3.1. Pattern- versus Effector-Triggered Immunity
Because of the conserved nature and broad occurrence of MAMPs in different microbes, sensing of MAMPs to activate PTI enables the host to detect and efficiently control a wide range of microbes [
4]. Adapted pathogens employ effectors to dampen PTI and to modulate host cell metabolism for their own needs. Plants, in turn, evolved immune sensors, called resistance (R) proteins that detect microbial effectors directly or indirectly by monitoring the effector targets, thus turning effectors into avirulence factors. This results in effector-triggered immunity (ETI), which includes overall similar defense responses like PTI but usually develops faster and in a stronger fashion and is mostly accompanied by an HR [
5]. Since effector genes are typically specific to certain pathogen races, while
R genes are limited to certain plant cultivars, this form of resistance is called race-specific or gene-for-gene resistance. In a co-evolutionary arms-race with their hosts, pathogens may be able to overcome
R-gene mediated ETI relatively easy [
5,
52]. Mostly, recognized effectors are not essential and/or functional redundant allowing microbes to evade recognition by diversifying or even losing them. Alternatively, microbes may produce novel effectors that e.g. suppress the same or other PTI components or R protein signaling [
5,
52]. To date, plant resistance breeding widely relies on race-specific resistance mechanisms, largely because effectors and
R genes typically result in complete or near-complete resistance. The selective pressure exerted on pathogen populations by ETI, however, may result in rapid resistance breaching in the field by the appearance of novel pathogen races/strains [
52]. PTI, by contrast, enables defense against a broad spectrum of pathogens and presumably is more durable because of the evolutionary constraints on MAMP modulation and the quantitative resistance conferred by PRRs which reduces the selective pressure on the pathogen.
3.2. PRR Transfer Between Plants Species
One feature makes PRRs particularly attractive targets for genetic engineering of plant immunity: signaling networks downstream of PRRs are sufficiently conserved (
Figure 1), not only within but also between plant families and even between monocot and dicot classes, to facilitate functional transfer of PRRs between them [
53,
54,
55]. Apparently, PRRs can plug into the existing signaling network by engaging with conserved interacting signaling partners (e.g., SOBIR, BAK1 or CERK1) (
Figure 2) [
54,
55]. Because PRRs as the ligand binding components of PTI receptor complexes determine the epitope specificity, introducing a PRR with a novel epitope specificity can confer recognition of this epitope onto previously insensitive plant species. In this way, plants can be equipped with additional MAMP sensing capacities to enhance disease resistance to pathogen classes harboring the respective MAMPs (
Figure 2). These can either be PRRs for MAMPs not yet sensed by the recipient plant or for additional epitopes within a given MAMP (e.g., flgII-28/FLS3). A breakthrough study utilizing the
Brassicaceae-specific EFR receptor has proven the general feasibility of this strategy [
53], which was subsequently shown for multiple PRRs in various plant species (summarized in [
6,
56]). EFR-transgenic tomato (
Solanum lycopersicum) and
Nicotiana benthamiana plants gained immune responsiveness to the elf18 epitope and were more resistant to several major bacterial pathogens carrying this epitope including
Ralstonia solanacearum,
P. syringae,
Xanthomonas perforans, and
Agrobacterium tumefaciens [
53]. EFR was subsequently also successfully transferred into monocots, such as wheat (
Triticum aestivum) and rice (
Oryza sativa) [
55,
57]. Similarly, transfer of the rice PRR XA21, which detects the RaxX protein from
Xanthomonas oryzae pv.
oryzae, to banana (
Musa sp.) and sweet orange (
Citrus sinensis) confers increased resistance against different
Xanthomonas spp. [
58,
59] and expression of the PRR ELR from a wild potato species renders cultivated potato (
Solanum tuberosum) more resistant to the oomycete pathogen
Phytophthora infestans [
60]. Taken together, these results demonstrate that addition of only a single PRR can already significantly enhance resistance of plants to adapted pathogens. Given that these pathogens are equipped with an arsenal of effectors to dampen PTI, this is quite astonishing and suggests that increased pathogen sensing provides a substantial advantage to the plant, possibly because of a stronger and/or faster onset of defense reactions.
