Biotechnological

Communication

Biosci. Biotech. Res. Comm. 10(4): 631-644 (2017)

Manipulating disease and pest resistance pathways in

plants for enhanced crop improvement

Paramita Ghosh, Anjanabha Bhattacharya* and Bharat Char

Maharashtra Hybrid Seeds Company Private Limited, Jalna-Aurangabad

Road, Dawalwadi, Jalna, Maharashtra, India

ABSTRACT

Plants are sessile organisms, therefore cannot escape challenges of their surrounding environment. The rich source

of nutrients plant possesses attracts various organisms. Biotic stress results from array of organisms such as bacteria,

fungi to various insects, pests and herbivores. Plants have evolved sophisticated mechanisms to protect themselves

against invaders. In this review, we explore the plant surveillance system, different nodes in the defence pathways

involved in plant protection and how it can be manipulated to get a resistant crop. Emerging technologies have

provided us with a vast number of potential candidate genes from plants, pathogens and other organisms. We here,

illustrate examples of technically useful solutions to make crops tolerant to pathogens and pests.

KEY WORDS: PLANT DEFENCE, BIOTIC STRESS, R GENE, DEFENCE SIGNALING TRANSDUCTION, NPR1, MAPK, GENETIC ENGINEERING

631

ARTICLE INFORMATION:

*Corresponding Author: anjanabha.bhattacharya@mahyco.com

Received 12

Nov, 2017

Accepted after revision 29

Dec, 2017

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

Thomson Reuters ISI ESC and Crossref Indexed Journal

NAAS Journal Score 2017: 4.31 Cosmos IF: 4.006

reserved.

Online Contents Available at:

http//www.bbrc.in/

DOI: 10.21786/bbrc/10.4/5

ABBREVIATIONS

MAMPs - Microbe Associated Molecular Patterns,SAR -

Systemic Acquired Resistance, VOCS – Volatile Organic

Compound, R gene – Resistance Gene, HR – Hyper-

sensitive Reaction, ROS – Reactive Oxygen Species,

MAPK – Mitogen Activated Protein Kinase, avr – Aviru-

lence, ETI – Effector Triggered Immunity, NBS – Nuclear

Binding Site, LRR – Leucine Rich Repeat , pv. – pathover,

NO – Nitric Oxide, SA – Salicylic Acid, JA – Jasmonic

Acid, ET- Ethylene, NPR1 – Non Expressor of PR Genes

1,PR – Pathogenesis Related, LTP –Lipid Transfer Pro-

tein, PPO – Polyphenol Oxidase, POD – Peroxidase,

UV- Ultraviolet, HIPV – Herbivore Induced Plant Vola-

tile, QTL – Qualitative Trait Loci, SIPK - Salicylic Acid

Induced Protein Kinase, OS – Oral Secretion, FACs –

Fatty Acid –Amino Acid Conjugates, WIPK – Wound

Induced Protein Kinase, MEKK- Mitogen-Activated

Protein Kinase Kinase, ISR – Induced Systemic Resist-

ance, PRSV - Papaya Ring Spot Virus, ZFN – Zinc

Finger Nucleases, TALENs – TAL Effector Nucleases,

GM – Genetically Modi ed, RPP2 – Recognition of per-

onospora parasitica 2

632 MANIPULATING DISEASE AND PEST RESISTANCE PATHWAYS IN PLANTS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Paramita Ghosh et al.

INTRODUCTION

Plants are nutrient rich organisms and therefore many

invaders prey on their food reserve. Some of the invad-

ers are signi cant threats to crop production, world-

wide. In the process of co-evolution (Seidl and Thomma,

2017); plants, pathogens and insects have evolved vari-

ous strategies to avoid each others’ defence system. The

goal of producing crops with durable and increased

resistance to a broad spectrum of diseases and insects is

therefore, a major focus in plant research.

In nature, plants are continuously challenged by differ-

ent organisms, whereas, only few are successful in gain-

ing entry into a prospective host. Plants have developed

an elegant defence system with a wide variety of consti-

tutive and inducible defences to protect themselves from

damages of different biotic factors. Constitutive defences

include many preformed barriers such as waxy epider-

mal cuticles, cell walls and bark (specialized morphologi-

cal structures). Inducible defences include production of

repellents, toxic chemicals, pathogen-degrading enzymes,

anti-nutritional effects and deliberate cell suicide (Free-

man and Beattie, 2008). Plants often do not produce toxic

compounds or defence-related proteins until pathogens

are detected due to the metabolic cost associated with the

production and maintenance of such compounds. Plants

have evolved to live in environments where they are

very often exposed to different stress factors in combi-

nation. Plants have developed various mechanisms that

allow them to detect precise environmental changes and

respond to complex stress conditions, minimizing dam-

age (Saskia and Jorunn, 2011).

NATURE OF ATTACKERS

Plant pathogens can be broadly divided into biotrops and

necrotrophs. Bacteria and fungi can adapt to both lifestyles

(Freeman and Beattie, 2008).Viruses are quintessential bio-

trophs, although they eventually kill the host cell. Insects,

on other hand, cause damage by chewing and sucking.

