Enhancing crop resilience to combined abiotic and biotic stress through the dissection of physiological and molecular crosstalk

1Plants are sessile and cannot escape stressful conditions originating from the physical environment (abiotic stress) and from interactions with insects and microorganisms such as fungi and bacteria (biotic stress). The on-going change in climate conditions due to mostly anthropogenic causes such as the increase in CO2 emissions exaggerates agricultural land deterioration due to temperature rise. This results in increased evapotranspiration, intensifying drought episodes and increasing soil salinization, augmenting the 7% of the total and 30% of the irrigated agricultural land already affected by salinity. Available data and projections on the effect of climate change on pathogen spread are not conclusive, although the evidence points to increased reproductive potential and geographic expansion that will lead to interactions with both more hosts and different pathogen strains, increasing the chances for the rise of more virulent strains. Therefore, the chances of plants encountering abiotic and/or biotic stress in the future are likely to be higher, with more frequent stress interactions.

2Plants have developed a multitude of defense responses that allow them to adapt, survive and reproduce under stress conditions. With the advancement of ~omics technologies and on-going functional characterizations of individual genes, it has become apparent that environmental adaptation is under tight regulation, which is critical for plant survival. Many components of this regulatory network are involved in responses to different stresses but may function antagonistically or some responses are prioritized over others, compromising plant resistance to multiple stresses simultaneously.

3Major components of the regulatory networks underlying environmental stress adaptation, pathogen recognition, and defense include reactive oxygen species (ROS) signaling, plant hormones, changes in redox status, and inorganic ion fluxes, such as Ca2+. Based on ~omics data analyses these components appear to be at least partly shared between both abiotic and biotic stress signaling, indicating crosstalk and convergence of mechanisms in these pathways and the existence of a general stress response.

4The nature of pathogen perception dictates that physical barriers such as the cuticle, stomata, and cell walls are also critical for timely pathogen recognition and interception. As data generated by ~omics analyses derive from a mixture of different cell types and tissues, these spatially important interactions may be missed and these datasets may lead to erroneous conclusions about components shared and their significance in abiotic and biotic stress crosstalk. Moreover, as combinatorial stress potentially results in novel interactions between signaling components, extrapolation of results from studies with single stress conditions should be done with care.

5Here we will elaborate on the mechanisms involved in adaptation and tolerance to combinatorial abiotic and biotic stress, with a focus on dehydration/salt stress and fungal and bacterial pathogens interaction. This review will particularly emphasize interactions that potentially arise during the pathogenesis timeline and were as yet given little attention. We will discuss molecular components with potentially critical roles in abiotic and biotic stress tolerance crosstalk, and propose breeding approaches toward effective crop improvement against combinatorial stress.

6Studies on the commonly occurring combination of drought and heat stress have revealed that physiological and molecular responses of plants exposed to both stresses are markedly different from their response to the individual stresses. Similarly, there are numerous reports about abiotic stress (mostly drought and salinity) affecting pathogen resistance, which is indicative of interaction between abiotic and biotic stress. There are reports of disease resistance attenuation by high humidity and high temperature. In most cases, abiotic stress predisposes plants to subsequent pathogen infection, although positive effects on resistance to foliar pathogens have also been reported.

7There is evidence that different levels of abiotic stress have a significantly different impact on disease susceptibility. Salinity stress, in particular, exerts its damaging effect through both osmotic effects and ion toxicity resulting from ion accumulation (mainly Na+ and Cl-). As NaCl is an antifungal agent it could potentially exert a direct toxic effect on fungal growth after accumulation inside the plants (Figure 1). In line with this argument are the many examples of reduction of fungal pathogenicity by metal accumulation, and a similar trend is observed for NaCl accumulation. Therefore salt stress–pathogen interactions may be highly influenced by stress intensity, which affects the degree of accumulation of salt in the plant. The different tolerance strategies of the host against ion toxicity (ion exclusion at the roots and/or ion compartmentalization in the above ground organs inside the vacuoles) can impact on the outcome of plant–pathogen interactions under salt stress. Therefore, it appears that the outcome of the interaction in most occasions is plant, genotype, pathogen, and stress intensity dependent. Moreover abiotic stress, except for potentially dampening or strengthening signaling responses for pathogen defense deployment, could create more or less favorable conditions for pathogen growth by additionally influencing the physiological status of the plant such as water and ion content.

8Vice versa, plant responses to abiotic stress can be affected by prior interactions with pathogenic fungi. Pathogen infection has been shown to reduce photosynthesis and water use efficiency (WUE) and induce abnormal stomata opening patterns, and all of these are critical for plant tolerance to abiotic stress. Salicylic acid (SA) signaling, induced after infection with biotrophic fungi, can attenuate abscisic acid (ABA) signaling that is orchestrating plant adaptive responses to abiotic stress. Infection by a root pathogen was shown to increase shoot Na+ and Cl- content under saline conditions in Phaseolus vulgaris (; Figure 1). Finally genetically heightened resistance to pathogens is often accompanied by a fitness cost that may generally affect the plant performance under both abiotic stress and stress-free conditions.

9A direct interaction of pathogen virulence factors with stress tolerance components of the plant host was demonstrated for the Pseudomonas syringae type III effector HopAM1 that targets HSP70 involved in heat tolerance and stomata closure under stress. Overexpression of HopAM1 in Arabidopsis thaliana results in increased sensitivity to ABA and salt stress, providing proof of direct manipulation of abiotic stress signaling components.

10Interaction of plants with microorganisms can also be beneficial to abiotic tress tolerance. For instance, infection of plants with RNA viruses improved tolerance to drought. Infection with the vascular pathogen Verticillium spp. increased Arabidopsis thaliana drought tolerance due to de novo xylem formation, which enhances water flow. Symbiosis with fungal endophytes as well as association of plant roots with non-pathogenic rhizobacteria and mycorrhizal fungi increases plant vigor under stress conditions through, among others, interactions with hormonal pathways and the sustainment of water and source-sink relations. Remarkably, rhizobacteria colonization is also shown to enhance plant resistance to fungal pathogens and insects, via systemic signaling that triggers immunity (induced systemic resistance, ISR; ).

