Department Of Agricultural Biotechnology

(Centre Of Excellence In Biotechnology)

Anand Agricultural University

Anand-388110.

‘Prime-ome’ : A Molecular Approach Towards Defense Modulation

Speaker : Dhanya A J

Degree : M. Sc. (Agri.) Plant Molecular

Biology and Biotechnology

Major Guide : Dr. G. B. Patil

Minor Guide : Dr. Sasidharan N

Course No : MBB 591

Reg. No. : 04-2348-2014

Date : 17/10/2015

Time : 16:00

Contents

Introduction

SAR and ISR

Priming

Prime-omics

Prime-omics in defense against pathogens and pest

Conclusion

Future thrust

1

5

6

7

2

3

4

2

Biotic stressAbiotic stress

Crop loss

>50% Crop loss

Insects- 25%

Pathogens- 20%

Vertebrate pests

- 6-8%

Plants have evolved various strategies to defend themselves

against stresses. Although some of these strategies are

constitutive, i.e. present at all times, others are induced only

in response to herbivore feeding or pathogen infection.

As early as 1933, priming called ‘sensitization’ at the time,

was widely accepted to be the pivotal phenomenon in

systemic plant immunity. [Chester., 1933]

Priming has now proven true as a critical process in various

types of systemic plant immunity [Conrath et al., 2002;

Conrath et al., 2006; Conrath et al., 2009; Jung et al.,

2009]. These include systemic acquired resistance (SAR),

induced systemic resistance (ISR), the resistance provided

by symbiotic fungi, b-aminobutyric acid-induced resistance

(BABA- IR) and wound-induced resistance.

Introduction

3

Induced systemic resistance (ISR): type of systemic,

broad spectrum immunity in plants. Induced systemic

resistance is elicited by colonization with selected

strains of non-pathogenic, plant growth-promoting

rhizobacteria and depends on the plant hormones

ethylene and jasmonic acid.

Systemic acquired resistance (SAR): type of

systemic, broad-spectrum immunity in plants. Systemic

acquired resistance is induced by local contact with a

MAMP*, PAMP*, or effector and depends on the plant

hormone salicylic acid.

* Microbe-associated molecular pattern (MAMP): molecular signatures typical of whole classes of microbes.

Their recognition plays a key role in innate immunity in plants and animals.* Pathogen-associated molecular pattern (PAMP): molecular signatures typical for potential microbial

pathogens of a given host organism.

Fig. 1

4

* Microbe-associated molecular pattern (MAMP): molecular signatures typical of whole classes of microbes.

Their recognition plays a key role in innate immunity in plants and animals.* Damage-associated molecular pattern (DAMP): signals arising from plants because of damage caused by

microbes; originally referred to as ‘endogenous elicitors’.

It is the induction of a

physiological state that

enables cells to respond to

very low levels of a

stimulus in a more rapid and

robust manner than non-

primed cells. In plants,

priming plays a role in

defense (‘defense priming’)

and seed germination (‘seed

priming’).

PRIMING

Fig. 2

5

Primed state: It is the physiological state of a plant that has been subjected to

priming. Usually starts on exposure of such a plant to a stress.

Fig. 36

Priming events can occur as a result of inter-individual or inter-species communication,

such as induced resistance mediated by rhizobacteria, mycorrhizal fungi, or virulent or a

virulent pathogens or by natural or axenic compounds. Plants ‘remember’ such events

(Priming memory) and, depending on the type of primary stimulus or priming

stimulus(initial trigger for priming) and the pathosystem involved (target of priming),

primed plants can deploy a diverse set of defense mechanisms.

Types based on the stimuli which

induces priming

Natural priming (by microorganisms)

Chemical priming (treatment with

chemicals)

Constitutive priming (alterations of specific

defense-repressive constitutive genes)

7

Fig. 4 8

Prime-ome:

Biotic Stress

Gene Transcript

Protein

Metabolite

Transcriptomics

Proteomics

Metabolomics

Prime-omics

Abiotic Stress

Prime-omics: the totality of transcriptional, proteomic, and metabolic data available to

describe the priming of plants or it’s the study of prime-ome.

