Under biotic or abiotic stress, plants produce a large number of reactive oxygen species (ROS), including superoxide anion (O²⁻), hydroxyl radical (OH), hydrogen peroxide (H₂O₂), and so on [71]. The induction of ROS is a significant signaling in control of various processes including immunity against pathogens, programmed cell death, and stomatal closure [72]. In Arabidopsis, the perception of MAMPs leads to a rapid, specific, and strong production of RBOHD-mediated ROS. RBOHD, a plant NADPH oxidase, is mainly controlled by Ca²⁺ via direct binding to EF hand motifs and phosphorylation by Ca²⁺-dependent protein kinases [73,74]. However, the accumulation of ROS also causes tissue cell damage [75]. Therefore, efficient scavenging of ROS by enzymatic and non-enzymatic reactions is necessary. Enzymatic ROS scavenging mechanisms in plants rely on peroxidase (POX), polyphenol oxidase (PPO), superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione peroxidase (GPX), and catalase (CAT), which are essential to the defense against ROS by reducing superoxide to H₂O.

B. cereus AR156 activates plant defense response by inducing the accumulation of hydrogen peroxide and callose in plants and the activation of POD and SOD enzymes, mainly through SA and MAPK signaling pathways [76]. Pseudomonas aeruginosa 7NSK2 produced pyocyanin increases H₂O₂ in both local and distal leaves and induces resistance to blast disease (Magnaporthe grisea) but not sheath blight (Rhizoctonia solani). The opposite effect can be alleviated by co-application of pyocyanin and the antioxidant sodium ascorbate, suggesting that the reactive oxygen species can act as a double-edged sword in resistance against different diseases [56].

Callose is a β-1, 3 glucan polymer that accumulates in weak or compromised sections of plant cell walls under pathogen attack and plays an important role in plant sieve tube metabolism. Its synthesis and decomposition are directly related to the normal growth and metabolism of plants. Aniline blue staining was used to detect callose response to identify particular induced resistance-related genes involved in callose deposition. A study in 2009 illustrated the significance of PEN2 and PEN3 genes for pathogen resistance, required for callose deposition and consequently [77]. MAMPs released by PGPR generate ROS and increase the level of SA. High level of SA triggers callose deposition by regulating the PDLP5-dependent expression of callose synthase gene (CALS10) [78]. Endophytic bacterium Pseudomonas fluorescens strain 63–28 enhanced resistance to Fusarium oxysporum in tomato, through the rapid accumulation of callose and chitinases [79]. Trichoderma harzianum T-203 triggered plant systemic defense responses by increasing peroxidase and chitinase activities and forming barriers of callose [80].

Ion fluxes are immediately induced by elicitors, such as K⁺/H⁺ exchange, Cl⁻ effluxes, and Ca²⁺ influx, which play an important role in cell development and signal transportation, as well as in plant immunity [81]. Among these ion fluxes, Ca²⁺ influx is regarded as one of the most significant events, because of its role of a second messenger for many diverse physiological changes and cellular processes [81]. Some reports show that elicitor-induced Ca²⁺ influx not only mediates subsequent events, but also further amplifies Ca²⁺ signaling through Ca²⁺-dependent production of H₂O₂, which is able to increase Ca²⁺ influx from extracellular sources [82,83]. Pretreatment on bean (Phaseolus vulgaris) with forskolin, dibutyryl cAMP or Ca²⁺ ionophore A23187 enhanced the production of ROS to antagonize Colletotrichum lindemuthianum. In contrast, the Ca²⁺ channel blocker decreased the oxidative burst [84], suggesting that Ca²⁺ influx is required for ROS.

