Age Related Resistance in Crop Plants

ManjappaPAL 0203Sr. M. Sc.

Dept. of Genetics & Plant BreedingUAS, GKVK, Bangalore, India

University of Agricultural Sciences, GKVK, Bangalore-65

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Host factors that affect development of epidemics

Level of Genetic Resistance or Susceptibility of host Degree of Genetic uniformity of host plantsType of cropAge of host plants

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Host Defense Mechanisms

1. Basal resistance

Recognition of microbe-associated molecular patterns (MAMPs), such as bacterial flagellin

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2. R gene-mediated resistance: (HR)

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SAR and ISR Response Pathway

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Life span of the plant

Susc

eptib

ility

Growth period Adult period

3. Age related resistance (ARR)

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(Change of susceptibility of plant parts with age)

Effect of Crop age rate of infection

Cassava planting of different ages exposed to African Cassava mosaic geminivirus show increased resistance to infection as they age.

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Age Related Resistance

Increase or acquisition of resistance to pathogenic infections as a function of plant development.

Eg: rice/Pyricularia oryzae & rice/Xanthomonas compestris pv oryzae

Syn: ontogenic resistance, developmental resistance, mature seedling resistance, adult seedling resistance.

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Positive correlation between increasing plant age & glyceollin production - Phytophthora megasperma var. sojae (Soybean)Cotton phytoalexin in response to Verticillium albo-atrumConstitutive accumulation of terpenoids in cottonCapsidol- phytophthora capsiciToxic compoundsDefense associated compounds

- In tobacco PR proteins – PR1, PR2 & PR3 against virus & fungal pathogens - Salicylic acid in Tobacco & Arabidopsis

ARR mechanisms will differ with crop/pathogen interaction

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Age-related Resistance in Arabidopsis Is a Developmentally Regulated Defense Response to

Pseudomonas syringae

• Arabidopsis thaliana ecotype Colombia (Col-0), mutants npr1-1, etr1-4,pad3-1, eds7-1, sid1 and sid2 & transgenic NahG line

• Avirulent & virulent strain of Pseudomonas syringae pv tomato (Pst) strain DC3000 & P. maculicola(Psm) strain 4326

Julianne et al. (2002) The Plant Cell12

Col-0 plant leaves 8 & 16 were inoculated with Pst @ 106 cfu/mL from 26-57 dag at 1 wk internal

Old plants become more resistant to Pst i.e. 10 fold reduction in bacterial growth between 30 & 40 dag.

There was no significant difference in leaf 8 & 16 bacterial levels- differences in leaf morphology do not affect ARR

50 dag

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Defective for SARMore susceptible

Accumulates very lessCamalexin, wild type

Susceptible to Pst & Psm, Wild type to SAR

Defective in ethylenesignaling & ISR ARR is not require ethylene signaling & not an ISR response to PGPR

SAR not requires to ARR

No effect on ARR

In Planta Bacterial Growth In Young and Mature Arabidopsis Mutants

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Then which factor is responsible ?...

• Transgenic NahG & mutant sid1 and sid2 plants (accumulate little SA) were tested to determine whether SA accumulation is required for ARR response.

Both sid1 & sid2 supported vigorous in planta bacterial growth in young & mature plants in a manner similar to NahG, unlike wild-type Col-0.

SA accumulation is required for ARR15

Accumulation of SA act as a signaling molecule, stimulating the production and secretion of heat stable anti-microbial compound(s) into the intercellular space

Antimicrobial activity was detected only in IWFs from mature plants inoculated with Pst, not in IWF from mature mock-inoculated plants

ARR is developmentally regulated and pathogen induced response in Arabidopsis.

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Stress induces an ARR-Like responses in young plants

Mild nutrient limitation can affect the level of in planta growth of Pst.

In young rice, wounding of one leaf induces accumulation of Jasmonate & local activation of PR genes. This is correlated with ISR to subsequent blast disease.

Mild drought- reduced in planta bacterial growth (2-6 fold) compared to control plants of same age

Constantly wet soil sometimes supports algal growth & that plants grown under these conditions exhibit reduced growth of Pst.

