Plant Immunity - deeper dive
How do plant pathogens cause disease?
Disease caused by plant pathogens, usually fungi or bacteria, is a multi-step process (Pfeilmeier et al., 2016). For the aboveground route, it is first necessary that both groups of microorganisms survive on leaves – often an inhospitable environment for microorganisms due to UV radiation and the ever-present danger of desiccation among other stresses. Fungi produce spores – microscopic particles analogous to seeds in plants – that are then spread by the elements (water, wind) or other organisms (insects as vectors). The spores then secrete adhesive substances that allow them to firmly adhere to the plant’s surface. This initial adherence is then followed by the development of fungal filaments on the leaf surface or, after entry into the leaf interior, ramification of filaments between plant cells (Doehlemann et al., 2017). Bacteria can migrate across the leaf surface and release substances that ensure that pores on the leaf surface remain open or that alter the leaf surface, allowing the bacteria to get inside. Stomata are openings in plant leaves that regulate gas exchange and water transpiration in response to changing environmental conditions. As a potential vulnerable site for infection, plants close their stomata upon detection of potential microbial pathogens. However, pathogenic bacteria have evolved strategies to suppress the closure of stomata, tricking the plant to allow them access to the leaf interior (Melotto et al., 2006). Many fungi, meanwhile, assemble a dome-shaped cell that breaches the outermost “skin” on the plant leaf. Once they have successfully breached the leaf surface, microscopic pathogens then deploy a range of strategies to subvert the normal functioning of plant cells and tissues to their benefit. Bacteria and fungi produce toxins that can directly damage plant cells or else hijack plant signaling pathways through mimicry. Plant-pathogenic fungi form additional specialized infection structures, so-called haustoria, that redirect nutrients from the infected host cells towards the invading fungus.
One strategy used by many pathogens is the delivery of different molecules either into the space around cells or directly into host cells. Effectors are often secreted proteins or specialized metabolites that manipulate the structure or function of host components and entire signaling pathways. This explains why effectors often suppress the host defense. However, some effectors also reprogram host cell metabolism. In case of infectious fungi that require living host material, the intruder keeps the invaded cells alive until it is able to move on to another host or, in the case of necrotrophic fungi, kills plant cells for nutrient acquisition.
How do plants block pathogen infection?
We know that plant immune responses are responsible for halting pathogen growth. How exactly these immune responses stop the pathogen remains unclear (Jian et al., 2024). Indeed, the study of plant immunity has almost entirely focused on pathogen recognition; once the plant recognizes a pathogen and activates immunity, this is generally sufficient to restrict growth of very different classes of microbial pathogens, including bacteria, fungi, oomycetes, viruses and nematodes – but we know little about the processes involved.
It is now well established that plants kill their own cells in an attempt to block pathogen spread and alert nearby cells, in a process that is known as the hypersensitive response (HR). It is thought that the HR restricts the spread and proliferation of pathogens by shutting off the supply of plant nutrients, by forming a structural barrier, and by reducing the space available to pathogens for growth (Balint-Kurti, 2019). However, this is only plausible for pathogens that need to obtain their nutrients from living host cells.
The HR itself is accompanied by other responses, complicating interpretation of what it is that is killing the pathogen. Bursts of reactive oxygen species (ROS) – unstable, highly reactive chemicals derived from oxygen, water, and hydrogen peroxide – could help to accomplish this. These ROS mediate activation of immune responses against pathogens. Due to their high reactivity, they can be directly toxic to invading microbes. However, at certain concentrations ROS act as signaling molecules that stimulate immune responses (Qi et al., 2017). Further, the production and release of specialized plant metabolites with antimicrobial activities (so-called phytoalexins; see the discovery of MAMPs below), cell wall fortification, and calcium signaling also likely contribute to terminating pathogen growth.
The issue is further complicated by the fact that resistance and HR can also be uncoupled, i.e., host cell death per se seems to not be a strict requirement for plants to be resistant to pathogen infection.
The zig-zag model of plant immunity
To understand the zig-zag or zig-zag-zig model of plant immunity, first conceptualized in 2006 (Jones and Dangl, 2006), it is first important to appreciate that the plant immune system consists of two branches, defined by two plant–pathogen interaction events.
In the first branch, whose arena is outside the plant cell, receptors known as pattern recognition receptors (PRRs) that span plant cell membranes make initial contact with pathogenic microbes through recognition of the pattern-containing molecules (elicitors) that are characteristic for the microbes. These molecules are known as microbe- or pathogen-associated molecular patterns (MAMPs or PAMPs). One example of a MAMP is flagellin, a protein which forms hair-like appendages on many microbial cells and is needed for bacterial motility. Essentially, there is very little the pathogen can do to avoid initial triggering of this first layer. However, if that was all there was to it, then there would be no plant disease. Therefore, in the second branch or layer, which takes place largely inside the plant cell, pathogens deliver effectors inside plant cells that help the pathogen overcome this first layer of immunity – either by blocking the initial perception event or, more commonly, the plant response to the perception. If the plant–pathogen interaction ended here, then no plants would survive. Thus, in phase three, a given pathogen effector is recognized by a plant R protein, which triggers a second layer of immunity that provides disease resistance. This resistance usually involves the demise of host cells at the site of attempted infection as a strategy to make sure the pathogen does not gain a foothold. Intriguingly, how exactly this response results in termination of pathogen growth is not yet clear. In phase four, pathogens attempt to circumvent this second layer of immunity by changing up the effector repertoire that they deploy in phase two, allowing pathogen strains to drop the effectors that are recognized by the host.
