Xpsr2021 (Rcg2) - C - - - - T - T C
Fr-A1 C C T - C C T T T C
Xcdo504 C C T C C C T T T C
Xwg644 C C T C C C T T T C
Xpsr426 C C T C C C T T T C
Vrn-A1 C C T C C C T T T C
Xpsr805 C T T C T C C T T C
Q - T - - - - C - T C
Xpsr370 T T T C T C C T T C
Xpsr164 T T T C T C C T T C
b-amy-A1 T C T C T C T T T C
Cbf conserve domain
Cbfs are differentially expressed in resistant vs susceptible plants
Cbf15
Cbf16
Cbf14
Selected Cbfs have been used for association mapping in a population of about 130 european barley cultiuvars
Knox et al., 2008
The ratio of variable to maximal fluorescence (Fv/Fm) in dark-adapted state measures the maximum quantum yieldfor PSII photochemistry and represents a diagnosticprobe for measuring low temperature stress-inducedinjury of photosynthesis
HvCbf14
HvCbf6 HvCbf3 HvCbf9
Linkage disequilibrium at CBF loci in 130 European barley cultivarsThe 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 SNP Allele F-value P-valueHvCbf14 SNP 10 (C/T) C 27.11 <0.0001
HvCbf14 SNP 7 (C/T) C 21.13 <0.0001
Vrn-H1 SNP1 (G/A) G 6.89 0.0016
HvCbf6 SNP 13 (G/C) C 5.64 0.0047
HvCbf14 SNP 10 (C/T) T 1.59 0.2106
HvCbf14 SNP 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
AcknowledgementsCRA – 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)