11Further evidence for abiotic and biotic stress resistance crosstalk comes from studies of the effects of exogenous application of chemicals that sensitize plant defense responses, a phenomenon called priming. For example, application in Arabidopsis thaliana of β-aminobutyric acid (β-ABA), a non-protein amino acid, results in enhanced resistance to a wide range of stresses including heat, drought, and salinity stress, as well as enhanced resistance to biotrophic as well as necrotrophic fungi. Exogenous application of SA renders many crop plants more tolerant to an extensive array of abiotic stresses, and similar observations have also been reported after treatment with jasmonates.

12Evidence for regulatory crosstalk between abiotic and biotic stress response at the molecular level comes mostly from observations of expression patterns of genes under independent imposition of the single stress conditions. In Arabidopsis thaliana, a significant number of genes up-regulated by salinity stress are also induced in response to biotic stresses. Whole genome expression meta-analysis experiments under different abiotic and biotic stress treatments revealed a significant number of genes that are commonly regulated under abiotic and biotic stress conditions. Functional categories enriched in the 197 commonly regulated genes identified by include response to ABA, SA, jasmonic acid (JA), and ethylene (ET), major stress hormones controlling adaptation to abiotic and biotic stress. Several members of signaling pathways involving mitogen-activated protein kinase (MAPK), Ca2+, ROS, phospholipids, mitochondrial functions, vesicle trafficking, and apoptosis were induced under biotic as well as abiotic stresses. Transcription factors (TFs) appear to be major orchestrators of stress crosstalk with members of WRKY, MYB, ERF, NAC, and HSF displaying similar induction patterns across stress treatments. On the other hand, another study using co-expression data to identify cis-regulatory elements (CREs) of stress responses identified distinct CREs for the response to abiotic and biotic stressors. In addition, a number of CREs identified for both types of stress appear to oppositely regulate the expression of downstream genes in response to abiotic or biotic stress.

13A different approach, yeast two-hybrid assays targeting major regulators of rice abiotic and biotic stress response, identified proteins that are present in multiple interactomes. These include OsMPK5, the wall-associated kinase 25 (WAK25), sucrose non-fermenting-1-related protein kinase-1 (SnRK1), and WRKY family TFs.

14Recently, examination of the transcriptional response of different Arabidopsis thaliana accessions to combinations of abiotic and biotic stressors revealed that across the treatments on average 60% of expression changes under combinatorial stress could not be predicted by the changes in response to the individual stresses. The functional categories enriched in the affected genes were similar to those discovered after transcriptome meta-analyses of individual stressors, i.e., stress hormone responses, ROS, and MAPK signaling and regulation of hypersensitivity response. The response of many of these transcripts was canceled or prioritized under stress combination in comparison with the individual stress pointing to potential antagonistic interactions with detrimental effects on plant adaptation under combinatorial stress. In a similar study, the increased susceptibility to a virus after simultaneous application of drought and heat stress was accompanied by down regulation of pathogenesis-related (PR) genes and R-genes, which were otherwise induced under single viral stress. This indicates a direct negative effect of abiotic stress on major defense executors, that adds up to the antagonistic regulation observed in other signaling pathways. These studies clearly emphasize that even though regulatory pathways overlap between stresses, combinatorial stress needs to be treated and studied as a unique condition. Further functional characterization of individual gene members playing key roles in these pathways is required to extract meaningful conclusions.

15As mentioned above, abiotic and biotic stress interactions can occur at multiple levels, depending on the type of the stress (osmotic, ionic), the lifestyle, and infection strategy of the pathogen (biotroph/necrotroph, infection by direct penetration/through stomata, etc.) as well as the pathogenesis stage. We will summarize molecular components that according to evidence mentioned above participate in stress crosstalk. We will follow the pathogenesis timeline highlighting first extracellular interactions taking place at the epidermis and the apoplast during the initial stages of pathogenesis and moving on to the interactions in the intracellular environment during pathogen colonization (Figure 2). As information under combined stress is limited, and a detailed coverage of all potential interactions is not possible, our intention is to provide leads for future research that will aid to further dissect plant adaptive responses and tolerance under combined abiotic and biotic stress.

16The cuticle and cell wall constitute the first layers of defense against microbial attackers. They not only serve as physical barriers against pathogen penetration, but also as sensitive sensors for the timely activation of the intracellular and systemic defense responses. Arabidopsis thaliana mutants in long-chain acyl-CoA synthetase 2 (LACS2), a gene that is involved in cuticle biosynthesis, exhibit increased permeability of the cuticular layer which leads to increased resistance to Botrytis cinerea. Interestingly, ABA deficiency causes similar cuticular defects and heightened resistance to B. cinerea through faster induction of defense responses and H2O2 production both in Arabidopsis and tomato, indicating a link between abiotic stress signaling, cuticle structure and defense responses. In the study by, however, lacs2 Arabidopsis mutants show no alteration in the resistance against the necrotroph Alternaria brassicicola and biotrophs, and even increased susceptibility against P. syringae. The latter observation points to a positive contribution of a thicker cuticle to resistance against P. syringae, indicating that the effects may be pathogen-specific. The well-documented increase in cuticular thickness under conditions of water deficiency may thus result in alteration in the deployment of the pathogen defense response. The cuticle does appear to be a sensor of the osmotic status and to be essential for the up-regulation of ABA biosynthesis genes under osmotic stress through a yet not clearly defined mechanism; cuticle disruption by pathogens may therefore affect osmotic stress acclimation.

17Cell walls similarly appear to be an integrated signaling component for the defense against pathogens. Changes in pectin properties and composition in the Arabidopsis powdery mildew-resistant (pmr) mutants pmr5 and pmr6 resulted in a SA, JA, and ET independent increase in resistance to powdery mildew species. Cellulose deficiency caused either by non-functional cellulose synthase genes or by chemical treatment enhances the synthesis of the defense hormones SA, JA, and ET and signaling and results in increased resistance to pathogens. Intriguingly, these responses were attenuated when plants were grown under high osmotic pressure which reduced the turgor pressure, suggesting that the defense response may be initiated by sensing the increased turgor pressure as a result of cell wall weakening. Osmotic stress, which is a common component of many abiotic stresses, may therefore interfere with the ability of plants to sense damage to the cell wall, due to already reduced turgor, resulting in inadequate activation of defense mechanisms.