Agrobacterium-mediated plant transformation
Certain plant diseases, like root neck (crown) gall, result from tumor induction that causes uncontrolled tissue proliferation and outgrowths at the crown, the structure at the junction between the root and the stem. These tumors are caused by a bacterium called Agrobacterium tumefaciens, which typically lives in the soil. When plants are injured close to the ground, A. tumefaciens can get into the plant tissue, triggering rapid tumor formation.
While studying crown galls in detail, scientists discovered that agrobacteria were capable of reprogramming plant cell metabolism and inducing proliferation, most likely by introducing foreign genetic material (DNA, deoxyribonucleic acid) into the plants.
Initially, the notion that Agrobacterium tumefaciens triggers plant tumor formation by integrating foreign DNA into the plant host’s genome was met with skepticism. However, scientists demonstrated that plant-pathogenic agrobacteria contain a circular DNA molecule, the tumor-inducing Ti-plasmid, which is absent from Agrobacterium strains incapable of tumor induction. When Agrobacterium infects the plant, it transfers a segment of its Ti-plasmid, the so-called T-DNA (transferred DNA), into the nuclear genome of the host cell, whose growth is reprogrammed by inter-species (horizontal) gene transfer so that the plant cells grow in an unregulated manner and form tumors. Researchers worked out an elegant technology to use Agrobacterium as a “gene ferry” to transform foreign genes of choice into plants. To apply this natural process to produce transgenic plants, it was important to avoid the formation of tumors. The scientists devised a way to remove the genes causing the tumor initiation and replace them with other genes without disturbing the T-DNA transfer mechanism. A “disarmed” T-DNA modified in this way is still incorporated into the plant genome. The transformed cells can then be regenerated to form fertile transgenic plants and if a “desired gene” was present on the “disarmed” T-DNA, pass it on to their offspring. Agrobacterium-mediated plant transformation was a phenomenal technological breakthrough. Until then, only genes between individuals of the same species could be combined through sexual crossing, and selection of a desired trait was a very time-consuming process. The disarmed T-DNA of optimized Ti-plasmids can now be used transfer any desired gene from one plant into the genome of different plant species, allowing the functions of these genes to be better studied and, in some cases, harnessed to improve plant performance. In addition, T-DNA-mediated gene transfer allows for the expression of foreign proteins in plants, furnishing plants with protection against herbicides, fungal infestations, or viral diseases.
The discovery of MAMPs
The concept of elicitors – molecules that induce defenses – was well established by the 1990s, but the nature and range of elicitors and their plant receptors remained uncharacterized. Elicitors were thought to be polysaccharides, and one major hallmark of their activity was the activation of antimicrobial molecules termed phytoalexins. The only well-known extracellular elicitors from bacteria at this point in time were known as harpins and scientists started off trying to characterize a harpin-like activity from Pseudomonas syringae pv tabaci, a bacterium that infects tobacco. Instead of harpin, though, the scientists found something else – flagellin, a protein that forms the hair-like projection conferring bacterial motility. Importantly, researchers could also see that a very conserved stretch of the flagellin protein, now known as a microbe-associated molecular pattern (MAMP) was being recognized by plant cells. But what was the plant receptor that recognized the flagellin? To do this, researchers performed random mutagenesis on plant cells and then scanned for mutants that were non-responsive. This led to the cloning of the first pattern recognition receptor (PRR) called FLS2 (in actual fact the candidates FLS1 and FLS3 turned out not to be receptors; Gómez-Gómez, 2000). What was the relevance for plant immunity? When scientists injected a bacterial pathogen inside plant leaves, there was no difference in resistance between WT plants and those mutated in FLS2. However, when the pathogen was sprayed onto plant leaves and thus able to enter through stomatal pores on the leaf surface, the plants that were mutant for FLS2 were more susceptible to disease, indicating that the receptor was active in recognizing flagellin at the stomatal pores. These landmark findings were published in the journal Nature in 2004 (Zipfel et al., 2004). Up to this point, many had been skeptical that flagellin could act as an immune-eliciting factor – from that point on, many working in the field of plant immunity began using flagellin to model pathogen attack.
