Inplant immunology, thehypersensitive response (HR) is a mechanism used by plants to prevent the spread of infection bymicrobialpathogens. HR is characterized by the rapiddeath of cells in the local region surrounding an infection and it serves to restrict the growth and spread of pathogens to other parts of the plant. It is analogous to theinnate immune system found in animals, and commonly precedes a slower systemic (whole plant) response, which ultimately leads tosystemic acquired resistance (SAR).[1] HR can be observed in the vast majority of plant species and is induced by a wide range of plant pathogens such asoomycetes,viruses, fungi and even insects.[2]
HR is commonly thought of as an effective defence strategy against biotrophic plant pathogens, which require living tissue to gainnutrients. In the case of necrotrophic pathogens, HR might even be beneficial to the pathogen, as they require dead plant cells to obtainnutrients. The situation becomes complicated when considering pathogens such asPhytophthora infestans which at the initial stages of the infection act as biotrophs but later switch to a necrotrophic lifestyle. It is proposed that in this case HR might be beneficial in the early stages of the infection but not in the later stages.[3]
The first idea of how the hypersensitive response occurs came fromHarold Henry Flor'sgene-for-gene model. He postulated that for everyresistance (R) gene encoded by the plant, there is a correspondingavirulence (Avr) gene encoded by themicrobe. The plant is resistant to thepathogen if both theAvr andR genes are present during the plant-pathogen interaction.[4] These genes, usually, have to be in a dominant state. Thegenes that are involved in the plant-pathogen interactions tend to evolve at a very rapid rate.[5]
Very often, the resistance mediated byR genes is due to them inducing HR, which leads toapoptosis. Most plantR genes encodeNOD-like receptor (NLR)proteins.[6] NLRprotein domain architecture consists of an NB-ARC domain which is anucleotide-binding domain, responsible for conformational changes associated with the activation of the NLRprotein. In the inactive form, the NB-ARC domain is bound toAdenosine diphosphate (ADP). When apathogen is sensed, theADP is exchanged forAdenosine triphosphate (ATP) and this induces a conformational change in the NLRprotein, which results in HR. At the N-terminus, the NLR either has aToll-Interleukin receptor (TIR) domain (also found in mammaliantoll-like receptors) or acoiled-coil (CC) motif. Both TIR and CC domains are implicated in causingcell death during HR. The C-terminus of the NLRs consists of aleucine-rich repeat (LRR) motif, which is involved in sensing thepathogenvirulence factors.[7]
HR is triggered by the plant when it recognizes apathogen. The identification of apathogen typically occurs when avirulence gene product, secreted by apathogen, binds to, or indirectly interacts with the product of a plantR gene.R genes are highlypolymorphic, and many plants produce several different types ofR gene products, enabling them to recognize virulence products produced by many differentpathogens.[8]
In phase one of the HR, the activation ofR genes triggers anion flux, involving anefflux ofhydroxide andpotassium to the outside the cells, and an influx ofcalcium andhydrogen ions into the cells.[9]
In phase two, the cells involved in the HR generate anoxidative burst by producingreactive oxygen species (ROS),superoxideanions,hydrogen peroxide,hydroxyl radicals andnitrous oxide. These compounds affectcellular membrane function, in part by inducinglipidperoxidation and by causing lipid damage.[9]
The alteration of ion components in the cell and the breakdown of cellular components in the presence of ROS result in the death of affected cells, as well as the formation of locallesions. Reactive oxygen species also trigger the deposition oflignin andcallose, as well as the cross-linking of pre-formedhydroxyproline-richglycoproteins such as P33 to the wall matrix via the tyrosine in the PPPPY motif.[9] These compounds serve to reinforce the walls of cells surrounding the infection, creating a barrier and inhibiting the spread of the infection.[10] Activation of HR also results in disruption of the cytoskeleton, mitochondrial function and metabolic changes, all of which might be implicated in causing cell death.[11][12][13]
HR can be activated in two main ways: directly and indirectly. Direct binding of thevirulence factors to the NLRs can result in the activation of HR. However, this seems to be quite rare. More commonly, thevirulence factors target certain cellularproteins that they modify and this modification is then sensed by NLRs. Indirect recognition seems to be more common as multiplevirulence factors can modify the same cellularprotein with the same modifications thus allowing one receptor to recognize multiplevirulence factors.[14] Sometimes, theprotein domains targeted by thevirulence factors are integrated into the NLRs. An example of this can be observed in plant resistance to therice blast pathogen, where the RGA5 NLR has aheavy-metal-associated (HMA) domain integrated into its structure, which is targeted by multipleeffector proteins.[15]
An example of indirect recognition: AvrPphB is atype III effector protein secreted byPseudomonas syringae. This is aprotease which cleaves a cellularkinase called PBS1. The modifiedkinase is sensed by RPS5 NLR.[16]
