What is this 'effector' thing, anyway?

In which I opine about the definition of (plant) pathogen effectors.

I took part in a Twitter discussion between plant pathologists recently, attempting to reach some kind of agreement on what an 'effector' is.  It's hard to make some points coherently in a 140-word limit, but in the end it seemed that everyone went away thinking that an understanding had been reached.  There were some things in there that I wasn't entirely happy with - I found it very difficult to be precise - so I thought I might expand a bit, here.

A quick intro for non-pathologists

Plants have an immune system that protects them (much of the time) from the onset or spread of disease, but it doesn't work quite like ours.  There's no real reason why it should - we diverged from plants a long time ago, and plants are essentially immobile: they don't have the opportunity to move away from danger.  Like the human immune system though, plants raise chemical defences against microbial invaders, and they need to assess the level of threat, convert it to a signal, and raise an appropriate level of response.  Too strong a response could be wasteful of resources and limiting of growth; too weak a response could leave the plant vulnerable.

MAMPs For The Memories

Picture (if you can) a microbial pathogen approaching a plant cell: a fairly flexible bag of living chemistry possibly thrusting itself through a fluid, or along a leaf (or root, or stem...) surface.  As it goes, it is likely to be producing and secreting a number of molecules that aren't produced by plants.  It is probably also shedding parts of itself, in the form of larger molecules, and fragments of larger molecules.  There is, then, a fairly sparse molecular cloud of non-plant chemistry accompanying the pathogen as it approaches the plant cell, which looms like a large shed (plant cells tend to be a bit more solidly-structured than pathogen cells).

The plant cell is not sitting there, entirely inert.  The outer surfaces of its cell walls are 'hairy' with detectors.  These are triggered by chemicals in the surrounding environment and are the eyes and ears of the plant cell.  The plant is also producing enzymes and pumping them into the surrounding locality: these can break down larger molecules in the vicinity into (more) smaller chemicals, which can then be detected more easily.  The overall effect is a bit like a chemical soup.

When the plant cell's surface detectors come across something unusually 'non-self' floating in the immediate chemical soup, this can be taken as a sign of imminent invasion or infection, and signals can propagate through the plant cell to raise up the appropriate defence.  The elements that come from an invading microbe that float through the soup are known as Microbe- (or Pathogen-) Associated Molecular Patterns: MAMPs or PAMPs.  The terms really only differ depending on whether we want to highlight the microbial or pathogenic nature of the organism being detected.

The Effects of Effectors

The invading microbe isn't usually passive, either, especially during interaction with the plant.  Microbes that interact with plants are variously able to interfere with the plant's normal operation to reduce the effectiveness of host defences, and to turn the plant's own chemistry to the microbe's ends.  They can do this by introducing into plant cells their own chemical species that either interfere with, replace, or otherwise subvert the biochemical systems of the host.  A number of strategies for introducing these species have evolved, including direct injection through a syringe-like structure (e.g. bacterial type III secretion).  These chemical species are typically what are referred to as 'effectors'.

So what's the issue?

Words are not equivalent to variables in computing languages, in the sense that they are not simple containers that just hold concepts.  We internalise the meanings associated with words and symbols in a way we simply don't do when we assign a value of 5 to the variable a, for example.  We can assign a value 3.14 to a variable x, but when we think of 'pi', we naturally associate many other concepts (transcendental numbers, randomness, circles, trigonometry and so on) and become psychologically 'primed' for those concepts.  The same is true when we talk about any other concept, including that of an 'effector' and, if we do not all share similar associations it is easy to talk at cross-purposes.  We need a common understanding of what we talk about when we talk about effectors.

I believe that, happily, the concept of an effector is pretty straightforward and easy to illustrate with some examples from the 'real world'.

Effectors modify plant behaviour.