Plants respond to the insects by producing protease inhibi-

tors and anti-feedants such as alkaloids [(Hanley et al.,

2007; Jeffery and Jonathan, 2001). Nematodes can adapt

to complex modes of parasitism by exhibiting variety of

parasitic modes affecting the development responses of

plants, causing galls, root knots or cysts (Jeffery and Jona-

than, 2001; Davis et al., 2004; Roland and Maurice,2011) .

Thus, plant immune system is highly polymorphic in their

capacity to recognize and respond to different stress fac-

tors (Jeffery and Jonathan, 2001).

PLANT SURVEILLANCE SYSTEMS

Although plants lack immune system comparable to

animals, they have developed sophisticated surveillance

mechanisms, which can respond rapidly before harmed.

These surveillance systems are linked to speci c pre-

programmed defence responses. Direct defences are

mechanical protection on the surface of the plants which

protects from all biotic factors (e.g., hairs, trichomes,

spines, thorns and thicker leaves) or toxic chemical pro-

duction.

Basal resistance is the  rst line of pre-formed and

inducible defences. It is also known as innate immunity

(Freeman and Beattie, 2008; Owen and Zamir, 2010), and

protect plants against entire groups of pathogens (Free-

man and Beattie, 2008). Basal resistance is triggered

when plants recognize microbe-associated molecular

patterns (MAMPs). MAMPs include speci c proteins,

lipopolysaccharides, and components of cell wall com-

monly found in microbes. During evolution pathogens

also have developed counter measures that are able to

suppress basal resistance in certain plant species. If the

basal defence is somehow suppressed, plants respond

with hypersensitive response (HR) (Freeman and Beattie,

2008). In HR plants limit the pathogen’s access to water

and nutrients thereby sacri cing few cells in the infec-

tion site i.e. deliberate cell suicide (programmed cell

death). HR is more pathogen speci c than basal resist-

ance. It is triggered in presence of disease-causing effec-

tor molecules. Once the hypersensitive response is trig-

gered, plant tissues become highly resistant to a broad

range of pathogens. This phenomenon is known as sys-

temic acquired resistance (SAR) (Freeman and Beattie,

2008; Nelson et al., 2017), which represents readiness of

plant metabolites to defend plants, in case of a height-

ened attack.

Mechanical damage caused by insects is not gener-

ally considered “true” plant disease although plants have

developed surveillance systems designed to not only

recognize insect pests, but also to respond with speci c

defence mechanisms. General wounding can be different

from insect feeding in a way that elicitors are present

in insect saliva. In response to insect chewing, plants

release volatile organic compounds (VOCs), second-

ary metabolites and proteins that have toxic, repellent,

and/or anti-nutritional effects on the herbivores (Free-

man and Beattie, 2008; Saskia and Jorunn, 2011; Abdul

Rashid War et al., 2012). Sometimes volatiles released by

plants also attract bene cial predators (natural enemies)

that prey on the destructive pests (Abdul Rashid War

et al., 2012; Walling, 2000; Rashid and Chung, 2017).

Plants become phenotypically plastic when induced

defence is triggered as a result it decreases the chances

of the attacking insects to adapt to the induced chemi-

cals (Abdul Rashid War et al., 2012).

In addition, plants can defend themselves against

viruses by a variety of mechanisms which include RNA

silencing (Novina and Sharp, 2004, Csorba and Burgyan,

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS MANIPULATING DISEASE AND PEST RESISTANCE PATHWAYS IN PLANTS 633

Paramita Ghosh et al.

2016). Plants can recognize the foreign double stranded

RNA or DNA, produced by viruses in the host cell dur-

ing replication, and respond by digesting the genetic

strands into non recognizable fragments and thereby

stopping the infection. The interaction of plants with

symbionts, pathogen, herbivores, and the natural ene-

mies, both above and below the ground is the focus of a

large amount of research effort and has great potential

for utilization in crop protection.

With cultivation of huge areas of genetically iden-

tical crops, protection relies on a small number of in-

bred disease resistance genes per crop species and on the

wide-spread application of pesticides. Unfortunately, an

absolute control is very dif cult to achieve through pes-

ticides (Cesari, 2017), as pathogens can overcome disease

resistance genes and/or become resistant to pesticides

(Nelson et al., 2017; Zhonghua et al., 2005). Genetic

manipulation can help solve the problem by inserting

multiple genes as transgenes by careful selection from

wild parent of the same plant species or from different

plant species (Campbell et al., 2002). Therefore a search

is on for genes that can confer a durable broad-spectrum

resistance against biotic factors. To make it more envi-

ronment friendly the gene product should be safe for all

organisms and also reduce the need of harmful pesti-

cides. However, the success so far achieved is very less.

In majority of cases the tansgene results in unpredict-

able expression in different parts of the plants, this phe-

nomenon is not due to the transgene itself, per se (Ham-

mond-Kosack and Parker, 2003; Stuiverand Custers,

2001) . Therefore, optimization of transgene expression

patterns needs close attention. Inducible expression of

such gene is essential (Hammond-Kosack and Parker,

2003; Michelmore, 2003). A highly inducible promoter

speci c for defence gene expression can help the plant

in directed recourse allocation by metabolic and tran-

scriptional adaption during stress. Plant can optimize

source sink relationship thus increasing yield or biologi-

cal harvest index (Hammond-Kosack and Parker, 2003).