18The above-mentioned alterations in plant pathogen interactions in cell wall component biosynthesis mutants may be the consequence of the erroneous activation of integral receptor proteins such as RLKs and RLPs (receptor-like kinases and receptor-like proteins, respectively) which survey the cell wall integrity and bind to MAMPs and DAMPs (microbial- and damage-associated molecular patterns, respectively). Upon activation these transmembrane proteins (e.g., the RLK family WAK), send signals for the elicitation of downstream defense responses. Changes of cell wall structure and adherence to the plasma membrane upon exposure to abiotic stresses may affect their functional integrity. This is emphasized by the observation that NDR1, an essential component of disease resistance mediated by CC-NB-LRR genes, is functioning in cell wall-plasma membrane adhesion. Down-regulation of NDR1 resulted in alterations in the cell wall-plasma membrane interaction and compromised resistance to virulent P. syringae. Abiotic stress may also affect the abundance of cell wall receptors by influencing their transcript levels. THE1 is a member of the CrRLK1L RLK family that is involved in cell wall damage sensing and subsequent control of the downstream accumulation of ROS, and its expression is down-regulated under abiotic stress but up-regulated after pathogen challenge, while similar expression patterns are observed for the WAK gene family.

19Pathogen recognition activates a battery of defense responses that target the apoplastic space. These include local cell wall enforcement, secretion of antifungal compounds at the site of intended penetration and up-regulation of enzymes with fungal cell wall degrading activities. These events are characterized and regulated by signature changes in pH, ROS homeostasis, and the redox state. Simultaneous exposure to abiotic stress can potentially impinge on the generation and decoding of these signatures, affecting subsequent responses. For example, apoplastic pH is transiently decreased following fungal infection, while an increase in pH is observed under salt stress. Moreover the downregulation of cell wall peroxidases under abiotic stress can potentially dampen the production of ROS signatures that trigger defense responses. Physical barriers enforcement after pathogen encounter through crosslinking of lignin monomers by ROS, which are produced by apoplastic peroxidases, NADPH oxidases and germin-like proteins, prevent pathogen penetration. Lignin content was found to be reduced under mild drought conditions to facilitate the maintenance of growth under conditions of decreased turgor pressure, but severe stress resulted in increased lignin content. These findings may provide insight on the mechanisms leading to differential responses under combined stress across different abiotic stress intensities.

20Another form of inducible defense response at the site of penetration is the formation of papillae that contain callose, antimicrobial secondary metabolites such as phenolic compounds, and ROS. Antimicrobial compounds are accumulating through vesicles originating from cellular compartments, such as the Golgi apparatus, which become polarized toward the site of infection. The significance of vesicle-mediated secretion in plant immunity has been demonstrated by the discovery of mutants defective in exocytosis of vesicles (with mutations in SNARE complex proteins HvROR2 and AtPEN1), which display diminished penetration resistance to powdery mildew pathogens. Vesicular trafficking appears to be rewired in an opposite way under salt stress, as vesicles containing Na+ are fused with the central vacuole to maximize compartmentalization of Na+. Interestingly, knockout of different SNARE proteins resulted in increased salt tolerance, indicating possible antagonistic interactions of salt stress and pathogen infection at the level of vesicle trafficking, although further comprehensive experiments are required to substantiate this hypothesis.

21Callose is a β-1,3-glucan polymer that is deposited at the sites of attempted fungal penetration in the form of papillae. It is an important inducible defense mechanism, with enhanced deposition being observed after exogenous application of priming chemicals like β-ABA. A mutant screen for plants defective in β-ABA-induced priming identified among others mutants in the ABA biosynthesis gene zeaxanthin epoxidase (ABA1; ). These mutants failed to exhibit both β-ABA-induced callose deposition against H. parasitica and increased tolerance to salt stress, thereby providing a link between the induction of abiotic and biotic stress responses by β-ABA. In accordance with these observations the callose-mediated increased resistance of the ocp3 Arabidopsis mutant to necrotrophic pathogens requires ABA. Moreover ocp3 mutants accumulate higher levels of ABA, and are more drought tolerant. Therefore ocp3, a homeodomain TF, appears to be a convergence point for ABA and callose regulation that can be manipulated to enhance resistance under combinatorial stress.

22Callose accumulation appears to be a point of convergence of abiotic and biotic signaling as variability in environmental conditions, which affect the redox state of the plant, such as light intensity, have a significant impact on the magnitude of callose deposition after pathogen elicitation. As callose deposition is a major component of the pre-invasion defense of plants, detailed characterization of the regulation of callose accumulation under simultaneous abiotic stress may be invaluable in building combined stress tolerance in crops.

23Changes in calcium fluxes and production of ROS are among the earliest plant responses to abiotic stress and pathogen challenge. The decoding of both signals relies on “signature” spatiotemporal patterns and oscillations specific to the stress encountered. Moreover, both components are highly interconnected: Ca2+ signaling components such as calmodulins (CaMs) and calcium-dependent protein kinases (CDPKs) regulate ROS production by NADPH-oxidases. ROS vice versa affect Ca2+ signaling through regulation of Ca2+ permeable channels. It is plausible that there are either unique signatures for combinations of stresses, or that there is interference between the abovementioned signals that potentially dampens or strengthens the downstream responses.

24Whole genome expression analyses coupled with promoter motif identification provided further evidence that Ca2+ orchestrates the early responses to both biotic and abiotic stress as the overrepresented motif “CGCGTT” identified in the promoters of the commonly regulated genes, contains the core “CGCG” Ca2+ responsive cis-element. The investigation of mutants defective in the induction of a hypersensitive response after pathogen infection has led to the identification of genes encoding for cyclic nucleotide gated channels (CNGCs) which are non-selective cation transporters. members of which are also involved in salt and heat stress tolerance through regulation of Ca2+ fluxes. Furthermore, Ca2+ downstream signaling components have been shown to mediate responses to both abiotic and biotic stress stimuli. The CAMTA3 TF is important for cold acclimation of Arabidopsis by stimulating the expression of CBF1, CBF2, and ZAT12 that are also involved in adaptation to dehydration and oxidative stress. Moreover, CAMTA3 negatively regulates SA accumulation and plant defenses through CaM binding. Other proteins interacting with CaM include TF families like NAM, WRKY, and MYB many members of which are involved in abiotic and biotic stress crosstalk.

