Plant Innate Immunity: What is it, How does it Operate Against Microbial Pathogens and Can it be Manipulated for Disease Control.

IntroductionThe plant immune system is described as an innate immune system. This is because the

plant immune system lacks the somatic (adaptive) element found in vertebrate immune

systems (Zipfel and Felix, 2005) and therefore relies on its innate cellular mediated immunity

to ward off pathogens (Goehre and Robatzek, 2008). The plant immune system can be

divided in to two constituent subsystems. The first being pattern triggered immunity (PTI),

which could be thought of as the first line of plant defence. The second being effector

triggered immunity (ETI)(Jones and Dangl, 2006). Both subsystems in the plant immune

system produce non-specific responses to impose conditions which inhibit pathogen growth

and proliferation. It would seem one of the determining factors for pathogen survival is the

ability of the plant host to detect it. Also it would seem in the majority of cases, due to the

broad target specificity of conserved plant immune responses, pathogen advancement can

be halted (Goehre and Robatzek, 2008). Therefore it could be that a pathogen’s success is

not necessarily because they show a particular resistance to an immune response, rather it

could be the fact that they avoid detection. With this in mind the review seeks to collate

current scientific thinking with regards to how the proteins involved with PTI and ETI function

to detect microbial pathogens and explain, with the aid of examples, how understanding the

ways PTI and ETI detect microbes can be manipulated to control disease in agricultural

systems.

PTIMicrobial pathogens conserve structural morphologies called pathogen associated molecular

patterns (PAMPs) (Jones and Dangl, 2006).Host cells have evolved ways to recognize

PAMPs and it involves specialized transmembrane proteins called pattern recognition

receptors (PRRs), example PAMPs include peptidoglycan (a conserved constituent of

bacterial cell walls) and flagellin (a constituent compound associated with microbial

locomotion) (Erbs et al., 2008; Zipfel and Felix, 2005). PRRs detect markers associated with

these PAMPs and signal for immune responses. Which can take the form of; cell wall

reinforcement, alterations in apoplastic pH and cellular efflux of antimicrobial molecules and

compounds (Goehre and Robatzek, 2008)..

PRRs can be broadly grouped into two classes, the receptor-like kinases (RLKs) and the

receptor like proteins (RLPs).RLKs feature an extra cellular ligand binding domain, followed

by a transmembrane domain and an intercellular juxtamembrane domain (Greeff et al.,

2012). RLPs, feature a similar morphology to RLKs, but possess a smaller cytoplasmic tail

on the cellular side of the plasma membrane (Goehre and Robatzek, 2008). RLKs have

been the most significantly studied group of plant PRRs and a lot of progress has been

made to determine their constituent member function (Greeff et al., 2012). RLPs however,

have not been so well studied. It has been found that RLPs are involved in PAMP

recognition, but their role with regards to initiating an immune response still remains unclear

(Goehre and Robatzek, 2008).

The recognition function conserved by RLKs is determined by their ligand binding domains,

which are located on the extracellular part of the protein (Greeff et al., 2012). RLKs are also

sub classified by their extracellular ligand binding domains and are involved in a wide range

recognition pathways. At least three of which, have been linked PTI - and are made up of the

LRR (Leucine Rich Repeat), LysM (Lysin Motif) and CrRLK1L (Caranthus roseus RLK1 –

Like) RLK subclasses. Examples of LRR RLKs can include the RLK known as: Xa21. Xa21

has been well studied and it functions to recognize a PAMP marker secreted from

xanthomonas oryzae.Xa21 initiates signalling by binding to a tyrosine sulfonation peptide

(AxYs22) with the aid of XB3 (an E3 ligase) acting as a substrate for Xa21 kinase activity. It

is thought that XB3 is (or involved with) a negative regulator of defense and its

phosphorylation causes a dissociation (of either itself or the down regulator(s)) which

initiates signal transduction via MAPK cascades and a subsequent immune response (Coll

et al., 2011; Greeff et al., 2012).