ENGINEERING PLANTS WITH INCREASED

RESISTANCE AGAINST PATHOGEN AND

INSECTS: TARGET GENES

(First generation strategies)

R gene

R genes (resistance genes) are important components

of plant surveillance system. A diverse array of defence

mechanism is triggered when R genes recognize patho-

gen or insects (Cesari, 2017). PR-gene induction, accu-

mulation of inhibitory metabolites and oxidative burst

response by production of reactive oxygen species, are

some of the downstream responses triggered by R genes

which lead to hypersensitive response (Owen and Zamir,

2010 ).

Pathogens possessing avr genes can overcome basal

immunity of plants by blocking perception of PAMP

or by inhibiting MAP kinase signaling cascade, which

is known as effector- triggered susceptibility. In case

of effector triggered immunity, the pathogen’s effec-

tor molecules are recognized by R proteins either by

direct or indirect interactions. Thus, enhancing the plant

resistance and it is faster than PTI. To trigger ETI, R pro-

teins must recognize speci c avirulence proteins (Avr)

in order to generate resistance. However, mutation in

either avr gene or R gene can change the scene i.e it will

result in compatibility and therefore loss of resistance. R

genes encode proteins which have nuclear binding sites

(NBS) and leucine rich repeat (LRR) domains (NBS-LRR

FIGURE 1. Shows pathogen triggered immunity when pathogen/herbivore associated

molecular patterns are recognized by the cell receptors. However in presence of effectors,

pathogens can surpass this immunity. In presence of R gene, effector triggered immunity

induces defence response in plants.

634 MANIPULATING DISEASE AND PEST RESISTANCE PATHWAYS IN PLANTS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Paramita Ghosh et al.

proteins) (Cesari, 2017; Jeffery and Jonathan, 2001; Mari

et al.,2013, Nelson et al., 2017).

Functional R genes conferring resistance against an

array of different biotic factors such as bacteria, virus,

fungus, nematodes and even insect pathogens have

been isolated (Cesari, 2017, Zhao et al., 2005; Vossen

et al., 2005; Reinink et al., 1989; Brotman et al., 2002).

Even though the mode of action as well as the effector

molecules of pathogens and insects are very different, R

genes encode only a few classes of proteins. NBS –LRR

class of proteins are the largest class of R gene which

encodes ‘nucleotide binding site with leucine rich repeat

(Jeffery and Jonathan, 2001). It is reported that NB-LRR

type R genes can confer resistance to multiple pathogens

even though the pathogens belong to taxonomically dis-

tinct families (Mari et al., 2013). It is also termed as MDR

or multiple disease resistance. In a maize recombinant

inbred line (RIL) a QTL, qMdr have been identi ed for

resistance to several diseases i.e, Norther blight, grey leaf

spot and souther leaf blight. The molecular mechanism

underlying the resistance is yet not known. In a research

it is found that a gene, ZmCCoAOMT2, which encodes

a caffeoyl-CoA O-methyltransferase is associated with

conferring quantitative resistance to both southern leaf

blight and gray leaf spot (Yang et al., 2017).

Bacterial effectors are delivered through type III

secretion system, which can be up to 30 per strain, and

by mimicking or inhibiting eukaryotic cellular functions

colonization is achieved (Abramovitch et al. 2006). An

example of a speci c R-gene, Rxo1 from maize con-

ferred resistance to bacterial streak disease caused by

Xanthomonas oryzae pv. Oryzicola(Zhao et al., 2005),

when introduced in rice. In another instance, R gene

RCT1 from Medicago truncatula expressed in alfalfa

conferred resistance to Colletotrichum trifolii (Yang et

al., 2008), RPI-BLB2 from wild potato gave resistance

to Phytophtohora infestans in day to day cultivated

potato (Vossen et al., 2005). Some of the R gene work

in pairs and are functional only when both genes are

present (Mari et al.,2013 ). Some of the examples of such

R gene pairs are RPP2A/RPP2( Sinapidou et al., 2004),

Pi5-1/Pi5-2 ( Lee et al., 2009) and Lr10/RGA2 (Loutre

et al., 2009). Examples in wheat rust, Sr31 from rye was

effective against all Pgt races for many years until the

appearance of Ug99 (Pretoriusetal.,2000).

Many single R genes responsible for resistance against

insects are mapped in cereal crops, including wheat con-

ferring resistance to Hessian  y (Hatchet et al., 1970).

For decades, R genes have been used to control Hessian

 y infestation in wheat. It is evident in support of gene-

for- gene model in plant- insect interactions.

Some of the insect resistant R genes that are effec-

tive against aphids include: the lettuce Nr gene which

gives resistance against aphid species Nasanova ribis-

nigri (Reinink et al., 1989), the Vat gene from melon

confers resistance against the melon/cotton Aphis gos-

sypii aphid (Brotman et al., 2002), in another instance,

the Sd1 gene gives resistance against Dysaphis devecta

aphid in apples (Walling, 2000; Roche et al. 1996), the

RAP1 gene gives resistance against the Pea Aphid in

Medicago truncatula (Stewart et al., 2009), and the Mi-1

gene in tomatoes (Rossi et al., 1998) found to be responsi-

ble for resistance against different organisms, the potato

aphid Macrosiphum euphorbiae, the root-knot nema-

todes Meloidogyne spp., and the white y Bemisia tabaci

(Nombela et al. 2003). The diverse resistance conferred

by the Mi-1 gene makes it is a very useful tool for inte-

grated pest management. While, Bph14 confers resist-

ance to the rice brown planthopper, Nilaparvata lugens

(Zhang et al., 2009). However, a constitutive expression

of a R gene can have a negative impact in absence of

attackers. Constitutive expression of R gene can be det-

rimental to plants and therefore needs to be expressed

with inducible promoters (Belbahri et al., 2001; Takakura

et al., 2004).