It is the entire set of messenger RNA (mRNA) molécules or transcripts, proteins and

metabolites produced or modified by an organism or system during the different stages

of priming in plants.

Fig. 5

9

The priming process consists of three clear stages

The initial phase of resistance induction, where the plant is preparing for a future attack but has not yet been challenged by a pathogen is called the priming phase (Conrath et al., 2002).

This phase lies between the perception of the priming cue and the first exposure to a future stress. During this time slot the plant generate and store information that will enable it to deploy faster and/or more accurate response to stress.

This phase starts with the exposure to a stress or challenge by the plant. In primed plants, cellular defense responses are not activated directly by the priming agent but are memorized and expressed in an accelerated manner after perception of a second biotic or abiotic stress signal.

During this time slot the plant has strong up- or downregulation of gene activity that will enable it to deploy faster and/or more accurate response to stress.

Priming for enhanced resistance also extends to next generations i.e. the progeny of the primed parental plants shows resistance to the stress against which it have been primed.

21 3

10

Pla

nt

react

ion

Priming stimulus Challenge Challenge

Priming phase Post – challenge primed state

Time

Transgenerational primed state

Stages of primingFig. 611

Seed collection & next generationInoculation

(12-oxo-phytodienoic acid)

12Gamir et al., 2014Fig. 7

(Indole-3-carboxylic acid)

Prime-omics in defenses against pathogens and pest

13

Plant Priming Tools Suggested mechanisms Refs

By Against

Priming phase Nicotiana

tabacum

Agrobacterium

tumefaciens

GV3101

– qRT-PCR Protein

immunoblotting

detection

SA, ROS, MAPK Sheikh et al., 2014

Solanum

tuberosum

Phosphite

BABA

GABA

Laminarin

INA

– Microarray

LC-MS/MS

SA, PR proteins, PTI, HR,

wall-associated kinase, primary

metabolism, TCA, ROS, Ca2+-

dependent

pathway redox- regulating

enzymes, sesquiterpene

phytoalexin biosynthesis

Bengtsson et al., 2014

Massoud et al., 2014

Lim et al., 2013

Jelonek et al., 2013

Hordeum

vulgare

Pseudomonas

fluorescens

– Microarray

RT-PCR

Detoxification, lipid transfer,

cell wall biosynthesis, JA

Petti et al., 2010

Zea mays Synthetic

indole

dispensers

– GC-flame

ionisation detector

(FID)

HIPVs, ABA, JA, JA-Ile Erb et al., 2015

Post-

challenge

primed state

Oomycetes

Vitis spp. Methionine Plasmopara viticola qRT-PCR H2O2

measurement

(FOX1 method)