25CDPKs have a unique feature to both bind calcium and functionally decode the message by target protein phosphorylation. They appear to represent a central node in the regulation of abiotic and biotic stress responses. For example, Arabidopsis CPK4 and CPK11 positively regulate ABA responses and their down-regulation renders plants salt-sensitive, and are important for the oxidative burst and defense responses. In addition, CDPKs regulate ROS production through phosphorylation-mediated regulation of RBOH activity. StCDPK4- and StCDPK5-mediated phosphorylation increases the activity of StRBOHs and the increased ROS production results in a stronger hypersensitivity response after pathogen challenge, favoring resistance against biotrophic pathogens but compromising resistance against necrotrophic fungi. Recently, the CDPK OsCPK12 was shown to increase salt stress tolerance and decrease blast disease resistance in rice through reduced ROS production as a result of down-regulation of RBOH expression, enhanced expression of antioxidant genes such as APX (ascorbate peroxidase), and increased sensitivity to ABA.

26Dissecting the spatiotemporal and molecular specificity of Ca2+ and ROS signaling components is crucial for determining their precise functions in stress responses, as is elegantly demonstrated by the identification of different Ca2+ binding affinities regulating the activation of two soybean CaMs.

27Mitogen-activated protein kinases are centrally positioned in Ca2+–ROS crosstalk and regulation as well as in the signal output after stress exposure. MAPK signaling cascades are relayed through MAPK kinase kinases (MAP3Ks) and MAPK kinases (MAP2Ks). Hydrogen peroxide (H2O2) has been shown to mediate activation of the three major and well-studied Arabidopsis MAPKs, MAPK3, 4, and 6, through MAP3Ks and other kinases. These MAPKs appear to have an overlapping function in signal transduction upon abiotic stress and pathogen challenge. Activation of Arabidopsis MAPK3 and MAPK6 as well as their homologs in tobacco WIPK and SIPK after PAMP recognition is essential for fungal and bacterial resistance. The importance of MAPK3 and MAPK6 in plant immune responses is highlighted by the discovery that the P. syringae effector HopAI1 directly interacts and inactivates both, promoting virulence. Additionally MAPK6 is directly involved in regulating ET biosynthesis in Arabidopsis by activation through phosphorylation of ACS2 and ACS6, which results in an increase in ET biosynthesis. MAPK4 acts as a negative regulator of defense responses and SA accumulation by phosphorylating MEKK2, a MAP3K protein.

28On the other hand, down-regulation of MAPK3 resulted in altered stomata opening patterns in response to ABA and H2O2 in Arabidopsis. Moreover, the ABA-induced expression of AtCAT1, which is involved in H2O2 homeostasis, is controlled by an AtMKK1–AtMAPK6 signaling cascade. Constitutive activation of AtMAPK4 and AtMAPK6 rendered plants more tolerant to cold and salt stress and CAT2 and tAPX, which are involved in H2O2 regulation, appear to be regulated by AtMAPK4. In rice, OsMAPK5 appears to be a convergence point of abiotic and biotic stress responses, as its silencing results in sensitized defense responses and resistance to fungal and bacterial pathogens at the expense of salt and drought tolerance.

29These examples emphasize the complexity of MAPK-mediated defense signaling with diverse and sometimes overlapping functions of different members of the signaling pathway. Downstream targets of MAPK6 overlapped 60% with MAPK3 targets, while a 50% overlap was observed between MAPK3 and MAPK4 targets. Probably, the one-dimensional overlap can be resolved by multidimensional regulation, such as different spatiotemporal transcription and protein subcellular localization, activation thresholds, feedback loops with phosphatases and scaffolding. Many of the above-mentioned components appear to be an integral part of broad stress tolerance priming by exogenous application of chemicals, and the detailed study of MAPK activation, localization, and substrate affinity under these conditions can increase our understanding of plant responses under stress combinations.

30Plant hormones are central to the integration of environmental stimuli in the coordination of growth under optimal and stress conditions, including the regulation of defense responses after pathogen attack. Plant hormones do not act independently, and extensive synergistic or antagonistic interaction between hormonal pathways is observed in development and stress responses after exogenous application, or through mutant analysis. Transcriptomic studies have aided in unveiling these interactions, and it was recently shown that hormonal pathways can be directly connected with each other by protein–protein interactions between their signaling components.

31Abscisic acid is the major hormone that positively contributes to adaptation to osmotic stress, a major component of several abiotic stresses. Its involvement in the regulation of defense responses has been a topic of recent comprehensive reviews. The consensus is that ABA negatively regulates defense responses against both biotrophic and necrotrophic pathogens through negative interactions with SA and JA/ET biosynthesis and signaling; ABA biosynthesis mutations show sensitization of these signaling pathways after pathogen challenge. Comprehensive analyses of ABA-deficient mutants revealed further pleiotropic alterations that may be part of ABA-defense crosstalk such as reduced cuticle thickness and sensitized H2O2 production in response to B. cinerea in tomato and altered cell wall composition in Arabidopsis. Moreover ABA compromised a chemically induced systemic acquired resistance (SAR) through suppression of SA biosynthesis in Arabidopsis, while genetically enhanced ABA catabolism reversed this effect.

32Nevertheless, ABA signaling can positively contribute to pre-invasive defense responses and to early defense signaling against certain necrotrophic pathogens. ABA positively contributes to resistance against pathogens that infect through stomata, such as P. syringae, as well as to other pre-invasion defense mechanisms such as callose deposition.

33Identification of downstream regulatory nodes that channel interactions between hormonal pathways is of great importance in fine-tuning resistance to both abiotic and biotic stress. Besides TFs, which will be discussed in a following section, other regulators of the transcriptional machinery have been uncovered to function in stress crosstalk. RNA chaperones such as RNA helicases are shown to regulate transcription in response to various stressors. MED25, a subunit of the mediator complex which is a component of the transcriptional machinery, is involved in the antagonistic crosstalk between ABA and JA. In a recent report, the Arabidopsis pathogenesis-related protein 2 (PR2), which encodes β-1,3-glucanase involved in callose degradation, was shown to be down regulated in response to ABA, partly elucidating ABA-mediated capacitation of callose deposition. The ahg2-1 mutant in Arabidopsis accumulates both ABA and SA and has increased expression of defense related genes, which is an indication that ABA and SA do not always act antagonistically. Transcriptome analysis of the ahg2-1 mutant revealed complex interactions between ABA and SA signaling involving altered mitochondrial and RNA metabolism, highlighting multilevel connections between the two signaling pathways that add to the complexity and hinder straightforward conclusions.