For decades, R genes have been used in conventional

breeding programme (Balconi et al., 2012) ; however, the

resistance is only against a strain of pathogen or a par-

ticular species of insect. Traditional breeding strategies

most often use only one R gene at a time. Pyramiding

multiple R genes can promise a long lasting resistance

as the pathogen has to accumulate mutation in multiple

Avr genes to escape resistance. Effective combinations of

R and APR gene by pyramiding or stacking can be con-

sidered for effective rust resistance (Jeffrey et al., 2014).

However, it is a lengthy process to introduce a R gene

into an elite cultivar by conventional breeding. R-genes

from unrelated plant species can be introduced through

genetic engineering, which often remain functional in

the new host plant (Collinge et al., 2008) . The limitation

of this technology being that resistance is conferred only

against a single pathogen similar to breeding (Balconi

et al., 2012). Additionally, R-gene only confers resist-

ance against pathogens that essentially act as a sink for

the host plant’s metabolism i.e. biotrophs.

Shuf ing of multiple R genes can also be consid-

ered rather than only pyramiding. Plant pathogen Clad-

osporium fulvum elicitors are recognition by Cf genes in

tomato which belongs to the Hcr9 gene clusters (Brande

et al., 2004). Studies have shown that Hcr9s are com-

posed of sequences that have been generated by sequence

exchange between individual homologues, Intra and

intergenic recombination, gene conversion, point muta-

tion, duplication and translocation. Therefore, shuf ing

multiple R genes might increase recognition speci ci-

ties and engineering R gene for novel disease resistance

speci cities in plants can be achieved (Cesar, 2017). For

example, gene shuf ing done in tomato Cf4 and Cf9 R

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS MANIPULATING DISEASE AND PEST RESISTANCE PATHWAYS IN PLANTS 635

Paramita Ghosh et al.

Table I. List of R gene against different pathogens

R gene Source (Donor)

Examples of

transgenic crop

Against Pathogen References

Rox1 Maize Rice Xanthomonas oryzae pv. oryzicola Zhao et al., 2005

RCT1 Medicago truncatula Alfalfa Colletotrichum trifolii Yang et al., 2008

RPI-BLB2

Potato

(Solanum bulbocastanum)

Potato Phytophtohora infestans Vossen et al., 2005

Bs2 Pepper Tomato Xanthomonas campestris Tai et al., 1999

Rpg1 Barley Barley Stem rust Brueggeman et al., 2002

Ve1 and Ve2 Tomato Potato Verticillium spp. Kawchuk et al., 2001

RRS1-R Arabidopsis Arabidopsis Ralstonia solanacearum Deslandes et al., 2002

Pi-d2 Rice Rice Chinese rice blast Chen et al., 2006

RPW8 Arabidopsis Arabidopsis, tobacco

Broad spectum resistance against

powdery mildew

Xiao et al., 2003

Pto Tomato Tomato Pseudomonas syringae Frederick et al., 1998

Table II. List of R gene against different insects

R gene Source (Donor) Against Insect Reference

Nr gene lettuce Aphid species Nasanova ribisnigri Reinink et al., 1989

melon Vat gene Melon Melon/cotton Aphis gossypii aphid Brotman et al., 2002

Sd1 gene Apple Dysaphis devecta aphid Roche et al., 1996; Walling, 2000

RAP1 gene Pea Pea Aphid in Medicago truncatula Stewart et al., 2009

Mi-1.2 gene Tomato

Potato aphid Macrosiphum euphorbiae, the

root-knot nematodes Meloidogyne spp., and

the white y Bemisia tabaci

Rossi et al., 1998

genes lead to the identi cation of sequences required for

the Avr-dependent HR in tomato (Brande et al., 2001).

SIGNAL TRANSDUCTION NETWORK

Plants can sense changes in their environment through

signaling pathways (Pankaj and Atle, 2013). When

pathogen elicitors interact with host receptors, signal

transduction cascades are likely to be activated includ-

ing oxidative burst (ROS), calcium  uxes, ion channel

 uxes, NO production (Bollwell et al., 1999) and vari-

ous protein kinases. Subsequently, transcriptional and/

or post transcriptional activation of transcription factors

takes place which lead to the induction of defence gene.

Plant hormones which play important role in defence

are SA, JA and ET. SA is primarily involved in the pro-

tective response against biotrophic and hemi-biotrophic

pathogens and systemic acquired resistance (SAR) (Grant

and lamb, 2006). Some mutants insensitive to SA shows

enhanced susceptibility to biotrophic pathogens. Methyl

salicylate is a mobile inducer of SAR and is induced

when the plant is infected with a pathogen in tobacco

plants (Park et al., 2007). After pathogen challenge the

elevated level of SA increases the expression of PR

genes, therefore increasing resistance. Whereas the level

of JA and ET are elevated against necrotrophic pathogen

and herbivorous insects (Park et al., 2007).