LysM and CrRLK1L RLKs have also been linked to PTI. However, it would seem the function

of CrRLK1Ls could be more linked to signalling rather than recognition. For instance, the

RLK FERONIA (FER) is thought to be involved with controlling reactive oxygen species

(ROS) production as a means of signalling a PTI response (Greeff et al., 2012). However,

these PRRs do have ligand binding domains so it could that its target PAMPs marker

remains unclear. LysM RLKs however, have been proven to show recognition function and

include RLKs such as the Chitin elicitor receptor kinase 1 (CERK 1). Which is involved in

recognizing chitin, a conserved component of fungal pathogen cell walls (Greeff et al., 2012).

PTI requires a wide variety of PRRs to detect PAMPs and signal an immune responses. PTI

responses can take the form of: cell wall reinforcement, alterations in apoplastic pH and

cellular efflux of antimicrobial molecules and compounds (Greeff et al., 2012). There has

been good progress made in understanding the underlying mechanisms involved with PRR

function, which is important and perhaps inserting or selecting for certain PRRs in crop

species may be a way of enhancing disease control in agricultural systems. However, it

could be worth noting that much still remains unclear and perhaps it could be prudent to

focus on some of the less well studied aspects of PTI, such as the significance of RLPs, and

whether CrRLK1L RLKs detect PAMP markers.

ETIThe second line of plant defence involves the recognition of molecules and compounds

called microbial effectors (Jones and Dangl, 2006). At some point during their evolutionary

history some plant pathogens have evolved to secrete inhibitory molecules and compounds

in order to bypass PAMP triggered immunity and effect cellular function (Deslandes and

Rivas, 2012; Feng and Zhou, 2012). These effectors can function to suppress immunity and

promote a pathogen friendly environment (Dou and Zhou, 2012; Hann et al., 2010).

Microbial effectors can be directly and indirectly recognized by a series of sophisticated

resistance proteins (R proteins) present throughout plant cells (Rafiqi et al., 2012). R

proteins are responsible for effector recognition and regulating cell immune response

(DeYoung and Innes, 2006; Dodds et al., 2006).This response can be regarded as effector

triggered immunity (ETI) and confers immune responses similar to those found with PAMP

triggered immunity, with the exception that ETI can trigger programmed cell death (Coll et

al., 2011) - also known as the hypersensitive response (HR)(Jones and Dangl, 2006).

R proteins are primarily made up of a class of proteins known as the nucleotide binding site -

leucine rich repeat (NBS-LRR) proteins. NBS LRR proteins consist of a LRR C terminal

domain and central NBS domain (Coll et al., 2011). Plant NBS LRR proteins can be further

divided into two groups, which are determined by the presence of a toll interleukin 1 receptor

(TIR) N-terminal domain (DeYoung and Innes, 2006).or a Coiled Coil (CC) N-terminal

domain (Coll et al., 2011). NBS LRR proteins function to detect pathogens using one of two

forms of detection. The first being detection by directly binding to a pathogen effector and

the second being via the guard hypothesis.

The guard hypothesis is the idea that NBS LRR proteins form as part of a complex with

target proteins and attack by microbial effectors infers a conformational change which then

allows the NBS LRR protein to initiate signalling for an immune response (Jones and Dangl,

2006). For instance, in Arabidopsis a plasma membrane associated protein: RIN4 acts as a

negative regulator of the NBS LRR proteins RPM1 and RPS2 with the aid of a NDR1 protein

substrate. The effectors (also known as avirulence proteins) AvrRpm 1 and AvrRpt 2 target

RIN4 at two different sites and infer a conformational change in RIN4 allowing for the release

of RPM1 and RPS2 to signal an immune response (Deslandes and Rivas, 2012).