34Recent research has highlighted the direct involvement of the growth hormones gibberellin, cytokinin, auxin, and brassinosteroid in responses to adverse growth conditions and pathogen attack. For example, GA signaling directly regulates JA signaling, mediated through direct binding of the GA repressor protein DELLA to JAZ proteins and relieving JA signaling repression. DELLA proteins appear to be central nodes in abiotic and biotic stress cross-talk. ABA and ET signaling promote DELLA stabilization which positively affects ROS detoxification (beneficial for acclimation to abiotic stress) through higher expression of ROS detoxification genes. DELLAs also sensitize JA signaling (through binding of DELLAs to JAZ) at the expense of SA signaling, enhancing resistance to necrotrophic pathogens. This may provide an explanation for the often-observed positive correlation between resistance to abiotic stress and resistance to necrotrophs.

35Cytokinins were shown to positively regulate defense responses to biotrophic pathogens via SA accumulation, and increased defense gene expression through interaction of the cytokinin response regulator ARR2 with TGA3, a TF central for defense gene activation. This suggests that the increased cytokinin catabolism observed under abiotic stress-induced senescence may potentially contribute to further down-regulation of SA responses and increased susceptibility to biotrophic pathogens.

36The roles of auxin and brassinosteroids in stress responses and their potential participation in stress crosstalk remains elusive. Auxin signaling shows antagonistic crosstalk with SA, although auxin contributes to reduced senescence which may be of great importance under exposure to a stress combination. Brassinosteroid (BR) signaling positively affects abiotic stress tolerance, as is evident by both BR exogenous application and genetic de-repression of the BR signaling pathway. BR signaling probably interacts synergistically with ABA signaling and stimulates ROS detoxification. BR’s involvement in defense signaling is rather complicated. In tobacco and rice exogenous application of BRs appeared to clearly enhance resistance to a wide range of pathogens. Similar results were obtained in cucumber, which showed heightened resistance to Fusarium oxysporum as a result of activated production of H2O2 by NADPH oxidase and expression of defense related genes. On the contrary BRs appear to be negatively regulating resistance to the root-infecting oomycete Pythium graminicola by antagonizing SA and GA related defense responses. BR signaling shares LRR–RLK and BAK1 proteins with PAMP immune signaling. Contradictory effects of BR signaling on immune responses have been recently reported in Arabidopsis, which require further study.

37It is clear that hormonal crosstalk is extensive and occurs in multiple combinations. Further understanding of plant responses under combined stress exposure is required to dissect the multilevel responses under these conditions. As an example of the underlying complexity, both drought stress and exogenous ABA application result in an increased endogenous ABA content in tomato, but they differentially affect resistance to powdery mildew and Botrytis, with drought enhancing and ABA application compromising resistance. Notably the ABA-deficient tomato mutant sitiens exhibited increased resistance similar to the effect of drought. The complexity of interactions under abiotic stress is further emphasized by transcriptome analyses under abiotic stress in which up-regulation of a significant number of JA/ET-responsive genes and accumulation of their transcripts was observed. Besides the effects of direct hormonal interactions on abiotic and biotic stress tolerance mechanisms additional indirect interactions should be considered, such as the alteration of developmental programs and the regulation of senescence which may be critical for evolutionary species fitness and yield performance in crop plants.

38The cellular redox state is the sum of reducing and oxidizing redox-active molecules and it acts both as a sensor of environmental perturbations (as most of them impose oxidative stress) and as a buffer against these perturbations to maintain cellular homeostasis. It acts as a central integrator of ROS, energy and metabolic regulation under stress as well as optimal conditions. Its major constituents are ascorbate, glutathione (GSH), NADP(H), small proteins acting as antioxidants like thioredoxin and glutaredoxins as well as many diverse metabolites such phenolics, amino acids, carotenoids, and tocopherols. The cellular redox state is dependent on both their accumulation and their reduction-oxidation state. Genetic manipulation of redox homeostasis results in altered hormone homeostasis and responses to pathogens and abiotic stresses, exemplifying its significance. As abiotic and biotic stress commonly impinge on the redox status (albeit not in a similar manner; ), redox homeostasis is potentially a central orchestrator of the phenotypic response to stress combinations. Redox perturbations after imposition of a stress factor may affect responses to subsequent challenges by additional stressors, thereby shaping the response to combined stresses. For example, a transient increase in GSH content drives the antagonistic crosstalk between SA and JA signaling and GSH oxidation appears to drive the induction of both SA and JA pathways.

39Plant hormone signaling can directly perturb the redox status by modifying the expression and activities of antioxidant enzymes. ABA induces the expression of catalase, activating also at the same time the production of the ROS hydrogen peroxide through AtMAPK6 signaling. SA inhibits the function of catalase and cytosolic ascorbate peroxidase (Corina ) and several glutathione transferases.

40Programed cell death (PCD) is a plant response to developmental and environmental stimuli (e.g., in senescence) and pathogen defense (in the form of HR) that is initiated and regulated by redox changes, like an increased oxidation ratio of GSH and ascorbate. APX appears to be central in the redox regulation leading to PCD. Decreased activity of APX isoforms was observed in heat-induced PCD, and overexpression or down-regulation in Arabidopsis of a thAPX increased or decreased, respectively, sensitivity to NO-induced cell death. APX isoforms are also commonly up-regulated under abiotic stress. Considering the important role of APX in the drought–heat stress interaction it is of great interest to explore APX enzyme regulation under combinatorial stress.

41Redox status changes can directly impact protein function through post-translational modifications. One pronounced example of post-translational modifications controlling protein activity and localization is the interplay of S-nitrosylation and thioredoxin-mediated reduction in the control of the oligomeric and monomeric state of NPR1, a master regulator of SA-mediated defense responses and recently proposed as a SA receptor. The function of many more proteins appears to be regulated by S-nitrosylation, among them AtRBOHD as mentioned above, SA binding protein 3 (SABP3), methionine adenosyltransferase 1, the metabolic enzymes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and glycine decarboxylase (GDC), as well as metacaspase 9. Identification of the dynamics of post-translational modifications on these and newly identified proteins under various stress combinations will shed light on their significance for plant adaptation responses to these conditions.

42NO was recently found to exhibit biphasic control over cell death triggered by pathogens and pro-oxidants in Arabidopsis. In initial stages S-nitrosothiol (SNO) accumulation results in enhanced and accelerated cell death. However, constitutively high SNO levels decreased cell death through S-nitrosylation-mediated reduction in AtRBOHD activity. This differential regulation might have implications in conditions of combined abiotic and biotic stress as both result in increased NO levels. At a certain plateau concentration of NO, signaling components may be desensitized or inversely regulated, as exemplified by AtRBOHD, with detrimental effects on stress acclimation.