Most often the SA and JA/ET defence pathways are

antagonistic, however reports of synergistic interaction

also exist (Kunkel and Brooks, 2002; Mur et al., 2006;

Schenk et al., 2000) . Speci c biotic factors regulate the

positive or negative cross talk between SA and JA/ET

pathways (Adie et al., 2007). In nature it is not one factor

that affects the plant but several attackers, here plants

have to employ complex regulatory mechanisms to cope

with the complex situation. The mechanism by which

plant is able to prioritize the responses is not known.

Non expressor of PR genes 1 (NPR1) is one of the

important components of SA signaling. NPR1 plays

an important role in SA-JA interaction (Dong, 2004).

Downstream of NPR1 are several WRKY transcription

factors which is also important is SA-dependent defence

response. WRKY70 maintains the balance between the

SA and JA pathways (Li et al., 2004; Li et al., 2006).

Another key component which is involved in mediating

the antagonism between SA and JA signaling in Arabi-

636 MANIPULATING DISEASE AND PEST RESISTANCE PATHWAYS IN PLANTS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Paramita Ghosh et al.

dopsis is mitogen activated protein kinases (Petersen

et al., 2000). In the second generation strategies, these

signaling nodes will be discussed. The goal of effective

and sustainable disease resistance can be achieved by

the knowledge of signal transduction pathways (David

et al., 2010), as the increased understanding has made

it clear that successful pathogen process through patho-

genicity factors (effectors). The disease resistance gene

are mostly downstream genes and often do not act as

speci c receptors produced by pathogens and insects.

A complex signaling network is also established when

herbivorous insects attack a plant. To identify new mol-

ecules important for  ne tuning of plant defence signal-

ing, there is a need of dynamic modeling and simula-

tion of signal transduction pathways (Beckers and Spoel,

2006; Erb et al., 2009).

Various plant protectant and defence gene are acti-

vated by the primary and secondary signals. The defence

gene products include glutathione S-transferases, per-

oxidases, cell wall proteins, proteinase inhibitors, hydro-

lytic enzymes (e.g., -1,3-glucanases and chitinases),

pathogenesis-related PR proteins (Balconi et al., 2012).

PR proteins

Other potential candidates for manipulation are patho-

genesis related (PR) genes, which shows promising activ-

ities against biotic factors i.e. pathogens as well as insect

pests. Pathogenesis related (PR) genes could increase the

level of pre-existing barriers (Owen and Zamir, 2010;

Hammond-Kosack and Parker, 2003). Naturally occur-

ring PR proteins are constitutively expressed at low lev-

els and are induced to high levels challenged by patho-

gens or application of either salicylic acid or jasmonic

acid (Ferreira et al., 2007) . PR proteins include several

groups of unrelated proteins. Seventeen classes of PR

protein have been examined, and numbered chronologi-

cally in order of discovery i.e. PR-1 to PR-17 (Balconi

et al., 2012). PR-2 ( b -1,3-glucanases), PR-3, -4, -8 and

-11 (chitinases) target the pathogen cell wall (Owen and

Zamir, 2010; Honee, 1999), PR-1 and PR-5 (thaumatin-

like proteins and osmotins) are termed as permatins as

they target the membrane, PR-10 has weak ribonuclease

activity therefore may target pathogen RNA or play a

role in defence against viruses, PR-6 proteins (protein-

ase inhibitors) may target nematodes, whereas the PR-7

protein (an endoproteinase) may be involved in micro-

bial cell wall dissolution (Jorda et al., 2000) . The PR-9

family may enhance resistance to multiple pathogens by

catalyzing ligni cations which helps in cell wall rein-

forcement (Passardi et al., 2004). Since PR-10 family has

weak ribonuclease activity it can be used against viruses

(Park et al., 2004), PR-12 (defensins), PR-13 (thionins)

and PR-14 (lipid transfer proteins) predicts antibacte-

rial and antifungal activities (Epple et al., 1997) , some

proteins generating hydrogen peroxide and are toxic to

pathogen and pest, PR-15 (oxalate oxidases) and PR-16

(oxalate oxidase-like proteins) belongs to this family

(Hu et al., 2003). PR-17 (uncharacterized) is detected in

infected tobacco, wheat and barley (Christensen et al.,

2002).

Most investigated PR proteins are chitinases and 1–3

glucanases (Owen and Zamir, 2010). Over-expression of

chitinase have been moderately successful against fun-

gal pathogens. Studies have found chitinase have role

in insect resistance as well. The combined expression of

chitinases and 1–3 glucanases have proven to enhance

resistance by synergistic effect (Anand et al., 2003; Jach

et al., 1995; Jongedijk et al., 1995; Zhu et al., 1994).

Chitinases originating from Trichoderma harzianum

(biocontrol agent), exhibit higher anti-fungal activity

(Dana et al., 2006; Baranski et al., 2008; Kumar et al.,

2009). Ectopic expression of thionins and defensins has

conferred broad spectrum disease resistance, though the

resistance is at low level (Punja, 2001). For example rad-

ish defensin RS-AFP2 (Kostov et al., 2009) when over-

expressed in tomato resulted in up to 90% reduction

in disease against agriculturally important pathogens.