Often, the isolation of MAMPs is the bottleneck in identifying PRRs [
6,
9]. However, it is not necessary to structurally identify a MAMP but sufficient to enrich it to a degree that facilitates genetic screenings for the respective PRR [
61,
62,
63]. Furthermore, the identification of various PRRs for chemically diverse types of MAMPs has revealed that plant PRRs are typically RLKs or RLPs. Thus, these protein families can now also be systematically tested for a putative role in plant immunity, for instance through screening for altered resistance against the pathogen of interest in loss- or gain-of-function mutants or natural accessions, or through heterologous expression in suitable model plants (e.g.,
Arabidopsis thaliana or
N. benthamiana) [
64,
65,
66,
67,
68,
69].
A major concern of introducing novel PRRs into plant species is that this might negatively affect beneficial interactions of these plant species with their natural microbiota and/or symbiotic interactions, such as legume-rhizobia or mycorrhizal interactions. Transgenic Medicago plants expressing EFR from Arabidopsis, however, are apparently not defective in symbiosis and are more resistant to the root pathogen
R. solanacearum. Although the EFR transgenic plants showed a delay in nodule formation the final extent of nodulation and nitrogen fixation of the EFR transgenics was comparable to the wild-type plants [
70]. Potentially, adapted symbionts have evolved efficient strategies to evade or suppress host PTI at various levels.
While to date mostly strong, constitutive promoters (e.g., viral promoters such as the 35S promoter of cauliflower mosaic virus, plant ubiquitin or actin promoters) are used to drive heterologous PRR expression in plants because of their universal functionality in diverse plant species and different plant tissues, this can result in unwanted side effects on general plant performance. Overexpression of RLKs, for instance, may trigger their activation in the absence of ligand and lead to growth defects because of constitutive activation of PTI or interference with developmental signaling [
71]. This can be overcome by using plant promoters from e.g. endogenous PRR genes that drive PRR expression in plant tissues preferentially targeted by the pathogen of interest (e.g., root) or at sites of pathogen entry (e.g. stomata guard cells) and are strongly induced above the basal level upon infection [
72,
73].
The growing interest in PTI in recent years already led to the identification of numerous PRRs from different model and wild plant species and there is more to come (summarized in [
6,
17]). Natural diversity provides plant breeders with a versatile genetic tool box for crop improvement. Relevant PRR genes can be introduced, for instance, from wild relatives through classical breeding strategies. However, this is usually a lengthy process, bears the risk of co-segregation of unfavorable traits (linkage drag), and is not applicable to all crop species (e.g., banana, which is sterile) [
56,
74]. Alternatively, modern genetic engineering tools facilitate direct transfer of PRR genes across plant families beyond the constraints of sexual compatibility with the advantage that PRR genes can be quickly introduced into elite crop varieties as single traits (
Figure 2). Additionally, this allows to utilize virtually any plant species as source of PRR genes. In conclusion, PRR transfer has great potential for conferring broad-spectrum and potentially durable resistance traits onto crop plants.
3.3. PRR Engineering
During co-evolution with host plants, pathogens modulate MAMP epitopes to evade host immunity whereas plants adapt their PRRs [
16]. Hence, there is a natural diversity of PRRs that recognize slightly different variants of a given MAMP epitope. Ecotype collections of plant species harboring a particular PRR are thus a rich source of PRR variants with enhanced sensing capacities for these epitope variants (
Figure 2) [
75,
76]. Additionally, in vitro evolution of PRRs can be applied to produce PRRs with altered ligand specificities [
77]. With the increasing availability of PRR ectodomain structures and computational modelling tools ligand binding sites of PRRs can be modified in a directed manner to perceive a desired epitope variant or may eventually even be designed
de novo (
Figure 2). Potentially, ligand binding sites can be engineered at the native gene locus through CRISPR/Cas-mediated genome editing.
Some PRRs may not be able to integrate optimally with the endogenous signaling adapters upon transfer in more distantly related plant species. In such cases, full signaling competence can be restored by exchanging the transmembrane and/or intracellular signaling domain with a related PRR from the recipient species (
Figure 2) [
54,
62]. Such chimeric PRRs have been shown to be fully functional and can be further exploited to combine different ligand binding specificities with different downstream signaling capacities (
Figure 2). Some PRRs naturally induce a stronger immune response including an HR, such as the rice RLKs XA21 [
78]. Sensing of chitin fragments by the rice PRR CEBiP, by contrast, does not result in HR [
79]. A chimeric receptor combining the chitin-sensing ectodomain from CEBiP with the intracellular signaling domain of Xa21 activates HR upon chitin sensing and enhances resistance to the rice pathogen
Magnaporthae oryzae, a pathogen that does not naturally activate Xa21 signaling [
80].