Direct binding involves the NBS LRR protein binding to an avirulence (Avr) protein. In flax

the NBS LRR proteins L5, L6 and L7 are involved in directly detecting Avr proteins released

by P.syringae and it is thought this is via binding in the LRR domain (DeYoung and Innes,

2006). Experiments were carried out to identify the crystal structures of two members of the

AvrL567 effector family (AvrL567 A and AvrL567 D), as released by the flax rust

Melampsora lini. It was discovered that the NBS LRR proteins forms a complex of multiple

bonds with their target Avr proteins. It was also observed that recognition is conserved to the

LRR domain because it forms the initial bond with a beta sheet on the Avr protein. The

multitude of other bonds form later as a means to stabilise the protein complex (Wang et al.,

2007). Therefore the ability of an NBS LRR protein to directly detect pathogen Avr proteins

could be thought of as a result of, and dependant on LRR domain specificity. So perhaps

plants have evolved the guard hypothesis as a means to bypass part of the evolutionary

arms race that would come with pathogen Avr proteins and LRR specificity. Unfortunately,

this idea does raise the question of which came first, but it is an interesting thought that may

be worth looking in to.

It would seem ETI is dependent on a large variety of sophisticated NBS LRR proteins that

work directly and indirectly to detect pathogen Avr proteins and signal an immune response.

These responses take the form of increased ion fluxes, extracellular oxidative bursts and

transcriptional responses and HR (Coll et al., 2011; DeYoung and Innes, 2006; Dodds et al.,

2006; Jones and Dangl, 2006).

Manipulating Pathogen Detection For Disease ControlIn this section the review will outline, with the use of example studies, how manipulating

plant PTI and ETI detection methods can have a positive effect on disease control in

agricultural systems.

Controlling disease in agricultural systems presents a way of maximising crop yields from

the majority of existing agricultural systems. Over the last 20 years there has been much

time and money spent developing transgenic crops to this end (Collinge et al., 2010). An

experiment carried out in 2003 (Song et al.) managed to successfully introduce an NBS LRR

encoding gene (known as the RB gene) into four potato cultivars. The gene was map-based

cloned from a relative potato species (Solanum bulbocastanum) and the subsequent

cultivars are now widely used in the USA. It was found that the NBS LRR protein which the

gene encodes for provides a high level of non-specific resistance that confers efficient

resistance to P. festans without the use of fungicides. Therefore the argument could be

made that pathogen detection is important because successful insertion of a detection

mechanism into a crop species can have a positive effect on disease control. Also it could be

prudent to consider the fact that using fungicides has an associated cost, so there may be

the potential for economic benefits to using transgenic crops. Although the benefits would be

largely dependent on the cost of the transgenic crop in question. It still could be expected

that there should still be potential for possible environmental benefits from reduced fungicide

usage.

However, by just focusing on inserting detection mechanisms into crops then the

evolutionary arms race between pathogens and plant host could be perpetuated. This is due

the ability of pathogens to rapidly adapt in response to selection pressures and could result

in a more short term resistance (Collinge et al., 2010). Therefore it could be prudent to point

out that transgenic crops that have been engineered to produce a wider array of immune

responses, such as; higher levels of antimicrobial compounds (Collinge et al., 2010). So this

side of transgenics may perhaps bypass this problem somewhat and confer a longer lasting

immunity. Although this remains to be proven, but does demonstrate the need for a multitude

of approaches when considering the use of transgenics in disease control.

Using transgenic crops to improve disease resistance represents a significant method for

controlling disease. However, it would seem that there are barriers with regards to

successfully introducing transgenic strains into agricultural systems. The main being the

associated controversy with using them. Out of the total amount of transgenic crops

approved for use, the component percentage that is disease resistant crops still remains low.

For instance, out of all transgenic crops approved for use in the USA only around 10% of

that number are disease resistant strains (Collinge et al., 2010). Therefore it could be argued

the underpinning problem with using disease resistant crops isn’t so much producing them,

it’s getting them introduced them into a wider amount of agricultural systems. . To this end

the review will provide some interesting non transgenic examples that represent a way of

employing detection mechanisms to help disease control in agricultural systems that may not

only avoid the socioeconomic hurdles associated with transgenics, but could also be used to

as a means of supplementing the use of transgenic crops.

A study carried out (Ding et al., 2015) intercropping pepper plants with maize, found that the

neighbouring maize plants exude defence compounds that restrict soil borne pathogens of

the pepper plants. A subsequent study was carried to see whether the maize plants receive

any benefits from the intercropping system. It was found that elicitors released by either the

pepper roots or soil microbes inferred an immune response in the maize plants, which were

then able to survive subsequent infection with southern corn leaf blight,. Which gives

credence to the idea of trying to incorporate intercropping into more agricultural systems

presents an indirect method of manipulating pathogen detection to increase control of

disease. Another interesting point is that due to the non-specific immune responses in plants

being applicable to many pathogens, perhaps the immune system can be primed in order to

increase disease control.