Lipid transfer proteins (LTP) are one of the important

PR proteins which act as a potential mobile signal for

systemic acquired resistance (SAR) in plants (Maldonado

et al., 2002). LTP activates SAR over-expression of LTP

might result in deleterious effect (Walters, 2007) , so far

no such effect is observed. A highly inducible promoter

can be used to over-express this gene in order to achieve

the goal of disease resistance.

The plant defensive metabolites are termed as second-

ary metabolites play an important rolein plant defense

against herbivore and other interspecies defense, thus

increasing the  tness of the plant. They can be either

constitutively stored (phytoanticipins) as inactive forms

or induced in response to the insect or microbe attack

(phytoalexins)(King et al., 2014). Herbivore induced plant

volatiles (HIPVs) play very important role in defense by

either attracting the natural enemies of the herbivores

or by acting as feeding and/or oviposition deterrent

(Rashid and Chung, 2017). HIPV are released by healthy

plants as well, however a different blend of volatiles is

produced in response to herbivory and is very speci c

for a particular insect-plant system (Liu et al., 2012). For

example, plants tend to release volatile compounds in

response to aphid –attack to attract parasitoid wasps.

In corn, plants release terpenoids in response to aphid

attack. Many other volatile compounds like MeSa, C

volatiles etc in uence plant-insect, pest and pathogen

interaction.

Metabolite engineering can play an important role

in developing plant with insect resistance. Increas-

ing the  ux of defence related secondary metabolites

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS MANIPULATING DISEASE AND PEST RESISTANCE PATHWAYS IN PLANTS 637

Paramita Ghosh et al.

by engineering the respective pathways can be of great

importance in developing crops with insect resistance

(Sanchez -Vallet et al., 2013). There are some reports of

metabolic engineering of dhurrin, a cyanogenic glyco-

side in transgenic A. thaliana plants which, resulted in

minor effects on the whole metabolome and transcrip-

tome (Dudareva et al., 2013 ). Resistance to green peach

aphid (Myzus persicae) feeding have been enhanced by

metabolic engineering of raf nose in the phloem of A.

thaliana (Jirschitzka et al., 2013). In another instance,

manipulation of plant volatile emissions has enhanced

the effectiveness of biological control agents. This can

be used as a strategy to  ght insect pests in an ecologi-

cally sound manner (Degenhardt et al., 2009).

SECOND GENERATION STRATEGIES

Master switch genes

Over-expression of a single defence-related gene is gen-

erally unable to provide high levels of resistance against

a broad range of biotic factors like pathogen and her-

bivores. The knowledge of pathogen-induced signal-

ing pathways in plants suggests that modi cations of

existing innate signaling pathways or expression of

‘masterswitch’ genes such as kinases and transcription

factors (Owen and Zamir, 2010; Hammond-Kosack and

Parker, 2003; Sarah and Paul 2005), which regulate a

large number of defence genes could increase resistance

against biotic factors (Owen and Zamir, 2010; Sarah

and Paul 2005). The disadvantage encountered by this

approach could be the harmful effect on plant develop-

ment, due to potential yield loss which is common with

over-expression of large number of genes at a time con-

stitutively (Owen and Zamir, 2010). Therefore, the ideal

candidates are the genes that activate partial pathways

or augment pathways.

Transcription Factors

Transcriptome and QTL data analysis suggested tran-

scription factors to be promising candidates for genetic

engineering to increase disease resistance characteristics

in plants (Sarah and Paul 2005) . They might behave as

master switch gene by taking care of the expression of

several genes in a single pathway. Therefore capable of

making large changes in single trait causing very few

disturbance on other traits (Doebley and Lukens, 1998).

A good example is WRKY transcription factors (Owen

and Zamir, 2010; Sarah and Paul 2005).

WRKY transcription factors are involved in SA- medi-

ated defence pathways. Several WRKYs have the poten-

tial for increasing disease resistance, among them the

most studied are WRKY70 from Arabidopsis [50].Sev-

eral other transcription factor families that have roles

in plant defence could yield useful master switch genes

like WRKY, ERF, TGA, MYB, Dof, GRAS, bHLH, GT1 and

the Whirly factor Why1(Desvaux et al., 2004). The only

limitation being, transcription factors mostly consist of

large multigene families and identifying the best candi-

date can be dif cult due to the functional redundancy

(Eulgem et al., 2000). However, several of Arabidop-

sis WRKY has been identi ed have good functionality

against pathogens (Sarah and Paul 2005).

MAP Kinase

Potential candidate master –switch genes which also

play vital roles under different stress are protein kinases

(Sarah and Paul 2005) . MAP kinase (MAPK) signaling is

a necessarypart of many defence-signalling pathways.