Recent structural studies of CC-NLRproteins have suggested that after thevirulence factors are sensed, the CC-NLRs assemble into a pentameric structure known as theresistosome.[17] The resistosome seems to have a high affinity for thecellular membrane. When the resistosome is assembled, ahelix sticks out from the N-terminus of each NLR and this creates a pore in the membrane which allows leakage ofions to occur and thus thecell dies. However, this mechanism is only inferred from the structure and there are currently no mechanistic studies to support this. It is still not known how the TIR-NLRproteins are activated. Recent research suggests that they require CC-NLRproteins downstream of them, which are then activated to form the resistosomes and induce HR.[18]
It is known that NLRs can function individually but there are also cases where the NLRproteins work in pairs. The pair consists of a sensor NLR and a helper NLR. The sensor NLR is responsible for recognizing thepathogen secretedeffector protein and activating the helper NLR which then executes thecell death. Thegenes of both the sensor and the respective helper NLR are usually paired in thegenome and theirexpression could be controlled by the samepromoter. This allows the functional pair, instead of individual components, to besegregated duringcell division and also ensures that equal amounts of both NLRs are made in the cell.[19]
The receptor pairs work through two main mechanisms: negative regulation or cooperation.
In the negative regulation scenario, the sensor NLR is responsible for negatively regulating the helper NLR and preventingcell death under normal conditions. However, when theeffector protein is introduced and recognized by the sensor NLR, the negative regulation of the helper NLR is relieved and HR is induced.[20]
In the cooperation mechanisms, when the sensor NLR recognizes theeffector protein it signals to the helper NLR, thus activating it.[21]
Recently, it was discovered that in addition to acting as singletons or pairs, the plant NLRs can act in networks. In these networks, there are usually many sensor NLRs paired to relatively few helper NLRs.[21]
One example ofproteins involved in NLR networks are those belonging to the NRC superclade. It seems that the networks evolved from a duplication event of agenetically linked NLR pair into an unlinked locus which allowed the new pair to evolve to respond to a newpathogen. This separation seems to provide plasticity to the system, as it allows the sensor NLRs to evolve more rapidly in response to the fast evolution ofpathogen effectors whereas the helper NLR can evolve much slower to maintain its ability to induce HR. However, it seems that during evolution new helper NLRs also evolved, supposedly, because certain sensor NLRs require specific helper NLRs to function optimally.[21]
Bioinformatic analysis of plant NLRs has shown that there is a conserved MADA motif at the N-terminus of helper NLRs but not sensor NLRs. Around 20% of all CC-NLRs have the MADA motif, implying the motif's importance for the execution of HR.[22]
Accidental activation of HR through the NLRproteins could cause vast destruction of the plant tissue, thus, the NLRs are kept in an inactive form through tight negative regulation at bothtranscriptional andpost-translational levels. Under normal conditions, themRNA of NLRs aretranscribed at very low levels, which results in low levels ofprotein in the cell. The NLRs also require a considerable number ofchaperone proteins for their folding. Misfoldedproteins are immediatelyubiquitinated and degraded by theproteasome.[23] It has been observed that in many cases, if the chaperone proteins involved in NLR biosynthesis areknocked-out, HR is abolished and NLR levels are significantly reduced.[24]
Intramolecular interactions are also essential for the regulation of HR. The NLRproteins are not linear: the NB-ARC domain is sandwiched in between theLRR andTIR/CC domains. Under normal conditions, there is a lot moreATP present in the cytoplasm thanADP, and this arrangement of the NLRproteins prevents the spontaneous exchange ofADP forATP and thus activation of HR. Only when avirulence factor is sensed, theADP is exchanged forATP.[14]
Mutations in certain components of plant defence machinery result in HR being activated without the presence ofpathogeneffector proteins. Some of these mutations are observed in NLRgenes and cause these NLRproteins to become auto-active due to disrupted intramolecular regulatory mechanisms. Other mutations causing spontaneous HR are present inproteins involved inROS production duringpathogen invasion.[3]
HR is also a temperature-sensitive process and it has been observed that in many cases plant-pathogen interactions do not induce HR at temperatures above 30 °C, which subsequently leads to increased susceptibility to thepathogen.[25] The mechanisms behind the influence of temperature on plant resistance topathogens are not understood in detail, however, research suggests that the NLRprotein levels might be important in this regulation.[26] It is also proposed that at higher temperatures the NLR proteins are less likely to formoligomeric complexes, thus inhibiting their ability to induce HR.[27]
It has also been shown that HR is dependent on the light conditions, which could be linked to the activity ofchloroplasts and mainly their ability to generateROS.[28]
Severalenzymes have been shown to be involved in generation ofROS. For example, copperamineoxidase,catalyzes theoxidativedeamination ofpolyamines, especiallyputrescine, and releases theROS mediatorshydrogen peroxide andammonia.[29] Other enzymes thought to play a role inROS production includexanthine oxidase,NADPH oxidase,oxalate oxidase,peroxidases, andflavin containing amine oxidases.[9]
In some cases, the cells surrounding the lesion synthesizeantimicrobial compounds, includingphenolics,phytoalexins, andpathogenesis related (PR)proteins, includingβ-glucanases andchitinases. These compounds may act by puncturingbacterialcell walls; or by delaying maturation, disruptingmetabolism, or preventingreproduction of thepathogen in question.