43Redox changes and post-translational modification appear to be integral in priming for stress tolerance after exogenous application of chemicals. This provides a potential explanation of the mechanism of action of diverse chemicals in plant defense sensitization. H2O2 and NO priming for salt tolerance in citrus moderately increased the abundance of oxidized and S-nitrosylated proteins, which then remained relatively similar after the application of stress. Non-treated plants were more stress sensitive and exhibited increased protein carbonylation and oxidation. As both compounds provide increased tolerance to both abiotic and biotic stress, further characterization including the timing and magnitude of these post-translational modifications under different stress treatments and under stress combination may help to better understand the redox changes leading to stress cross-tolerance.

44Metabolites are the end products of gene expression and protein activities and therefore are the penultimate regulatory component for the phenotypic expression under stress conditions. As metabolites can have multiple functions such as being energy carriers, structural molecules and redox regulators or exerting direct antimicrobial activity against pathogens, uncovering their regulation and homeostasis under combined stress is of great significance.

45Adaptation to both abiotic and biotic stress impinges significantly on primary metabolism homeostasis. Synthesis of antimicrobial metabolites and defense proteins is energy demanding, while abiotic stress potentially leads to energy deprivation as photosynthesis is reduced under abiotic stress. As a result, it is fair to assume that under stress combinations these strong antagonistic effects will result in disturbed energy balance. However, recent results challenge the carbohydrate deprivation notion under mild dehydration stress and further experimental data under combined stress are required for firm conclusions. More evidence that sugar homeostasis and signaling drives defense responses are demonstrated by the down regulation of cell wall invertases. This results in dampening of defense responses and increased susceptibility to pathogens as a result of decreased availability of carbohydrates to fuel the defense responses at the site of infection. Cell wall invertases appear to be down regulated under abiotic stress and as the regulation of their activity is a convergence point of hormonal and sugar signals for stress tolerance and senescence progression, fine tuning of their expression might be a focal point in enhancing combined stress tolerance. The metabolic status of the host is also crucial for pathogen growth as it appears that pathogens manipulate different aspects of plant metabolism to achieve optimal conditions for their growth.

46The significance of amino acid homeostasis for the induction and regulation of defense responses was recently highlighted. Amino acids may function as precursors in hormone biosynthesis and affect the redox state through their chemical properties or as precursors of redox regulators such as GSH. Amino acid abundance can impact hormone signaling through conjugation-mediated regulation of hormone activity. Amino acid concentration appears to be significantly perturbed by abiotic stress as is revealed by metabolomics studies. On the other hand a direct link between amino acid abundance and activation of SA-induced defense responses was recently demonstrated with heat-shock factor HsfB1, the translation of which is initiated under conditions of phenylalanine starvation. Phenylalanine appears to be accumulated under abiotic stress conditions and its potential as a molecular switch between abiotic and biotic stress responses should be explored.

47Metabolic alterations under abiotic stress include the accumulation of compounds such as the raffinose family oligosaccharides raffinose and galactinol and the amino acid proline. These exhibit osmoprotective and antioxidant functions and have been positively correlated with abiotic stress tolerance. Galactinol overproduction was recently associated with increased resistance to necrotrophic pathogens. Moreover, proline metabolic regulation at the site of pathogen infection is important for both HR deployment and containment, probably through modulation of ROS levels as shown by expression and functional studies of proline dehydrogenase. Myo-inositol metabolic regulation appears to be a convergence point for abiotic and biotic stress responses. Myo-inositol is accumulating under most abiotic stress conditions and is positively contributing to tolerance as a compatible solute. A negative relationship between myo-inositol accumulation and pathogen resistance and PCD initiation was found in Arabidopsis, with a positive correlation between myo-inositol depletion and increased SA production and cell death.

48Analysis of mutants that exhibit qualitative and quantitative alterations in the accumulation of fatty acid metabolites demonstrated that fatty acids are not only structural components of the cellular membranes, but they also exert a multitude of signaling functions. Fatty acid release from the membranes after pathogen encounter triggers the defense response. Linolenic acid (18:3) is a precursor for the production of the major cellular signaling components JA and oxylipins. A reduction of the levels of oleic acid (18:1) triggers constitutive defense responses that are independent of SA signaling, but dependent on NO production. Fatty acid homeostasis is disturbed under abiotic stress, as membrane composition changes are vital for the maintenance of membrane rigidity and functionality. Dehydration stress is shown to result in a reduction in 18:3 and increase in 18:1 lipid levels, and increased 18:3 levels by FAD3 or FAD8 overexpression enhanced drought tolerance in tobacco. Manipulation of fatty acid composition can provide further insight into their function under stress combination.

49Regulatory modules like MAPKs-based pathways and core hormone signaling modules control the expression of a vast number of genes and therefore their manipulation in most cases have severe pleiotropic effects. Identification of downstream regulators involved in abiotic and biotic stress crosstalk such as TFs is important for more targeted manipulation and adaptation of plants to multiple stresses. The appropriate fine-tuning of their expression is an important aspect toward translation of scientific knowledge in crop plant improvement.

50Bioinformatics and functional analyses have demonstrated that TFs involved in stress crosstalk comprise a diverse collection of members of the largest TF families in plants, such as NAC, MYB, AP2/ERF, WRKY, and others, reflecting the complexity of the genetic regulatory networks underlying stress crosstalk. Many members of these families are involved in regulation of leaf senescence, an integral component of both abiotic and biotic stress. Moreover, in most cases the TFs identified are stress hormone-regulated, and therefore potentially act as molecular switches for the fine-tuning of hormonal responses.

51Characterization of the mechanism of action of the candidate TFs involved in stress crosstalk is of great importance. For example, a TF with positive contribution to both abiotic and biotic stress tolerance can be directly useful for breeding combined stress tolerance. Functional characterization of several TFs has revealed various members that confer both abiotic and biotic stress tolerance. Overexpression of the rice OsNAC6 conferred tolerance to salt and dehydration stress as well as resistance to blast disease. Similarly in wheat, overexpression of the R2R3MYB gene TaPIMP1 results in drought stress tolerance and resistance to Bipolaris sorokiniana through increased expression of abiotic stress (many of them ABA inducible) and defense-related genes. Members of the AP2/ERF TF family have been shown to be positive regulators of both abiotic and biotic stress. DREB TFs are also members of the AP2/ERF family and important contributors to abiotic stress tolerance that may have additional signaling functions for biotic stress tolerance. AtDREB2A was upregulated in plants overexpressing the CC-NB-LRR gene ADR1 which conferred pathogen resistance and drought tolerance. Overexpression of OsDREB1B in tobacco resulted in increased resistance to abiotic stress and also virus infection.