When tobacco MAPK, SIPK is over-expressed it led to

activation of defence responses and HR-like cell death

showing the potential role of these genes (Zhang and

Liu, 2001) . Enhanced resistance to virulent P. syringae

and Botrytis cinerea was observed when MKK4a, MKK5a

were over expressed transiently and MEKK1 was acti-

vated constitutively (Asai et al., 2002) . Other potential

protein kinases are calcium dependent sensor proteins

that changes Ca

defence response (Romeis et al., 2001)

. In response to herbivore-induced cues such as insect

oral secretions (OS) and oviposition  uid compounds,

plants undergo a change in transcriptomes, proteomes,

and metabolomes. The major components of the oral

secretion of insects are fatty acid-amino acid conju-

gates (FACs) which activate the mitogen-activated pro-

tein kinase (MAPK) pathway. The MAPK pathway not

only play an important role in signaling transduction

in responses to a number of stresses including cold,

heat, ROS, UV, drought, pathogen and insect attack but

also regulate plant growth and development (Wu et al.,

2007). On application of FACs in oral secretion of M.

sexta leads to activation of several compounds/mol-

ecules of MAPKs, salicylic acid induced protein kinase

(SIPK) and wound-induced protein kinase (WIPK), JA,

SA and ethylene. In another case brown plant hopper N.

lugens induces expression of putative OmMKKI (MAPK).

Several FAC elicitors have been isolated from various

lepidopteran species (Wu et al., 2007; von Dahl et al.,

2007) .

NPR1

One of the most promising candidates of second gen-

eration strategy is NPR1 (Cao et al., 1994). Pathogen or

insect pest resistance can be achieved through signaling

modi cation. The Npr1 gene was discovered originally

from various independent genetic screens. The Arabi-

dopsis mutants npr‐1 do not respond to inducers of sys-

temic acquired resistance (SAR) such as salicylic acid

(Cao et al., 1994; Delaney et al., 1995; Shah et al., 1997)

638 MANIPULATING DISEASE AND PEST RESISTANCE PATHWAYS IN PLANTS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Paramita Ghosh et al.

or lost the ability to accumulate PR transcripts and were

also hypersensitive to biotrophic pathogens (Pieterse et

al., 2004). NPR1 acts as a switch between the signal-

ing pathways involving ethylene/jasmonic acid (ET/JA)

(ISR) and salicylic pathway (SAR), therefore resistance

to both necrotrophic and biotrophic pathogens depends

on modulation of NPR1 gene (Li et al., 2004; Cao et

al., 1994; Pieterse et al., 2004) . NPR1 is the key master

switch as it constitutes a node which links SAR, ISR, SA,

JA, ethylene, and also R gene-mediated resistance (Piet-

erse et al., 2004). The activation of NPR1 gene is through

redox pathways by SA accumulation in the cytosol and

then translocated to the nucleus, however without bind-

ing to DNA directly it acts through transcription factors,

which in turn induces expression of several PR genes

(Pieterse et al., 2004). NPR1 is constitutively expressed

at low levels, when challenged by pathogen or treated

with SA, transcript accumulation increases up to two-

fold. SA gives better defence against piercing and suck-

ing insect pests than the chewing pests (Zhao et al.,

2009).

SA-mediated expression of proteins by NPR1 include

the WRK70 transcription factors this lead to suppres-

sion of JA-dependent signaling events (Li et al., 2004;

Ndamukong et al., 2007). However, nuclear localization

of NPR1is not required for direct regulation of JA-path-

ways which indicates a dual function between the cyto-

solic and nuclear located NPR1 (Glazebrook et al., 2003;

Spoe et al., 2003; Yuan et al., 2007).

As both SA and JA dependant pathways are controlled

by NPR1, it can be targeted to achieve broad spectrum

disease resistance through genetic engineering. There

are several instances where over-expression of NPR1

has resulted in resistance against both biotrophic (Cao

et al., 1994; Lin et al., 2004). Necrotrophic (Lin et al.,

2004; Makandar et al., 2006; Wally et al., 2009) patho-

gen in several plant species as well as against insect pest

in tobacco plants. Over-expression of NPR1 resulted in

quicker and higher intensity of PR proteins for longer

duration. The function of NPR1 remained unchange

when AtNPR1 was expressed in different crop like rice

(Fitzgerald et al., 2004) , wheat (Makandar et al., 2006),

carrot (Wally et al., 2009) tobacco ( Meur et al., 2008)

and tomato (Lin et al., 2004) indicating the conserved

functionality of the signaling system as well as the

NPR1 like proteins.

However, when AtNPR1 or the rice ortholog OsNH1

was expressed in transgenic rice, the constitutive expres-

sion of PR genes lead to stunted growth of plants and

more light sensitivity apart from desired increase in dis-

ease resistance (Chern et al., 2005). Green tissue speci c

expression of AtNPR1 in rice reduced such developmen-

FIGURE 2. A summary of role of MAPK, NPR1 and transcription factors in plant defence.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS MANIPULATING DISEASE AND PEST RESISTANCE PATHWAYS IN PLANTS 639

Paramita Ghosh et al.

tal abnormalities and conferred resistance to the sheath

blight pathogen without compromising the growth and

yield parameters (Molla et al., 2016)

MANIPULATION THE EXPRESSION OF

TARGET GENES

Expressing Avr protein

De Wit (1992) proposed an interesting exploitation of R

gene response, where a plant can be designed to express

an active Avr protein under the control of a pathogen-

responsive promoter for which the plant has a R gene.