Calmodulin is a ubiquitous Ca²⁺ sensor, which can be activated by Ca²⁺ binding. Ca²⁺ and activated calmodulin further activate Ca²⁺/calmodulin-dependent protein kinase and protein phosphatase, membrane-bound enzymes, or transcription factors [85]. A large kinase family, known as Ca²⁺-dependent protein kinases (CDPK), with essential roles in plant defense responses, is regulated by binding of Ca²⁺. The application and colonization of PGPR, Pseudomonas putida MTCC 5279, activated calcium-dependent signaling by upregulating the expression of calcium-dependent protein kinase (CPK32) [86]. The Ca²⁺ signal can be non-linearly amplified upon binding of Ca²⁺, Ca²⁺ sensor relay proteins, calmodulin-binding transcription activators, and regulated transcription in plants [87]. Besides the functions on ROS, protein kinase cascades further the transfer of lipid signaling messengers and amplification of the elicitor signals to downstream reactions; another significant effect of Ca²⁺ spiking is differential activation of transcription factors, which directly regulate extensive defense gene expression [87,88,89]. A regulatory mechanism linking Ca²⁺ signaling to salicylic acid level is EDS1, an established regulator of salicylic acid level modulated by Ca²⁺/calmodulin-binding transcription factors [90]. The beneficial root-colonizing fungus Mortierella hyalina activated a Ca²⁺-dependent signaling pathway to resist Alternaria brassicae [64]. Cell wall extract of Piriformospora indica, a growth-promoting root endosymbiont, transiently alleviated cytosolic Ca²⁺ in Arabidopsis and tobacco through activating an important Ca²⁺ channel encoded by CYCLIC NUCLEOTIDE GATED CHANNEL 19 (CNGC19) in the mutualistic interaction between beneficial microbe and plant [91,92].

Several crucial transcriptional factors are involved in the regulation network of ISR through JA or/and ET signaling pathway. WRKY transcription factors are implicated in the responses to plant–microbes interactions. The Arabidopsis thaliana WRKY genes are differentially expressed in a time-dependent manner during the plant interaction with beneficial fungus T. atroviride. The expression of positive regulators in JA-mediated pathway, such as AtWRKY8 and AtWRKY33, was more anticipated than the expression of the WRKY genes regulated by SA pathway [67]. WRKY11 and WRKY70 were involved in the regulation of B. cereus strain AR156-triggered ISR in Arabidopsis, through the JA and SA signaling pathways, respectively [93]. MYB family proteins function as transcriptional factors regulating plants development and responses to biotic and abiotic stress [94]. MYB72 was activated upon colonization of P. fluorescens WCS417r and was required in the early signaling steps of beneficial microbe-mediated ISR by acting upstream of ethylene in the signaling pathway [95]. The basic helix-loop-helix (bHLH) transcription factor MYC2 was required for beneficial microbe-triggered ISR, while its function was targeted by pathogens through effector-mediated suppression of host immunity [96]. Ethylene response factor1 (ERF1) encodes a transcription factor that regulates the expression of pathogen response genes that prevent disease progression. The expression of ERF1 can be activated rapidly and synergistically by both JA and ET [97]. There are two branches, the MYC branch and the ERF branch, in the JA signaling pathway responding to wounding stress and necrotrophic pathogen attack, regulated by MYC-type transcriptional regulator and APETALA2/ethylene response factor (AP2/ERF) family of transcriptional regulator, such as ERF1 and ORA59, respectively [98]. Future attempts to unravel more detailed regulatory mechanisms on transcription factors involved in beneficial microorganism-mediated ISR will improve the understanding of the formation and regulation of ISR.

Defense mechanisms of ISR depend on an accurate and context-specific regulation of gene expression. Interactions between genes and their products result in complex circuits and form a regulatory network. Timmermann et al. explored the regulatory mechanism of the ISR defense response triggered by the beneficial bacterium Paraburkholderia phytofirmans PsJN and drew a regulatory network according to gene expression and time series data [99]. The Plant Defensin 1.2 (encoded by PDF1.2; AT5G44420) has previously been proved to accumulate systemically via a SA-independent pathway in leaves of Arabidopsis upon challenge by fungal pathogens and play a role as a marker of the JA signaling pathway [100,101]. As previously mentioned, some SA-dependent PR genes express antimicrobial proteins. Notably, the activation of PR1, PR2, and PR5 depend on SA signaling, whereas PDF1.2, as well as PR3 and PR4 genes, are activated via an SA-independent and JA-dependent pathway [102]. Although it was proposed that PR genes were irrelevant with ISR after certain beneficial microbe treatment [19,103], pretreatment with non-pathogenic B. cereus AR156 triggered expression of PR1, PR2, PR5, and PDF1.2 of Arabidopsis thaliana, which indicated the activation of SA and JA/ET signaling pathways, respectively [21,104,105]. The loss-function mutant of NPR1, an important regulatory factor in the SA-dependent pathway [106,107], was able to express neither ISR nor SAR [103]. Based on the previous research results, NPR1 coordinates SA and JA signaling pathway and regulate downstream defense response genes [108].