ROS Boubakri et al., 2013

S. Tuberosum Phosphite Phytophthora

infestans

LC-MS/MS Callose deposition, HR, TCA Lim et al., 2013

Overview of the omics involved in various stages of priming in plantsTable 1

Balmer et al., 201514

Post-

challenge

primed

state

Plant Priming Tools Suggested

mechanisms

Refs

By Against

Bacteria

N. tabacum A. tumefaciens

GV3101

Pipecolic acid

P. syringae pv. tabaci

P. syringae pv. tomato DC3000

qRT-PCR, Protein

immunoblotting

detection

Callose deposition, SA,

nicotine

Vogel et al., 2013

Sheikh et al., 2014

Rico et al., 2010

Solanum

lycopersicum

Hexanoic acid P. syringae pv. tomato

DC3000

qRT-PCR

LC/MS

JA biosynthesis,

SA-responsive genes

Scalschi et al., 2013

Capsicum

annuum

VOC 3-pentanol Xanthomonas axonopodis

pv. vesicatoria

qRT-PCR SA, JA Choi et al., 2014

Fungi

Brassicia carinata BABA Alternaria brassicicola Enzyme activity

assay

ROS Chavan et al., 2013

Triticum spp. line

PmA/var. Sahara

H2O2

Z-3-HAC

Blumeria graminis

Fusarium graminearum

Deep sequencing

qRT-PCR

U-HPLC-MS

JA and/or Et signalling

pathways, lipid

metabolism

JA

Li et al., 2011

Ameye et al., 2015

Hordeum vulgare P. fluorescens

Piriformospora

indica

Fusarium culmorum

B. graminis

RT-PCR ELISA

qPCR

Microarray

LC/MS

IAA, JA, ABA, PR

genes, sugar cycling,

TCA, detoxification,

lipid transfer, cell wall

biosynthesis

Molitor et al., 2011

Petti et al., 2010

Petti et al., 2012

S. lycopersicum Trichoderma

harzianum

B. cinerea qRT-PCR JA, SA, ABA Medina et al., 2013

Cucumis sativus Pseudomonas

azotoformans

Paenibacillus

Elgii, BABA

Colletotrichum orbiculare Enzyme activity

assay

HR, H2O2 defence-

related enzyme

accumulation

Sang et al., 2014

Balmer et al., 2015

Table 1.1

15

Plant Priming Tools Suggested mechanisms Refs

By Against

Post-

challenge

primed state

Nematodes

S. lycopersicum Arbuscular

mycorrhizal fungi

(AMF)

Meloidogyne

incognitaRT-PCR Suppression

subtractive

hybridisation

Phenylpropanoid pathway,

ROS metabolism

Vos et al., 2013

Vitis spp. Arbuscular

mycorrhizal fungi

Xiphinema index RT-PCR Suppression

subtractive

hybridisation

Chitinase, PR genes,

shikimate enzyme pathway

Hao et al., 2012

Arthropods

Phaseolus

lunatus

JA

(E)-b-Ocimene

Tetranychus urticae RT-PCR GC/MS

Olfactory choices

PIOS, volatile emission, JA,

predator attraction

Muroi et al., 2011

Gols et al., 2003

A. thaliana MeSA + feeding

larvae

Caterpillar feeding

Pieris brassicae RT-PCR

Choice assays

Northern blot

ABA, JA, oviposition

deterrence

Groux et al., 2014

Vos et al., 2013

S. lycopersicum AMF

Aphid feeding

Helicoverpa

arimigera

Bemisia tabaci

RT-PCR

Olfactory choices

Larval deterrence, JA

signalling, systemin signalling,

HIPVs – indirect defence

Song et al., 2013

Ammopiptanthus

mongolicus

Conspecifics

HIPVs

Orgyia ericae NMR- metabolomics TCA, amino acids, lipids,

glycolate, sugars

Sun et al., 2014

Oryza sativa Silicon Cnaphalocrocis

medinalis

RT-PCR JA signalling Ye et al., 2013

Z. mays (E)-b-Ocimene Mythimna separata GC/MS

Olfactory choices

Parasitoid attraction Muroi et al., 2011

Transgenera-

tional primed

state

S. lycopersicum MeJA/herbivory Pieris raphae qPCR JA signalling Rasmann et al., 2012

N. tabacum Tobacco mosaic

virus

Tobacco mosaic virus NMR GC/MS Sugars, amino acids Mandal et al., 2012

Table 1.2

Balmer et al., 201516

Prime-omics in defense against bacteria

Vogel et al., 2013Germany

Also proved that exogenous application of Pipecolic acid to tobacco plants provides significant

protection to infection by Pstb and hypersensitive cell death-inducing P. syringae pv maculicola

(Psm).

Pipecolic acid thereby primes tobacco for rapid and strong accumulation of SA and nicotine

following bacterial infection.

L-Pipecolic acid is a Lys-derived non-protein amino acid of plants whose levels increase upon

plant treatment with growth-affecting chemicals, in response to osmotic stress, and in the course

of pathogen infections.

Tobacco plants respond to leaf infection by the compatible bacterial pathogen Pseudomonas

syringae pv tabaci (Pstb) with a significant accumulation of several amino acids, including Lys,

branched-chain, aromatic, and amide group amino acids. Moreover, Pstb strongly triggers,

alongside the biosynthesis of SA and increases in the defensive alkaloid nicotine, the production

of the Lys catabolites Pipecolic acid (Pip) and α-aminoadipic acid (Aad).