The idea of priming plant immunity could be a good way to increase disease control without

necessarily relying on the use of transgenic crops. A study was carried out (Ravensdale et

al., 2014) to see whether using two non-pathogenic mutants of Fusarium graminearum (Tri6

and noxAB) could prime immune responses in wheat plants to resist infection with a

pathogenic strain of F. graminearum (fusarium head blight). The results were very promising

and the study found that priming the wheat plants with these non-pathogenic strains did

indeed lead to fusarium head blight resistance phenotypes and they did find an overall

reduction in the amount of fungal mycotoxins in the primed plants. Therefore priming plant

immunity could also be another method to manipulate pathogen detection in plants to

increase disease control in agricultural systems.

Conceptually, plant detection mechanisms can be potentially manipulated both directly and

indirectly in many ways. Directly with the use of transgenics to insert additional detection

methods (Collinge et al., 2010) and with the use of microbial primers to induce non-specific

immune responses (Ravensdale et al., 2014). Indirectly with the use of intercropping

systems (Ding et al., 2015). There are associated problems with all of these concepts. With

transgenics it’s getting them approved for use and selling them (Collinge et al., 2010), with

priming it could be argued that it may not confer long lasting immunity because plants lack a

somatic immune system, and with intercropping it could be expected that are issues with

nutrient availability for the primary crop and it also that it would perhaps make the task of

harvesting the crop somewhat more difficult and less efficient. Therefore these concepts

may not be applicable to every situation. The underlying point could be the fact that neither

one of these concepts represents the “one true” method for controlling disease in agricultural

systems and therefore it could be prudent to consider a combination of these methods to

produce a more rounded approach to controlling disease in agricultural systems. This is

because it would reduce the over reliance on one particular method which could be a way of

side stepping the hurdles associated with a particular method because staggering each

method with other methods, in addition to existing practises, may be a way to reduce the

rate to which pathogens can adapt to anyone method. This is because utilising many

disease control methods could push selection in a multitude of ways as opposed towards

resistance to one method in particular.

ConclusionPlants have an innate immune system that can be subdivided into two constituent

subsystems. The PTI and ETI responses. The proteins involved with PTI and ETI function to

detect microbial pathogens and initiate an immune response. The associated responses are

non-specific and can inhibit pathogen growth in the majority of cases. The proteins involved

with PTI and ETI are of importance because they can be directly manipulated to control

disease in agricultural systems with the use of transgenics and microbial primers. They can

also be manipulated indirectly with the use of intercropping systems to detect pathogens and

activate inter plant signalling to confer immune responses to surrounding plants. It would be

prudent to consider approaches that utilize two or more of these methods to control disease

in agricultural systems as it may present a way to reduce overreliance on anyone method.

This could perhaps side step potential hurdles associated with each one.

Future DirectionsFirstly it may be prudent to carry out further investigations to fully understand the

mechanisms involved with PTI and ETI with aim of discovering the detection and signalling

pathways of more PRRs and R proteins. This information could then be amalgamated into

some form of proteomic/genetic library to be used as a tool for selecting favourable genes

and proteins for transgenic insertion. Also, it would be interesting to see whether

intercropping existing crop plants with transgenic strains has a positive effect like the ones

witnessed in previous studies. Which if true, could represent a means of disease control that

is not over-reliant on a particular transgenic strain and which has the potential to increase

biodiversity in agricultural systems. Plus intercropping with a multitude of different transgenic

strains could be a way of reducing pathogen selection to overcome one type of transgenic

crop. Finally, to preserve yields agricultural systems are reliant on pesticide/fungicide

treatments. If primer molecules and compounds can be isolated perhaps they could be

utilised as part of a pesticide/fungicide treatment in order to not only supplement disease

control, but as a means to reduce the amount of the harmful ingredients found in pesticides

and fungicides.

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