Recent breakthroughs in our understanding of immune receptors
The last five years have seen a burst of fresh insights into how plant immune receptors are activated and how they convert the perception of pathogens into effective immune outputs (Chai et al., 2023). Pathogen effector-activated forms of intracellular plant immune receptors are called resistosomes, which often signal as large protein scaffolds. A detailed 3D structure of the first plant resistosome, ZAR1 from the model plant Arabidopsis, was revealed in 2019 (Wang et al., 2019a; Wang et al., 2019b) using cryogenic electron microscopy (cryo-EM for short), a Nobel Prize-winning method in which specimens are embedded in an amorphous form of ice that preserves their biological structure. This technique allows scientists to resolve biomolecules down to the level of individual atoms. The breakthrough was also enabled by developments in cryo-EM during the 2010s which made it much easier to elucidate the structures of large multi-domain proteins as well as “multimeric” complexes with several different protein members. The active ZAR1 resistosome is a stunning ring-like oligomer of five ZAR1 monomers that becomes incorporated into plasma membranes to form a channel which allows the influx of ions into cells. The change in cellular calcium then rapidly turns on defenses that slow or prevent pathogen growth.
A flurry of studies followed from the initial descriptions of the ZAR1 resistosome assembly and signaling mechanism. Soon after, the structure of a wheat immune receptor (like the ZAR1 resistosome, a pentamer) that recognizes an effector from a wheat stem rust pathogen was resolved (Förderer et al., 2022). One key – and striking – takeaway from these studies is that there is a shared general working principle for immune receptor activation between plants and animals: in both kingdoms of life activated receptors often assemble into higher-order structures called oligomers, which set in train crucial defense programs leading to host cell death and pathogen resistance (Hu and Chai, 2023). In plants, an emerging theme for resistosomes of one particular receptor class with terminal channel-forming domains is their action as pathogen-induced ion channels at the plasma membrane that stimulate immunity. Studies show that these channels let calcium ions, charged atoms that are widely used across evolution for signal transduction, into host cells. Scientists are currently working to understand how resistosome-mediated increases in intracellular calcium levels of plant cells translate into mobilization of an immune responses. Current thinking is that there is a common calcium decoding system which turns resistance pathways on.
A second major class of resistosomes instead function as enzymes that catalyze the production of small nucleotide-based signaling molecules. In this case, the generated small molecule messengers activate downstream immune responses by binding to and altering the conformation of their host receptors. The small molecule-modified host receptors are then recognized by a set of so-called “helper” immune receptors which are thought in turn to form resistosome-like oligomeric channels for calcium ion flux across host cell membranes (Jacob et al., 2021; Huang et al., 2022; Jia et al., 2022). Thus, in this case of plant host–pathogen recognition, a key downstream step for turning on defense after initial immune receptor detection of a pathogen effector is further recognition of “modified” host proteins to amplify defense. Intriguingly, recent 3D structural comparisons and modelling of pathogen-activated vs. host self-activated immune receptors suggest they work by a common set of molecular rules. This provides further leads in the design of new receptor activation and signaling modules to combat diseases in crops.
Remarkably, key components of the cell-autonomous innate immune system of plants and animals have ancient evolutionary roots in prokaryotic genes that protect bacteria against infection by bacteriophages. Wide sampling of bacterial genomes in nature and elegant functional studies have shown that many of the building blocks for effective immunity we see in plants and animals were already selected and are operational in bacterial cells and define an important part of the immune system of bacteria (Wein and Sorek, 2022; Li et al., 2023). Delving into these ancient immune systems tells us a lot about immunity logic and robustness across kingdoms.
The CRISPR/Cas9 method for genome editing
CRISPR stands for clustered regularly interspaced short palindromic repeat DNA sequences. The CRISPR system for genome editing comprises an endonuclease – the cutting enzyme Cas9 (or others) – that can be programmed by a so-called “short guide RNA”. Gene editing tools have existed for decades, what sets the CRISPR system apart are its extreme flexibility and editing efficiency – researchers from diverse fields can now edit practically any site in the genome, and since its invention as a gene-editing tool it has been enthusiastically adopted by researchers the world over (Adli et al., 2018).
The CRISPR system was first described in bacteria in the late 1980s, but it wasn’t until the 2000s that biologists could fathom what it did. It turns out that it’s an immune system used by microbes to help protect themselves from invading viruses. To stop the invaders, the microbes use CRISPR to recognize and eliminate specific trespassers. How does it do this? When a virus enters a bacterial cell, the bacterium snatches some of the intruder’s DNA and incorporates it into its own genome. This is so it can recognize and efficiently protect itself against the virus should it ever encounter it again. In this way, it’s similar to the human immune system where antibodies represent the immune memory. (image credits)
Most pre-CRISPR gene-editing tools were single proteins that needed to be reprogrammed by changing the peptide sequence for each gene target. This was time-consuming and also didn’t always work. By incorporating another element into the system – the guide RNA – it actually becomes simpler to target genes of interest. This is because redesigning RNA sequences is technically very easy. Thus, CRISPR/Cas9 works as a GPS system where the guide RNA brings the Cas9 nuclease to a desired location to cut a gene of interest. Having cut through both strands of the DNA strand, DNA repair pathways serve to introduce different sequences into the cut genes, thus inactivating them or changing their function.
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