Pipecolic acid enhances resistance to bacterial infection and primes salicylic acid and nicotine accumulation in tobacco

18

Mean values of 3 to 5 replicate samples are given in μg g-1 fresh weight (FW) ± SD. Mock-treatments were performed by infiltration of

leaves with a 10 mM MgCl2 solution. Asterisks denote statistically significant differences between Pstb- and MgCl2- samples (2-tailed t-

test; ***: p < 0.001; **: p < 0.01; *: p < 0.05). Ratios of the values of the Pstb (P) and the MgCl2 (M)-samples (P/M) are also given.

Changes in the levels of free amino acids and amines in N. tabacum cv Xanthi leaves(4 week old) upon

inoculation with compatible P. syringae pv tabaci (Pstb) 2 d post inoculation (dpi). Table 2

19

Time course of (A) pipecolic acid (Pip) and (B) α-aminoadipic acid (Aad) accumulation in tobacco leaves inoculated with

compatible Pstb at indicated times after inoculation.

Time course of (A) salicylic acid (SA ) accumulation and (B) nicotine production in tobacco leaves inoculated with compatible

Pstb at indicated times after inoculation.

Fig. 8

Fig. 9

20

Exogenous Pip primes tobacco plants for effective SA and nicotine production

upon Pstb inoculation. H2O or 10 μmol Pip were applied to plants through the soil. Leaves were infiltrated 1 d later with Pstb (dark bars) or MgCl2 (light bars), and leaf

metabolite levels were scored 8 h later. (A) SA contents in leaves. (B) Leaf nicotine levels. Bars represent the mean ± SD of 3 replicate samples. Different

letters above the bars denote statistically significant differences between pairwise compared samples (p < 0.05, 2-tailed t-test).

Fig. 10

21

Exogenous Pip enhances disease resistance of tobacco plants to Pstb and Psm.

Plant pots were supplied with 10 ml of H2O or 10 ml of 1 mM (10 μmol) Pip 1 d prior to bacterial inoculation. (A) Bacterial numbers of Pstb (applied in titers

of OD 0.001) in leaves at 0 dpi and 5 dpi. The y-axis is depicted in a logarithmic scale. Bars represent the mean ± SD of at least 7 replicate samples. Asterisks

denote statistically significant differences between leaf samples of control- and Pip-treated plants (***: p < 0.001; 2-tailed t-test). (C) Bacterial numbers of

Psm (applied in titers of OD 0.005) in leaves at 0 dpi and 5 dpi.

Fig. 11

22

Representative disease symptoms of Pstb-infected tobacco leaves from H2O and

Pip pre-treated plants.

Pstb Psm

Fig. 12

0 dpi 0 dpi5 dpi 5 dpi

23

Prime-omics in defense against oomycetes

Burra et al., 2014Sweden

.

Phosphite-induced changes of the transcriptome and secretome in Solanum tuberosum leading to resistance against Phytophthora infestans

Investigated the transcriptome of Solanum tubersoum (cv. Desiree) and characterized the

secretome by quantitative proteomics after foliar application of the protective agent

phosphite.

It seems that multiple defense pathways are rapidly induced by phosphite treatment that

causes heightened defense leading to enhanced resistance after pathogen infection in local

tissue.

Phosphite had a rapid and transient effect on the transcriptome, with a clear response after

3 h of treatment. This effect lasted less than 24 h, whereas protection was observed

throughout all time points tested.

It activates the genes associated to both biotic and abiotic stress response.

In field applications, the dual nature of the phosphite molecule both being an inducer of

plant resistance and having a direct toxic effect on oomycetes might explain the high

efficacy.

25

Detached leaflet assay of potato plants

Potatoes (cv. Desiree) were foliar sprayed either with 36 mM proalexin (Potassium phosphite; phosphite treated) or tap water

(Water treated). “Covered leaves” leaflets were obtained by covering two leaves per plant during phosphite spray. Washed

leaflets were obtained by spraying leaves with 36 mM proalexin, washing and drying away the phosphite present on the

leaves. Infection was measured as lesion size 7 days after inoculation with P. infestans. Data corresponds to mean ± SD

obtained from 12 biological replicates.

