It is often presumed that activation is associated with a change in oligomerization state that imposes or induces proximity or conformational changes on the N-terminal signaling domain . However, knowledge of whether this occurs, and of ensuing steps in the process, is inadequate. There is a need for research on NLR mechanisms in multiple pathosystems. Although we cannot yet design new disease resistance genes, foundational knowledge has enabled some new recognition capacities to be created. For example, changing a protease recognition sequence in the PBS1 “guardee” protein enabled its guard, the RPS5 NLR protein, to recognize different protease effectors . Knowledge of which pathogen proteases are important players in plant-pathogen interactions will facilitate the development of multiple novel R genes. A major constraint on obtaining novel recognition abilities is the capacity to screen for R proteins that provide a useful phenotype without constitutive activation. If clones could be transiently delivered and tested for capacity to recognize specific effectors, for example with a defense promoter:luciferase reporter fusion, thousands of clones could be evaluated in a high-throughput manner. Synthetic biology and genome editing tools can also be used to develop rules for assembly and engineering of novel NLRs. Signaling from cell surface PRRs is slightly better understood than signaling from NLRs . We are, however, again not yet at the stage where PRRs can be designed with novel recognition capacities. As with NLRs, more detailed structural information is required before this will be possible. In the interim, pipp vertical racks identification of additional natural diversity in PRR recognition capacity would impact crop improvement. A promising approach is to screen diverse plants for novel PRR recognition capacities and to transfer useful corresponding receptors between taxa.

For example, species in the Brassicaceae can detect the apoplastic bacterial translation factor EF-Tu via the RLK EFR, but Solanaceous species cannot; transfer of EFR to species in the Solanaceae elevates resistance to several bacterial diseases . There is an urgent need to discover novel PRR ligands from a broad spectrum of pathogens/pests, including nematodes and aphids . PRR ligands will be useful for direct identification of new PRRs, screening for natural variation in strongly responding PRRs, and engineering new PRRs. Prospecting for novel recognition capabilities should involve biochemical exploration of pathogen components that trigger defense responses, searching for natural or induced genetic variation in such recognition capacity, cloning the corresponding receptor, and inter-generic transfer. Sequence capture targeted to RLKs and RLPs could enhance the efficiency of identification of novel PRR genes. Development of methods to engineer effector-insensitivity into PRR response pathways that are disrupted by pathogen effectors is an additional opportunity.In addition to canonical plant immune receptors such as NLRs and PRRs, genes encoding other types of resistance are important for adding diversity and potential durability to resistance. One source of useful genes will be quantitative disease resistance loci. QDR determine host resistance that results in a reduction, but not complete absence of disease. QDR can be controlled by quantitative variation in NLR or PRR activation or by completely different mechanisms . QDR is frequently controlled by multiple quantitative trait loci that interact with each other and are influenced by the environment . Some QTL may encode modifiers that enhance immunity; others may encode genes that are not components of the immune system. Emerging opportunities for engineering enhanced resistance includes a better understanding of the mechanisms underlying QDR, including the role of chloroplasts and other organelles in plant defense. Genes have been identified that confer partial resistance to multiple diseases, including several rust species, and even to broad ranges of pathogens .

Pyramiding multiple QDR loci, either through marker-assisted breeding or the 8 / Molecular Plant-Microbe Interactions application of genomic selection, can provide broad spectrum resistance; for example, four QDR loci, each controlling a different aspect of resistance to the blast fungus, have been pyramided in rice . Natural variability at QDR loci can be identified using classical genetic approaches, pathogen phenotyping, and analyzing molecular markers of defense. Characterization of QDR loci can determine at which step during infection resistance is acting and if weak activation of classical defense signaling is induced. Transfer of existing, evolutionarily unique resistance mechanisms to other plant species is likely to be feasible in many instances. Pyramiding multiple sources of QDR with canonical immune receptor loci is a desirable strategy to achieve durable resistance. There is great interest in the identification of plant susceptibility genes that facilitate pathogen development and their manipulation for durable disease control . S genes that act during different stages of infection and against different pathogens and insects have been identified . Recent advances in genome editing technologies greatly enhance our capacity to manipulate multiple S genes in crops. This approach is exemplified by S genes that control viral replication and translation in their hosts. Potyviruses require the host translation initiation complex including the cap-binding protein eIF4E . Natural variants in eIF4E and eIF4E have been identified in multiple plant species that abolish susceptibility to potyviruses . Importantly, plants possess more than one initiation factor complex isoform; isoforms seem to function redundantly and mutation of one isoform does not affect plant vigor . A natural knockout of eIF4E in Brassica resulted in broad-spectrum potyvirus resistance . CRISPR/Cas9-mediated mutations of eIF4E have been shown to be a viable strategy for engineering resistance to multiple potyviruses in cucumber . Similarly, knockout of eIF4E in tomato provided resistance to two potyviruses; however, plants remained susceptible to other potyvirus strains , indicating further research is needed to understand potyvirus-eIF4E/eIF4E interactions to inform exploitation and development of durable resistance.