52Overexpression of AtHSFA1b provided stress hormone independent, but H2O2 signaling dependent increased tolerance to drought and resistance to bacterial and oomycete pathogens. It appears that the HSF TF gene family has broad biological functions in ROS signaling and defense responses and SAR regulation, which can be further exploited for building broad stress tolerance into crops. Whole genome expression meta analyses can provide evidence of potential antagonistic regulation in different stress responses for a given TF, by analyzing expression patterns under different stress conditions. Detailed characterization of spatiotemporal expression and cis-element binding patterns is, however, required for the understanding of the underlying mode of regulation. This was recently elegantly demonstrated in the characterization of OsWRKY13 which exhibits tissue specific expression and condition specific binding to cis-elements of downstream genes and thereby inversely regulated resistance to drought and bacterial infection of rice.

53Functional conservation of TF functions across species can be exploited to take advantage of the wealth of experimental data generated in the model plant Arabidopsis thaliana. For example, the Arabidopsis AtBOS1, an R2R3MYB TF, as well as its homolog in tomato SlAIM1 appear to regulate tolerance to abiotic and biotic stress in the same way, as mutant plants exhibit reduced tolerance to salt stress as well as to Botrytis infection. Further similar efforts should be undertaken to accelerate the translation of experimental observations obtained in model plants species to crops.

54The results obtained by the functional characterization of TFs are encouraging as many of them appear to regulate cross-resistance in a unidirectional manner, in contrast to the observations at the level of hormonal regulation that point to antagonistic relationships. Therefore, their manipulation offers many opportunities to bypass the antagonistic effects on abiotic and biotic stress tolerance observed in the more upstream regulatory nodes.

55Epigenetic modifications such as DNA cytosine methylation and histone residues methylation and acetylation contribute to the transcriptional control of amongst others adaptive responses to environmental stimuli. A significant portion of these modifications appears to be persistent across generations and significantly contributes to phenotypic variation. While cytosine methylation generally has repressive effects on gene transcription, leading to gene silencing, histone modifications can lead to transcriptional activation through local chromatin de-condensation which facilitates the accessibility of TFs. Recently, epigenetic modifications and specifically chromatin-regulated gene activation have been proposed to govern priming responses. Genome wide approaches studying DNA methylation under abiotic and biotic stress have demonstrated widespread methylation alterations. It would be of particular interest to further examine the occurrence of differential alterations and their impact under combinatorial stress.

56Functional studies of chromatin remodeling enzymes have revealed a functional involvement of these enzymes in the regulation of both abiotic and biotic stress responses. Histone deacetylase 19 (HDA19) mutants exhibit enhanced basal expression of many SA-responsive genes but decreased expression of ABA and JA/ET-responsive genes, and the mutants are hypersensitive to salt stress. The histone lysine methyltransferase ATX1 is likely to be involved in dehydration stress signaling, as atx1 mutants were sensitive to drought and ATX1 methyltransferase activity positively regulated the expression of the ABA biosynthesis enzyme NCED3. Interestingly, down-regulation of the TF WRKY70 during dehydration stress coincided with decreased presence of ATX1 at the WRKY70 gene locus.

57Chromatin structure can also be altered by the active deposition of variants of the canonical histones. Deposition of one of the these variants, H2A.Z, is linked to transcriptional activation in response to environmental stimuli, and disruption of this mechanism leads to misregulated responses to both pathogens and elevated temperature.

58It would be highly interesting to investigate how a previously imposed stress predisposes plants at the methylation and chromatin level for the encounter of a subsequent stress, (de)sensitizing subsequent responses. This type of acclimation/predisposition may even be a useful tool for preparing seeds and propagated material for stressful environments.

59The plant immune system consists of successive layers counteracting suppression of defense responses by pathogens through secretion of effector proteins. Recognition of the effectors by corresponding R-genes belonging to NB-LRR protein family or the effect of effectors on intracellular host proteins (guarded proteins) results in effector-triggered immunity (ETI). This is usually but not always manifested by localized cell death, termed the hypersensitivity response. The complexity in the regulation of ETI is outlined by network analyses of individual and combined hormone mutants, which revealed compensatory interactions in contrast to synergistic interaction observed in PTI (PAMP-triggered immunity; ), and which may explain the robustness of ETI to genetic perturbations. This robustness may be ideal in building tolerance to combinatorial stress through pyramiding R-genes with genes conferring abiotic stress tolerance.

60However, it is becoming clear that there are multiple aspects of regulation at the NB-LRR protein level that are indispensable for the deployment of R-gene resistance. These include spatial regulation of NB-LRR accumulation in cellular compartments (e.g., the nucleus). Reduction of nuclear NB-LRR accumulation was shown to be responsible for the heat stress attenuation of disease resistance conferred by the proteins SNC1 and RPS4 in Arabidopsis. Interestingly, mutants with reduced sensitivity to heat-induced defense inhibition were found to be based on changes in among others ABA biosynthesis enzymes, indicating that abiotic stress factors may affect R-gene compartmentation through ABA biosynthesis and signaling, although no further evidence is available. In addition, chaperone-mediated transport and folding of NB-LRR protein is important for their activity. The heat shock protein HSP90 is a component of this chaperone machinery. HSP90 is also required for the maintenance of folding of other proteins under stress conditions, and could potentially become limiting for proper R-gene signaling or stress protection under combined stress conditions. The recent discovery that NB-LRR protein accumulation is controlled by microRNAs adds a novel layer of regulation that would be interesting to investigate under different stress conditions.

61Initial pathogen perception and interception through PTI or ETI triggers systemic signals that prime plant defense responses to effectively counter subsequent infection attempts and limit spreading of the disease. This is referred to as SAR. Many compounds and genes have been identified that function in mobile signal generation and transport. Conversion of MeSA produced at the infection site to SA at the systemic tissues appears to be a prerequisite for SAR manifestation. Additional metabolites such as pipecolic acid, dehydroabietinal, azelaic acid, and glyceraldehyde 3-phosphate probably function in the amplification of the signal, with no clear conclusions yet on their precise placement in the SAR circuit pathway. SAR has been shown to be affected by environmental conditions such as exposure to light and abiotic stresses such as salinity, through ABA suppression of SA biosynthesis. The further investigation of the patterns of accumulation and transport of these metabolites under conditions of combined abiotic and biotic stress may reveal potential connections between their regulation and plant phenotypic responses to combined stress.