Studies have suggested that the actual mode and sequence of the dismantling of plant cellular components depends on each individual plant-pathogen interaction, but all HR seem to require the involvement ofcysteine proteases. The induction of cell death and the clearance ofpathogens also requires activeprotein synthesis, an intactactincytoskeleton, and the presence ofsalicylic acid.[8]
Pathogens have evolved several strategies to suppress plant defense responses. Host processes usually targeted by bacteria include; alterations toprogrammed cell death pathways, inhibiting cell wall-based defenses, and alteringplant hormone signaling and expression of defensegenes.[30]
Local initiation of HR in response to certain necrotrophicpathogens has been shown to allow the plants to develop systemic immunity against thepathogen.[31] Scientists have been trying to exploit the ability of HR to induce systemic resistance in plants in order to createtransgenic plants resistant to certainpathogens. Pathogen-induciblepromoters have been linked to auto-active NLRgenes to induce HR response only when thepathogen is present but not at any other time. This approach, however, has been mostly unfeasible as the modification also leads to a substantial reduction in plant yields.[3]
It has been noticed inArabidopsis that sometimes when two different plant lines are crossed together, the offspring show signs ofhybrid necrosis. This is due to the parent plants containing incompatible NLRs, which when expressed together in the same cell, induce spontaneous HR.[32]
This observation raised a hypothesis that plantpathogens can lead to thespeciation of plants – if plantpopulations from the samespecies develop incompatible NLRs in response to differentpathogen effectors, this can lead tohybrid necrosis in theF1 offspring, which substantially reduces thefitness of theoffspring andgene flow to subsequent generations.[33]
Both plants and animals have NLRproteins which seem to have the same biological function – to inducecell death. The N-termini of plant and animal NLRs vary but it seems that both haveLRR domains at the C-terminus.[34]
A big difference between animal and plant NLRs is in what they recognise. Animal NLRs mainly recognisepathogen-associated molecular patterns (PAMPs), while plant NLRs mostly recognisepathogeneffector proteins. This makes sense as NLRs are present inside of thecell and plants rarely haveintracellular pathogens, except forviruses and viruses do not havePAMPs as they are rapidly evolving. Animals, on the other hand, haveintracellular pathogens.[35]
The vast majority of plant lineages, except for certainalgae, such asChlamydomonas, have NLRs. NLRs are also present in many animalspecies, however, they are not present in, for example,Drosophila melanogaster andArthropods.[34]
Upon recognition ofPAMPs by NLRs in animals, the NLRsoligomerise to form a structure known as theinflammasome, which activatespyroptosis. In plants, structural studies have suggested that the NLRs alsooligomerise to form a structure called the resistosome, which also leads tocell death. It seems that in both plants and animals, the formation of the resistosome or theinflammasome, respectively, leads tocell death by forming pores in themembrane. It is inferred fromprotein structures that in plants the NLRs themselves are responsible for forming pores in themembrane, while in the case of theinflammasome, the pore-forming activity arises fromgasdermin D which is cleaved bycaspases as a result of theoligomerisation of the NLRs.[36][37] Plant cells do not havecaspases.[38]