However, these pathosystems illustrate the potential of S loci as sources of resistance. The identification of effector targets also provides opportunities for detection and targeting of new plant S genes. Multiple Xanthomonas transcription activator-like effectors enhance the expression of genes encoding SWEET sugar transporters, which are attractive targets for genome editing . The wild type MLO gene in barley suppresses defenses against powdery mildew disease and is conserved across the plant kingdom. Natural and induced loss-of-function mlo alleles have been generated in multiple species using a variety of approaches including radiation and genome editing . However, mutation of MLO can have deleterious physiological consequences requiring analysis over multiple environments and possibly introgression into an appropriate genetic background . Pathogen lifestyle should also be taken into account when targeting S genes and stacking different resistance genes. An R gene against a biotrophic pathogen can function as an S gene during infection by necrotrophic pathogen . Enhancing the foundational understanding of QDR and S genes provides an opportunity to expand our understanding of the mechanisms controlling both resistance and susceptibility. This information can then be translated into effective disease control strategies, especially with the advent of genome editing.Small RNAs are central players of RNA silencing, which is a universal and fundamental mechanism of gene regulation in eukaryotes. Extensive studies have established small RNAs as essential regulators of growth and development; moreover, accumulating evidence implicates small RNAs as having an integral role during plant-pathogen interactions that influences the outcome of pathogen challenge . Specific plant and pathogen small RNAs are activated during infection and there is bi-directional trafficking of silencing RNAs between multiple filamentous pathogens and their hosts . The importance of host small RNA pathways in plant defenses is evidenced by the multiplicity of effectors produced by viral, bacterial and oomycete pathogens that target host RNA silencing pathways . Our understanding of the involvement of small RNAs in pathogen/pest interactions is far from complete; for example, additional foundational studies are needed to address the regulation of immune-related host genes via endogenous microRNAs and small interfering RNAs or other silencing pathways, with potential implications in epigenetics . There is also an urgent need to understand the mechanisms by which small RNAs are transferred from pathogens/pests to host cells and vice versa. As our understanding of small RNA function and evolution advances, the number of novel opportunities to deploy this knowledge to safeguard plant health will increase. Pathogen suppression of host silencing pathways may be mitigated to maintain or enhance endogenous resistance. Host-induced gene silencing and RNA interference are being demonstrated in an increasing number of biotrophic, hemibiotrophic, and necrotrophic interactions . The efficacy of these approaches should be tested in numerous pathosystems, pipp drying racks particularly against insects, pests, and parasitic weeds for which there are currently few alternative control measures. Constitutive ectopic expression of small RNAs can profoundly affect endogenous small RNA profiles with potentially deleterious consequences; research is needed to fine tune approaches such as HIGS. Research is also needed to determine if exogenous application of small RNAs is an efficacious approach to pathogen control and if so, what is the most effective way to deliver small RNAs exogenously. Because HIGS and RNAi can be targeted against vital pathogen/pest processes, they are anticipated to be durable; however, research is necessary to investigate the potential of pathogens and pests to counteract control strategies based on small RNA-centric approaches and to identify optimal targets to reduce the chances of evolution of resistance.

Multiple technological advances can facilitate a greater foundational understanding of small RNAs as well as aid in the deployment of translational approaches to utilize small RNAs for crop improvement. High-resolution imaging will enable investigations of transfer and localization of RNAs, both in vitro and in vivo, at tissue and subcellular levels, throughout the dynamic process of infection. Similarly, sequencing and quantification of small RNA, mRNAs, and small RNA targets in single cells will allow informative dissection of small RNA biology in plant-pathogen/pest interactions. Continued increases in genome sequences of both crops, models, and their pathogens, coupled with detailed molecular and biochemical experiments, will enable studies of the diversity of mechanisms by which plants and pathogens deploy and manipulate small RNA pathways to enhance resistance or avoid disease.These compounds can collectively be considered metabolite immuno modulators. Their characterization could lead to breeding or engineering efforts to enhance plant health; also, some modulators may be useful for direct application to plants either as sprays or soil additives. Both beneficial and pathogenic microbes and pests also produce chemical-based effectors/toxins that might be exploited. Examples of activities of potentially useful metabolites include direct antimicrobials/antipests , signaling intermediates and pathway modulators , secreted compounds that can impact the phyllosphere or rhizosphere microbiomes , and pest/microbe chemical effectors that modulate plant behavior and resistance . To successfully exploit chemical immunomodulators, we need to define the chemical repertoires of plants and interacting organisms under diverse conditions, infer processes impacted by diverse sets of metabolic outputs, identify biosynthetic and regulatory mechanisms, and identify targets and modes of action. Furthermore, plants have diverse chemistries, some of which are family- or species-specific. Therefore, screening broad taxonomic groups is warranted. This will require collaborations with analytical chemists for natural products analysis and synthesis for proof of concept and/or deployment. A finer understanding of the roles of these metabolites will be gained when single cell metabolic analyses are feasible in order to dissect their roles in space and time during the infection process. An important goal will be to identify pathways for synthesis and action using biochemical genetic screens, metabolite-based genetic mapping, and expression based analyses; however, to fully realize the opportunities for identifying metabolites with potential value as control agents, additional assays may need to be developed. Opportunities also exist to generate novel compounds through using combinations of biosynthetic enzymes that may not occur naturally together . 10 / Molecular Plant-Microbe InteractionsAt present, comprehensive metabolite analysis is not routine, especially when mixtures are complex, chemical libraries are limited, and there are many unknown compounds. Investments in national/international repositories for plant/microbe/pest metabolite identification and analysis are needed. Several approaches can be used and combined to identify immunomodulating chemicals. These include exploiting differing chemistries among diverse genetic backgrounds and mutant collections, informaticsled searches for novel predicted enzymes and activities, and bio-assay-based approaches for discovery of new activities . As the sensitivity of instruments for chemical analysis improves and chemical libraries expand, it will become increasingly feasible to survey root exudates, vascular exudates, apoplastic extracts, plant-pathogen interface sampling, secreted molecules from microbes and pests to identify high value metabolites. As new activities and compounds are inferred, partitioning can be used to reduce the complexity of metabolite extracts. For this, it will be important to utilize multiple and complementary methods of extraction, derivatization, separations, and analysis.