The induced Avr product would induce responses which

would result in incompatible reaction to a wide range of

pathogens (Dewit, 1992). The pathogen inducible pro-

moter (hsr203J) in tobacco resulted in successful exploi-

tation of HR elicitor cryptogein (Keller et al., 1999). The

main bene t is resistance against a wide range of patho-

gens. However the real value of this strategy is yet to be

exploited.

Synthetic modi cations of PR proteins

To enhance the effectiveness of PR proteins, synthetic

modi cations such as linking a single chain antibody

gene against a particular pathogen can be done (Peschen

et al., 2004). The antibodies would then attach to the

invading pathogen’s cell wall and the antimicrobial pro-

teins would effectively degrade the fungi. It has been

demonstrated against Fusarium graminearum. It was

highly effective against nine different species of the

Fusarium genus, in Arabidopsis, however, not effective

against unrelated pathogens (Peschen et al., 2004). This

method has been also implemented in transgenic wheat,

which reduced the disease symptoms against Fusarium

head blight (Li et al., 2008).

Toxic gene products to engineer local cell death

One of the  rst strategies applied for increased dis-

ease resistance in plants was generation of an ‘HR-like’

local cell death arti cially by expressing a toxic gene

(Li et al., 2008). This strategy is only successful when

‘HR’ is restricted to infection sites otherwise uncon-

trolled cell death will occur even in uninfected tissues

which is undesirable. Components of the pathogen can

be expressed as toxic genes. But the promoters used so

far have undesired background expression in uninfected

tissues. Moreover, the toxicity level of the gene product

needs to be studied well before the product is marketed.

RNAi

A useful tool inhibiting pathogen expression is through

RNAi (Csorba and Burgyan, 2016; Novina and Sharp,

2004) technology. It inhibits the expression at both the

transcriptional and post transcriptional levels in plants.

RNAi has been exploited to develop many virus resist-

ant plants (Fuentes et al.,2016). For example, papaya

ringspot virus (PRSV) coat protein protected papaya in

Hawaii has already been commercialized.

Stacking antimicrobial compounds

Expressing antimicrobial proteins, phytoalexins and

enzymes in plant cell reinforcement or in the breakdown

of pathogen infection structures has also been tried. The

limitation of this strategy is resistance towards a speci c

pathogen. However to broaden the spectrum of resist-

ance, stacking of antimicrobial peptides could be a rea-

sonable approach (Van der Biezen, 2001).

Targeting inducible promoters

With the signi cant advances in sequencing technolo-

gies for transcriptome analysis, number of important

crop genomes have been sequenced, which make it fea-

sible for high throughput recognition of promoters and

putative cis elements. Cis regulatory elements function

as molecular switches in respose to various stress signals

(Kazuko and Kazuo, 2005). Transcription factors inter-

act with cis acting elements in the promoter region and

forms a complex to initiate transcription thus can help

in formation of initiation complex when activated and

act as molecular switches to determine transcription ini-

tiation events. Therefore, it is important to determine the

elements in the stress responsive promoters to under-

stand the molecular switches of stress inducible genes.

Apart from this, plant pathogen molecular interaction

has shown that the promoter region also plays an impor-

tant role in pathogen recognition (Patrick et al., 2009) .

In gene for gene interaction pathogen effector interacts

with the promoter region for activation of R gene. For

example, some bacterial effectors like TAL effectors Avr

BS3 and AVR Xa27 interact with the promoter region

and activate the corresponding R genes (Patrick et al.,

2009).

The current limitation of development of resistant

transgenic crops using genetic transformation is una-

vailability of the right kind of promoter. Strong syn-

thetic inducible promoters can be designed to address

the issue of biotic stress. Promoters can be designed to

not only recognize speci c predators but also effector

molecules from different pathogen and pests, thus giv-

ing a broad spectrum resistance against several biotic

factors. It is also possible to use bidirectional promoters

to activate two genes at the same time.

CONCLUSION

Durable pest and disease resistance so far has been

achieved by traditional breeding and chemical appli-

cations. However, conventional breeding has prioritize

640 MANIPULATING DISEASE AND PEST RESISTANCE PATHWAYS IN PLANTS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Paramita Ghosh et al.

quality parameters and agronomic adaptation over

resistant breeding. Therefore, new improved genomic

tools are required to empower the process of genetic

analysis and crop improvement. High through put

sequencing and complete genome sequencing of many

crops allows understanding of many metabolic path-

ways and disease resistance mechanisms. Understanding

of omics are shedding light on the different compounds

associated with plant defense. Using new technologies,

it might be possible to achieve more durable and long

term resistance through various genetic approaches.

The wide spread application of pesticides can also be

reduced through this technology. There are several suc-

cess stories of plant genetic engineering which include

herbicide resistant for weed control and insect resistance

for lepidopteran insect control. However, transgenic dis-

ease resistance crop and resistance against sap suck-

ing insects represent a very small portion of transgenic

crops. Also the scope is wide with the advancement of

genome editing tools like CRISPR-Cas9 and new digital

phenotyping technologies, to develop a more sustain-

able agriculture that involves adaptation to changing

climates. The global food demand needs to be ful lled

and therefore, it is the need of the hour to combat yield

losses caused by diseases and sap sucking insect pests

on a global scale. Also, an increased and stable yield is

required to address decreasing land availability issues.

Engineering disease resistance with new tools available

needs to be made a priority.

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