This seems a natural place to start.  By behaviour I mean any manifestation of 'behaviour' from changes to chemical responses upwards.  We wouldn't be thinking about the subject at all if there hadn't been some noticeable effect on a plant, somewhere.  That's not to say that an effector always modifies plant behaviour.  Sometimes an effector might need the plant to be in a particular state to be able to act.  Sometimes the effector might need to act together with some other molecule (perhaps another effector) to have an effect.  Sometimes the plant may be able to block the effector's action.  But, essentially, the clue is in the name: effector.

Effectors are molecules produced by microbes.

Not everything that has an effect on plant behaviour is a molecule: plants also respond to changes in temperature, the location of a light source, local pH or other ionic concentrations, and mechanical stress or damage, for example.  We can state a definition that effectors should be identifiable molecules, whether small molecules, proteins, or something else.  This does not preclude the action of effectors requiring dimerisation (or multimerisation), or simple association with other molecules.

However, not everything that a microbe produces is a molecule, or simple association of molecules.  For example, bacteria may produce biofilms, which are complex aggregates of the bacteria embedded within a matrix of slimy extracellular polymers.  These biofilms can result in changes to the local environment, to which a plant may in turn respond.  As a complex structure, biofilms do not count as simple associations of readily definable molecules and so do not - by this definition - count as effectors.

Not every molecule derived from a microbe that modifies plant behaviour is an effector.

MAMPs/PAMPs modify plant responses, but they are not effectors.  They are, rather, tell-tale indicators of a microbe's presence.  The difference between a MAMP and an effector is one of function or (if you find this approach easier to follow) agency.  

Often, MAMPs are fragments of larger molecules with no direct functional activity of their own, such as flg22 - a fragment of the flagellin protein - or fragments of chitin (the polymer that constitutes much of fungal cell walls).  Plant cellular behaviour is modified on MAMP detection, but that is not the function of the MAMP.  This is especially so if the MAMP results from the action of the plant on the microbe: there is no aspect of agency on behalf of the pathogen in the production of the MAMP.  This is what I'm aiming for in the distinction between 'produced by' and 'derived from'.

An analogy might be the detection of an aeroplane by a defence system's radar, and the launch of a missile in response.  Here, the designed purpose of the aircraft is neither to reflect radar waves, nor to trigger the missile launch.  Likewise, the evolved 'purpose' of the MAMP is not to trigger a plant defence response.  Hence MAMPs are not effectors.

Modification of plant behaviour may look like normal plant behaviour.  

If the action of an effector is to disrupt normal plant behaviour, that also includes things like disruption of MAMP detection: essentially, restoring 'normal' plant operation when the 'natural' behaviour could be to raise some kind of defence.

Effectors may act within, or outwith the plant cell.

The type III secretion system mentioned above involves the injection of microbial proteins into the host plant cell, and this is how we (or, at least, I) most commonly think of effectors - as species that act within the host's cell.  This doesn't need always to be so.

To extend the aircraft analogy from above, we can imagine a stealth paint coating that absorbs the radar signal, thus preventing detection.  The stealth paint coating would be considered to have the purpose of disrupting the defence the usual chain of signals that would otherwise result in the launch of missile defence.  By virtue of its action on the 'normal' operation of the defence system, it could be considered to act like an effector.  Notably, this action takes place 'in the open', away from the detection system itself.

There is a protein produced by the fungal plant pathogen Cladosporium fulvum that acts like this stealth paint.  This protein, called Avr4, protects the pathogen's cell wall (specifically, the chitin component) against being broken down by a protein produced by the plant (a chitinase).  Here, the plant's chitinase is like an outgoing radar signal; normally it would elicit a chitin fragment from the fungus (the return signal), which would be picked up by a plant surface protein (the radar dish).  By preventing the generation of a chitin fragment, the Avr4 protein essentially absorbs the outgoing signal, disrupting the 'normal' plant detection system.  This happens outside the plant.  By our definition so far, Avr4 is an effector.

Being required for observable modification of plant behaviour is not equivalent to being an effector.