Fig. 13

26

Gene ontology (GO) analysis: representation of processes and associated

example transcripts (in brackets) significantly regulated at each time point.Fig. 14

27

Time(h)

No

: of

sign

ific

ant

tran

scri

pts

Differentially expressed genes: A

comparison of number of transcripts

induced and repressed at each time

point.

Area proportional Venn

diagram depicting overlap of

transcripts significantly altered

at all the time points .

Fig. 15Fig. 16

28

Molitor et al., 2011Germany

• Colonization of barley roots with the basidiomycete fungus Piriformospora indica

(Sebacinales) induces systemic resistance against the biotrophic leaf pathogen Blumeria

graminis f. sp. hordei (B. graminis).

• In plants that were more B. graminis resistant due to P. indica root colonization, 22

transcripts, including those of pathogenesis related genes and genes encoding heat-

shock proteins were differentially expressed.

• Detailed expression analysis revealed a faster induction after B. graminis inoculation

between 8 and 16 hpi, suggesting that priming of these genes is an important

mechanism of P. indica-induced systemic disease resistance.

Barley leaf transcriptome and metabolite analysis reveals new aspects of compatibility and Piriformospora indica–mediated systemic induced

resistance to powdery mildew

30

Upregulated

Downregulated

Powdery mildew-regulated

transcripts in barley leaves. Venn diagram showing numbers of Blumeria

graminis f. sp. hordei-responsive transcripts (false

discovery rate < 0.05) in 3-week-old P. indica

colonized barley plants 12, 24, and 96 h post

inoculation (hpi) with the pathogen. Mean fold-inductions of transcript levels were calculated from

levels detected in three independent experiments by

hybridization to the Affymetrix Barley1 Gene Chip.

Fig. 17

31

Systemic effect of

Piriformospora indica on

expression of Blumeria

graminis f. sp. hordei (B.

graminis)–induced genes in

barley. • Transcript levels determined by quantitative real-

time polymerase chain reaction were calculated

relative to the mean of three constitutively expressed

genes.

• The gray frame highlights the 12-hpi time point for

which differential induction was originally detected

in the Gene Chip experiment.

Fig. 18

32

Systemic effect of Piriformospora indica on expression of defense-associated BCI-7

(barley chemically induced 7 ). • Determined by quantitative real-time polymerase chain reaction, were calculated relative to the mean of three constitutively expressed genes.

Fig. 19

33

Lower

Higher

(compared with mock control)

• Arrow thickness correlates with the proposed metabolic

flux relative to the other depicted metabolic pathways.

Only significantly altered metabolites and transcripts

with a fold change >2 relative to mock control are

included in the model.

• Abbreviations• Metabolites: ADPglc (ADP-glucose), aro-aa

(aromatic amino acids), aKG ( ketoglutarate), bc-aa

(branched-chain amino acid),Cit (citrate), Hex

(hexoses), HexP (hexose phosphates), Icit (isocitrate),

PEP (phosphoenol pyruvate), 3PGA (3

phosphoglycerate), Suc (sucrose), TP (triose

phosphates), UDPglc (UDP-glucose).

• Transcripts: AsnS (asparagine synthetase), CS

(chorismate synthase), cwINV (cell wall invertase),

GDH (glutamate dehydrogenase),

GS (glutamine synthetase), IPMS

(isopropylmalate synthase), P5CDH ( ’1-

pyrroline 5-carboxylate dehydrogenase),

PGM (phosphoglycerate mutase), PPT

(phosphoenolpyruvate / phosphate translocator),

SuSy (sucrose synthase).

Based on the results of the combined metabolome and transcriptome analysis, a model illustrating the major

redirection of metabolism in B. graminis–infected barley leaves was developed that integrates the dynamics in

central metabolism observed at 24 and 96 h post inoculation (hpi) (changes observed only at 96 hpi are depicted

in gray).

Fig. 20

34

Prime-omics in defense against nematode

Arbuscular mycorrhizal fungi (AMF) have great potential as biocontrol organisms against

the root-knot nematode Meloidogyne incognita which causes severe gall formation in plants.