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Forms of ARR

1. Resistance & developmental transition2. Resistance & tissue maturity3. Increased or acquired resistance & plant development4. specific & broad spectrum resistance

In Turnip and Arabidopsis, the long-distance movement of CaMV is influenced by the developmental stage of the invaded leaves & progressively more restricted to basal portion of lamina during growth

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1. Resistance & developmental transition

Postembryonic /vegetative in ArabidopsisCol-O ecotype develop resistance in true leaves against DM fungi but not so in Ws-O ecotype (it lacks RPP31)

Juvenile/adult transition during vegetative growthIn maize Corngrass1 mutant (Cgm1), the juvenile-vegetative phase is extended & adult resistance to common rust (Puccinia sorghi) is delayed. There fore expression of adult characteristics are necessary for leaf resistance.In Cabbage adult stage is resistant to DM than cotyledony stage

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Cont…

Correlation between floral transition & resistanceArabidopsis resistance to CaMV & Pseudomonas syringae.Confirmed by terminal flower 1 (tfm) mutant. (TFL1 Protein floral induction & maintenance of floral identity in apical meristem)PR1 & PR2 are specifically expressed in tobacco floral tissue

Resistance may correlated with senescence. (Not so in Arabidopsis/Pst to SAG13)

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Developmentally regulated mechanisms affect the ability of a fungal pathogen to infect and colonize

tobacco leaves

Karine Hogot et al. (1999), The Plant Journal, 20(2)

Vegetative phase Flowering phase

Control infection effectiveness (intercellular fluid, not SA)

Restriction of fungal hyphae expansion(PR1 & SA accumulation)

Material & methods: Nicotiana tabacum cv Xanthi nc., transgenic lines NahG-8 & NahG-9 expressing or not expressing NahG genes (codes for Salicylate dehydrogenase, converts SA to catechol)Phytophthora parasitica isolate 329, infiltrated 50µl of suspension (100 zoospores)

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Tobacco plants begin to express developmental resistance at 75-85 das

An average of 60% of the inoculated zones showed disease symptoms, but spreading was reduced by 80% not only control of fungal hyphae expansion but also decrease in infection efficiency during flowering phase

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SA-dependent & SA-independent mechanisms

Expression of mechanisms which lead to control of fungal devt. after floral transition require SA accumulation.Increase in inoculated zones without any symptoms of diseases in NahG-8 shows, induction of events that affect the ability of fungus to infect leaf tissue do not require SA accumulation 24

Acquired resistance during development & apoplastic PR1 protein accumulation

Establishment of SAR leads to systemic transcriptional activation of a subset of PRs genes

Immunoblotting expt’s were undertaken to study accumulation of PR1 protein in apoplasm during plant devt.

Xanthi nc plants were treated with cryptgein to induce SAR. (induced PR proteins)

PR1 Protein expression was correlated with the ability of tobacco to inhibit the fungal devt. in planta rather than infection effectiveness during flowering

IFs (Hammond-Kosak)- Protein extract

15% SDS-PAGE electrophoresis

Incubate Nitrocellulose membrane with IgG antibody

Goat peroxidase cojugated IgG

Detection system (Amersham)

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Which controls Infection effectiveness during flowering ?

• Intercellular fluid (cytotoxic activity)

Survived cells

germinated cells::::

The influence of Ifs on in vitro germination of zoospores

Zoospores (5x105) were incubated for 2 h in the presence of different IFs

Cytotoxic activity on fungal cells was not detected in IFs from cryptgein treated Xanthi plants.

The expression of an in vitro cytotoxic activity on fungal cells in IFs is from plant committed to flowering

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2. Resistance & Tissue maturity

• Several plant species develop resistance that is restricted to a given tissue or organ, as a function of maturity.

• Soybean: Resistance to Phytoophthora sojae in hypocotyl varies with tissue maturity.

• Apple tree: Leaf maturity is positively correlated with resistance to Venturia inequalis

• Maize: During vegetative growth, leaves with juvenile traits are susceptible & leaves with adult traits are resistant (>8th node) to Puccinia sorghi

• Rice: Leaf maturity has no effect on the degree of resistance to Xanthomonas compestris pv. oryzae, where as leaf rank does have an effect.