Under natural conditions, plants produce a vast array of secondary metabolites, which are critical for plant adaptation to abiotic and/or biotic stresses. Plant secondary metabolites are able to interact with beneficial microbes and modulate plant growth and immune process, and inhibit growth or metabolism of pathogenic microorganisms. PGPR can be recruited by root exudates, which structure a special community of rhizosphere microorganisms and enhance biofilm formation of beneficial microbes [109]. Biochemical evidence showed that plant roots secreted L-malic acid (L-MA) to selectively recruit beneficial rhizobacteria, such as B. subtilis FB17 [110]. Metabolites derived from the tryptophan and phenylpropanoid pathways, such as flavonoids, play roles in plant interactions with beneficial and pathogenic microbes, and these pathways are regulated by nutrient availability [111]. The relative abundance of root-associated Acidobacteria, Gaiellales, Nocardioidaceae, and Thermomonosporaceae in the soil can be affected by the flavonoid (7,40-dihydroxyflavone) excreted from Medicago sativa [112]. Moreover, the flavonoids, such as luteolin, from the leguminous plants can act as growth regulators as well as signaling molecules for Rhizobium bacteria to initiate symbiosis [113]. Plants also release strigolactones that stimulate the branching of hyphae of arbuscular mycorrhizal fungi to establish beneficial symbiosis [114]. Camalexin and glucosinolates are required for the P. fluorescens SS101-induced SA signaling-dependent resistance against Pst [58].

In turn, the secondary metabolites secreted by beneficial microorganisms can directly antagonize pathogenic bacteria and act as immune elicitors to raise ISR [115]. Phenazines produced by beneficial Pseudomonas bacteria showed antifungal activity and were able to elicit ISR [116]. B. cereus AR156 extracellular polysaccharides (EPS) could induce systemic resistance to Pst DC3000 in Arabidopsis [76]. Lipopolysaccharides (LPS), as MAMP molecules, triggered the activation of signal transduction pathways involved in phytohormones SA and JA, and the associated methyl esters and sugar conjugates [117]. Harzianic acid produced by Trichoderma harzianum M10-induced, modulated signaling pathway and differentially expressed genes (DEGs) involving JA/ET- and SA-mediated signaling pathways, and increased reactive oxygen species (ROS) [69]. Microbial volatile compounds (MVCs) have been shown to promote plant growth via improved photosynthesis rates, enhanced immune system, and activated phytohormone signaling pathways [118]. Critical reviews have shown the effects of VOCs on ISR and their interactions with SA, JA/ET, and auxin signaling pathways [119,120,121]. Cyclic lipopeptides surfactin and VOC 2,3-butanediol, produced by Bacillus spp., have been identified as elicitors of ISR [46,122]. These results illustrate the network of interaction between plant and beneficial microorganisms, in which plants generate metabolites to recruit beneficial microbes and inhibit harmful microbes, and beneficial microbes secrete secondary metabolites to enhance resistance of host plants.

Stomata play an important role in plant photosynthesis, respiration, and transpiration. Although stomatal closure decreases gas exchange, resulting in the reduction in photosynthetic activity, this reaction is actually a part of a plant innate immune response to restrict bacterial invasion [123]. Abscisic acid (ABA) plays significant roles in the regulation of stomatal aperture. ABA is produced under stress. The cellular ABA receptors bind to ABA and interact with a group of type 2C protein phosphatases (PP2C) [124,125], inactivating the inhibitory regulatory function of PP2C, but activating SnRK2 protein kinase OST1 [126]. Activated OST1 binds directly to and phosphorylates the anion channel slow anion channel-associated1 (SLAC1), mediating anion release from the guard cells and promoting stomatal closure [127,128,129]. ROS play a key role in ABA-controlled, hyperpolarization-activated Ca²⁺ channels in the plasma membrane of guard cells [130]. The production of H₂O₂ can be catalyzed by OST1 [131,132]. Lipoxygenase encoding gene LOX1, also known as a JA-responsive gene, is expressed in guard cells in response to PAMPs and is required to trigger stomatal defense [133], indicating the JA signaling pathway participates in the regulation of stomatal defense. PGPR B. amyloliquefaciens FZB42 mediates ABA and JA pathways and produce acetoin and 2,3-butanediol to induce stomatal closure in response to biotic stress [45,134,135], which suggests multiple signaling components coordinate in stomatal regulation.