Suppression subtractive hybridization (SSH) was used to investigate plant genes that are

specifically up-regulated in tomato roots (Solanum lycopersicum cv. Marmande) pre-colonized

by the AMF Glomus mosseae (BEG 12) after 12 days of soil inoculation with M. incognita

juveniles.

The higher expression of a selection of defense-related plant genes specifically in the

biocontrol interaction compared to in plants that were only mycorrhizal or only nematode

infected was confirmed, which pleads for the existence of mycorrhiza-induced priming of

plant defense responses.

In particular, the involvement of the phenylpropanoid pathway and reactive oxygen species

(ROS) metabolism could explain the reduced root-knot nematode infection in mycorrhizal

tomato roots, processes that have also been reported to play a pivotal role in plant resistance to

nematodes.

Vos et al., 2013Belgium

Mycorrhiza-induced resistance against the root-knot nematode Meloidogyne incognita involves priming of defense gene responses in tomato.

36

Genes responsible for secondary and hormone metabolism

Transcript abundance relative to the control treatment in roots of G. mosseae colonized

tomato plants, M. incognita-infected plants, and nematode-infected mycorrhizal plants,

12 days after nematode inoculation.

Fig. 21

37

Genes responsible for secondary and hormone metabolism

Transcript abundance relative to the control treatment in roots of G. mosseae colonized

tomato plants, M. incognita-infected plants, and nematode-infected mycorrhizal plants,

12 days after nematode inoculation.

Fig. 22

38

Fig. 2339

Nematode infection parameters in tomato plants colonized or not by the AMF G.

mosseae, 12 days after M. incognita inoculation (Pi ¼ 1000).

Treatment Number

of J2 & J3

Number

of J4

Total number

of nematodes

per root system

Gall indexa

- G. mosseae 118 ± 24 b 34 ± 9 b 151 ± 27 b 2.3 ± 0.2 b

+ G. mosseae 48 ± 13 a 9 ± 2 a 58 ± 14 a 1.7 ± 0.2 a

P (treatment) 0.024 0.013 0.008 0.048

Data represent mean standard error (n ¼ 8). Within each column, values followed by different letters are significantly different (P 0.05) according to one-

way ANOVA and Tukey’s HSD test. Statistical analysis was performed on log(x þ 1) transformed nematode infection data: n.s. ¼ not significant; J2, 2nd

stage juveniles; J3, 3rd stage juveniles; J4, 4th stage juveniles. a Gall index was rated on a scale of 1e10, according to Bridge and Page (1980).

Table 3

40

Prime-omics in defense against arthropods

Stress

severity

Time

Defense against arthropods

Fig. 24Niinemets et al., 201342

Defense against arthropods

Direct defense priming can be achieved through either chemical or natural stimuli. Natural stimuli can stem from arbuscular mycorrhiza, nonpathogenic rhizobacteria, or various arthropod cues such as oviposition or insect wounding. In priming against arthropods, abscisic acid (ABA) is a key regulator that activates defenses coordinately with jasmonic acid (JA)- and systemin-dependent signaling.

Indirect defense priming is more diverse. It involves the enhancement of defense responses in neighbouring plants and also more efficient attraction of predators. Primed defenses in surrounding plants activate ocymene synthase (OS), which catalyzes the accumulation of b-ocimene.

43

Pest

Parasitoid

Predator

(12-oxo-phytodienoic acid)

Balmer et al., 2015Fig. 25

Pieris rapae

Helicoverpa zeae

Tetranychus urticae

Encarsia Formosa

Bemisia tabaci

Nesidiocoris tenuis

Phytoseiulus persimilis44

Prime-omics of direct defense against arthropods

Mao et al., 2013China

• These results suggest a strong interaction between Si and JA in defense against insect herbivores

involving priming of JA-mediated defense responses by Si and the promotion of Si accumulation by JA.