62In accordance with individual abiotic and biotic stressors, each abiotic stress/pathogen/host combination should be treated independently as, despite the potential universal applicability of some interactions that were characterized in Arabidopsis, many unique interactions may be crucial for the phenotypic response. As a result improving crops to these complex stress conditions first requires an extensive phenotypic characterization at different levels of cellular regulation, i.e., transcription, translation, post-translation, and metabolites, as well as at different stages of plant development. As evidence from research on individual abiotic and biotic stress responses points to a strong dependency on developmental as well as environmental factors, the environmental conditions and developmental stages of the plants should be appropriately defined before any interpretation of the phenotypic and molecular response can be done. Finally the different layers of defense can be differentially affected by abiotic stress imposition (Figure 2); therefore, the outcome of the interaction will vary with the defense mechanisms employed and on the pathogens involved.

63Breeding for resistance to combinatorial stress is challenging. However, various novel approaches can aid in dissecting interactions between various types of stressors and identifying genetic components that can be breeding targets. The combination of different ~omics technologies has enabled the molecular dissection of plant phenotypes. They provide information about the biological function of the whole gene set of an organism, and overlapping expression patterns might imply participation in common pathways, enabling more efficient reverse genetic approaches. Utilization of‘~omics in combination with forward genetic approaches like association mapping may narrow down the candidate genes responsible for the observed phenotypes and provide targets for functional characterization, further manipulation and improvement of crops through breeding. As mentioned previously, currently there are limited studies on the ~omics characterization of combined abiotic and biotic stress tolerance, however, functional characterization of differentially regulated genes is starting to provide interesting candidates for combined stress tolerance and their mode of action.

64Manipulations that induce resistance to abiotic and biotic stress such as application of priming chemicals, followed by comprehensive phenotypic characterization can be used for candidate gene identification and molecular processes underlying stress cross-tolerance. Utilization of pre-existing chemical libraries for compounds that can prime abiotic and/or biotic stress tolerance and identification of their mode of action through chemical genetics approaches can both provide biotechnological targets for crop stress improvement and an opportunity to directly use the identified chemical in agricultural practice if no unintended side effects are observed. Moreover as the effects of chemical priming are shown to, in part, be exerted through induction of phosphorylation and other post-translational modifications, probing these modifications and genetically manipulating the underlying codons to constitutively mimic them can result in altered responses under combinatorial stress.

65Breeding for resistance to exposure to combined abiotic and biotic stress by incorporation of genetic components regulating the response to both stresses faces various challenges. For example, TFs can have thousands of binding sites across the genome, increasing the chance of unwanted pleiotropic effects and therefore more sophisticated deployment should be employed. Both expression regulation and binding specificity can be altered through promoter and binding domain engineering which can be aided by comparative genomic approaches and applied through novel site-specific mutagenesis techniques. As selective and stimulus specific TF binding drives stress responses regulation, implementation of the above methods will aid to fine-tune downstream targets toward the desired phenotypic response. A potential drawback of TF utilization is that resistance typically achieved by this approach is partial, and potentially prone to numerous antagonistic effects between stresses that cannot be predicted and can hinder efficient deployment for crop improvement to combined stresses.

66Pyramiding genes that provide increased tolerance to either stress and do not (negatively) interact with each other offers an alternative route. Strong resistance mediated by R-genes, that appear to be robust to perturbations, can be pyramided with well-characterized genes conferring abiotic stress tolerance (; Kissoudis et al., unpublished data). R-gene robustness can be assessed by testing resistance responses under different abiotic stressors prior to pyramiding. The drawback of this approach is the quick breakdown of resistance due to evolving pathogens, and the fact that necrotrophic fungi resistance cannot be acquired with these genes. R-gene stacking aided by novel biotechnological approaches can reduce the risk of breakdown of R-gene-mediated resistance.

67Pre-invasion defense mechanisms can be exploited, especially the one that is conferred by preformed or inducible physical barriers such as callose and antimicrobial compound deposition at the site of attempted penetration. As discussed earlier, callose deposition appears to be positively regulated by ABA signaling, therefore positive or no interaction should be expected under abiotic stress. Genes such as the OCP3 TF can be utilized, and for instance pyramiding abiotic stress tolerance with resistance conferred by mlo loss of function which sensitizes callose deposition at the site of infection for resistance against powdery mildew may be a viable route (Kissoudis et al. unpublished data). However, pleiotropic effects reported in mlo mutants such as compromised resistance against necrotrophic pathogens and accelerated senescence can have adverse consequences under stress combination.

68The mechanisms through which abiotic stress tolerance is conferred can have a differential effect on disease resistance. As mentioned earlier, drought tolerance through ABA upregulation at the whole-plant level is expected to have antagonistic effects with SA signaling and therefore compromises resistance to biotrophs. Localized ABA sensitization in stomata can overcome these drawbacks and offer an advantage for resistance against pathogen that infect through stomata. Manipulation of developmental traits such as root system architecture can be beneficial for drought tolerance with potentially no adverse effects on disease resistance, as they employ cell type specific signaling. Deployment of genes that have a protective function on proteins and cellular components under abiotic stress, such as dehydrins, LEA proteins or RNA chaperones that apparently are downstream components of abiotic stress adaptation and mostly function through their structural properties, can minimize interaction with biotic stress signaling. Moreover, under salt stress, increased tolerance through Na+ compartmentalization in the vacuoles may offer an advantage in comparison with Na+ exclusion, as Na+ at high concentrations can have adverse effects on pathogen feeding and development.

69Approaches that result in greater antioxidant capacity such as the accumulation of flavonoids appear to confer resistance to abiotic and oxidative stress while overproduction of their derivatives, anthocyanins, increase resistance to the necrotrophic pathogen B. cinerea in tomato by minimizing ROS burst. Therefore engineering for increased flavonoid accumulation can be promising in conferring resistance to multiple stressors, however, it is unknown how it can affect the deployment of hypersensitivity response due to disturbed ROS homeostasis and thus resistance against biotrophic pathogens.

70Exploitation and deployment of different strategies (Figure 3) under different abiotic stress/pathogen combinations will demonstrate their feasibility and applicability, further leading toward the goal of breeding for crops that maintain their robustness and yield performance under diverse environmental conditions.

71The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.