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)