Priming of jasmonate-mediated anti-herbivore defense responses in rice by silicon

• To explore the role of JA in Si-enhanced resistance, the expression of allene oxide synthase (OsAOS;

active in JA biosynthesis) and CORONATINE INSENSITIVE1 (OsCOI1; active in JA perception)

genes are silenced in transgenic rice plants via RNAi and examined resulting changes in Si accumulation

and defense responses against caterpillar Cnaphalocrocis medinalis (rice leaffolder, LF) infestation.

• Reduced Si deposition and Si cell expansion were observed in leaves of OsAOS and OsCOI1 RNAi

plants in comparison with wild-type (WT) plants, and reduced steady-state transcript levels of the Si

transporters OsLsi1, OsLsi2, and OsLsi6 were observed in Si-pretreated plants after LF attack.

• Upon LF attack, wild-type plants subjected to Si pretreatment exhibited enhanced defense responses relative to

untreated controls, including higher levels of JA accumulation; increased levels of transcripts encoding defense

marker genes; and elevated activities of peroxidase, polyphenol oxidase, and trypsin protease inhibitor.

46

Gain in mass of LF larvae fed on WT rice plants and the OsCOI1 RNAi and OsAOS RNAi lines

treated with Si and MeJA. • Seven-day-old seedlings were transplanted to nutrient solution containing 2 mM K2SiO3. In the Si-deficient treatment, potassium chloride was used to

replenish potassium.

• OsCOI1 and OsAOS RNAi lines and WT rice plants were sprayed with 1 mM MeJA or buffer (control) 20 d after transplanting.

• Two days later all plants were infected by third-instar LF larvae at leaf node 3.

• The individual larvae were measured 3 d later, and the mean percentage of gain in mass was calculated. Values are mean ± SE (n ( 20). Letters above

bars indicate significant differences among treatments (P < 0.05 according to Tukey’s multiple range test).

Fig. 26

47

Steady-state levels of OsAOS (A and C) and OsCOI1 (B and D) transcripts in the leaves of WT rice plants

not treated with Si or treated with Si with or without LF infestation (A and B) or treatment with MeJA (C

and D). For gene-expression experiments the treatments included no Si added (Si−); no Si added followed by MeJA treatment (Si−MeJA+) or LF infestation

(Si−LF+); 2 mM potassium silicate added (Si+); and 2 mM potassium silicate added followed by treatment with 1 mM MeJA (Si+MeJA+) or LF

infestation (Si+LF+). Real-time qRT-PCR analysis was used to determine the relative steady-state transcript levels shown. Values shown are mean ±

SE (n ( 3).

Fig. 27

Time after treatment (h)

Rel

ati

ve g

ene

exp

ress

ion

48

Steady-state levels of JA levels (E) in the leaves of WT rice plantsFor JA analysis the six treatments were Si−, Si+, Si−MeJA+, Si−LF+, Si+MeJA+, and Si+LF+; values are mean ± SE (n ( 6). For each time point,

letters above bars indicate significant difference among treatments (P < 0.05 according to Tukey’s multiple range test).

Fig. 28

49

Scanning electron micrographs of rice

leaf cross-sections. Bilobed (“dumbbell”-

shaped) Si cells from node 3 leaves of

WT, OsAOS RNAi, and OsCOI1 RNAi

plants are shown.(Scale bar: 20 μm.) Silica cells were quantified at a magnification of 2,000×. The

length and width of the Si cell were measured; then the Si area was calculated.

SC, silica cell.

Fig. 29

50

Steady-state levels of OsLsi1

and OsLsi2 transcripts in

roots and of OsLsi6 in the

leaf sheath of WT rice

plants, OsCOI1 RNAi and

OsAOS RNAi plants treated

with Si plus MeJA or LF

infestation. • Transcripts levels of OsLsi1 (A), OsLsi2 (B), and

OsLsi6 (C) in OsAOS RNAi, OsCOI1 RNAi, and

WT plants were analyzed 24 h after MeJA

application or LF infestation.

• Real-time qRT-PCR analysis was used to

determine the relative steady-state transcript

levels shown. Values are mean ± SE (n ( 3). For each time point, letters above

bars indicate significant difference among treatments (P < 0.05

according to Tukey’s multiple range test).