There are two issues here.  Firstly, many molecules and systems that play no direct part in modification of plant behaviour may be essential for microbial well-being - but microbial well-being may be essential for modification of plant behaviour.  Removing all the enzymes of glycolysis from a microbe may prevent microbe-plant interaction, but they are not effectors because of that.  Secondly, some molecules that affect plant behaviour may be redundant, in that they may be deleted or removed without affecting the change to plant behaviour (e.g. Redundant Effector Groups).  Either case is sufficient to show that being an effector and being required for modification of plant behaviour are not equivalent.

Also, effectors may have a host-specific action: they may disrupt behaviour in some plants, but not others.

Avirulence proteins are not equivalent to effectors.

Avirulence proteins are proteins detected by the plant, and whose detection results in plant resistance (or, at least, a defined resistance response) to the microbial invasion.  Many of the best-understood avirulence proteins are effectors but, since there is no obvious requirement that the detected protein be an effector, or that effectors be detected and result in resistance reponses, the two definitions are clearly not equivalent.

Effectors are not restricted to pathogens.

I have tried not to give definitions that lean too heavily on the biology of pathogens, as disease is a concept with a distinctly human bias.  The effect of a disease is - quite obviously - negative.  But there are many associations between plants and microbes that do not result in what we would, as humans, call disease.  An obvious example is the symbiosis of nitrogen-fixing bacteria with plant hosts.  But even these beneficial (or any number of benign) interactions can involve complex molecular interactions between the microbe and host plant, in which the microbe seeks to modify host behaviour using effectors.  

I consider pathogens to lie at one extreme of a continuum of plant-microbe interaction in which the pathogen gains all the benefit of the interaction and the plant none.  At the other end, the plant gains all the benefit, and the pathogen none.  Through the rest of this continuum, which includes many shades of symbiotic interaction, the picture is not so clear, and an interaction that is apparently mutually beneficial or benign may suddenly turn pathogenic if the environment changes (such as the emergence of pathogenesis under stress).

I think this definition of 'effector' applies quite readily beyond plant-microbe systems, too... 

Effectors act directly.

This is the part of my definition that gives me most trouble.  Where we know how effectors work, they tend to interact directly with a host plant molecule.  Where they do not (such as Avr4), their role in disrupting a chain of communication or signalling is typically quite clear, and we can consider such direct disruption to normal information transfer to be direct action.

The picture becomes less clear when proteins (such as Ace1 from Magnaporthe oryzae, or AvrD from Pseudomonas syringae) act to synthesise chemical species that disrupt the host.  By this definition, the synthesised molecule is the effector, and the synthesising proteins are not effectors, because they 'act at a distance', the same way a ribosomal protein is not an effector simply because it plays a role in synthesising effectors.  However, AvrD is (judging from the name), an avirulence protein.  

The picture is also less clear when we think of proteins that might modify the environment of a host cell to disrupt behaviour.  With Avr4 we can make a reasonable claim that it is disrupting the normal operation of a signalling pathway; it is acting precisely.  Modifications to a bulk property of the environment (e.g. ionic concentrations, temperature, viscosity) do not count by this definition, as they are not direct action, but action mediated by, well... the medium.

Effectors need not benefit the microbe.  At least, not right now.

Although it is difficult to think a reason why an effector would persist indefinitely if it never provided any benefit to the invading microbe, since we are always observing a snapshot of evolution, there is no reason to impose an absolute cost or benefit requirement as part of the definition of an effector.

In Conclusion

I hope I have been able to make a case for the following one sentence definition of an effector (with the caveats above):

"An effector is a molecule produced by a microbe that acts directly to disrupt the normal operation of host biochemistry."

Or, more generally:

"An effector is a simple entity produced by system A that acts directly to disrupt the normal operation of system B."

(where 'simple' is defined appropriately for the two systems).

As ever, brickbats, bouquets, and better definitions are welcomed in the comments.