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Effect of Age & Leaf Maturity On the Quantitative Resistance of Rice Cultivars to Xanthomonas campestris Pv,oryzae

• Materials: Cisadane, BR51-282-8(BR51), IR28, IR40 are moderately resistant.

• TN1 & IR9101-46 (9101)- susceptible checks• Xanthomonas campestris ov. oryzae race2 strain PXO86 &

race6 strain PXO99 (1 X 109 cfu/ml)

} Largest decrease in lesion length (cm)

Koch et al. (1991), American Phytopath. Soc. 28

Cont…Decrease in lesion length was less evident after maximum tillering, but flag leaves were more resistant than leaves at booting stage

No significant age X cultivar interaction was foundPlant age does not greatly affect our ability to distinguish among

intermediately resistant cultivarsDifference in lesion length between immature and mature leaves

was similar in all cultivarsScreening for quantitative resistance to X.c. oryzae can be done at

all stages of growth

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3.Increased or acquired resistance & plant development

Rice: Xoo & X. compestris pv. Oryzae Pyricularia oryzae

Tobacco: TMV, Peranospora tabacina

Soybean: Phytophthora sojae

Arabidopsis: Hyaloperanospora parasitica

Wheat: P. recondita f.sp. tritici

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4a. Developmental effect on Specific resistance

• ARR may be effective against several pathogens, a particular pathovar, strain or race of pathogen

Crop Gene(s) PathogenRice Xa6 X. Campestris pv. oryzae

Xa21 X. oyzae pv. oryzaeWheat Lr gene fam. (few) P. recondita f.sp. tritici

Sr gene fam. (few) P. graminis f.pv. TriticiTomato Hcr9-9A, Hcr9-9B,

Hcr9-9thCladosporium falvum

Maize Cg1 Puccinia sorghi

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Developmental control of Xa21-mediated disease resistance in rice

Reproducible means of infecting plants at full leaf expansion

Xa21-resistance progressively increases from the susceptible juvenile leaf 2 stage.

Resistance (%)= 1 Xa21 line mean lesion length susceptible line lesion length - X 100

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Cont.. Xa21 expression is independent of plant

developmental stage, infection with Xoo, or wounding

Expression of the Xa21 gene transcript is not correlated with expression of Xa21 disease resistance

Developmental regulation of Xa21-resistance is either controlled post-transcriptionally or by other factorsXA21 has an intracellular serine–threonine kinase domain, it is a likely possibility that XA21 activity is controlled by phosphorylation statusRice Xa21 binding protein 3 is a ubiquitin ligase (HB3) required for full Xa21-mediated disease resistance

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4b. Developmental effect on Broad spectrum resistance

Flowering growth in tobacco: express resistance to Peranospora tabacina, Phytophthora parasitica & TMV

Mature Arabidopsis: Pseudomonas syringae pv. tomato and pv. maculicola as well as Hyaloperanospora parasitica

After onset of berry ripening in grape: express resistance to 3 ascomycetes

Control of viral migration (CaMV) in Turnip & Arabidopsis.

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Genetic Analysis of Developmentally Regulated Resistance to DM (Hyaloperonospora parasitica) in

Arabidopsis thaliana ARR to H. parasitica Emco5

is activated in true leaves of Arabidopsis Col-O but not Ws-O:

Ws-O is highly susceptible to Emco5 throughout the devt., in contrast to Col-O (shows delayed HR)

McDowell et al. (2005), American Phytopath. Soc.

S-Sporangiospore, O-oospore,TN- Trailing necrosis, H-hyphae 35

Adult resistance in Col-O is race-specific & is suppressed by defense signal transduction mutants

Adult Col-O plants are susceptible to H. parasitica isolate Noco2 & Ahco2

Trailing necrosis/HR & H2O2 production in true leaves indicates, it is results from active defense response of host

Each of isogenic Col lines is deficient in SA-mediated signaling their by impairing basal resistance, SAR & certain R genes

Adult resistance in Col-O requires variety of regulatory components previously associated with inducible defense

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Genetic analysis of Adult resistance

Phenotypes in F1 indicates susceptibility is incompletely dominant over resistance.