Fig. 30

51

Prime-omics of indirect defense by and against arthropods

Predatory mites

(Dicke & Sabelis, 1988)

Parasitoid wasps

(Turlings et al., 1990)

Predatory bugs

(Drukker et al., 1995)

Predatory lady beetles

(Ninkovic et al., 2001) Herbivorous moths, which are repelled

(de Moraes et al., 2001)Nematodes

(Rasmann et al., 2005)

Parasitic plants

(Runyon et al., 2006)

Predatory birds

(Mantyla et al., 2008)

Resistance to pathogens

(Yi et al., 2009)

Other plants

(Baldwin & Schultz, 1983;

Rhoades,1983)

Depolarization events and Ca2+ influxes in

membranes exposed to HI-VOCs (Zebelo et

al., 2012) it is likely that the next discovery

will concern their perception by local cells

that are surrounding the injured tissues.

Milestones in the research of herbivore induced-volatile organic

compounds(HI-VOCs)

Fig. 31 Martin., 2014

53

Reports on herbivore-induced volatile organic compounds (HI-VOCs) published

between 1983-2013.

Martin., 2014Fig. 32

54

Vieira et al., 2012Brazil

• It induce indirect defenses in soybean plants

against stink bugs by egg parasitoid Telenomus

podisi.

• Spraying cis-jasmone increased number of

Scelionidae egg parasitoids in soybean plots.

• Their results suggest that treatment with cis-

jasmone effectively attracted and enhanced the

population of Scelionid parasitoids, but had no

effect on the occurrence and intensity of parasitism

and in the number of stink bugs.

• The secondary metabolite cis-Jasmone activates the metabolic pathway that produces

volatile organic compounds attractive to natural enemies and, sometimes, repellent to

herbivores. cis-Jasmone is an herbivore induced plant volatile.

cis-Jasmone indirect action on egg parasitoids (Hymenoptera: Scelionidae) and its application in biological control of soybean stink bugs (Hemiptera:

Pentatomidae)

Constitutive

volatiles

Selective attraction

HIPVs

Spraying

cis-jasmone

Soybean field plotsFig. 33 55

Abundance of adult parasitoids sampled in soybean plots, with yellow sticky

traps for nine weeks in control and cis-jasmone treated plots in two areas. Legend: Aph ( Aphelinidae), Bet ( Bethylidae), Bra ( Braconidae), Cer ( Ceraphronidae), Chal ( Chalcididae), Chalni ( Chalcidoidea unidentified), Chr (

Chrysididae), Dia ( Diapriidae), Dry ( Dryinidae), Euc ( Eucoilidae), Eul ( Eulophidae), Eup ( Eupelmidae), Eur ( Eurytomidae), Eva ( Evaniidae), Fig (

Figitidae), Ich (Ichneumonidae), Meg ( Megaspilidae), Mma ( Mymarommatidae), Mym ( Mymaridae), Per ( Perilampidae), Pla ( Platygastridae), Pro (

Proctrupidae), Sce ( Scelionidae), Sig ( Signiphoridae), Tet ( Tetracampidae), Tor ( Torimidae), Tri ( Trichogrammatidae).Indicate significant difference (P

< 0,05).

Fig. 34

Ab

un

dan

ce

Family

d

56

The costs and benefits of priming

Fig. 3557

Conclusion

Prime-omics is an integrated approach for defense priming which involves different techniques spanning the fields of transcriptomics, proteomics, and metabolomics together with adequate bioinformatics tools.

Primed plants show faster and stronger defense responses when subsequently challenged by microbes, insects, or abiotic stress, and this is frequently linked to development of local and systemic immunity and stress tolerance.

The availability of rapidly growing transcriptomic, proteomic, and metabolomicdata sets – the prime-ome – describes the state of primed plants.

The knowledge of prime-omics can be exploited for a better defence modulation in plants.

58

Future thrust

Studies on adaptation occurring in the attacker stresses in response to priming

and priming response at different developmental stages of plants will suffice the

strategies to manage any deviations in response.

Research on the priming response in combination with two or more stress

should be investigated.

The work on development of metadata will speed up further research in this

area.59