F2 segregation ratio: 24% Col-like adult resistance (1):76% full/intermediate susceptible (3)- indicates single recessive gene controls

Segregation of adult resistance to Hyaloperanospora parasitica Emco5Cross F1 F2

Sus Int Res Res

rpm1-3 (Col- 5) x Ws-O 0 8 1 30 89 1.09 (P>0.3)

Col-O X Ws-O 0 6 3Ws-O X Col-O 0 2 4

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Recessivity of resistance in Col-O x Ws-O hybrid could be a gene-dosage effect !

XWs-O (2x) Col (CS3151, CS3432-4x)

F1 (3x) Col/Col/Ws-O Susceptible

But supported less sporulation than Ws-O parent & diploid F1 Ws-O susceptibility phenotype is incompletely dominant to adult resistance in Col-O.Genetic mapping in a Col x Ws F2 population revealed a major locus on the bottom arm of the chromosome 5, named as RPP31

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Molecular Mechanisms of Developmental Resistance

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Cont…

Crop Developmental control

Response Pathogen

Tobacco FloweringPR1a,PR2Chitinase, ß- (1-3) glucanase, peroxidase

TMVPeranospora tabacina

Grape Berry ripening Chitinase, PR5, LTP U. necatorHordeum vulgare

Embryo developingBefore grain desiccation

9-LOX (Lipoxigenase) pathwayPR

Pathogens

Developmental resistance also involves up-regulation of gene involved in modification /strengthening of cell wall along with defense genes

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Defense mechanisms in Arabidopsis

• SA has antimicrobial activity & partially rescues the iap1-1 ARR defect.

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Suggesting iap1-1 lies up-stream of SA accumulation in ARR pathway.

Prediction: iap1-1 accumulates little intercellular SA.

Leaves of 5 wk old plants inoculated with Pstat 5 & 24 h post infiltration

Level of SA in Pst or mock inoculated mature plantsmeasured by Gas chromatography-mass spectrometry

Cont…

Addition of SA in the intercellular space of eds1-1 plants did not result in the rescue of the eds1-1 ARR defect. EDS1-1 at down-stream of SA

Combining microarray analysis with reverse genetics using T-DNA insertion lines, 4 additional genes were identified, UGT85A1, CDA1, ANAC055 and ANAC092

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iap1-1

Salicylic acid

eds1-1

UGT85A1, CDA1, ANAC055 and ANAC092

Defense to infectionCarviel, J. L., et al.,2009, Mol. Plant Path., 10(5): 621-634

PR proteins (PR3, PR5, nsLTP) are express constitutively at low level in cells and accumulate in response to fungal attack or inducers of acquired resistance with the exception to reproductive organs (developmentally regulated expression).

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A. Pattern of protein accumulation in grape berry during ripening.

B. Comparison of total protein profile of susceptible Vs resistant variety to Botrytis cinerea

Developmental accumulation of antifungal proteins like – thoumatin like protein (GO), chitinase (CBC & AC forms) & ns LTP and hexoses in grape berry provide resistance against Guignardia bidwellii (fruit rot) & Botrytis cinerea

GO

nsLTP

ACCBC

Seyval Blanc

Concord

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Sugar molecules may act as a signal molecule to regulate the expression of antifungal proteins genes during fruit ripening.Sugar repress genes which codes for C-assimilatory enzymesUp-regulates genes for protease inhibitors, chalcone synthase, PR3, PR1b & PR-Q

Sugar and antifungal proteins interact to mediate host plant defense against phytopathogens either byDisruption of fungal gene regulation by sugar repressionSolute mediated preservation of native protein structure (CBC, GO)

Relationship between defense & development in plants

Expression of resistance to disease during host devt.Involvement of plant hormones in devt. & P-P interaction

Eg: SA, JA, Ethylene, ABAMolecular conservation of transduction pathways governing various process

Eg: Arabidopsis- In csa1 mutant TIR-NBS-LRR protein involved in photomorphogenic devt. is complemented by RPS4 (homologue of TIR-NBS-LRR confers resistance to Pst)

Tobacco transcription factors of the TGA family: TGA2.1- SA inducible gene expression & TGA 2.2- regulatory role in correct stamen devt.

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Con clusion

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